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 .
Claims Status
Claims 1-23 are pending.
Claims 1 and 9 are amended.
Election/Restrictions
Applicant’s election without traverse of Group 1 (claims 1-8) in the reply filed on 06/25/2026 is acknowledged.
Applicant’s election without traverse of:
Species Group 1: TDHi engineered promoter
Species Group 2: S. cerevisiae Y294
Species Group 3: filtration
in the reply filed on 06/25/2026 is acknowledged.
Claims 9-23 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 06/25/2026.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 04/12/2024 is acknowledged. The submission is in compliance with the provision of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1 and 3-8 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1 (and claims 3-8 dependent on) is directed to a method for producing all possible cutinase-like enzymes (CLE1) in a S. cerevisiae cell, the method comprising a nucleic acid encoding the CLE1 in the cell, wherein the nucleic acid is codon optimized for expression in S. cerevisiae, further wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5’UTR intron; (claim 3) wherein the nucleic acid encodes all possible CLE1 that have a nucleotide sequence substantially identical to SEQ ID NO: 1 and (claim 4) wherein the engineered promoter is all possible TDHi engineered promoters having a nucleotide sequence substantially identical to SEQ ID NO: 9. The examiner notes that the specification defines “substantially identical” sequence as a nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions that do not destroy or substantially reduce the activity of one or more of the expressed polypeptides (see pg. 16-17). There is no disclosure of any particular structure to function/activity relationship of the disclosed species.
Cutinases and cutinase-like enzymes have recently been the subject of intense and increasing research because of their substrate diversity. A substantial number of cutinase genes have been identified and an increasing number of three-dimensional structures have been solved, however, most of the genes identified have been those of fungal cutinases; identification of cutinase genes from bacterial and other sources have been relatively limited. With this growing body of knowledge, it has become clear that there are substantial differences in structure and function between different sources Chen et al. (Biotechnology Advances, Vol. 31, pg. 1754-1767; published 2013, see pg. 1764, conclusion paragraph).
Regarding the level and skill and knowledge in the art of amino acid mutation, the reference of Singh et al. (Curr. Protein Pept. Sci. 18:1-11, 2017) reviews various protein engineering methods and discloses that despite the availability of an ever-growing database of protein structures and highly sophisticated computational algorithms, protein engineering is still limited by the incomplete understanding of protein functions, folding, flexibility, and conformational changes (see. pg. 7, column 1, top). Also, the unpredictability associated with amino acid mutations is exemplified by the reference of Zhang et al. (Structure 26: 1474-1485, 2018) which discloses that even a mutation of a surface residue that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide (p. 1475, column 1).
Describing the relationship between nucleotide sequence (genotype) and function (phenotype) lies at the heart of understanding biology. Currently, this is possible only locally in a narrow mutational neighborhood around a wild-type sequence rather than globally from any sequence. Direct experimental characterization of genotype-phenotype mapping has been demonstrated. However, existing technology limits experimental exploration to only a tiny fraction of all possible sequences (see Sarkisyan et al., Nature, Vol. 533, pg. 397-401; published 2016; PMID: 27193686 and Lagator et al. eLife, 11:e64543, published January 26, 2022, PMDI: 350980492).
Regarding the level of skill and knowledge in the art of mutagenesis of polynucleotides/promoters and effects on gene expression, gene expression is one of the most fundamental processes of life and tuning expression levels underpins complex biological functions. Computational and theoretical attempts to predict the relationship between genotype (polynucleotide/promoter sequence) and its phenotype (gene expression levels) have adopted broad approaches. Bioinformatics identifies promoters based on sequence homology but does not predict gene expression from them (see Lagator et al, eLife, 11:e64543, published January 26, 2022, PMID: 35080492). In summary, there is a lack of generalizable and predictive theoretical and biological understanding between promoter genotype and its function (gene expression phenotype) (see Lagator et al, eLife, 11:e64543, published January 26, 2022, PMID: 35080492).
Given this lack of additional representative species as encompassed by the claims, Applicants have failed to sufficiently describe the claimed invention, in such full, clear, concise, and exact terms that a skilled artisan would recognize Applicants were in possession of the claimed invention.
