DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of Claims
Receipt of Arguments/Remarks filed on 9/2/2025 is acknowledged. Claims 1-5 and 7-20 are pending. Claim 1 was amended. Claim 6 was cancelled. Claims 9-15 and 18-20 are withdrawn as being directed to a nonelected invention. Claims 1-5, 7-8, and 16-17 are being examined on the merits herein.
Withdrawn Rejections
The rejection of claims 2 and 4 under 35 U.S.C. § 112(a) is withdrawn in view of the receipts of deposit for the biological materials and the statement in the response dated 9/2/2025 that the deposits were made under the terms of the Budapest Treaty and that the strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent.
New, maintained, and modified rejections necessitated by amendment
Claim Rejections - 35 USC § 112
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 7 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 7 depends on claim 6, which is now canceled. Therefore, the scope of claim 7 is unclear. Claim 7 should be amended to depend upon an existing claim. For the purposes of examination, claim 7 is interpreted as being dependent upon claim 1, which has been amended to incorporate the limitations of previous claim 6.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 2, and 7-8 are rejected under 35 U.S.C. 103 as being unpatentable over Pedersen (US 11,608,504 B2) in view of Jennewein (US 11,713,475 B2), hereinafter Jennewein ‘475, Jennewein (US 2021/0212335 A1), hereinafter Jennewein ‘335, and Puchart et al., Biotechnology Advances; 33(2):261-76.
Regarding claim 1, Pedersen teaches a bacterial strain, MDO, for oligosaccharide production. This strain is derived from Escherichia coli K12 DH1, which has inactivated gyrA96, recA1, relA1, endA1, thi-1, glnV44, and hsdR17 (Pedersen col. 54 lines 15-25; Table 6). Strain MDO additionally has inactivated lacZ, lacA, nanKETA, melA, wcaJ, and mdoH (Pedersen Table 6).
Pedersen does not teach a strain with inactivated ptsG, manX, manY, or pfkA or an expression vector of a glycoside phosphorylase as recited in claim 1, selected from the group recited in claim 7.
Regarding claim 1, Jennewein ‘475 teaches an E. coli strain for oligosaccharide production with ptsG, pkfA, lacZ, and wcaJ, deleted (Jennewein ‘475 col. 20 Ex. 3).
Regarding claim 1, Jennewein ‘335 teaches an E. coli strain for oligosaccharide production with manXYZ deleted (Jennewein ‘335 para. 47 Ex. 1).
Regarding claims 1 and 7, Puchart teaches that enzymes such as glycoside phosphorylases and glycosyltransferases are used in oligosaccharide production by catalyzing formation of glycosidic bonds (Puchart pg. 263 Fig. 1; pg. 269 “Biotechnological use of glycoside phosphorylases”). Puchart teaches many α- and β-phosphorylases for oligosaccharide production, including α-D-Glucopyranosyl-1,3-L-rhamnose phosphorylase, trehalose phosphorylases, and laminaribose phosphorylases (Puchart pg. 264 Table 1). Puchart teaches that glycoside phosphorylases can be heterologously expressed for industrial applications in microorganisms (Puchart pg. 269 “Biotechnological use of glycoside phosphorylases”). Puchart teaches the use of GPs for high-yield and economic synthesis of oligosaccharides and teaches that engineered GPs are promising efficient and cheap producers of valuable substances, including oligosaccharides (Puchart p. 273 Section 9 para. 1).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of these references to create an E. coli strain with inactivated recA1, gyrA96, thi-1, glnV44, relA1, hsdR17, endA1, lacZ, nanKETA, lacA, melA, wcaJ, mdoH as taught by Pedersen, with additional inactivation of ptsG, manX, manY, and pfkA as taught by Jennewein ‘475 and Jennewein ‘335. All of these references teach genetically engineered E. coli strains for the production of oligosaccharides by inactivating genes involved in oligosaccharide biosynthesis pathways. It would additionally have been obvious to incorporate an expression vector with a glycoside phosphorylase as taught by Puchart. All of the references are directed to bacterial production of oligosaccharides. Given the teachings of Puchart that glycoside phosphorylases are useful in industrial production of oligosaccharides, it would be obvious to combine these teachings with the other references that are directed to bacterial strains engineered for industrial production of oligosaccharides.
