DETAILED OFFICE ACTION
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendment to the claims, filed June 27, 2025, is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Claims 1-11, 13-15, and 18-23 are pending and are being examined on the merits.
Claims 12 and 16-17 are cancelled.
Applicant’s amendment to the specification, filed June 27, 2025, is acknowledged.
Applicant’s remarks filed June 27, 2025 in response to the non-final rejection mailed April 8, 2025, is acknowledged and have been fully considered.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Previous rejections of claims 12, 16, and 17 are withdrawn in view of the instant amendment to cancel these claims.
Sequence Compliance
The objection of the specification for the disclosing the file size of the sequence incorporation statement in kilobytes is withdrawn in view of amendments to the specification.
Specification/Informalities
The objection of the specification for reciting “ATCC”, a trade name or a mark used in commerce, is withdrawn in view of amendments to the specification.
The objection of the specification for containing an embedded hyperlink and/or other form of browser-executable code is withdrawn in view of amendments to the specification.
Claim Objections
Claims 1 and 21-23 are objected to for reciting “is at least 98% identical” and “is 100% identical.” The Office recommends modifying the phrases to “has at least 98% sequence identity” and “has 100% sequence identity.”
Claim Rejections - 35 USC § 112
The rejection under 35 U.S.C. 112(b) of claims 3, 6-7, and 9-10 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matter which the inventor or a joint inventor regards as the invention because of the recitation of “turnover rate” is withdrawn in view of amendments to claims 3, 6, and 9 to recite “MHET” and a turnover rate.
Claims 3, 6-7, 9-10, and 19 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. The new rejection is necessitated by amendment.
Claims 3, 6-7, and 9-10 are indefinite because the term “about” is a term of degree (see MPEP 2173.05(b). The specification does not provide examples or teachings that can be used for ascertaining the variance intended by the recited term of degree. Moreover, there is nothing in the specification or prior art of record to indicate that one of skill in the art could have ascertained the scope of the recited degree. It is suggested that applicant clarify the meaning of the claims.
Claim 19 is rejected as indefinite because the claim recites the name "Escherichia coli". Claim 18 does not recite a genus from which Escherichia coli is a member. Since claim 19 depends upon claim 18, there is insufficient antecedent basis for this limitation and therefore “Escherichia coli” lacks antecedence.
Claim Rejections - 35 USC § 101
The rejection of claim 18 under 35 U.S.C. 101 as being directed to or encompassing a human organism is withdrawn in view of amendments to the claim.
Claim Rejections - 35 USC § 102
The rejection of claims 1 and 11 are under 35 U.S.C. 102(a)(1) as being anticipated by OBJECTIVE #1: Engineer E. coli to degrade PET, 2018 iGEM Yale team web page Team Yale results, December 7, 2018, pages 1-4 (cited on the IDS filed on 02/22/2023) as evidenced by Yoshida et al. (A bacterium that degrades and assimilates poly(ethylene terephthalate), Science, published March 11, 2016, Vol. 351, No. 6275, p. 1196-1199) is withdrawn in view of amendments to the claims because Yale does not teach or suggest a sequence that is at least 98% identical to SEQ ID NO: 36, 38, or 40.
Claim Rejections - 35 USC § 103
Claims 1-2, 4, 11, and 13-14 are rejected under 35 U.S.C. 103 as being unpatentable over OBJECTIVE #1: Engineer E. coli to degrade PET (OBJECTIVE #1: Engineer E. coli to degrade PET, 2018 iGEM Yale team web page Team Yale results, December 7, 2018, pages 1-4; cited on the IDS filed on 02/22/2023; hereafter “Yale”) in view of
Yoshida et al. (A bacterium that degrades and assimilates poly(ethylene terephthalate), Science, published March 11, 2016, Vol. 351, No. 6275, p. 1196-1199; cited on the Form PTO-892 mailed on April 8, 2025; hereafter “Yoshida”),
PETH_IDESA (PETH_IDESA, Accession A0A0K8P6T7, entry version 5
, Uniprot, published May 11, 2016, accessed from < https://rest .uniprot. org/unisave/ A0A0K8P6T7?format=txt&versions=5> on March 26, 2025; cited on the attached Form PTO-892; hereafter “PETH_IDESA”),
MHETH_IDESA (Uniprot, published May 11, 2016, accessed from < https://rest .uniprot. org/unisave/ A0A0K8P8E7?format=txt&versions=5 > on March 26, 2025; cited on the Form PTO-892 mailed on April 8, 2025; hereafter “MHETH_IDESA”),
LQ290181 (European Bioinformatics Institute (EBI), published October 4, 2016, accessed from < https://www.ebi.ac. uk/Tools/dbfetch/ dbfetch?db=epo_prt&id=LQ290181> on March 26, 2025; cited on the Form PTO-892 mailed on April 8, 2025; hereafter LQ290181),
Chen et al. (Fusion protein linkers: Property, design and functionality, Advanced Drug Delivery Reviews, published 2013, Vol. 65, No. 10, p. 1357-1369; cited on the Form PTO-892 mailed on April 8, 2025; hereafter “Chen”),
Yoshida et al. (Supplementary Material for A bacterium that degrades and assimilates poly(ethylene terephthalate, Science, published April 26, 2016, obtained from https://www.science.org/doi/10.1126/science.aad6359; cited on the IDS filed on 02/22/2023; hereafter “Yoshida-Supplementary”), and
Amet et al. (Insertion of the Designed Helical Linker Led to Increased Expression of Tf-Based Fusion Proteins, Pharmaceutical Research, published March 2009, Vol. 26, No. 3, p. 523-528; cited on the attached Form PTO-892; hereafter “Amet”). The new rejection is necessitated by amendments to the claims.