Claim 1 (and claims 3-8 dependent on) is rejected, because the specification, while being enabling for a method for producing a cutinase-like enzymes (CLE1) in a S. cerevisiae cell, the method comprising a nucleic acid encoding the CLE1 in the cell, wherein the nucleic acid is codon optimized for expression in S. cerevisiae, further wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5’UTR intron; wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5’UTR intron , (claim 3) wherein the nucleic acid encoding the CLE1 has a nucleotide sequence identical to SEQ ID NO: 1 and (claim 4) wherein the engineered promoter is a TDHi engineered promoter having a nucleotide sequence identical to SEQ ID NO: 9, does not reasonably provide enablement for a method for producing all possible cutinase-like enzymes (CLE1) in a S. cerevisiae cell, the method comprising a nucleic acid encoding the CLE1 in the cell, wherein the nucleic acid is codon optimized for expression in S. cerevisiae, further wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5’UTR intron; (claim 3) wherein the nucleic acid encodes all possible CLE1 that have a nucleotide sequence substantially identical to SEQ ID NO: 1 and (claim 4) wherein the engineered promoter is all possible TDHi engineered promoters having a nucleotide sequence substantially identical to SEQ ID NO: 9. (The examiner notes that the specification defines “substantially identical” sequence as a nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions that do not destroy or substantially reduce the activity of one or more of the expressed polypeptides (see pg. 16-17). The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required, are summarized in In re Wands (858 F.2d 731, 8 USPQ 2nd 1400 (Fed. Cir. 1988)) as follows: (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art , (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claim(s).
Claim 1 (and claims 3-8 dependent on) is so broad as to encompass a method for producing all possible cutinase-like enzymes (CLE1) in a S. cerevisiae cell, the method comprising a nucleic acid encoding the CLE1 in the cell, wherein the nucleic acid is codon optimized for expression in S. cerevisiae, further wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5’UTR intron; (claim 3) wherein the nucleic acid encodes all possible CLE1 that have a nucleotide sequence substantially identical to SEQ ID NO: 1 and (claim 4) wherein the engineered promoter is all possible TDHi engineered promoters having a nucleotide sequence substantially identical to SEQ ID NO: 9.
The claims rejected under this section of U.S.C. 112, first paragraph, place minimal structural limits on the required variant nucleotide sequences encompassed by the claims. Since the nucleotide sequence of a gene expressing a polypeptide or a promoter has an effect on gene expression, its sequence determines its structural (genotype) and functional properties (phenotype), predictability of which changes can be tolerated in a gene or promoters nucleotide sequence and obtain the desired activity requires a knowledge of and guidance with regard to which nucleotides in the sequence, if any, are tolerant of modification and which are conserved (i.e. expectedly intolerant to modification) and detailed knowledge of the ways in which the genes and promoters genotype relates to its function (phenotype). However, in this case the disclosure is limited to:
(claim 1 and claims 3-8 dependent on) a method for producing a cutinase-like enzymes (CLE1) in a S. cerevisiae cell, the method comprising a nucleic acid encoding the CLE1 in the cell, wherein the nucleic acid is codon optimized for expression in S. cerevisiae, further wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5’UTR intron, (claim 3) wherein the nucleic acid encoding the CLE1 has a nucleotide sequence identical to SEQ ID NO: 1 and (claim 4) wherein the engineered promoter is a TDHi engineered promoter having a nucleotide sequence identical to SEQ ID NO: 9.
While recombinant and mutagenesis techniques are known, it is not routine in the art to screen for multiple substitutions or multiple modifications, as encompassed by the instant claims, and the positions with a polynucleotide or promoter where nucleotide modifications can be made with a reasonable expectation of success in obtaining the desired activity/utility are limited in any polynucleotide or promoter and the result of such modifications is unpredictable. In addition, one skilled in the art would expect any tolerance to modification for a given polynucleotide or promoter to diminish with each further and additional modification, e.g. multiple substitutions, deletions, or insertions.