A person of ordinary skill in the art would have been motivated to combine the teachings of these references and create a strain with all the recited genes inactivated because strains with all of these genes deleted are associated with production of human milk oligosaccharides (HMOs), which are of significant commercial value (Pedersen col. 1 lines 25-45). HMOs have limited natural availability and efficient large-scale fermentative production is in high demand (Jennewein ‘475 col. 2 lines 5-26). A skilled artisan would have been motivated include an expression vector with a glycoside phosphorylase because production of oligosaccharides such as HMOs by recombinant bacteria has commercial value and Puchart teaches a number of glycoside phosphorylases that can be used in large-scale biotechnological production processes (Pedersen col. 1 lines 25-45 and Puchart pg. 269 “Biotechnological use of glycoside phosphorylases”).
A skilled artisan would have a reasonable expectation of success in combining these teachings to obtain a modified E. coli strain for oligosaccharide production, given the teachings by Pedersen, Jennewein ‘475, and Jennewein ‘435 that deletions of the recited genes yield strains useful for oligosaccharide production; and the teachings of Puchart that glycoside phosphorylases are known in the art to be heterologously expressed in microorganisms and are useful in production of oligosaccharides.
Regarding claim 2, Pedersen, Jennewein ‘475, Jennewein ‘335, and Puchart teach the strain of claim 1. The deposited strain appears to have the same characteristics as the strain of claim 1, which is rendered obvious as set forth above. Therefore, the strain as recited in claim 2 is also obvious in view of the prior art.
Regarding claim 8, Pedersen, Jennewein ‘475, Jennewein ‘335, and Puchart teach the strain of claim 1. Claim 8 recites the intended use limitation “for use for producing oligosaccharides or in a process for producing oligosaccharides”. This intended use does not limit the product structurally, and therefore any composition with this same structure is capable of performing the intended use. However, Pedersen additionally teaches that this strain is used for oligosaccharide, lacto-N-neotetraose/LnNT, production (Pedersen col. 70-71 Ex. 12).
Claims 3, 4, 16, and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Pedersen in view of Jennewein ‘475, Jennewein ‘335, and Puchart as applied to claims 1 and 7-8 above, and further in view of Jennewein (US 2022/0145342 A1), hereinafter Jennewein ‘342, and Lo Leggio et al., Biochemistry; 42(18):5225-35.
Pedersen, Jennewein ‘475, Jennewein ‘335, and Puchart teach the strain of claim 1 as set forth above.
Regarding claims 16 and 17, Puchart teaches that enzymes such as glycoside phosphorylases are used in oligosaccharide production by catalyzing formation of glycosidic bonds (Puchart pg. 263 Fig. 1; pg. 269 “Biotechnological use of glycoside phosphorylases”). Puchart teaches many α- and β-phosphorylases for oligosaccharide production, including α-D-Glucopyranosyl-1,3-L-rhamnose phosphorylase, trehalose phosphorylases, and laminaribose phosphorylases (Puchart pg. 264 Table 1). Puchart teaches that glycoside phosphorylases can be heterologously expressed for industrial applications in microorganisms (Puchart pg. 269 “Biotechnological use of glycoside phosphorylases”).
These references do not teach a strain further comprising maa or manA gene deletions as recited in claim 3.
Regarding claim 3, Jennewein ‘342 teaches an E. coli strain for oligosaccharide production with deletions of manA, lacZ, and wcaJ for production of the oligosaccharide 2’3-difucosyllactose (Jennewein ‘342 pg. 15 para. 123). Jennewein ‘342 further teaches that deletion of manA allows for mannose-6-phosphate availability within the cell, which can be utilized for generating GDP-Man or GDP-Fuc (Jennewein ‘342 pg. 16 para. 32).
Regarding claim 3, Lo Leggio teaches that the maa gene in E. coli encodes maltose O-acetyltransferase (MAT), which acetylates sugars including glucose and maltose (Lo Leggio pg. 5225 Abstract and Introduction).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of these references to create a strain that additionally has a deletion of manA, as Jennewein ‘342 teaches that a strain with a deletion of manA is used for oligosaccharide production. It would further have been obvious to combine the teachings of these references with the teachings of Lo Leggio, creating a strain with an additional deletion of maa, given the role of maa in sugar acetylation.