As amended, the claims are drawn to a non-naturally occurring enzyme comprising:
a first polypeptide that catalyzes the hydrolysis of a polyester to produce mono-(2- hydroxyethyl) terephthalate (MHET);
a second polypeptide that catalyzes the cleavage of MHET to produce at least one of terephthalic acid or ethylene glycol;
and a third polypeptide that links the first polypeptide with the second polypeptide,
wherein the enzyme is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40.
Regarding claims 1-2, 4, 11, 13-14, Yale teaches a fusion protein comprising PETase-linker-MHETase (p. 4, para 6 - p. 4, para 7 – “… to improve functionality of PETase and MHETase; the main way we decided to approach this issue was by combining the PETase and MHETase proteins into either a fusion protein… construct via Gibson Assembly. Using the PETase containing construct as a foundation, we inserted MHETase directly after PETase using Gibson Assembly; this was in the hopes of creating a PETase-MHETase fusion protein to increase PET degradation efficiency. Additionally, we believed that creating a PETase-MHETase fusion protein to increase PET degradation efficiency.”; Figure at bottom of p. 4). Based on these teachings, an ordinary artisan would immediately recognize that the linker of the fusion protein of Yale is a peptide linker.
Yoshida teaches that poly(ethylene terephthalate) (PET) hydrolase (termed PETase) converts the polyester PET to mono(2-hydroxyethyl) terephthalic acid (MHET) (p. 1197, col 3, para 3 – p. 1198, col 1, para 2). Furthermore, MHET hydrolases (MHETases) convert MHET to terephthalic acid TPA and ethylene glycol (p. 1199, col 1, para 2; Figure 3). Also, Yoshida teaches many plastics, and specifically polyester, comprise a high amount of poly(ethylene terephthalate) (PET) (p. 1196, col 1, para 1 – p. 1196, col 2, para 2). Therefore, the PETase of Yale’s fusion protein catalyzes the hydrolysis of a polyester comprising PET to produce MHET and is considered to be the “first polypeptide” in claim 1; the MHETase of Yale’s fusion protein catalyzes the cleavage of MHET to produce TPA and ethylene glycol and is considered to be the “second polypeptide” in claim 1; and the peptide linker of Yale’s fusion protein is considered to be the “third polypeptide” of claim 1.
The differences between Yale and the claimed invention are:
Yale does not teach the enzyme has least 98% identity to a sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40; and
Yale does not teach a specific length and composition of a linker peptide.
Yoshida teaches many plastics, such as polyester, comprises a high amount of aromatic compounds like poly(ethylene terephthalate) (PET) (p. 1196, col 1, para 1 – p. 1196, col 2, para 2). Production and disposal of large quantities of PET have been introduced into and contaminated the environment (p. 1196, col 2, para 1). Yoshida teaches a method of using microorganisms able to degrade PET as a means to remediate environmental PET strategies or to degrade and recycle PET waste (p. 1196, col 2, para 2 – p. 1196, col 3, para 1).
Yoshida teaches a bacterium, Ideonella sakaiensis (I. sakaiensis) 201-F6, that degrades PET into environmentally benign monomers terephthalic acid (TPA) and ethylene glycol; PET is degraded into mono(2-hydroxyethyl) terephthalic acid (MHET) via a PET hydrolase (termed PETase; encoded by ISF6_4831) and MHET is metabolized into TPA and ethylene glycol via MHET hydrolase (termed MHETase; encoded by ISF6_0224) (Abstract; p. 1196, col 2, para 1; p. 1197, col 3, para 3 – p. 1198, col 1, para 3; p. 1199, col 1, para 2; Figure 3).
Yoshida teaches the PETase encoded by ISF6_4831 and purified from E. coli recombinantly expressing the hydrolase converts PET to MHET as well as TPA and bis(2-hydroxyethyl) TPA (BHET) (p. 1197, col 3, para 3 – p. 1198, col 1, para 2; Supplemental p. 7, para 2 – supplemental p. 8, para 1). The MHETase encoded by ISF6_0224 and purified from E. coli recombinantly expressing the hydrolase converts MHET to TPA and ethylene glycol (p. 1198, col 3, para 3; p. 1199, col 1, para 2; Supplemental p. 7, para 2 – supplemental p. 8, para 1).
PETH_IDESA teaches the amino acid sequence of ISF6_4831; the recommended name for ISF6_4831 is poly(ethylene terephthalate) (PET) hydrolase (termed PETase). ISF6_4831 comprises 290 amino acids in total wherein the amino acids 1-27 comprise a signal sequence and amino acids 28-290 comprise the PETase (note, amino acid residues 28-290 of ISF6-4831 has 100% sequence identity with amino acid residues 612-874, 616-878, and 624-886 of SEQ ID NOs: 36, 38, and 40, respectively, of the instant application; see alignment with SEQ NO: 36 below).