The specification does not support the broad scope of the claims which encompass any possible:
(claim 1 and claims 3-8 dependent on) method for producing all possible cutinase-like enzymes (CLE1) in a S. cerevisiae cell, the method comprising a nucleic acid encoding the CLE1 in the cell, wherein the nucleic acid is codon optimized for expression in S. cerevisiae, further wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5’UTR intron; (claim 3) wherein the nucleic acid encodes all possible CLE1 that have a nucleotide sequence substantially identical to SEQ ID NO: 1, and (claim 4) wherein the engineered promoter is all possible TDHi engineered promoters having a nucleotide sequence substantially identical to SEQ ID NO: 9 because the specification does not establish: (A) regions of the polynucleotide/promoter which may be modified affecting the polynucleotide of claims 1, 3, and 4: (B) the general tolerance of the polynucleotide and promoters of claims 1, 3, and 4 to modification and extent of such tolerance; (c) a rational and predictable scheme for modifying any nucleotide of the polynucleotide or promoter in claims 1, 3, and 4 with an expectation of obtaining the desired biological function; and (D) the specification provides insufficient guidance as to which of the essentially infinite possible choices is likely to be successful. Because of this lack of guidance, the extended experimentation that would be required to determine which substitutions would be acceptable to retain the function of the CLE1 expressed by the polynucleotide of claim 1 and 3 or the engineered promoter of claim 1 and 4 and the fact that the relationship between the sequence of a polynucleotide or promoter (genotype) and tis phenotype are not well understood are not predictable (see Ngo et al. in The Protein Folding Problem and Tertiary Structure Prediction, 1994, Merz et al. (ed.), Birkhause, Boston, MA, pg. 433 and 492-495; Fanceus et al., J. Ind. Microbiol. Biotechnol. Vol, 44, pp 687-695, 2017 and Lagator et al. eLife, 11:e64543, published January 26, 2022, PMID: 35080492), it would require undue experimentation for one skilled in the art to arrive at the majority of polynucleotides having the function of CLE1 in claim 1 and 3 and the engineered promoter in claim 1 and 4 of the claimed genus.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1 and 7-8 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017), hereinafter referred to as Zhang, in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016), hereinafter referred to as Hoshida.
With regards to claim 1, Zhang teaches a method for production of T. fusca cutinase in engineered S. cerevisiae where four different constitutive promoters, ADH1, HXT1, TEF1, and TDH3 were comparatively investigated in regards to cutinase production (operably linked) (see pg. 538, Abstract). Zhang further teaches that tfu gene encoding cutinase from T. fusca was firstly optimized and synthesized according to codon usage bias (codon optimized) of S. cerevisiae (see. pg. 541, left panel, top paragraph). Zhang teaches that the recombinant ScTDH3 accumulated cutinase to 9.04 U/mL and produced cutinase faster than other promoters confirming that TDH3 is a strong promoter in S. cerevisiae (see pg. 542, left panel, top paragraph).
With regards to claim 7, Zhang teaches culture cultivation conditions for the yeast (see pg. 539, Medium and culture conditions).
With regards to claim 8, Zhang teaches that to purify the recombinant cutinase, the fermentation culture was collected and centrifuged at 10,000 rmp for 20 min (see pg. 540, left panel, top paragraph). Zhang teaches that ammonium sulfate was added to the supernatant and the precipitated protein was collected by centrifugation. Zhang further teaches that the precipitated protein was dissolved in buffer, and dialyzed (see pg. 540, left panel, 2nd paragraph).
Zhang does that the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5’UTR intron.
However, Hoshida teaches that Saccharomyces cerevisiae is one of the most suitable microorganisms for recombinant protein production (see Abstract, pg. 241). Hoshida teaches that the effect of some introns on protein expression were analyzed (see Abstract, pg. 241). Hoshida teaches that RPS25A, RPS26A, and RPS26B contain single introns within the 5’-untransalted regions (5’UTRs) (see Abstract, pg. 241).. Hoshida teaches that intron promoters showed higher expression than the TDH3 promoter which is one of the strongest promoters in S. cerevisiae (see Abstract, pg. 241). Deletion of the introns from these promoters decreased the luciferase expression indicating that these promoters have role in enhancing protein expression (see Abstract, pg. 241). Hoshida teaches that a construct containing TDH3p followed by the RPS25A intron showed 50 fold higher expression than the TDPH3p (see Abstract, pg. 241).
It would have been obvious to one of ordinary skill in the art of protein engineering before the effective filing date of the current instant application to modify the engineered S. cerevisiae host that expresses T. fusca cutinase taught by Zhang by replacing the TDH3 promoter that is operably linked to the cutinase with the chimeric TDH3p-RPS25A intron taught by Hoshida since Hoshida teaches that the chimeric TDH3p-RPS25A shows 50 fold higher expression than the TDPH3p promoter. One of ordinary skill in the art would be motivated to make such changes to the S. cerevisiae host taught by Zhang since Hoshida teaches that TDH3p-RPS25A (comprising a 5’UTR intron) significantly improves protein expression and production in S. cerevisiae. One of ordinary skill in the art of protein engineering would have expectations of success in doing so since the combined teachings of Zhang and Hoshida provide all the methods and guidance to do so.