A person of ordinary skill in the art would have been motivated to combine the teachings of these references and create a strain with additional inactivation of manA because Jennewein ‘342 teaches the deletion of manA in a strain for HMO production, and HMOs are of significant commercial value (Pedersen col. 1 lines 25-45). HMOs have limited natural availability and efficient large-scale fermentative production is in high demand (Jennewein ‘475 col. 2 lines 5-26). Additionally, Puchart teaches a number of glycoside phosphorylases that can be used in large-scale biotechnological production processes, and thus a skilled artisan would have been motivated to create a strain expressing glycoside phosphorylases (Puchart pg. 269 “Biotechnological use of glycoside phosphorylases”).
A skilled artisan would have further been motivated to combine the teachings of these references to create a strain with a deletion of maa, because Puchart teaches that oligosaccharide synthesis is catalyzed by carbohydrate catabolic enzymes, such as glycoside phosphorylases (Puchart pg. 262 “Introduction”). This involves formation of glycosidic bonds, which requires a hydroxyl group. Thus, modifications such as acetylation of sugars, which replace hydroxyl groups with acetyl groups, would impact enzyme substrate recognition and oligosaccharide production (Puchart pg. 268 “Substrate recognition”). As maa acetylates sugars, a skilled artisan would recognize that it would be desirable to delete this gene in order to reduce these modifications to the sugar building blocks of oligosaccharides.
A skilled artisan would have a reasonable expectation of success in combining these teachings to achieve the predictable outcome of an E. coli strain for oligosaccharide production, given the teachings of Jennewein ‘342 that a manA deletion yields a strain useful for HMO production, and the teachings of Lo Leggio and Puchart that sugar acetylation would be undesirable for oligosaccharide production and that glycoside phosphorylases can be heterologously expressed in bacteria and are used for oligosaccharide production.
Regarding claim 4, Jennewein ‘475, Jennewein ‘335, Puchart, Jennewein ‘342, and Lo Leggio teach the strain of claim 3. The deposited strain as claimed appears to have the same characteristics as the strain of claim 3, which is rendered obvious as set forth above. Therefore, the strain as recited in claim 4 is also obvious in view of the prior art.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Pedersen in view Jennewein ‘475, Jennewein ‘335, Puchart, Jennewein ‘342, and Lo Leggio, as applied to claim 3 above, and further in view of Lu et al., Applied microbiology and biotechnology; 93:2455-62 and Flores et al., Metabolic engineering; 7(2):70-87.
Pedersen, Jennewein ‘475, Jennewein ‘335, Puchart, Jennewein ‘342, and Lo Leggio teach the strain of claims 1 and 3 as set forth above. These references do not teach that a promoter of the GalP gene is an HIF promoter as recited in claim 5.
Regarding claim 5, Lu teaches increasing the expression of galP in an E. coli strain with an inactive PEP/carbohydrate phosphotransferase system (PTS) to improve glucose utilization in this strain (Lu pg. 2455-2456 Introduction). Lu teaches that expression of galP with an inducible promoter is problematic because inducers such as IPTG are expensive in large-scale fermentation processes, so it is preferable to constitutively express galP (Lu pg. 2456 para. 2-3).
Regarding claim 5, Flores teaches that the integration host factor (IHF or HIF) gene is used as an internal control or housekeeping gene to measure gene expression in an E. coli strain with inactivated PTS (Flores pg. 73-74 “Real-time PCR”). The same expression level of the ihf gene was detected in all growth conditions, indicating that this gene is constitutively expressed (Flores pg. 74 “Real-time PCR”).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of these references to create a strain with the gene deletions as instantly claimed and incorporation of an IHF/HIF promoter for galP. All of these references are directed to carbohydrate transport and oligosaccharide biosynthesis. Both Lu and Flores teach galP expression for the regulation of glucose utilization in strains that lack PTS, particularly relevant to the deletion strain as instantly claimed which lacks ptsG.
A skilled artisan would have been motivated to combine the teachings of these references to create a strain with an IHF/HIF promoter for galP expression because the expression of galP is important for regulating glucose utilization, which is necessary for oligosaccharide production (Lu pg. 2455-2456 Introduction). Additionally, Lu teaches that it is desirable to constitutively express galP because this does not require the use of expensive inducers, which are a problem for large-scale fermentation processes, so a skilled artisan would have been motivated to utilize the IHF/HIF promoter, which is a constitutive promoter as taught by Flores (Lu pg. 2456 para. 2-3 and Flores pg. 73-74 “Real-time PCR”).