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MHETH_IDESA teaches the amino acid sequence of ISF6_0224 the recommended name for ISF6_0224 is mono(2-hydroxyethyl) terephthalate (MHET) hydrolase (termed MHETase). ISF6_0224 comprises 603 amino acids in total (note, amino acid residues 1-603 of ISF6_0224 has 100% sequence identity with amino acid residues 1-603 of SEQ ID NOs: 36, 38, and 40 of the instant application; see alignment below).
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LQ290181 teaches an eight amino acid residue glycine- and serine-based protein linker: GGGSGGSG (note, LQ290181 has 100% sequence identity with amino acid residues 604-611 of SEQ ID NO: 36; see alignment below).
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Chen teaches the generation of multi-functional fusion proteins wherein two or more protein domains are fused together such as with peptide linkers (Abstract; p. 1357, col 2, para 1). Glycine- and serine-rich linkers are shown to improve fusion protein folding, stability, and biological activity in fusion proteins (p. 1363, col 1, para 3). Chen teaches creating fusion proteins comprising in all possible directions (i.e., (Protein A)-(Protein B) and (Protein B)-(Protein A)) in order to test the optimal orientation (p. 1363, col 2, para 3). Chen teaches fusion protein wherein the domains are fused via a dipeptide linker comprising leucine (L) -glutamic acid (Q) (p. 1366, col 2, para 2).
Amet teaches fusing domains of a fusion protein with a leucine (L) and glutamic acid (E) dipeptide which is a product of a XhoI restriction (Abstract; p. 524, col 1, para 3).
In view of the teaches of Yale, Yoshida, and PETH_IDESA, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the PETase-linker-MHETase fusion protein taught by Yale such that the PETase comprises the ISF6_4831 PETase taught by PETH_IDESA.
An ordinary artisan would have been motivated to substitute the PETase in the PETase-linker-MHETase fusion protein taught by Yale for the amino acids 28-290 of ISF6_4831 PETase from PETH_IDESA. This because the simple substitution of one known PETase for another known PETase in the prior art would generate predictable results because the enzymes share the same activity. Furthermore, PETH_IDESA taught amino acids 1-27 of ISF6_4831 are a signal sequence and amino acids 28-290 are the PETase enzyme. Additionally, recombinant ISF6_4831 PETase, derived from Ideonella sakaiensis, is taught by Yoshida to degrade PET into environmentally benign monomers terephthalic acid (TPA) and ethylene glycol.
An ordinary artisan would have had a reasonable expectation of success of substituting the PETase in the PETase-linker-MHETase fusion taught by Yale for the amino acids 28-290 of ISF6_4831 PETase from PETH_IDESA. This because the simple substitution of one known PETase for another known PETase in the prior art would generate predictable results because the enzymes share the same activity.
In view of the teaches of Yale, Yoshida, and MHETH_IDESA, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the PETase-linker-MHETase fusion protein connected by a linker taught by such that the PETase comprises the ISF6_0224 MHETHase taught by MHETH_IDESA.
An ordinary artisan would have been motivated to substitute the MHETase in the PETase-linker-MHETase fusion protein connected by a linker taught by Yale for the ISF6_0224 MHETase from MHETH_IDESA. This because the simple substitution of one known MHETase for another known MHETase in the prior art would generate predictable results because the enzymes share the same activity. Additionally, recombinant ISF6_0224 MHETase, derived from Ideonella sakaiensis, is taught by Yoshida to metabolize MHET into TPA and ethylene glycol.
An ordinary artisan would have had a reasonable expectation of success of substituting to substitute the MHETase in the PETase-linker-MHETase fusion protein connected by a linker taught by Yale for the ISF6_0224 MHETase from MHETH_IDESA. This because the simple substitution of one known MHETase for another known MHETase in the prior art would generate predictable results because the enzymes share the same activity.
In view of the combined teachings of Yale, Chen, and LQ290181, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to substitute the linker in the PETase-linker-MHETase fusion protein taught by Yale for the eight amino acid glycine- and serine-rich linker GGGSGGSG from LQ290181.
An ordinary artisan would have been motivated to substitute the linker in the PETase-linker-MHETase fusion protein by Yale for the GGGSGGSG linker from LQ290181. This because the simple substitution of one known fusion protein linker for another known fusion protein linker in the prior art would generate predictable results because the linkers share the same function. Furthermore, Chen taught glycine- and serine-rich linkers are shown to improve fusion protein folding, stability, and biological activity in fusion proteins.
An ordinary artisan would have had a reasonable expectation of success of substituting the linker in the PETase-linker-MHETase fusion protein by Yale for the GGGSGGSG linker from LQ290181. This because the simple substitution of one known fusion protein linker for another known fusion protein linker in the prior art would generate predictable results because the linkers share the same function.
In view of the combined teachings of Yale and Chen, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention modify the PETase-linker-MHETase fusion protein taught by Yale such that the order of the fusion protein comprises MHETase-linker-PETase. This because Chen taught creating fusion proteins comprising in all possible directions (i.e., (Protein A)-(Protein B) and (Protein B)-(Protein A)) in order to test the optimal orientation.