Therefore, claims 1, and 7-8 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017) in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016).
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017), hereinafter referred to as Zhang, in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016), hereinafter referred to as Hoshida, as applied to claim 1 above, and further in view of Masaki et al. (Applied and Environmental Microbiology, Vol. 71, pg. 7548-7550; published November 2005), hereinafter referred to as Masaki.
The teachings of Zhang and Hoshida as applied to claim 1 are summarized above.
With regards to claim 2, neither Zhang or Hoshida teach a cutinase-like enzyme that has the amino acid sequence of SEQ ID NO: 2 of the instant application.
However, Masaki teaches a purified lipase from the yeast Crytopcoccus sp. strain S-2 that exhibits remote homology to proteins belonging to the cutinase family (see pg. 7548, Abstract). Masaki teaches a nucleic acid sequence for the enzyme (see nucleotide sequence accession numbers, DDBJ accession number of CLE is AB10202945, pg. 7550) (see nucleotide sequence below) whose translated amino acid sequence has 100% sequence identity to SEQ ID NO: 2 of the current instant application (see amino acid sequence alignment below). Masaki teaches that the cutinase-like enzyme could effectively degrade biodegradable plastics (see pg. 7548, Abstract).
OCUS AB102945 720 bp mRNA linear PLN 05-OCT-2006
DEFINITION Cryptococcus sp. S-2 mRNA for cutinase-like protein, complete cds.
ACCESSION AB102945
VERSION AB102945.1
KEYWORDS .
SOURCE Cryptococcus sp. S-2
ORGANISM Cryptococcus sp. S-2
Eukaryota; Fungi; Dikarya; Basidiomycota; Agaricomycotina;
Tremellomycetes; Tremellales; Cryptococcaceae; Cryptococcus.
REFERENCE 1 (bases 1 to 720)
AUTHORS Masaki,K. and Iefuji,H.
TITLE Direct Submission
JOURNAL Submitted (07-FEB-2003)
Contact:Kazuo Masaki
National Research Institute of Brewing, Environmental research
division; 3-7-1 kagamiyama, Higashi-hiroshima, Hirosima 739-0046,
Japan
REFERENCE 2
AUTHORS Masaki,K., Kamini,N.R., Ikeda,H. and Iefuji,H.
TITLE Cutinase-Like Enzyme from the Yeast Cryptococcus sp. Strain S-2
Hydrolyzes Polylactic Acid and Other Biodegradable Plastics
JOURNAL Appl. Environ. Microbiol. 71 (11), 7548-7550 (2005)
COMMENT
FEATURES Location/Qualifiers
source 1..720
/db_xref="taxon:87049"
/mol_type="mRNA"
/organism="Cryptococcus sp. S-2"
/strain="S-2"
CDS 1..720
/codon_start=1
/product="cutinase-like protein"
/protein_id="BAC67242.1"
/transl_table=1
/translation="MLVSALALAVLSAASLGRAAPTPESAEAHELEARATSSACPQYV
LINTRGTGEPQGQSAGFRTMNSQITAALSGGTIYNTVYTADFSQNSAAGTADIIRRIN
SGLAANPNVCYILQGYSQGAAATVVALQQLGTSGAAFNAVKGVFLIGNPDHKSGLTCN
VDSNGGTTTRNVNGLSVAYQGSVPSGWVSKTLDVCAYGDGVCDTAHGFGINAQHLSYP
SDQGVQTMGYKFAVNKLGGSA"
BASE COUNT 136 a 253 c 212 g 119 t
ORIGIN
1 atgctcgtct ccgctctcgc tctcgcggtg ctgtccgctg cttctctcgg ccgagccgca
61 ccaacgcccg agtccgccga ggcgcacgag ctcgaggccc gcgccacgtc cagcgcttgt
121 ccgcagtacg tcctgatcaa cacgcgaggc acgggcgagc cgcaaggcca gtcggccggc
181 ttccgaacga tgaacagcca gatcaccgcc gcgctgtcgg gtggcaccat ctacaacact
241 gtctacaccg ccgatttcag ccagaacagc gcggccggca cggccgacat catccgccgg
301 atcaactcgg gtctcgcggc caacccgaac gtgtgctaca tcctccaagg gtacagccag
361 ggcgcggctg ctaccgtcgt cgcgctgcaa cagctcggca cgagtggagc ggcgttcaac
421 gccgtcaagg gtgtgttcct cattggcaac ccggaccaca agtcgggcct gacttgcaac
481 gtcgactcga acggcggcac taccacacgc aatgtcaacg gcctgtcggt cgcgtaccag
541 ggctcggtcc cctcaggatg ggtcagcaag actctcgatg tctgcgctta tggcgacggc
