DETAILED CORRESPONDENCE
Status of the Application
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on February 26, 2026 has been entered.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 121-131, 133-139, 141, 143-148, 150, and 151 are pending in the application.
Applicant’s amendment to the claims, filed February 26, 2026, is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s remarks filed February 26, 2026 in response to the final rejection filed August 29, 2025 have been fully considered.
Claims 132, 140, 142, and 149 have been canceled in the amendment filed February 26, 2026 and rejections previously applied to these claims are withdrawn.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Restriction/Election
In response to a requirement for restriction/election filed September 13, 2023, applicant elected without traverse the invention of Group II, corresponding to pending claims 131, 133-139, 141, 143-148, 150, and 151, drawn to a method for forming a product stream having a benzylisoquinoline alkaloid product that is downstream of canadine, Species D4), benzylisoquinoline alkaloid product is noscapine, Species E8), noscapine synthase, Species F7), norcoclaurine synthase, and
Species G4), latex, in the response filed November 13, 2023.
Claims 121-130 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claim 134 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim.
Claims 131, 133, 135-139, 141, 143-148, 150, and 151 are being examined on the merits with claims 131, 135, 137, 141, 144, and 146 being examined only to the extent the claims read on the elected subject matter.
Claim Objections
Claims 131 and 141 are objected to because of the following informalities:
Claim 131 is objected to in the recitation of “(CXE2);,” in line 12 and in the interest of improving claim form, it is suggested that the comma be deleted.
Claims 131 and 141 are objected to in the recitation of “4;-O-desmethyl-3-O-acetylpapveroxine synthase” in line 10 and in the interest of improving claim form, it is suggested that the semicolon be deleted.
Claims 131 and 141 are objected to in the recitation of “narcotinehemiacetal synthase II CXE2” in line 16 and in the interest of improving claim form, it is suggested that the term “narcotinehemiacetal synthase II” be deleted.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 131, 133, 135-139, 141, 143-148, 150, and 151 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claims 131 (claims 133, 135-139, and 150 dependent therefrom) and 141 (claims 143-148, and 151 dependent therefrom) are confusing in the recitation of “wherein the cell increases production of the noscapinoid product within the engineered microbial cell relative to a control cell” in lines 24-25 of claim 131 and lines 23-24 of claim 141. The recitation of “wherein the cell increases production of the noscapinoid product within the engineered microbial cell” is confusing and it is unclear as to the scope of “a control cell.” Applicant may consider an amendment to recite “wherein expression of the heterologous enzymes in the engineered microbial cell increases production of the noscapinoid product relative to an otherwise identical microbial cell that does not express the heterologous enzymes.”
Claim Rejections - 35 USC § 103
Claims 131, 133, 135-139, and 150 are rejected under 35 U.S.C. 103 as being unpatentable over Smolke et al. (U.S. 2008/0176754 A1; cited on the IDS filed on March 14, 2023; hereafter “Smolke”) in view of
Liscombe et al. (J. Biol. Chem. 282:14741-14751; 2007; cited on the IDS filed on March 14, 2023; hereafter “Liscombe”),
Winzer (WO 2013/136057 A2; cited on the IDS filed on March 14, 2023; hereafter “Winzer”),
Facchini (WO 2015/021561 A1; cited on the IDS filed on March 14, 2023; hereafter “Facchini”), and
Dang et al. (Plant Physiology 159:618-631, 2012; cited on Form PTO-892 mailed on January 31, 2025; hereafter “Dang”) and
as evidenced by Li et al. (Nat. Commun. 7:12137, 14 pages, 2016; cited on the IDS filed on March 14, 2023; hereafter “Li”).
This rejection has been modified from its previous version in order to address applicant’s amendments to the claims.
As amended, the claims are drawn to (in relevant part) a method for preparing a noscapinoid product, the method comprising:
culturing an engineered microbial cell that converts canadine to a noscapinoid product within the engineered cell, wherein the noscapinoid product comprises
noscapine, wherein the engineered microbial cell comprises at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2);
wherein the engineered microbial cell comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of narcotoline-4’-O-methyltransferase 1 (MT2) + narcotoline-4’-O-methyltransferase 2 (MT3);
wherein the cell increases production of the noscapinoid product within the engineered microbial cell relative to a control cell and
separating the noscapinoid product from cellular material of the engineered microbial cell (claim 131),
wherein the noscapinoid product is a phthalideisoquinoline product (claim 133),
wherein the engineered microbial cell is a canadine-producing cell comprising a coding sequence for producing norcoclaurine synthase (claim 135),
wherein the engineered microbial cell is selected from the group consisting of bacterial cells and yeast cells (claim 136),
wherein the product stream does not contain more than 5ppm of latex (claim 137),
wherein the noscapinoid product contains at least one portion of a microbial cell (claim 138),
wherein the at least one portion of the microbial cell is present in the noscapinoid product in a detectable amount which is detectable by a method selected from the group consisting of: liquid chromatography-mass spectrometry, mass spectrometry, and spectroscopy (claim 139), and
wherein the engineered microbial cell comprises heterologous enzymes of TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, and SDR1, and wherein the engineered microbial cell comprises at least one of: CXE1 and CXE2 (claim 150).
Regarding claims 131 and 150, the reference of Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]).
Smolke does not teach engineering the bacteria or yeast to add the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1 or CXE2, MT2, and MT3 for the conversion of canadine to noscapine.
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, Facchini, and Dang collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine.
Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
Dang shows that silencing of SOMT2 or SOMT3 (hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131) reduces noscapine content in opium poppy (p. 625, Figure 6C). Dang teaches bacterial recombinant production of MT2 and MT3 (p. 628, column 2, top).
In view of the combined teachings of Smolke, Facchini, Liscombe, Winzer, and Dang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the bacteria or yeast of Smolke’s method to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and MT3 for the conversion of canadine to noscapine within the modified bacteria or yeast. One would have been motivated and would have expected success to so because Smolke teaches a metabolic pathway for the production of canadine and teaches adding additional metabolic steps for the production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine within a bacteria or yeast cell, and Liscombe, Winzer, Facchini and Dang collectively disclose enzymes recited in claims 131 and 150 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + MT3. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests the bacteria or yeast of Smolke modified to co-express enzymes for the conversion of canadine to noscapine, including MT2 and MT3. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing MT2 and MT3 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that MT2 forms a heterodimer with MT3 in the bacteria or yeast of Smolke modified to co-express MT2 and MT3 genes.
Regarding claim 133, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 135, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 136, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (e.g., Facchini at paragraphs [000187] and [000188] and Winzer at p. 8, lines 24-30).
Regarding claim 137, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 138, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 138.
Regarding claim 139, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 131, 133, 135-139, and 150 would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 141, 143-148, and 151 are rejected under 35 U.S.C. 103 as being unpatentable over Smolke in view of Liscombe, Winzer, and Facchini and as evidenced by Li.
This rejection has been modified from its previous version in order to address applicant’s amendments to the claims.
As amended, the claims are drawn to (in relevant part) a method for preparing a noscapinoid product, the method comprising:
culturing an engineered microbial cell that converts canadine to a noscapinoid product within the engineered cell, wherein the noscapinoid product comprises
noscapine, wherein the engineered microbial cell comprises at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2);
wherein the engineered microbial cell comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + P. somniferum 6-O-methyltransferase (Ps6OMT);
wherein the cell increases production of the noscapinoid product within the engineered microbial cell relative to a control cell and
separating the noscapinoid product from cellular material of the engineered microbial cell (claim 141),
wherein the noscapinoid product is a phthalideisoquinoline product (claim 143),
wherein the engineered microbial cell is a canadine-producing cell comprising a coding sequence for producing norcoclaurine synthase (claim 144),
wherein the engineered microbial cell is selected from the group consisting of bacterial cells and yeast cells (claim 145),
wherein the product stream does not contain more than 5ppm of latex (claim 146),
wherein the noscapinoid product contains at least one portion of a microbial cell (claim 147),
wherein the at least one portion of the microbial cell is present in the noscapinoid product in a detectable amount which is detectable by a method selected from the group consisting of: liquid chromatography-mass spectrometry, mass spectrometry, and spectroscopy (claim 148), and
wherein the engineered microbial cell comprises heterologous enzymes of TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, and SDR1, and wherein the engineered microbial cell comprises at least one of: CXE1 and CXE2 (claim 151).
Regarding claims 141 and 151, the reference of Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (NCS) and 6-O-methyltransferase (6OMT) (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]). Figure 12 of Smolke shows that canadine is a downstream metabolite of reticuline and according to Smolke, for production of metabolites beyond reticuline, yeast strains with chromosomal integrations of 6OMT, CNMT, and 4′OMT were used when possible (paragraph [0068]), noting that the P. somniferum 6OMT and CNMT were selected with either the P. somniferum or T. flavum 4′OMT as the best enzyme combinations (paragraph [0067]).
Smolke does not teach engineering the bacteria or yeast to add the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1 or CXE2, and MT2 for the conversion of canadine to noscapine.
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, and Facchini collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine. Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
In view of the combined teachings of Smolke, Liscombe, Winzer, and Facchini, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the bacteria or yeast of Smolke’s method to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, and MT2 for the conversion of canadine to noscapine within the modified bacteria or yeast. One would have been motivated and would have expected success to do so because Smolke teaches a metabolic pathway for the production of canadine using enzymes including Ps6OMT and teaches adding additional metabolic steps for production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine, and Liscombe, Winzer, and Facchini collectively disclose enzymes recited in claims 141 and 151 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + Ps6OMT. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests the bacteria or yeast of Smolke modified to co-express enzymes for the conversion of canadine to noscapine, including Ps6OMT and MT2. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing Ps6OMT and MT2 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that Ps6OMT forms a heterodimer with MT2 in the bacteria or yeast of Smolke modified to co-express Ps6OMT and MT2 genes.
Regarding claim 143, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 144, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 145, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (e.g., Facchini at paragraphs [000187] and [000188] and Winzer at p. 8, lines 24-30).
Regarding claim 146, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 147, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 147.
Regarding claim 148, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 141, 143-148, and 151 would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
RESPONSE TO REMARKS: Beginning at p. 9 of applicant’s remarks, applicant argues the combination of cited prior art fails to teach or suggest every element of the claims for the same reasons presented in the response and declaration filed July 25, 2025.
Applicant’s arguments and declaration filed July 25, 2025 have been fully addressed in the Office action filed August 29, 2025. In the interest of brevity, applicant’s arguments and declaration filed July 25, 2025 will not be further addressed in this Office action unless specifically reiterated in the remarks filed February 26, 2026.
Beginning at p. 10 of applicant’s remarks, applicant argues the combination of cited prior art fails to establish a reasonable expectation of success to practice the claimed methods.