A skilled artisan would have a reasonable expectation of success in combining these teachings to achieve the predictable outcome of an E. coli strain for oligosaccharide production with galP under an IHF/HIF promoter given the teachings of Lu and Flores that galP expression is beneficial for glucose utilization and that IHF is constitutively expressed.
Response to Arguments
Applicant's arguments filed 9/2/2025 have been fully considered but they are not persuasive.
Rejections Under 35 U.S.C. § 103
Applicant argues that this combination of four distinct references represents an a posteriori (hindsight) reconstruction of the claimed invention, rather than a teaching or suggestion arising naturally from the cited references. Applicant argues that the cited references each address different genetic modifications or enzyme applications in different contexts and there is no teaching, suggestion, or motivation in the cited references that would have led a person of ordinary skill in the art, without the knowledge of Applicant's invention, to selectively extract and combine particular gene deletions from Pedersen, Jennewein '475 and Jennewein '335, and the general enzyme teachings of Puchart.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, 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. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In the instant case, the prior art teaches E. coli strains modified for oligosaccharide production. All of the inactivated genes as recited in claim 1 are known in the art to be involved in oligosaccharide production, and have been modified in bacterial strains for the purpose of improving oligosaccharide production, as discussed above regarding the teachings of Pedersen, Jennewein ‘475, and Jennewein ‘335. Thus, it is considered that the knowledge required to create a strain as instantly claimed was within the level of ordinary skill in the art at the time the invention was made.
In response to applicant's argument that the examiner has combined an excessive number of references, reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991).
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007).
In this case, as set forth above, a skilled artisan would have been motivated create a strain with all the recited genes inactivated because strains with all of these genes deleted are associated with production of human milk oligosaccharides (HMOs), which are of significant commercial value, and it is of interest to develop efficient large-scale fermentative production techniques (Pedersen col. 1 lines 25-45 and Jennewein ‘475 col. 2 lines 5-26). A skilled artisan would have been motivated include an expression vector with a glycoside phosphorylase because production of oligosaccharides such as HMOs by recombinant bacteria has commercial value and Puchart teaches a number of glycoside phosphorylases that can be used in large-scale biotechnological production processes (Pedersen col. 1 lines 25-45 and Puchart pg. 269 “Biotechnological use of glycoside phosphorylases”). The references are all directed to biotechnological production of oligosaccharides, making it obvious for a skilled artisan to combine these teachings. Further, creation of recombinant bacterial strains with modifications to numerous genes involved in oligosaccharide production or related pathways are established in the art as a method of efficient oligosaccharide production, as taught by the cited references, and therefore it would be obvious to a skilled artisan to create a strain having multiple known oligosaccharide-related genes modified for the purpose of enhancing oligosaccharide production industrially
Applicant argues that Pedersen is silent about an Escherichia coli strain whose recA1, gyrA96, thi-1, glnV44, relA1, hsdR17, endA1, lacZ, nanKETA, lacA, melA, wcaJ, mdoH, ptsG, manX, manY, and pfkA genes are inactivated, and that the claimed strain comprises 4 further genetic modifications. Applicant argues that a strain according to the invention allows the production of oligosaccharides via glycoside phosphorylases and that such a strain not only allows the accumulation of the non-phosphorylated carbohydrates required for the synthesis of oligosaccharides by glycoside phosphorylases, but also the excretion of said oligosaccharides into the culture medium, and that the diffusion or excretion of oligosaccharides into the extracellular medium could allow a reaction catalyzed by glycoside phosphorylases (GP) to be obtained, favoring reverse phosphorolysis. Applicant argues that the additional deletion of the maa and manA genes both eliminates the production of contaminating acetylated sugar and maximizes the conversion of mannose to mannobiose and that substitution of the galP gene promoter by the strong constitutive HIF promoter in strain CS1 allows a high conversion yield (>49%) to be obtained.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., accumulation of non-phosphorylated carbohydrates, excretion of oligosaccharides, reverse phosphorolysis, high conversion yield of mannose to mannobiose) are not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). It is noted that the instant invention is directed to a modified E. coli strain. As discussed above, the strain as claimed with inactivated recA1, gyrA96, thi-1, glnV44, relA1, hsdR17, endA1, lacZ, nanKETA, lacA, melA, wcaJ, mdoH, ptsG, manX, manY, and pfkA is obvious in view of Pedersen, Jennewein ‘475, and Jennewein ‘335.