601 gtgtgcgaca ccgcgcacgg attcggtatc aacgcacagc acctgtcgta ccctagtgac
661 caaggcgtcc agaccatggg atacaagttt gccgtcaaca agcttggcgg gtcggcctaa
//
SEQUENCE ALIGNMENT OF AMINO ACID RESIDUES
Title: US-18-701-026-2
Perfect score: 1223
Sequence: 1 MLVSALALAVLSAASLGRAA..........DQGVQTMGYKFAVNKLGGSA 239
Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5
Searched: 1 seqs, 239 residues
Total number of hits satisfying chosen parameters: 1
Minimum DB seq length: 0
Maximum DB seq length: inf
Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 50 summaries
Database : NASEQ2_07022026_154252.fasta:*
SUMMARIES
%
Result Query
No. Score Match Length DB ID Description
----------------------------------------------------------------------------
1 1223 100.0 239 1 NASEQ2_07022026_154252
ALIGNMENTS
RESULT 1
NASEQ2_07022026_154252
Query Match 100.0%; Score 1223; DB 1; Length 239;
Best Local Similarity 100.0%;
Matches 239; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MLVSALALAVLSAASLGRAAPTPESAEAHELEARATSSACPQYVLINTRGTGEPQGQSAG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MLVSALALAVLSAASLGRAAPTPESAEAHELEARATSSACPQYVLINTRGTGEPQGQSAG 60
Qy 61 FRTMNSQITAALSGGTIYNTVYTADFSQNSAAGTADIIRRINSGLAANPNVCYILQGYSQ 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 FRTMNSQITAALSGGTIYNTVYTADFSQNSAAGTADIIRRINSGLAANPNVCYILQGYSQ 120
Qy 121 GAAATVVALQQLGTSGAAFNAVKGVFLIGNPDHKSGLTCNVDSNGGTTTRNVNGLSVAYQ 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 GAAATVVALQQLGTSGAAFNAVKGVFLIGNPDHKSGLTCNVDSNGGTTTRNVNGLSVAYQ 180
Qy 181 GSVPSGWVSKTLDVCAYGDGVCDTAHGFGINAQHLSYPSDQGVQTMGYKFAVNKLGGSA 239
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GSVPSGWVSKTLDVCAYGDGVCDTAHGFGINAQHLSYPSDQGVQTMGYKFAVNKLGGSA 239
It would have been obvious to one of ordinary skill in the art of protein engineering before the effective filing date of the current instant application to use the cutinase-like enzyme taught by Masaki as a cutinase-like enzyme in expression method in the S. cerevisiae host taught by the combined teachings of Zhang and Hoshida for heterologous expression of the cutinase-like enzyme in order achieve improved protein expression. One of ordinary skill in the art would further be motivated to express the cutinase-like enzyme in S. cerevisiae since Masaki teaches a cutinase-like enzyme from yeast that hydrolyzes polylactic acid and other biodegradable plastics (see pg. 7548, Abstract) and therefore one would benefit from higher expression levels. One of ordinary skill in the art of protein engineering would have expectations of success in doing so from the guidance from the combined teachings of Zhang, Hoshida, and Masaki who teach all the methods and materials needed to do so.
Therefore, claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017) in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016) as applied to claim 1 above, and further in view of Masaki et al. (Applied and Environmental Microbiology, Vol. 71, pg. 7548-7550; published November 2005).
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017), hereinafter referred to as Zhang, in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016 ), hereinafter referred to as Hoshida. as applied to claim 1 above, and further in view of Iefuji et al. (JP2004073123A; published March 03, 2004), hereinafter referred to as Iefuji.
The teachings of Zhang and Hoshida as applied to claim 1 are summarized above.
With regards to claim 3, neither Zhang or Hoshida teach a cutinase-like enzyme (CLE1) that has a nucleotide sequence substantially identical to SEQ ID NO.1 of the instant application.