Addressing the reference of Winzer, applicant argues that while Winzer suggests expressing enzymes that convert canadine to noscapine in a microbial cell, Winzer does not teach production within an engineered cell, wherein the engineered microbial cell comprises at least three of the recited heterologous enzymes, and wherein the cell increases production of the noscapinoid product within the engineered microbial cell relative to a control cell.
Addressing the reference Liscombe, applicant argues Liscombe teaches recombinant bacterial expression of TNMT but does not teach production of a noscapinoid product.
Addressing the reference of Facchini, applicant argues that Facchini teaches enzyme activities with purified microsomes from yeast and does not teach production within an engineered cell, wherein the engineered microbial cell comprises at least three of the recited heterologous enzymes, and wherein the cell increases production of the noscapinoid product within the engineered microbial cell relative to a control cell.
Applicant’s arguments are not found persuasive. In this case, applicant's arguments are directed against the references individually, however, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See MPEP 2145.IV.
Also, it appears applicant is requiring absolute predictability to support obviousness. However, according to MPEP 2143.02.II, obviousness does not require absolute predictability, only some degree of predictability is required. For reasons stated above, in view of the combined teachings of the cited prior art, one of ordinary skill in the art would have had at least some degree of predictability to modify the bacteria or yeast of Smolke’s method to include the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, and MT2 as taught by Liscombe, Winzer, and Facchini for the conversion of canadine to noscapine within the modified bacteria or yeast.
At p. 12 of applicant’s remarks, applicant argues the obviousness rationale is based on improper hindsight reasoning.
Applicant’s argument is not found persuasive. It must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. In this case, the obviousness rationale is based only on knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, the obviousness rejections are proper. See MPEP 2145.X.A.
Beginning at p. 12 of applicant’s remarks, applicant argues the cited prior art fails to provide motivation to co-express MT2 and MT3 as recited in claim 131 and fails to provide motivation to co-express MT2 and Ps6OMT as recited in claim 141.
Applicant’s arguments are not found persuasive. Contrary to applicant’s arguments and for the reasons stated above, in view of the combined teachings of Smolke, Facchini, Liscombe, Winzer, and Dang, it would have been obvious to modify the bacteria or yeast of Smolke’s method to express the additional enzymes MT2 and MT3, and in view of the combined teachings of Smolke, Liscombe, Winzer, and Facchini, it would have been obvious to modify the bacteria or yeast of Smolke’s method to express the additional enzyme MT2.
For these reasons, it is the examiner’s position that the claimed invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claim Rejections - Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
U.S. Patent No. 8,975,063 B2
Claims 131, 133, 135-139, and 150 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 27 of U.S. Patent No. 8,975,063 B2 (cited on the IDS filed on March 14, 2023) in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
The difference between claim 27 of the patent and instant claims 131 and 150 is that claim 27 of the patent does not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + MT3.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, Facchini, and Dang collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine.
Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
Dang shows that silencing of SOMT2 or SOMT3 (hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131) reduces noscapine content in opium poppy (p. 625, Figure 6C). Dang teaches bacterial recombinant production of MT2 and MT3 (p. 628, column 2, top).
In view of Smolke, Liscombe, Winzer, Facchini, and Dang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast of the method of the claim of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and MT3 for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success because Smolke teaches a metabolic pathway for the production of canadine and teaches adding additional metabolic steps for the production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine within a bacteria or yeast cell, and Liscombe, Winzer, Facchini and Dang collectively disclose enzymes recited in claims 131 and 150 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + MT3. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests the yeast of the claim of the patent modified to co-express enzymes for the conversion of canadine to noscapine, including MT2 and MT3. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing MT2 and MT3 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that MT2 forms a heterodimer with MT3 in the yeast of the claim of the patent modified to co-express MT2 and MT3 genes.
Regarding instant claim 133, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding instant claim 135, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding instant claim 136, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding instant claim 137, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding instant claim 138, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 138.
Regarding instant claim 139, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 131, 133, 135-139, and 150 of this application are unpatentable over claim 27 of the patent in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
Claims 141, 143-148, and 151 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 27 of U.S. Patent No. 8,975,063 B2 (cited on the IDS filed on March 14, 2023) in view of Smolke, Liscombe, Winzer, and Facchini and as evidenced by Li.
The difference between claim 27 of the patent and instant claims 141 and 151 is that claim 27 of the patent does not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + Ps6OMT.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (NCS) and 6-O-methyltransferase (6OMT) (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]). Figure 12 of Smolke shows that canadine is a downstream metabolite of reticuline and according to Smolke, for production of metabolites beyond reticuline, yeast strains with chromosomal integrations of 6OMT, CNMT, and 4′OMT were used when possible (paragraph [0068]), noting that the P. somniferum 6OMT and CNMT were selected with either the P. somniferum or T. flavum 4′OMT as the best enzyme combinations (paragraph [0067]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, and Facchini collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine. Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
In view of Smolke, Liscombe, Winzer, and Facchini, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast of the method of claim 27 of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and Ps6OMT for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success to do so because Smolke teaches a metabolic pathway for the production of canadine using enzymes including Ps6OMT and teaches adding additional metabolic steps for production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine, and Liscombe, Winzer, and Facchini collectively disclose enzymes recited in claims 141 and 151 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + Ps6OMT. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests modifying the yeast of claim 27 of the patent to co-express enzymes for the conversion of canadine to noscapine, including Ps6OMT and MT2. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing Ps6OMT and MT2 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that Ps6OMT forms a heterodimer with MT2 in the yeast of claim 27 of the patent modified to co-express Ps6OMT and MT2 genes.