An ordinary artisan would have been motivated to reverse the order of the enzymatic domains in the PETase-linker-MHETase fusion protein such that the order of the fusion protein comprises MHETase-linker-PETase. This because Chen taught creating fusion proteins comprising in all possible directions (i.e., (Protein A)-(Protein B) and (Protein B)-(Protein A)) in order to test the optimal orientation.
An ordinary artisan would have had a reasonable expectation of success to reverse the order of the enzymatic domains in the PETase-linker-MHETase fusion protein such that the order of the fusion protein comprises MHETase-linker-PETase. This because Chen taught creating fusion proteins comprising in all possible directions (i.e., (Protein A)-(Protein B) and (Protein B)-(Protein A)) in order to test the optimal orientation.
In view of the combined teachings of Yale, Chen, Yoshida-Supplementary, and Amet, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention modify the modified MHETase-linker-PETase such that the C-terminus comprises a dipeptide LE linker followed by a C-terminal hexa-histidine tag.
An ordinary artisan would have been motivated to modify the MHETase-linker-PETase such that the C-terminus comprises a dipeptide LE linker followed by a C-terminal hexa-histidine tag. This is because Yoshida-Supplementary taught fusing a hexa-histidine tag to the C-terminus of PETase in order to purify the protein with an affinity column as well as that Chen and Amet taught fusion proteins comprising a LE linker between domains and Amet discloses is the result of XhoI restriction site modifying the fusion protein comprise such that the C-terminus comprises a dipeptide LE linker followed by a C-terminal hexa-histidine tag.
An ordinary artisan would have had a reasonable expectation of success of modifying the MHETase-linker-PETase such that the C-terminus comprises a dipeptide LE linker followed by a C-terminal hexa-histidine tag. This is because Yoshida-Supplementary taught fusing a hexa-histidine tag to the C-terminus of PETase in order to purify the protein with an affinity column as well as that Chen and Amet taught fusion proteins comprising a LE linker between domains and Amet discloses is the result of XhoI restriction site modifying the fusion protein comprise such that the C-terminus comprises a dipeptide LE linker followed by a C-terminal hexa-histidine tag.
Therefore, in view of the combined teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, Chen, LQ290181, Yoshida-Supplementary, and Amet, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the PETase-linker-MHETase fusion protein connected by a linker taught by Yale such that the fusion protein comprises from N-terminus to C-terminus: the ISF6_0224 MHETase from MHETH_IDESA, the eight amino acid glycine- and serine-rich linker GGGSGGSG from LQ290181, amino acids 28-290 of ISF6_4831 PETase from PETH_IDESA, the LE dipeptide linker from Chen and Amet, and a hexa-histidine tag taught by Yoshida (hereafter referred to as “MHETase-GGGSGGSG-PETase-LE-6xH”) wherein the sequence is identical to SEQ ID NO: 36 (see alignment below).
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Thereby arriving at the invention of claims 1-2, 4, 11, and 13-14.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Yale in view of Yoshida, MHETH_IDESA, PETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, and Amet as applied to claims 1-2, 4, 11, 13-14 above, and further in view of Palm et al. (Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate, Nature Communications, published April 12, 2019, Vol. 10, No. 1717; cited on the Form PTO-892 mailed on April 8, 2025; hereafter “Palm”). The new rejection is necessitated by amendments to the claims.
The combined teaches teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, and Amet as applied to claims 1-2, 4, 11, 13-14 are discussed above and incorporated herein.
Regarding claim 3,
The difference between the combination of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, and Amet and the enzyme of claim 3 is that the combination does not explicitly teach wherein the fusion protein has a MHET turnover rate of about 60.25 s-1 to about 77.57 s-1.
Palm teaches wild-type I. sakaiensis MHETase has a turnover rate of MHET of about 10-12.5 s-1 (Figure 4; Meeting limitation regarding turnover rate in dependent claim 4).
Since Yoshida teaches the ISF6_0224 MHETase is the natural MHETase from Ideonella sakaiensis, in view of the combined teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Amet, Palm, and Yoshida-Supplementary, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention that the MHETase turnover rate in the MHETase-GGGSGGSG-PETase-LE-6xH fusion protein is increased over the natural free MHETase from Ideonella sakaiensis. It is the examiner’s position MHETase turnover rate in the MHETase-GGGSGGSG-PETase-LE-6xH fusion protein necessarily has the MHETase turnover rate of the invention of claim 3 because the MHETase-GGGSGGSG-PETase-LE-6xH fusion protein is identical to the structure of the protein of instant claim 3. Since the Office does not have the facilities for examining and comparing applicant’s protein with the modified chimeric protein MHETase-GGGSGGSG-PETase-LE-6xH, the burden is on the applicant to show a novel or unobvious difference between the properties of the protein of claim 3 and the recited protein of the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald et al., 205 USPQ 594.