However, Iefuji teaches a lipase CS2 enzyme that has a nucleotide sequence (SEQ ID NO. 2) substantially identical (shares 50.9% sequence identity) to SEQ ID NO.1 of the instant application. Iefuji teaches that the enzyme is a cutinase that can be widely used for a decomposer for biodegradable plastics (see pg. 1 of translated document) (see sequence alignment below). Note that the examiner is interpreting the meaning of “substantially identical” as what is set forth in the current instant application specification, pg. 16-17, which states that “herein a substantially identical sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substituti9ons, or by one or more non-conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions at positions of the sequence that do not destroy or substantially reduce the activity of one or more of the expressed polypeptides or of the polypeptides encoded by the nucleic acid molecules” (see specification, pg. 16-17).
RESULT 3
ADN00232
(NOTE: this sequence has 4 duplicates in the database searched.
See complete list at the end of this report)
ID ADN00232 standard; DNA; 720 BP.
XX
AC ADN00232;
XX
DT 11-JUN-2007 (revised)
DT 17-JUN-2004 (first entry)
XX
DE Lipase CS2 enzyme DNA.
XX
KW lipase CS2; enzyme; cDNA*pG-1; liquid decomposition agent;
KW biodegradable plastic; polylactic acid; PLA; polybutylene succinate; PBS;
KW polycaprolactone; PCL; biodiesel fuel; food; beverage; detergent;
KW cosmetics; organic solvent; environmental pollution; ds; gene.
XX
OS Cryptococcus sp. S-2.
XX
FH Key Location/Qualifiers
FT CDS 1..720
FT /*tag= a
FT /product= "Lipase CS2 enzyme"
XX
CC PN JP2004073123-A.
XX
CC PD 11-MAR-2004.
XX
CC PF 20-AUG-2002; 2002JP-00239842.
XX
PR 20-AUG-2002; 2002JP-00239842.
XX
CC PA (DOKU-) DOKURITSU GYOSEI HOJIN SHURUI SOGO KENKY.
XX
DR WPI; 2004-233086/22.
DR P-PSDB; ADN00231.
DR PC:NCBI; gi29467702.
DR PC_ENCPRO:NCBI; gi29467703.
XX
CC PT New lipase CS2 enzyme derived from Cryptococcus species S-2 strain,
CC PT useful as decomposition agent of biodegradable plastics e.g., polylactic
CC PT acid.
XX
CC PS Claim 3; SEQ ID NO 2; 10pp; Japanese.
XX
CC The invention relates to a novel lipase CS2 enzyme having a fully defined
CC sequence of 239 amino acids as given in the specification. The invention
CC further comprises: a DNA of the gene encoding the lipase CS2 enzyme,
CC comprising a fully defined sequence of 720 nucleotides as given in the
CC specification; and a recombinant vector CS2 lipase cDNA*pG-1 comprising
CC at least a coding region of the lipase CS2 enzyme gene. The recombinant
CC vector is useful in producing the protein which has lipase CS2 activity,
CC which involves introducing the vector into a microorganism, and
CC recovering the enzyme from the cells. The lipase CS2 enzyme is useful as
CC a liquid decomposition agent of biodegradable plastics such as polylactic
CC acid (PLA), polybutylene succinate (PBS) and polycaprolactone (PCL). The
CC lipase CS2 enzyme is also useful in the industrial manufacturing of
CC biodiesel fuel. The lipase CS2 enzyme is useful as a pharmaceutical
CC component, a food/beverage-products component and also useful in
CC detergents and in cosmetics. The lipase CS2 enzyme has efficient lipase
CC CS2 activity and has excellent plastic decomposing activity. The lipase
CC CS2 enzyme is more stable in organic solvents and effective in preventing
CC environmental pollution. This polynucleotide sequence represents the DNA
CC encoding the lipase CS2 enzyme protein of the invention
CC
CC Revised record issued on 11-JUN-2007 : Enhanced with precomputed
CC information from BOND.