Regarding instant claim 143, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding instant claim 144, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding instant claim 145, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding instant claim 146, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding instant claim 147, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 147.
Regarding instant claim 148, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 141, 143-148, and 151 of this application is unpatentable over claim 27 of the patent in view of Smolke, Liscombe, Winzer, and Facchini, and as evidenced by Li.
U.S. Patent No. 9,322,039 B2
Claims 131, 133, 135-139, and 150 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 3 of U.S. Patent No. 9,322,039 B2 (cited on the IDS filed on March 14, 2023) in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
The difference between claims 1 and 3 of the patent and instant claims 131 and 150 is that claims 1 and 3 of the patent does not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + MT3.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, Facchini, and Dang collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine.
Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
Dang shows that silencing of SOMT2 or SOMT3 (hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131) reduces noscapine content in opium poppy (p. 625, Figure 6C). Dang teaches bacterial recombinant production of MT2 and MT3 (p. 628, column 2, top).
In view of Smolke, Liscombe, Winzer, Facchini, and Dang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and MT3 for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success because Smolke teaches a metabolic pathway for the production of canadine and teaches adding additional metabolic steps for the production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine within a bacteria or yeast cell, and Liscombe, Winzer, Facchini and Dang collectively disclose enzymes recited in claims 131 and 150 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + MT3. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests the yeast or bacterial cell of the claims of the patent modified to co-express enzymes for the conversion of canadine to noscapine, including MT2 and MT3. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing MT2 and MT3 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that MT2 forms a heterodimer with MT3 in the yeast or bacterial cell of the claims of the patent modified to co-express MT2 and MT3 genes.
Regarding claim 133, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 135, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 136, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 137, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 138, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 138.
Regarding claim 139, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 131, 133, 135-139, and 150 of this application are unpatentable over claims 1 and 3 of the patent in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
Claims 141, 143-148, and 151 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 3 of U.S. Patent No. 9,322,039 B2 (cited on the IDS filed on March 14, 2023) in view of Smolke, Liscombe, Winzer, and Facchini and as evidenced by Li.
The difference between claims 1 and 3 of the patent and instant claims 141 and 151 is that claims 1 and 3 of the patent do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + Ps6OMT.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (NCS) and 6-O-methyltransferase (6OMT) (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]). Figure 12 of Smolke shows that canadine is a downstream metabolite of reticuline and according to Smolke, for production of metabolites beyond reticuline, yeast strains with chromosomal integrations of 6OMT, CNMT, and 4′OMT were used when possible (paragraph [0068]), noting that the P. somniferum 6OMT and CNMT were selected with either the P. somniferum or T. flavum 4′OMT as the best enzyme combinations (paragraph [0067]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, and Facchini collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine. Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
In view of Smolke, Liscombe, Winzer, and Facchini, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast or bacterial cell of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and Ps6OMT for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success to do so because Smolke teaches a metabolic pathway for the production of canadine using enzymes including Ps6OMT and teaches adding additional metabolic steps for production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine, and Liscombe, Winzer, and Facchini collectively disclose enzymes recited in claims 141 and 151 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + Ps6OMT. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests modifying the yeast or bacterial cell of the claims of the patent to co-express enzymes for the conversion of canadine to noscapine, including Ps6OMT and MT2. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing Ps6OMT and MT2 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that Ps6OMT forms a heterodimer with MT2 in the yeast or bacterial cell of the claims of the patent modified to co-express Ps6OMT and MT2 genes.
Regarding claim 143, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 144, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 145, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 146, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 147, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 147.
Regarding claim 148, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 141, 143-148, and 151 of this application is unpatentable over claims 1 and 3 of the patent in view of Smolke, Liscombe, Winzer, and Facchini, and as evidenced by Li.
U.S. Patent No. 10,240,175 B2
Claims 131, 133, 135-139, and 150 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 2 of U.S. Patent No. 10,240,175 B2 (cited on Form PTO-892 mailed on January 9, 2024) in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
The differences between claims 1 and 2 of the patent and instant claims 131 and 150 is that claims 1 and 2 of the patent do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + MT3.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, Facchini, and Dang collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine.
Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
Dang shows that silencing of SOMT2 or SOMT3 (hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131) reduces noscapine content in opium poppy (p. 625, Figure 6C). Dang teaches bacterial recombinant production of MT2 and MT3 (p. 628, column 2, top).
In view of Smolke, Liscombe, Winzer, Facchini, and Dang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and MT3 for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success because Smolke teaches a metabolic pathway for the production of canadine and teaches adding additional metabolic steps for the production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine within a bacteria or yeast cell, and Liscombe, Winzer, Facchini and Dang collectively disclose enzymes recited in claims 131 and 150 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + MT3. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests the yeast or bacterial cell of the claims of the patent modified to co-express enzymes for the conversion of canadine to noscapine, including MT2 and MT3. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing MT2 and MT3 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that MT2 forms a heterodimer with MT3 in the yeast or bacterial cell of the claims of the patent modified to co-express MT2 and MT3 genes.
Regarding claim 133, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 135, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 136, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 137, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 138, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 138.
Regarding claim 139, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 131, 133, 135-139, and 150 of this application are unpatentable over claims 1 and 2 of the patent in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
Claims 141, 143-148, and 151 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 2 of U.S. Patent No. 10,240,175 B2 (cited on Form PTO-892 mailed on January 9, 2024) in view of Smolke, Liscombe, Winzer, and Facchini and as evidenced by Li.