Applicant argues that Jennewein '475 does not cure the deficiencies of Pedersen, and that contrary to the examiner's argument, the E. coli mentioned in example 3 comprises more than only pfkA- and ptsG- and also mentioned 4 more inactivated genes, lacZ-, wcaJ-, glk, gcd-.
In response to this argument, it is noted that lacZ and wcaJ are both recited as genes which are inactivated in the claimed strain. While the strain in Example 3 of Jennewein ‘475 does also have deletions of glk and gcd which are not in the instant claims, Jennewein ‘475 clearly teaches deletion of pfkA and ptsG (as well as lacZ and wcaJ, as instantly claimed) for the production of oligosaccharides. Despite the deletion of additional genes that are not recited in the instant claims, a skilled artisan would be aware based on these teachings that pfkA and ptsG are important for oligosaccharide production and would have found it obvious to make deletions of these genes in addition to the gene deletions taught by Pedersen for enhancing oligosaccharide production. It is further noted that as written, the claims do not exclude the additional modification of other genes aside from those recited, and therefore the teachings of Jennewein ‘475 regarding deletions of additional unclaimed genes are not relevant.
Applicant argues that Jennewein '335 does not cure the deficiencies of the above-mentioned documents and that contrary to the examiner's argument, the E. coli mentioned in example comprise more gene inactivations than manXYZ. Applicant argues that the subject matter of Jennewein '335 is a purification process and one looking for the claimed invention would not consider Jennewein '335. Applicant argues that unless an ex post facto analysis, one in the art cannot arrive to the claimed invention since it has to make a very particular selection which is not suggested nor disclosed in the cited documents and nothing in the cited documents suggest the unexpected effects disclosed in the examples.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., the disclosed unexpected effects) are not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
It is noted that the claimed invention is directed to a modified E. coli strain product. As discussed above, Jennewein ‘335 teaches an E. coli strain with a deletion of manXYZ, for the production of oligosaccharides. Thus, a skilled artisan would have found it obvious to combine the teachings of Jennewein ‘335 with Pedersen, Jennewein ‘475, and Puchart, as all are directed to improving biotechnological production of oligosaccharides. Despite the deletion of additional genes that are not recited in the instant claims, a skilled artisan would be aware based on these teachings that manXYZ are important for oligosaccharide production and would have found it obvious to make deletions of these genes in addition to the gene deletions taught by Pedersen for enhancing oligosaccharide production. It is further noted that as written, the claims do not exclude the additional modification of other genes aside from those recited, and therefore the teachings of Jennewein ‘335 regarding deletions of additional unclaimed genes are not relevant.
Applicant argues that Jennewein '342 does not disclose nor suggest the particular strain defined in the claim set, and for some of the strains taught by Jennewein ‘342, overexpression of particular genes are needed. Applicant argues that Jennewein '342 does not cure the deficiencies of the above-mentioned documents and does not suggest a particular strain comprising 17 particular genes inactivated as defined in the claimed invention.
In response to this argument, it is again noted that despite the modification of additional genes that are not recited in the instant claims, a skilled artisan would be aware based on the teachings of Jennewein ‘342 that manA is important for oligosaccharide production and would have found it obvious to make deletions of this gene in addition to the gene deletions taught by Pedersen, Jennewein ‘475, and Jennewein ‘335 for enhancing oligosaccharide production. It is further noted that as written, the claims do not exclude the additional modification of other genes aside from those recited, and therefore the teachings of Jennewein ‘342 regarding deletions of additional unclaimed genes are not relevant.
Applicant argues that Puchart is silent about particular mutations in a strain to optimize the activity of Glycoside phosphorylases and about any mutation of a strain that could allow the production of oligosaccharides via glycoside phosphorylases, the accumulation of the non-phosphorylated carbohydrates required for the synthesis of oligosaccharides by glycoside phosphorylases, but also the excretion of said oligosaccharides into the culture medium.
Applicant argues that Puchart does not cure the deficiencies of the above-mentioned documents and does not suggest a particular strain comprising 17 particular genes inactivated as defined in the claimed invention.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., accumulation of non-phosphorylated carbohydrates, excretion of oligosaccharides) are not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). It is again noted that the instant claims are directed to a modified E. coli strain product, and particular functional features are not required by the claims.