XX
SQ Sequence 720 BP; 136 A; 253 C; 212 G; 119 T; 0 U; 0 Other;
Query Match 50.9%; Score 366.4; Length 720;
Best Local Similarity 69.3%;
Matches 499; Conservative 0; Mismatches 221; Indels 0; Gaps 0;
Qy 1 ATGTTGGTTTCAGCATTGGCTTTAGCAGTTTTGTCTGCTGCATCATTAGGTAGAGCTGCA 60
||| | || || || | ||| | || || |||| ||||| || | || |||| |||
Db 1 ATGCTCGTCTCCGCTCTCGCTCTCGCGGTGCTGTCCGCTGCTTCTCTCGGCCGAGCCGCA 60
Qy 61 CCAACACCAGAATCTGCTGAAGCACATGAATTGGAAGCTAGAGCAACTTCTTCAGCTTGT 120
||||| || || || || || || || || | || || | || || || ||||||
Db 61 CCAACGCCCGAGTCCGCCGAGGCGCACGAGCTCGAGGCCCGCGCCACGTCCAGCGCTTGT 120
Qy 121 CCACAATACGTTTTGATTAATACAAGAGGTACTGGTGAACCACAAGGTCAATCAGCTGGT 180
|| || ||||| |||| || || |||| || || || || ||||| || || || ||
Db 121 CCGCAGTACGTCCTGATCAACACGCGAGGCACGGGCGAGCCGCAAGGCCAGTCGGCCGGC 180
Qy 181 TTTAGAACAATGAACTCTCAAATTACTGCTGCATTATCAGGTGGTACAATCTATAACACA 240
|| |||| |||||| || || || || || | || ||||| || ||||| |||||
Db 181 TTCCGAACGATGAACAGCCAGATCACCGCCGCGCTGTCGGGTGGCACCATCTACAACACT 240
Qy 241 GTTTACACTGCTGATTTCTCTCAAAATTCAGCTGCAGGTACTGCAGATATCATCAGAAGA 300
|| ||||| || |||||| || || || || || || || || |||||| | |
Db 241 GTCTACACCGCCGATTTCAGCCAGAACAGCGCGGCCGGCACGGCCGACATCATCCGCCGG 300
Qy 301 ATTAATTCTGGTTTGGCTGCAAACCCAAACGTTTGTTACATCTTGCAAGGTTACTCACAA 360
|| || || ||| | || || ||||| ||||| || |||||| | ||||| ||| ||
Db 301 ATCAACTCGGGTCTCGCGGCCAACCCGAACGTGTGCTACATCCTCCAAGGGTACAGCCAG 360
Qy 361 GGTGCTGCAGCTACAGTTGTTGCTTTGCAACAATTAGGTACTTCTGGTGCAGCTTTTAAT 420
|| || || ||||| || || || ||||||| | || || ||| || || || ||
Db 361 GGCGCGGCTGCTACCGTCGTCGCGCTGCAACAGCTCGGCACGAGTGGAGCGGCGTTCAAC 420
Qy 421 GCAGTTAAGGGTGTTTTCTTGATCGGTAACCCAGATCATAAGTCTGGTTTGACATGTAAC 480
|| || |||||||| ||| | || || ||||| || || ||||| || |||| || |||
Db 421 GCCGTCAAGGGTGTGTTCCTCATTGGCAACCCGGACCACAAGTCGGGCCTGACTTGCAAC 480
Qy 481 GTTGATTCAAATGGTGGTACTACAACTAGAAATGTTAATGGTTTGTCTGTTGCTTATCAA 540
|| || || || || || ||||| || | ||||| || || |||| || || || ||
Db 481 GTCGACTCGAACGGCGGCACTACCACACGCAATGTCAACGGCCTGTCGGTCGCGTACCAG 540
Qy 541 GGTTCTGTTCCATCAGGTTGGGTTTCAAAAACATTAGATGTTTGTGCTTACGGTGACGGT 600
|| || || || ||||| ||||| || || | ||||| || ||||| || |||||
Db 541 GGCTCGGTCCCCTCAGGATGGGTCAGCAAGACTCTCGATGTCTGCGCTTATGGCGACGGC 600
Qy 601 GTTTGTGATACTGCTCATGGTTTCGGTATTAATGCACAACATTTGTCTTATCCATCAGAT 660
|| || || || || || || |||||||| || ||||| || |||| || || ||
Db 601 GTGTGCGACACCGCGCACGGATTCGGTATCAACGCACAGCACCTGTCGTACCCTAGTGAC 660
Qy 661 CAAGGTGTTCAAACTATGGGTTACAAGTTCGCTGTTAATAAGTTGGGTGGTTCTGCATAA 720
||||| || || || ||||| |||||||| || || || ||| | || || || || |||
Db 661 CAAGGCGTCCAGACCATGGGATACAAGTTTGCCGTCAACAAGCTTGGCGGGTCGGCCTAA 720
Therefore, it would have been obvious to one of ordinary skill in the art of protein engineering before the effective filing date of the current instant application to use the lipase CS2 enzyme (SEQ ID NO.2) taught by Iefuji as an optional cutinase-like enzyme for expression in the S. cerevisiae cell taught by the combined teachings of Zhang and Hoshida. One of ordinary skill in the art would be motivated to do so since Iefuji teaches a cutinase-like enzyme that can be used as a decomposer of biodegradable plastic. One of ordinary skill in the art of protein engineering would have expectations of success in doing so as the combined teachings of Zhang, Hoshida, and Iefuji provide all the guidance and methods needed to do so.