Consequently, the invention of claim 3 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 5 and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Yale in view of Yoshida, MHETH_IDESA, PETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, and Amet as applied to claims 1-2, 4, 11, and 13-14 above, and further in view of van Rosmalen et al. (Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins, Biochemistry, published November 23, 2017, Vol. 56, No. 50; cited on the attached Form PTO-892; hereafter “Rosmalen”) and Zhang et al. (Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration, Eng Life Sci, published May 17, 2017, Vol. 17, No. 9, p. 989-996; cited on the Form PTO-892 mailed on April 8, 2025; hereafter “Zhang”). The new rejection is necessitated by amendments to the claims.
The combined teaches teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, and Zhang as applied to claims 1-2, 4, 11, and 13-14 are discussed above and incorporated herein.
Regarding claim 5, Chen further teaches the length and composition of linkers impact the function of domains in proteins (p. 1359, col 2 para 3; p. 1368, col 1, para 2). Chen also teaches 5-20 amino acid long glycine- and serine-peptide linkers for fusion proteins (Table 3).
The difference between the combination of Yale, Yoshida, Chen, MHETH_IDESA, PETH_IDESA, LQ290181, Yoshida-Supplementary, and Amet and the enzyme of claim 5 is that the combination does not explicitly teach wherein the enzyme sequence is SEQ ID NO: 38.
Rosmalen teaches modifying the length and amino composition of a glycine- and serine-linker in a multi-domain protein impacts the function and properties of fusion proteins (Abstract; p. 6565, col 1, para 1). Altering the length of the linker impacts the ability of the fusion protein domains to interact and function together (Abstract). Altering the linker amino acid composition to comprise less glycine decreased the flexibility of the linker and also decreased the ability of the fusion protein domains to interact and function together (Abstract).
Zhang teaches a bi-functional fusion protein comprising two metabolic enzymes, formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH), linked by a peptide linker expressed in E. coli cells (Abstract; p. 990, col 1, para 3). Incorporation of two catalytic entities into one complex permits a proximity effect which can accelerate cofactor regeneration, catalytic efficiency, and production rate of the complex (Abstract; p. 991, col 2, para 4). Zhang teaches LeuDH-FDH and FDH-LeuDH fusion proteins wherein the C-terminus of the first enzyme is fused to the N-terminus of the second protein (p. 991, col 2, para 4). FDH-linker-LeuDH fusion protein complexes retained around 70-100% activity of LeuDH and around 25-140% as compared to free enzymes (Figure 2). Zhang teaches FDH-LeuDH fusion complexes with a flexible peptide linker comprising glycine and serine amino acid residues (i.e., GSSSS) between 5 and 15 amino acids in length, and the fusion enzymatic complex increased the production rate of the complex of up to 1.2 times compared to a free enzyme mixture (Abstract; Table S1).
In view of the combined teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, Amet, Rosmalen, and Zhang, it would have been obvious for a person of ordinary skill in the art to modify the MHETase-GGGSGGSG-PETase-LE-6xH to comprise a 12 amino acid residue long glycine- and serine-linker comprising a sequence of GGGSGGSGGGSG instead of the GGGSGGSG linker. Thereby arriving at the claimed invention of claim 5.
Since Rosmalen teaches glycine- and serine-linkers in fusion proteins between 5 and 15 amino acids in length as well as teaches that amino acid length and amino acid composition are results-effective variables that impact the ability of the fusion protein domains to interact and function together, an ordinary artisan would have been motivated to optimize the linker protein to comprise a sequence of GGGSGGSGGGSG in order to increase the catalytic efficiency and turnover rate of the fusion protein due to enhancing the proximity effect between domains of a fusion protein taught by Zhang (See MPEP 2144.05.II).
Therefore, a sequence comprising the fusion protein comprises from N-terminus to C-terminus: the ISF6_0224 MHETase from MHETH_IDESA, the 12 amino acid glycine- and serine-rich linker GGGSGGSGGGSG, amino acids 28-290 of ISF6_4831 PETase from PETH_IDESA, the LE dipeptide linker from Chen and Amet, and the hexa-histidine tag from Yoshida, wherein the fusion is identical to SEQ ID NO: 38 (hereafter “MHETase-GGGSGGSGGGSG -PETase-LE-6xH”;see alignment below).
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An ordinary artisan would have had a reasonable expectation of success of substituting one glycine- and serine-peptide linker for another (e.g., GGGSGGSGGGSG) and generated predictable results because the linkers have similar properties and functions.
Regarding claim 8, Chen further teaches the length and composition of linkers impact the function of domains in proteins (p. 1359, col 2 para 3; p. 1368, col 1, para 2). Chen also teaches a Chen teaches a 20 amino acid long glycine- and serine-linker peptide linker (e.g., (GGGGS)4) (p. 1361, col 1, para 2).
The difference between the combination of Yale, Yoshida, Chen, MHETH_IDESA, PETH_IDESA, LQ290181, Yoshida-Supplementary, and Amet and the enzyme of claims 8 is that the combination does not explicitly teach wherein the enzyme sequence is SEQ ID NO: 40.
Rosmalen teaches modifying the length and amino composition of a glycine- and serine-linker in a multi-domain protein impacts the function and properties of fusion proteins (Abstract; p. 6565, col 1, para 1). Altering the length of the linker impacts the ability of the fusion protein domains to interact and function together (Abstract). Altering the linker amino acid composition to comprise less glycine decreased the flexibility of the linker and also decreased the ability of the fusion protein domains to interact and function together (Abstract).