Applicant argues that, regarding the teachings of the Puchart reference, those skilled in the art are not encouraged to use GPs to carry out the reactions of the invention; GPs described in the prior art act as enzymes that degrade glycosides to force the reaction towards phosphorolysis; without continuous removal of the reverse phosphorolysis product or the released inorganic phosphate to modify the reaction equilibrium, the yields of oligosaccharide synthesis are low; the specificity of the invention's process is to affect the reaction equilibrium and remove the reverse phosphorolysis product from the intracellular environment; the art does not encourage the use of GPs in a simple process that does not involve complex steps; Puchart teaches away from the solution of the invention; Puchart is silent about genetically modified strain and suggests other solutions; and that the strain of the invention allows the accumulation of non-phosphorylated carbohydrates required for the synthesis of oligosaccharides by GPs, enables the excretion of oligosaccharides into the culture medium, and allows a GP-catalyzed reaction to be obtained that is favored in the direction of reverse phosphorolysis.
In response to this argument, it is noted that Puchart teaches the use of GPs for high-yield and economic synthesis of oligosaccharides and teaches that engineered GPs are promising efficient and cheap producers of valuable substances, including oligosaccharides (Puchart p. 273 Section 9 para. 1). Thus, Puchart clearly teaches that GPs are useful for oligosaccharide production and are a promising area of future research, providing motivation for a skilled artisan to utilize such an enzyme when developing an efficient process for oligosaccharide production using a recombinant E. coli strain. Further, Puchart teaches heterologous expression of GPs in bacteria including E. coli (Puchart pg. 269 “Biotechnological use of glycoside phosphorylases”). Additionally, it is noted that the instant claims are directed to a modified E. coli strain product, and particular functional features (such as accumulation of non-phosphorylated carbohydrates or excretion of oligosaccharides) are not required by the claims.
Applicant argues that Leila Lo Leggio is silent about any mutation of a strain that could
allow the production of oligosaccharides via glycoside phosphorylases, the accumulation of the non-phosphorylated carbohydrates required for the synthesis of oligosaccharides by glycoside
phosphorylases, but also the excretion of said oligosaccharides into the culture medium and
that Leila Lo Leggio does not cure the deficiencies of the above-mentioned documents and does not suggest a particular strain comprising 17 particular genes inactivated as defined in the claimed invention.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., accumulation of non-phosphorylated carbohydrates, excretion of oligosaccharides) are not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). It is again noted that the instant claims are directed to a modified E. coli strain product, and these particular functional features are not required by the claims.
Applicant argues that concerning Flores and Lu, it appears to be a clear ex-post facto analysis of the documents. These documents do not cure the deficiencies of the above-mentioned documents. In addition, these documents do not provide any motivation to one skilled in the art and Lu does not indicate that it allows, in combination with GPs expression, a surprising increase in the oligosaccharide production yield by GPs and that nothing in the document would lead to the conclusion that galP expression would result in a favored catalyzed reaction in the direction of reverse phosphorolysis for a specific type of enzyme: GPs.
Applicant argues that the strain according to the invention and its combination with an expression vector of a glycoside-phosphorylase selected from a β-glycoside- or an α-glycoside-phosphorylases the allows: the production of oligosaccharides via glycoside-phosphorylases and also advantageously both an internalization of precursors for the synthesis, in particular of non-phosphorylated carbohydrates; intracellular accumulation of phosphorylated carbohydrates; intracellular accumulation of phosphorylated carbohydrates; recovery and facilitated purification of the produced oligosaccharides due to their excretion in the culture medium; and reaction catalyzed by the glycoside phosphorylases favored in the direction of reverse phosphorolysis. Applicant argues that the expression of GalP gene in strain with inactivated pfkA allows to restore the growth capacity.
In response to the argument regarding the ex-post facto analysis of Lu and Flores, Applicant does not provide sufficient detail to describe why the analysis is considered ex-post facto or improper hindsight. The references were applied to teach that galP is preferably expressed with a constitutive promoter (Lu, as discussed above) and that HIF is a known constitutive promoter (Flores, as discussed above), thus providing motivation to utilize HIF as the promoter for galP in a recombinant strain.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., accumulation of non-phosphorylated carbohydrates, excretion of oligosaccharides, surprising increase in oligosaccharide production yield, favored catalyzed reaction in the direction of reverse phosphorolysis, intracellular accumulation of phosphorylated carbohydrates) are not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). It is again noted that the instant claims are directed to a modified E. coli strain product, and these particular functional features are not required by the claims.
Conclusion
Claims 1-5, 7-8, and 16-17 are rejected.
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653