The difference between claims 1 and 2 of the patent and instant claims 141 and 151 is that claims 1 and 2 of the patent do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + Ps6OMT.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (NCS) and 6-O-methyltransferase (6OMT) (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]). Figure 12 of Smolke shows that canadine is a downstream metabolite of reticuline and according to Smolke, for production of metabolites beyond reticuline, yeast strains with chromosomal integrations of 6OMT, CNMT, and 4′OMT were used when possible (paragraph [0068]), noting that the P. somniferum 6OMT and CNMT were selected with either the P. somniferum or T. flavum 4′OMT as the best enzyme combinations (paragraph [0067]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, and Facchini collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine. Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
In view of Smolke, Liscombe, Winzer, and Facchini, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast or bacterial cell of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and Ps6OMT for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success to do so because Smolke teaches a metabolic pathway for the production of canadine using enzymes including Ps6OMT and teaches adding additional metabolic steps for production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine, and Liscombe, Winzer, and Facchini collectively disclose enzymes recited in claims 141 and 151 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + Ps6OMT. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests modifying the yeast or bacterial cell of the claims of the patent to co-express enzymes for the conversion of canadine to noscapine, including Ps6OMT and MT2. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing Ps6OMT and MT2 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that Ps6OMT forms a heterodimer with MT2 in the yeast or bacterial cell of the claims of the patent modified to co-express Ps6OMT and MT2 genes.
Regarding claim 143, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 144, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 145, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 146, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 147, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 147.
Regarding claim 148, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 141, 143-148, and 151 of this application is unpatentable over claims 1 and 2 of the patent in view of Smolke, Liscombe, Winzer, and Facchini, and as evidenced by Li.
U.S. Patent No. 10,240,176 B2
Claims 131, 133, 135-139, and 150 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 7 of U.S. Patent No. 10,240,176 B2 (cited on Form PTO-892 mailed on January 9, 2024) in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
The differences between claims 1 and 7 of the patent and instant claims 131 and 150 is that claims 1 and 7 of the patent do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + MT3.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, Facchini, and Dang collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine.
Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
Dang shows that silencing of SOMT2 or SOMT3 (hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131) reduces noscapine content in opium poppy (p. 625, Figure 6C). Dang teaches bacterial recombinant production of MT2 and MT3 (p. 628, column 2, top).
In view of Smolke, Liscombe, Winzer, Facchini, and Dang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast or bacterial cell of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and MT3 for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success because Smolke teaches a metabolic pathway for the production of canadine and teaches adding additional metabolic steps for the production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine within a bacteria or yeast cell, and Liscombe, Winzer, Facchini and Dang collectively disclose enzymes recited in claims 131 and 150 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + MT3. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests the yeast or bacterial cell of the claims of the patent modified to co-express enzymes for the conversion of canadine to noscapine, including MT2 and MT3. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing MT2 and MT3 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that MT2 forms a heterodimer with MT3 in the yeast or bacterial cell of the claim of the patent modified to co-express MT2 and MT3 genes.
Regarding claim 133, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 135, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 136, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 137, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 138, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 138.
Regarding claim 139, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 131, 133, 135-139, and 150 of this application is unpatentable over claims 1 and 7 of the patent in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
Claims 141, 143-148, and 151 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 7 of U.S. Patent No. 10,240,176 B2 (cited on Form PTO-892 mailed on January 9, 2024) in view of Smolke, Liscombe, Winzer, and Facchini and as evidenced by Li.
The difference between claims 1 and 7 of the patent and instant claims 141 and 151 is that claims 1 and 7 of the patent do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + Ps6OMT.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (NCS) and 6-O-methyltransferase (6OMT) (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]). Figure 12 of Smolke shows that canadine is a downstream metabolite of reticuline and according to Smolke, for production of metabolites beyond reticuline, yeast strains with chromosomal integrations of 6OMT, CNMT, and 4′OMT were used when possible (paragraph [0068]), noting that the P. somniferum 6OMT and CNMT were selected with either the P. somniferum or T. flavum 4′OMT as the best enzyme combinations (paragraph [0067]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, and Facchini collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine. Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
In view of Smolke, Liscombe, Winzer, and Facchini, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast or bacterial cell of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and Ps6OMT for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success to do so because Smolke teaches a metabolic pathway for the production of canadine using enzymes including Ps6OMT and teaches adding additional metabolic steps for production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine, and Liscombe, Winzer, and Facchini collectively disclose enzymes recited in claims 141 and 151 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + Ps6OMT. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests modifying the yeast or bacterial cell of the claims of the patent to co-express enzymes for the conversion of canadine to noscapine, including Ps6OMT and MT2. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing Ps6OMT and MT2 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that Ps6OMT forms a heterodimer with MT2 in the yeast or bacterial cell of the claims of the patent modified to co-express Ps6OMT and MT2 genes.
Regarding claim 143, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 144, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 145, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 146, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 147, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 147.
Regarding claim 148, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 141, 143-148, and 151 of this application is unpatentable over claims 1 and 7 of the patent in view of Smolke, Liscombe, Winzer, and Facchini, and as evidenced by Li.