Zhang teaches a bi-functional fusion protein comprising two metabolic enzymes, formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH), linked by a peptide linker expressed in E. coli cells (Abstract; p. 990, col 1, para 3). Incorporation of two catalytic entities into one complex permits a proximity effect which can accelerate cofactor regeneration, catalytic efficiency, and production rate of the complex (Abstract; p. 991, col 2, para 4). Zhang teaches LeuDH-FDH and FDH-LeuDH fusion proteins wherein the C-terminus of the first enzyme is fused to the N-terminus of the second protein (p. 991, col 2, para 4). FDH-linker-LeuDH fusion protein complexes retained around 70-100% activity of LeuDH and around 25-140% as compared to free enzymes (Figure 2). Zhang teaches FDH-LeuDH fusion complexes with a flexible peptide linker comprising glycine and serine amino acid residues (i.e., GSSSS) between 5 and 15 amino acids in length, and the fusion enzymatic complex increased the production rate of the complex of up to 1.2 times compared to a free enzyme mixture (Abstract; Table S1).
In view of the combined teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, Amet, Rosmalen, and Zhang, it would have been obvious for a person of ordinary skill in the art to modify the modified fusion protein to comprise a 12 amino acid residue long glycine- and serine-linker comprising a sequence of GGGSGGSGGGSGGGGSGGSG. Thereby arriving at the claimed invention of claim 8.
Since Chen teaches glycine- and serine-linkers in fusion proteins between 5 and 20 amino acids in length and Rosmalen teaches that amino acid length and amino acid composition are results-effective variables that impact the ability of the fusion protein domains to interact and function together, an ordinary artisan would have been motivated to optimize the linker protein to comprise a sequence of GGGSGGSGGGSGGGGSGGSG in order to increase the catalytic efficiency and turnover rate of the fusion protein due to enhancing the proximity effect between domains of a fusion protein taught by Zhang (See MPEP 2144.05.II).
Therefore, a sequence comprising the fusion protein comprises from N-terminus to C-terminus: the ISF6_0224 MHETase from MHETH_IDESA, the 20 amino acid glycine- and serine-rich linker GGGSGGSGGGSGGGGSGGSG, amino acids 28-290 of ISF6_4831 PETase from PETH_IDESA, the LE dipeptide linker from Chen and Amet, and the hexa-histidine tag from Yoshida, wherein the fusion is identical to SEQ ID NO: 40 (hereafter “MHETase-GGGSGGSGGGSGGGGSGGSG-PETase-LE-6xH”; see alignment below).
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An ordinary artisan would have had a reasonable expectation of success of substituting one glycine- and serine-peptide linker for another (e.g., GGGSGGSGGGSGGGGSGGSG) and generated predictable results because the linkers have similar properties and functions.
Consequently, the invention of claims 5 and 8 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 6-7 and 9-10 are rejected under 35 U.S.C. 103 as being unpatentable over Yale in view of Yoshida, MHETH_IDESA, PETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, Amet, Rosmalen, and Zhang as applied to claims 1-2, 4-5, 8, 11, and 13-14 above, and further in view of Palm. The new rejection is necessitated by amendments to the claims.
The combined teaches teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, Amet, Rosmalen, and Zhang as applied to claims 1-2, 4-5, 8, 11, and 13-14 are discussed above and incorporated herein.
Regarding claims 6-7,
The difference between the combination of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, Amet, Rosmalen, and Zhang and the enzyme of claims 6-7 is that the combination does not explicitly teach wherein the fusion protein has a MHET turnover rate of about 64.05 s-1 to about 89.93 s-1.
Palm teaches wild-type I. sakaiensis MHETase has a turnover rate of MHET of about 10-12.5 s-1 (Figure 4; Meeting limitation regarding turnover rate in dependent claim 4).
Since Yoshida teaches the ISF6_0224 MHETase is the natural MHETase from Ideonella sakaiensis, in view of the combined teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, Amet, Rosmalen, Zhang, and Palm, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention that the MHETase turnover rate in the MHETase-GGGSGGSGGGSG-PETase-LE-6xH fusion protein is increased over the natural free MHETase from Ideonella sakaiensis. It is the examiner’s position MHETase turnover rate in the MHETase-GGGSGGSGGGSG-PETase-LE-6xH fusion protein necessarily has the MHETase turnover rate of the invention of claim 6-7 because the MHETase-GGGSGGSGGGSG-PETase-LE-6xH fusion protein is identical to the structure of the protein of instant claim 5 which claims 6-7 are dependent therefrom. Since the Office does not have the facilities for examining and comparing applicant’s protein with the modified chimeric protein MHETase-GGGSGGSGGGSG-PETase-LE-6xH, the burden is on the applicant to show a novel or unobvious difference between the properties of the protein of claims 5-7 and the recited protein of the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald et al., 205 USPQ 594.
Regarding claims 9-10,
The difference between the combination of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, Amet, Rosmalen, and Zhang and the enzyme of claims 9-10 is that the combination does not explicitly teach wherein the fusion protein has a MHET turnover rate of about 51.98 s-1 to about 60.52 s-1.
Palm teaches wild-type I. sakaiensis MHETase has a turnover rate of MHET of about 10-12.5 s-1 (Figure 4; Meeting limitation regarding turnover rate in dependent claim 4).