Therefore, claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017) in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016) as applied to claim 1 above, and further in view of Iefuji et al. (JP2004073123A; published March 03, 2004).
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017), hereinafter referred to as Zhang, in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016 ), hereinafter referred to as Hoshida, as applied to claim 1 above, and further in view of West et al. (The FASEB Journal, Vol. 23, pg. 1694-704; published February 18, 2009; PMID: 19225166), hereinafter referred to as West.
The teachings of Zhang and Hoshida as applied to claim 1 are summarized above.
With regards to claim 5, neither Zhang or Hoshida teach a CLE1 that comprises a secretion signal.
However, West teaches a Culp1 cutinase-like protein which has a putative secretion signal (see pg. 1995, left panel, top paragraph) and has enzymatic activity for for short-chain fatty acids (see pg. 1702, left panel, 2nd paragraph). West teaches that Culp2 was detected in the cytoplasm and the majority of the protein is destined for immediate secretion (see pg. 1700, right panel, top paragraph).
It would have been obvious to one of ordinary skill in the art of protein engineering before the effective filing date of the current instant application to select the Culp1 taught by West as a cutinase-like enzyme for expression in the S. cerevisiae host of the combined teachings of Zhang and Hoshida. One of ordinary skill in the art would be motivated to do so by the teachings of West who teaches that Culp1 is a cutinase-like enzyme having a secretion signal and by the teachings of Zhang , and West whose combined teaching provide a S. cerevisiae host for improved recombinant protein expression. One of ordinary skill in the art of protein engineering would have expectations of success in doing so as the combined teachings of Zhang, Hoshida, and West provide all the guidance and materials to do so.
Therefore, claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017) in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016 ) as applied to claim 1 above, and further in view of West et al. (The FASEB Journal, Vol. 23, pg. 1694-704; published February 18, 2009; PMID: 19225166).
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017), hereinafter referred to as Zhang, in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016), hereinafter referred to as Hoshida, as applied to claim 1 above, and further in view Cripwell et al. (FEMS Yeast Research, Vol. 19(2); published March 1, 2019; PMID: 30535120), hereinafter referred to as Cripwell.
The teachings of Zhang and Hoshida as applied to claim 1 are summarized above.
With regards to claim 6, neither Zhang or Hoshida teach that the S. cerevisiae cell is the strain S. cerevisiae Y294.
However, Cripwell teaches that codon-optimized glucoamylase variants from T. emersonii expressed in S. cerevisiae Y294 laboratory strain yielded higher extracellular activity. (see pg. 1, Abstract).
It would have been obvious to one of ordinary skill in the art of protein engineering before the effective filing date of the current instant application that the S. cerevisiae Y294 strain taught by Cripwell can serve as an optional S. cerevisiae strain in the expression method of the codon-optimized cutinase-like enzymes described by the combined teachings of Zhang and Hoshida to achieve heterologous protein expression. One of ordinary skill in the art of protein engineering would be motivated by the teachings of Cripwell to use the S. cerevisiae cell for heterologous protein expression since Cripwell demonstrates its utility for expression of codon-optimized recombinant genes. One of ordinary skill in the art of protein engineering would have expectations of success in doing so from the combined teachings of Zhang, Hoshida, and Cripwell who provide all the teachings and guidance needed to do so.
Therefore, claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Journal of Microbiology, Vol. 55, pg. 538-544; published 2017), in view of Hoshida et al. (Applied Microbiol. Biotechnol. Vol. 101, pg. 241-251; published online October 12, 2016) as applied to claim 1 above, and further in view Cripwell et al. (FEMS Yeast Research, Vol. 19(2); published March 1, 2019; PMID: 30535120).
Conclusion
No claims are allowed.
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/GEORGE THEMISTOCLIS LOUNTOS/ Examiner, Art Unit 1652
/ROBERT B MONDESI/ Supervisory Patent Examiner, Art Unit 1652
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