U.S. Patent No. 11,124,814 B2
Claims 131, 133, 135-139, and 150 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 24-26 of U.S. Patent No. 11,124,814 B2 (cited on the IDS filed on March 14, 2023) in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
The differences between claims 24-26 of the patent and instant claims 131 and 150 is that claims 24-26 of the patent do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + MT3.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, Facchini, and Dang collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine.
Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
Dang shows that silencing of SOMT2 or SOMT3 (hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131) reduces noscapine content in opium poppy (p. 625, Figure 6C). Dang teaches bacterial recombinant production of MT2 and MT3 (p. 628, column 2, top).
In view of Smolke, Liscombe, Winzer, Facchini, and Dang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast or microbial cell of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and MT3 for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success because Smolke teaches a metabolic pathway for the production of canadine and teaches adding additional metabolic steps for the production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine within a bacteria or yeast cell, and Liscombe, Winzer, Facchini and Dang collectively disclose enzymes recited in claims 131 and 150 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + MT3. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests the yeast or microbial cell of the claim of the patent modified to co-express enzymes for the conversion of canadine to noscapine, including MT2 and MT3. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing MT2 and MT3 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that MT2 forms a heterodimer with MT3 in the yeast or microbial cell of the claims of the patent modified to co-express MT2 and MT3 genes.
Regarding claim 133, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 135, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 136, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 137, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 138, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 138.
Regarding claim 139, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 131, 133, 135-139, and 150 of this application is unpatentable over claims 24-26 of the patent in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
Claims 141, 143-148, and 151 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 24-26 of U.S. Patent No. 11,124,814 B2 (cited on the IDS filed on March 14, 2023) in view of Smolke, Liscombe, Winzer, and Facchini and as evidenced by Li.
The difference between claims 24-26 of the patent and instant claims 141 and 151 is that claims 24-26 of the patent do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + Ps6OMT.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (NCS) and 6-O-methyltransferase (6OMT) (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]). Figure 12 of Smolke shows that canadine is a downstream metabolite of reticuline and according to Smolke, for production of metabolites beyond reticuline, yeast strains with chromosomal integrations of 6OMT, CNMT, and 4′OMT were used when possible (paragraph [0068]), noting that the P. somniferum 6OMT and CNMT were selected with either the P. somniferum or T. flavum 4′OMT as the best enzyme combinations (paragraph [0067]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, and Facchini collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine. Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
In view of Smolke, Liscombe, Winzer, and Facchini, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast or microbial cell of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and Ps6OMT for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success to do so because Smolke teaches a metabolic pathway for the production of canadine using enzymes including Ps6OMT and teaches adding additional metabolic steps for production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine, and Liscombe, Winzer, and Facchini collectively disclose enzymes recited in claims 141 and 151 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + Ps6OMT. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests modifying the yeast or microbial cell of the claims of the patent to co-express enzymes for the conversion of canadine to noscapine, including Ps6OMT and MT2. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing Ps6OMT and MT2 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that Ps6OMT forms a heterodimer with MT2 in the yeast or microbial cell of the claims of the patent modified to co-express Ps6OMT and MT2 genes.
Regarding claim 143, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 144, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 145, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 146, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 147, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 147.
Regarding claim 148, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 141, 143-148, and 151 of this application is unpatentable over claims 24-26 of the patent in view of Smolke, Liscombe, Winzer, and Facchini, and as evidenced by Li.
U.S. Patent No. 11,859,225 B2
Claims 131, 133, 135-139, and 150 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 10 and 11 of U.S. Patent No. 11,859,225 B2 (cited on Form PTO-892 mailed on January 9, 2024) in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
The differences between claims 10 and 11 of the patent and instant claims 131 and 150 is that claims 10 and 11 of the patent do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + MT3.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, Facchini, and Dang collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine.
Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
Dang shows that silencing of SOMT2 or SOMT3 (hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131) reduces noscapine content in opium poppy (p. 625, Figure 6C). Dang teaches bacterial recombinant production of MT2 and MT3 (p. 628, column 2, top).
In view of Smolke, Liscombe, Winzer, Facchini, and Dang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and MT3 for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success because Smolke teaches a metabolic pathway for the production of canadine and teaches adding additional metabolic steps for the production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine within a bacteria or yeast cell, and Liscombe, Winzer, Facchini and Dang collectively disclose enzymes recited in claims 131 and 150 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + MT3. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests the yeast or bacteria cell of the claims of the patent modified to co-express enzymes for the conversion of canadine to noscapine, including MT2 and MT3. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing MT2 and MT3 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that MT2 forms a heterodimer with MT3 in the yeast or bacteria cell of the claims of the patent modified to co-express MT2 and MT3 genes.
Regarding claim 133, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 135, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 136, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 137, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 138, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 138.
Regarding claim 139, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 131, 133, 135-139, and 150 of this application is unpatentable over claims 10 and 11 of the patent in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
Claims 141, 143-148, and 151 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 10 and 11 of U.S. Patent No. 11,859,225 B2 (cited on Form PTO-892 mailed on January 9, 2024) in view of Smolke, Liscombe, Winzer, and Facchini and as evidenced by Li.