Since Yoshida teaches the ISF6_0224 MHETase is the natural MHETase from Ideonella sakaiensis, in view of the combined teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Amet, Zhang, and Palm, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention that the MHETase turnover rate in the MHETase-GGGSGGSGGGSGGGGSGGSG-PETase-LE-6xH fusion protein is increased over the natural free MHETase from Ideonella sakaiensis. It is the examiner’s position MHETase turnover rate in the MHETase-GGGSGGSGGGSGGGGSGGSG-PETase-LE-6xH fusion protein necessarily has the MHETase turnover rate of the invention of claims 9-10 because the MHETase-GGGSGGSGGGSGGGGSGGSG-PETase-LE-6xH fusion protein is identical to the structure of the protein of instant claim 8 which claims 9-10 are dependent therefrom. Since the Office does not have the facilities for examining and comparing applicant’s protein with the modified chimeric protein MHETase-GGGSGGSGGGSGGGGSGGSG-PETase-LE-6xH, the burden is on the applicant to show a novel or unobvious difference between the properties of the protein of claim 8-10 and the recited protein of the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald et al., 205 USPQ 594.
Consequently, the invention of claim 6-7 and 9-10 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, and Amet as applied to claims 1-2, 4, 11, and 13-14 above, and further in view of Zhang. The new rejection is necessitated by amendments to the claims.
Regarding claim 15, Yoshida further teaches a plurality of enzymes that hydrolyze PET (Table S2). Some of those PET hydrolases also exhibit hydrolytic activity against PET degradation products like MHET and BHET (Table S2). Enzymes exhibiting additional activity comprising BHET hydrolytic activity include TfH, FsC, HiC, and BsEstB; TfH, FsC, and BsEstB also exhibit MHETase activity (Table S2).
The difference between the combination of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, and Amet and the enzyme of claim 15 is that the combination does not explicitly teach wherein the enzyme further comprises a fourth polypeptide capable of catalyzing hydrolysis of a polyester to produce mono-(2- hydroxyethyl) terephthalate (MHET); and a fifth polypeptide, wherein: the fifth polypeptide covalently links the fourth polypeptide with the second polypeptide.
Zhang teaches a bi-functional fusion protein comprising two metabolic enzymes, formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH), linked by a peptide linker expressed in E. coli cells (Abstract; p. 990, col 1, para 3). Incorporation of two catalytic entities into one complex permits a proximity effect which can accelerate cofactor regeneration, catalytic efficiency, and production rate of the complex (Abstract; p. 991, col 2, para 4). Zhang teaches LeuDH-FDH and FDH-LeuDH fusion proteins wherein the C-terminus of the first enzyme is fused to the N-terminus of the second protein (p. 991, col 2, para 4). FDH-linker-LeuDH fusion protein complexes retained around 70-100% activity of LeuDH and around 25-140% as compared to free enzymes (Figure 2). Zhang teaches FDH-LeuDH fusion complexes with a flexible peptide linker comprising glycine and serine amino acid residues (i.e., GSSSS) between 5 and 15 amino acids in length, and the fusion enzymatic complex increased the production rate of the complex of up to 1.2 times compared to a free enzyme mixture (Abstract; Table S1).
Since Chen teaches linking two or more protein domains, in view of the combined teachings of Yale, Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Yoshida-Supplementary, Amet, and Zhang, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify MHETase-GGGSGGSG-PETase-LE-6xH to a second PETase that exhibits additional MHETase and/or BHET hydrolytic activity to allow degradation of MHET and BHET into TPA as taught by Yoshida. Thereby arriving at the claimed invention of claim 15.
An ordinary artisan would have been motivated to fuse via a peptide linker the MHETase-GGGSGGSG-PETase-LE-6xH a tri-functional PETase/MHET hydrolase/BHET hydrolase taught by Yoshida in order to produce a fusion protein complex that can degrade a plurality of PET degradation products (i.e., PET and BHET) and more completely remediate PET contamination or recycle PET waste as well as benefit from a proximity effect and increased production rate of the complex achieved when two metabolic enzymes a linker are fused together taught by Zhang.
An ordinary artisan would have had a reasonable expectation of success of producing a multi-functionalized fusion protein wherein protein domains are connected via peptide linkers because of the teachings of Chen multi-functional fusion proteins with two or more domains connected by peptide linkers.
Consequently, claim 15 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 18-23 are rejected under 35 U.S.C. 103 as being unpatentable over Yale in view of Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Palm, Amet, Palm, Rosmalen, and Zhang as applied to claims 1-11 and 13-14 above, and further in view of Vague et al. (Pseudomonas isolates degrade and form biofilms on polyethylene terephthalate (PET) plastic, bioRXiv, published May 24, 2019; cited on the attached Form PTO-892; hereafter “Vague”). The new rejection is necessitated by amendments to the claims.
The combined teaches teachings of Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Palm, Amet, Palm, Rosmalen, and Zhang as applied to claims 1-11 and 13-14 are discussed above and incorporated herein.
