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 11/10/2025 is acknowledged. Claims 1-14 are pending. Claim 1 was amended. New claims 13 and 14 were added. Claims 8-12 are withdrawn as being directed to a nonelected invention. Claims 1-7 are under examination herein.
Withdrawn Objections and Rejections
The sequence listing filed 11/10/2025 is acknowledged and accepted.
The rejection of claims 1-7 under 35 U.S.C. § 112(b) is withdrawn.
New and modified rejections necessitated by amendment
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 1-4 and 13-14 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al., US 2020/0232000 A1 in view of Zhang et al,. Amb Express; 7(1):105.
Regarding claim 1, Li teaches a recombinant microorganism for production of phenylacetic acid by culturing on a fermentable carbon substrate, i.e. glucose (Li “Filed of Invention” para. 1; pg. 8 para. 134). Li teaches that the microorganism can be engineered to overexpress genes of a midstream module to convert L-phenylalanine to styrene, comprising a phenylalanine ammonia lyase (AtPAL2) and a phenylacrylic acid decarboxylase (AnPAD or AnFDC) (Li pg. 7 para. 126; pg. 16 para. 169; Fig. 1). AnPAD and AnFDC catalyze decarboxylation of trans-cinnamic acid (Li Fig. 2; pg. 16 para. 169). Li additionally teaches that the microorganism is engineered to overexpress a downstream module to convert styrene to styrene oxide (SO) by styrene monooxygenase (SMO), which is then converted to phenylacetaldehyde (PA) by styrene oxide isomerase, which can be oxidized to phenylacetic acid (PAA) by an aldehyde dehydrogenase (Li pg. 7 para. 126; Fig. 1). Li further teaches that the aldehyde dehydrogenase to convert styrene to PAA is phenylacetaldehyde dehydrogenase (EcALDH) (Li pg. 10 para. 145). Li teaches that all the enzymes responsible for the reactions are co-expressed in one E. coli strain (Li pg. 8 para. 127).
Regarding claim 2, Li teaches that the organism is Escherichia coli (Li pg. 3 para. 56).
Regarding claim 3, Li teaches using a strain engineered for producing phenylalanine (L-Phe) in high concentrations (i.e. overproducing) by expressing the upstream shikimate pathway (Li pg. 7 para. 126, Fig. 1).
Regarding claim 4, Li teaches that the gene encoding a polypeptide having phenylalanine ammonia lyase activity, AtPAL2, is from Arabidopsis thaliana (Li pg. 2 para. 20).
Li does not teach that the organism is not capable of producing 2-phenylethanol by an enzyme having 2-phenylacetaldehyde reductase (PAR) activity (claim 1) or that the endogenous gene encoding an enzyme with PAR activity is deleted or lacking (claims 13-14).
Regarding claim 1, Zhang teaches metabolic engineering of E. coli for the production of phenylacetic acid, or PAA (Zhang “Abstract”). Zhang teaches a recombinant organism expressing phenylacetaldehyde dehydrogenase (Zhang p. 4 first partial para.). Zhang teaches that in the process of producing PAA in E. coli, 2-phenylethanol is converted from phenylacetaldehyde by phenylacetaldehyde reductase (PAR). Zhang teaches that a knock-out of phenylacetaldehyde reductase improves PAA production in E. coli (Zhang pp. 4-5 “Construction of the PAA biosynthesis pathway from l‑phenylalanine in E. coli” para. 2). Zhang teaches that PAA is an important compound in many industries and demand for PAA is high, and recombinant microorganisms are sustainable alternatives for the production of chemicals like PAA (Zhang p. 5 “Discussion para. 1-2).
Regarding claims 13 and 14, Zhang teaches an E. coli strain in which the endogenous gene encoding PAR is knocked out, or deleted (Zhang pp. 4-5 “Construction of the PAA biosynthesis pathway from l‑phenylalanine in E. coli” para. 2). The limitation in claim 14 “wherein the organism lacks an endogenous gene encoding an enzyme having PAR activity”, under broadest reasonable interpretation, reads on an organism wherein the endogenous gene has been deleted, because if the gene is deleted the organism would necessarily lack an endogenous gene having PAR activity. Therefore, the deleted PAR gene of Zhang reads on claims 13 and 14.
It would have been obvious to a skilled artisan, before the effective filing date, to create a recombinant microorganism comprising all of the heterologous genes (i)-(v) as disclosed by Li and which is not capable of producing 2-phenylethanol by an enzyme having PAR activity. Li teaches that the recombinant microorganism comprising the claimed genes is used for bioproduction of 2-Phenylethanol (2-PE), phenylacetaldehyde (PA), phenylacetic acid (PAA), and phenylethylamine (PEA) (Li p. 1 para. 2). Zhang teaches that knocking out or eliminating PAR in E. coli improves PAA production. Thus, a skilled artisan would have found it obvious to combine these teachings, which are both directed to engineered microorganisms for PAA production, and create a strain as taught by Li with no PAR and no phenylethanol production.