The difference between claims 10 and 11 of the patent and instant claims 141 and 151 is that claims 10 and 11 of the patent do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + Ps6OMT.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (NCS) and 6-O-methyltransferase (6OMT) (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]). Figure 12 of Smolke shows that canadine is a downstream metabolite of reticuline and according to Smolke, for production of metabolites beyond reticuline, yeast strains with chromosomal integrations of 6OMT, CNMT, and 4′OMT were used when possible (paragraph [0068]), noting that the P. somniferum 6OMT and CNMT were selected with either the P. somniferum or T. flavum 4′OMT as the best enzyme combinations (paragraph [0067]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, and Facchini collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine. Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
In view of Smolke, Liscombe, Winzer, and Facchini, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast or bacteria cell of the method of the claims of the patent to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and Ps6OMT for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success to do so because Smolke teaches a metabolic pathway for the production of canadine using enzymes including Ps6OMT and teaches adding additional metabolic steps for production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine, and Liscombe, Winzer, and Facchini collectively disclose enzymes recited in claims 141 and 151 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the patent do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + Ps6OMT. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests modifying the yeast or bacteria cell of the claims of the patent to co-express enzymes for the conversion of canadine to noscapine, including Ps6OMT and MT2. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing Ps6OMT and MT2 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that Ps6OMT forms a heterodimer with MT2 in the yeast or bacteria cell of the claims of the patent modified to co-express Ps6OMT and MT2 genes.
Regarding claim 143, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 144, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 145, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 146, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 147, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 147.
Regarding claim 148, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 141, 143-148, and 151 of this application is unpatentable over claims 10 and 11 of the patent in view of Smolke, Liscombe, Winzer, and Facchini, and as evidenced by Li.
Co-Pending Application No. 19/391,825
Claims 131, 133, 135-139, and 150 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 12, 13, and 15 of co-pending application no. 19/391,825 (reference application) in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li.
The differences between claims 12, 13, and 15 of the reference application and instant claims 131 and 150 is that claims 12, 13, and 15 of the reference application do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + MT3.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, Facchini, and Dang collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine.
Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
Dang shows that silencing of SOMT2 or SOMT3 (hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131) reduces noscapine content in opium poppy (p. 625, Figure 6C). Dang teaches bacterial recombinant production of MT2 and MT3 (p. 628, column 2, top).
In view of Smolke, Liscombe, Winzer, Facchini, and Dang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast cell of the claims of the reference application to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and MT3 for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success because Smolke teaches a metabolic pathway for the production of canadine and teaches adding additional metabolic steps for the production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine within a bacteria or yeast cell, and Liscombe, Winzer, Facchini and Dang collectively disclose enzymes recited in claims 131 and 150 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the reference application do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + MT3. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests the yeast of the claims of the reference application modified to co-express enzymes for the conversion of canadine to noscapine, including MT2 and MT3. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing MT2 and MT3 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that MT2 forms a heterodimer with MT3 in the yeast cell of the claims of the reference application modified to co-express MT2 and MT3 genes.
Regarding claim 133, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 135, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 136, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 137, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 138, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 138.
Regarding claim 139, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 131, 133, 135-139, and 150 of this application is unpatentable over claims 12, 13, and 15 of the reference application in view of Smolke, Liscombe, Winzer, Facchini, and Dang and as evidenced by Li. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 141, 143-148, and 151 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 12, 13, and 15 of co-pending application no. 19/391,825 (reference application) in view of Smolke, Liscombe, Winzer, and Facchini and as evidenced by Li.
The difference between claims 12, 13, and 15 of the reference application and instant claims 141 and 151 is that claims 12, 13, and 15 of the reference application do not recite engineering the yeast to convert canadine to noscapine within the yeast by at least three heterologous enzymes selected from the group consisting of tetrahydroprotoberberine N- methyltransferase (TNMT), N-methylcanadine 1-hydroxylase (CYP82Y1), 1-hydroxy-N- methylcanadine 13-hydroxylase (CYP82X2), 1, 13-dihydroxy-N-methylcanadine 13-O acetyl transferase (AT1), 4-O-desmethyl-3-O-acetylpapveroxine synthase (CYP82X1), and noscapine synthase (SDR1), and wherein the engineered microbial cell comprises at least one of narcotinehemiacetal synthase I (CXE1) and narcotinehemiacetal synthase II (CXE2), and comprises at least two heterologous sequences encoding a heterodimeric enzyme, wherein the heterodimeric enzyme is a heterodimeric enzyme of MT2 + Ps6OMT.
Smolke teaches that chemical synthesis of benzylisoquinoline alkaloids (BIAs) is normally a costly and time-consuming process (paragraph [0008]). Smolke teaches that microbial biosynthesis enables green synthesis and the production of these molecules without extreme reaction conditions and toxic waste streams (paragraph [0010]). Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a microbe such that L-tyrosine is converted to a BIA intermediate and the yeast can be engineered by adding additional metabolic steps for conversion of the BIA intermediate to a desired alkaloid end product (paragraph [0009]). Smolke discloses the microbe includes bacteria and yeast (paragraph [0029]). Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase (NCS) and 6-O-methyltransferase (6OMT) (Figures 1, 12, and 16B; Example 7). Smolke teaches integration of the heterologous coding sequences (paragraph [0033]). Figure 12 of Smolke shows that canadine is a downstream metabolite of reticuline and according to Smolke, for production of metabolites beyond reticuline, yeast strains with chromosomal integrations of 6OMT, CNMT, and 4′OMT were used when possible (paragraph [0068]), noting that the P. somniferum 6OMT and CNMT were selected with either the P. somniferum or T. flavum 4′OMT as the best enzyme combinations (paragraph [0067]).