Regarding claims 18-23, Yale teaches expressing PETase and MHETase in E. coli (p. 4, para 1 – “The goal of Objective #1 was to engineer E. coli to degrade PET by express and secreting PETase and MHETase, two genes originally found in Ideonella sakaiensis.”; p. 4, para 7 – “…we believed that creating a fusion protein would lessen the metabolic burden on E. Coli and also eliminate any concern about the regulation of each protein independently.”).
The combination of Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Palm, Amet, Palm, Rosmalen, and Zhang does not teach:
a genetically modified organism expressing the modified protein from claim 1 in a bacterial species from the genus Pseudomonas, a bacterial species from the genus Bacillus, a bacterial species from the genus Caulobacter, a bacterial species from the genus Lactoccocus, a bacterial species from the genus Streptomyces, a bacterial species from the genus Streptococcus, and a bacterial species from the genus Corynybacterium, a fungal species from the genus Trichoderma, a fungal species from the genus Penicillium, a fungal species from the genus Humicola, a fungal species from the genus Chrysosporium, a fungal species from the genus Gliocladium, a fungal species from the genus Aspergillus, a fungal species from the genus Fusarium, a fungal species from the genus Neurospora, a fungal species from the genus Hypocrea, and a fungal species from the genus Emericella, a fungal species from the genus Saccharomyces, a fungal species from the genus Pichia, and a fungal species from the genus Kluyveromyces (Claim 18);
wherein the organism is Pseudomonas putida (Claim 19);
a method of degrading a polyester comprising contacting the organism of claim 18 with the polyester (Claim 20)
wherein the enzyme of the method has 100% sequence identity to SEQ ID NOs: 21-23 (Claims 21-23, respectively).
Vague teaches Pseudomonas putida degrades plastics like PET (Abstract; p. 9, para 2 – p. 12, para 2; p. 14, para 1; p. 17, para 4 – p. 18, para 1; Table 1; Figures 5-6). Vague teaches biofilm formation is essential for colonization of plastic by microorganisms and without it bacteria cannot effectively degrade plastic, and Pseudomonas putida forms enhanced biofilms on PET (Abstract; p. 12, para 3 – p. 13, para 1; p. 15, para 2-3). Vague teaches PET degradation by bacteria generates a major product of mono(2-hydroxyethyl) terephthalic acid (MHET) and eventually bis(2-hydroxyethyl) terephthalic acid (BHET); PETase is secreted and breaks down PET to MHET, which is broken down by secreted MHETase to terephthalic acid (TPA) and ethylene glycol (p. 18, para 2). Vague teaches a bioaugmentation as plastic remediation strategy wherein bacteria are used to break down plastic waste (Abstract; p. 18, para – p. 19, para 1).
In view of the combined teachings of Yoshida, PETH_IDESA, MHETH_IDESA, LQ290181, Chen, Palm, Amet, Palm, Rosmalen, Zhang, and Vague, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to express the modified PETase-MHETase fusion protein identical to SEQ ID NOs: 36, 38, or 40 in Pseudomonas putida and use the modified bacteria to degrade polyester containing PET. Thereby arriving at the claimed invention of claims 20-23.
An ordinary artisan would have been motivated to express the modified MHETase-linker-PETase fusion proteins identical to SEQ ID NOs: 36, 38, or 40 in Pseudomonas putida and use the modified bacteria to degrade polyester containing PET. This is because Vague taught Pseudomonas putida grows on and degrades plastics like PET. Vague also taught PET degradation by bacteria generates a major product of mono(2-hydroxyethyl) terephthalic acid (MHET) and eventually bis(2-hydroxyethyl) terephthalic acid (BHET); PETase is secreted and breaks down PET to MHET, which is broken down by secreted MHETase to terephthalic acid (TPA) and ethylene glycol. bioaugmentation as plastic remediation strategy wherein bacteria are used to break down plastic waste. Additionally, Yoshida taught polyester contains high amounts of PET and teaches using microorganisms expressing of PETase and MHETase to degrade PET.
An ordinary artisan would have had a reasonable expectation of success of expressing the modified PETase-MHETase fusion protein in Pseudomonas putida because Yale and Yoshida taught the recombinant expression of PETase and MHETase in bacteria. An ordinary artisan would have had a reasonable expectation of success of using the recombinant Pseudomonas putida expressing modified PETase-MHETase fusion protein with sequence identity identical do any one of SEQ ID NOs: 36, 38, or 40 to degrade PET and therefore polyester because Vague taught non-recombinant Pseudomonas putida already degrades PET and Yale taught polyester contains high amounts of PET. Since Vague taught the degradation of PET by bacteria comprises secreted PETase that breaks down PET to MHET which is further broken down to terephthalic acid (TPA) and ethylene glycol by secreted MHETase, Pseudomonas putida expressing the modified PETase-MHETase fusion protein with PETase and MHETase activity would only enhance the degradation of PET/polyester.
Consequently, the invention of claims 18-23 would have been obvious to one of ordinary skill in the art before the effective filing date.
Conclusion
No claims are in condition for allowance.
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 SCOTT E. MULDER whose telephone number is (571)272-2372. The examiner can normally be reached Monday - Friday 7:30 AM - 3:30 PM.
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/SCOTT E. MULDER/
Examiner, Art Unit 1656
/David Steadman/Primary Examiner, Art Unit 1656