A person of ordinary skill in the art would have been motivated to modify the organism of Li to create an organism that is not capable of producing phenylethanol by eliminating PAR activity because Zhang teaches that knocking out PAR in E. coli is beneficial for PAA production. Zhang further teaches that PAA is in high demand industrially and sustainable and efficient production process are needed, and phenylethanol production by PAR leads to reduced PAA production (Zhang pp. 4-5 “Construction of the PAA biosynthesis pathway from l‑phenylalanine in E. coli” para. 2). Therefore, a skilled artisan would have been motivated to create a strain which does not produce phenylethanol via PAR, as this would be beneficial in using the strain to produce PAA.
A skilled artisan would have a reasonable expectation of success in creating a strain as taught by Li and additionally with no phenylethanol production, because Zhang teaches that deletion of PAR in E. coli leads to a lack of phenylethanol production and better PAA production. A skilled artisan could reasonably expect that in a strain taught by Li, which is also E. coli, the PAR gene could be successfully deleted with the same result as taught by Zhang.
Claims 5 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Li and Zhang as applied to claims 1-4 and 13-14 above, and further in view of Nielsen et al., US 2014/0057325 A1.
Li and Zhang teach the organism of claim 1 as set forth above. Li teaches an enzyme with trans-cinnamic acid decarboxylase activity from Aspergillus niger for converting trans-cinnamic acid to styrene, and an enzyme with styrene monooxygenase activity from Pseudomonas sp. VLB120 for converting styrene to styrene oxide (Li Fig. 2-3; pg. 2 para. 14 and 20).
Li does not teach that gene encoding the enzyme with trans-cinnamic acid decarboxylase activity is derived from Saccharomyces cerevisiae as set forth in claim 5, or that the gene encoding the enzyme having styrene monooxygenase activity is derived from Pseudomonas putida as set forth in claim 6.
Regarding claims 5 and 6, Nielsen teaches recombinant microorganisms for producing styrene and styrene oxide (Nielsen “Field of the Invention”; pg. 2 para. 11). Nielsen teaches that the microorganism expresses a gene encoding a polypeptide with trans-cinnamate decarboxylase (CADC) activity to convert trans-cinnamic acid to styrene; and a gene encoding a polypeptide with styrene monooxygenase activity to covert styrene to styrene oxide (Nielsen pg. 2 para. 11). Nielsen teaches that genes having CADC activity include phenylacrylic acid decarboxylases and ferulic acid decarboxylases, including PAD1 or FDC1 derived from Saccharomyces cerevisiae (Nielsen pg. 5 para. 139; pg. 6 para. 147; pg. 9 para. 169) and that the gene for styrene monooxygenase is derived from Pseudomonas putida (Nielsen para. 84).
It would have been obvious to a skilled artisan to combine the teachings of these references and use the trans-cinnamic acid decarboxylase from S. cerevisiae and the styrene monooxygenase from P. putida in the recombinant E. coli strain as taught by Li and Zhang. Li teaches an enzyme with trans-cinnamic acid decarboxylase activity from Aspergillus niger, and an enzyme with styrene monooxygenase activity from Pseudomonas sp. VLB120. It is considered prima facie obvious to use the enzymes taught by Nielsen in place of the enzymes taught by Li, as this would be a simple substitution of one known element for another of the same function, with a reasonable expectation of obtaining predictable results.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Li and Zhang as applied to claims 1-4 and 13-14 above, and further in view of Miyamoto et al., Tetrahedron letters. 2007; 48(18):3255-7.
Li and Zhang teach the organism of claim 1 as set forth above. Li teaches that the styrene oxide isomerase from Pseudomonas sp. VLB120 expressed in the recombinant strain catalyzes the conversion of styrene oxide to phenylacetaldehyde (PA) (Li Fig. 4).
Li does not teach that the gene encoding a polypeptide with styrene oxide isomerase activity is derived from Pseudomonas putida (claim 7).
Regarding claim 7, Miyamoto teaches that styrene oxide isomerase from Pseudomonas putida is an enzyme in the metabolic pathway of styrene which catalyzes the conversion of styrene oxide to phenylacetaldehyde (Miyamoto pg. 3255 para. 1-2; Scheme 2).
It would have been obvious to a skilled artisan to combine the teachings of these references and use the styrene oxide isomerase from P. putida in the recombinant strain as taught by Li and Zhang. Li teaches a styrene oxide isomerase from another Pseudomonas strain, sp. VLB120. It is considered prima facie obvious to use the enzyme taught by Miyamoto in place of the enzyme taught by Li, as this would be a simple substitution of one known element for another of the same function, with a reasonable expectation of obtaining predictable results.
Response to Arguments
In light of amendments to the claims, the rejection of claims 1-4 under 35 U.S.C. § 102 has been withdrawn. However, upon further consideration, new grounds of rejection of claims 1-4 and 13-14 are made under 35 U.S.C. § 103 in view of Li and Zhang as set forth above. Given these new grounds of rejection, the arguments presented regarding claims 1-4 rejected under 35 U.S.C. § 102 are moot.
Conclusion
Claims 1-7 and 13-14 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