Facchini teaches that noscapine is produced by opium poppy (Papaver somniferum) and may be used as a pharmaceutical agent, including in the treatment of cancer and as a cough suppressant (paragraph [0004]). Facchini teaches that noscapine is currently harvested from opium poppy or may be prepared synthetically, however, such methods suffer from low yields and/or great expense, and acknowledges a desire for biosynthetic production of noscapine (paragraph [0005]). Facchini teaches introducing and expressing noscapine synthesis enzymes in the bacteria or yeast cells (paragraphs [000187] and [000188]) to produce noscapine within the cell (paragraph [00084]).
The references of Liscombe, Winzer, and Facchini collectively teach nucleic acids encoding enzymes of a metabolic pathway for the production of noscapine. Liscombe teaches bacterial recombinant expression of a nucleic acid encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (p. 14743, paragraph bridging columns 1 and 2), noting that TNMT converts canadine to N-methylcanadine (p. 14741, Abstract) and participates in the biosynthesis of noscapine (p. 14750, column 1, top).
Winzer teaches genes encoding enzymes in a pathway that converts canadine to noscapine, including CYP82Y1, CYP82X2, AT1, CYP82X1, CXE1, SDR1, PSMT1, PSMT2, and PSMT3 (p. 19, lines 27 and 28; p. 36, Example 1). In the interest of clarity, PSMT2 and PSMT3 of Winzer are hereafter referred to as MT2 and MT3, respectively, for consistency with claim 131. Winzer teaches bacteria or yeast recombinantly expressing the genes to produce an alkaloid and isolating the alkaloid from the microbial cell (e.g., p. 8, lines 24-30 and claims 30 and 31).
Facchini teaches enzymes for synthesis of noscapine from a canadine derivative including CYP82Y1, CYP82X2, AT1, CYP82X1, OMT, CXE1, and NOS (SDR1) (p. 20, paragraph [00079] and p. 23, paragraph [00086]).
In view of Smolke, Liscombe, Winzer, and Facchini, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast cell of the method of the claims of the reference application to express the additional enzymes TNMT, CYP82Y1, CYP82X2, AT1, CYP82X1, SDR1, CXE1, MT2, and Ps6OMT for the conversion of canadine to noscapine within the modified yeast. One would have been motivated and would have expected success to do so because Smolke teaches a metabolic pathway for the production of canadine using enzymes including Ps6OMT and teaches adding additional metabolic steps for production of a desired alkaloid end product, Facchini acknowledges a desire for biosynthetic production of the alkaloid noscapine, and Liscombe, Winzer, and Facchini collectively disclose enzymes recited in claims 141 and 151 involved in metabolic steps for conversion of canadine to noscapine and their corresponding nucleic acids for recombinant expression.
The claims of the reference application do not recite and the combination of cited references does not teach or suggest a heterodimeric enzyme of MT2 + Ps6OMT. However, according to MPEP 2112, the inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. 103. As noted above, the combination of cited prior art teaches and/or suggests modifying the yeast cell of the claims of the reference application to co-express enzymes for the conversion of canadine to noscapine, including Ps6OMT and MT2. The evidentiary reference of Li is cited as providing extrinsic evidence that a yeast strain co-expressing Ps6OMT and MT2 forms a heterodimer (p. 3, Figure 1; p. 7, column 1, middle and column 2, middle). In view of the extrinsic evidence of Li, it is presumed that Ps6OMT forms a heterodimer with MT2 in the yeast cell of the claims of the reference application modified to co-express Ps6OMT and MT2 genes.
Regarding claim 143, Liscombe teaches noscapine is a phthalideisoquinoline (p. 14749, column 2, bottom).
Regarding claim 144, as noted above, Smolke teaches a metabolic pathway for production of (S)-canadine through the intermediate (S)-reticuline using enzymes including norcoclaurine synthase.
Regarding claim 145, as noted above, Smolke teaches production of BIAs by expressing cloned and synthetic DNAs expressing enzymes of a metabolic pathway in a yeast such that L-tyrosine is converted to a BIA intermediate and each of Facchini and Winzer teaches recombinant expression in yeast (Facchini at paragraph [000207] and Winzer at p. 8, lines 24-30).
Regarding claim 146, since bacteria and yeast do not produce latex and none of the cited references teaches or suggests culturing bacteria or yeast on latex, noscapine or an intermediate thereof produced by the bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine would not contain more than 5 ppm of latex.
Regarding claim 147, given a broadest reasonable interpretation, the “portion” of a microbial cell encompasses any molecule within the recited microbial cell. The bacteria or yeast of Smolke’s method modified to add additional metabolic steps for the conversion of canadine to noscapine is considered to produce noscapine that “contains at least one portion of a microbial cell” as recited by claim 147.
Regarding claim 148, Facchini teaches HPLC for separation of alkaloids (p. 68, lines 11-16) and teaches analyzing reaction product by LC-MS/MS (Example 8 at p. 67).
Therefore, the method of claims 141, 143-148, and 151 of this application is unpatentable over claims 12, 13, and 15 of the reference application in view of Smolke, Liscombe, Winzer, and Facchini, and as evidenced by Li. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
RESPONSE TO REMARKS: Applicant will address the obviousness double patenting rejections, to the extent necessary, upon an indication that the claims are otherwise in condition for allowance. Applicant’s remarks are acknowledged.
Conclusion
Status of the claims:
Claims 121-131, 133-139, 141, 143-148, 150, and 151 are pending.
Claims 121-130 and 134 are withdrawn from further consideration.
Claims 131, 133, 135-139, 141, 143-148, 150, and 151 are rejected.
No claim is in condition for allowance.
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/David Steadman/Primary Examiner, Art Unit 1656
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