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 6/3/2026 is acknowledged. Claims 1-16 are pending. Claims 1-14 were amended. New claim 16 was added. Claims 4-5 and 15 are withdrawn as being directed to a nonelected invention.
Information Disclosure Statement
The information disclosure statements (IDS) filed on 4/30/2026, 6/3/2026, and 8/11/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Withdrawn Rejections
The rejections of claim 14 under 35 U.S.C. § 112(a) and of claims 1-3 and 6-14 under 35 U.S.C. § 112(b) are withdrawn.
The rejection of claims 1-3, 6-11, and 13-14 under 35 U.S.C. § 102 is withdrawn, as claim 1 has been amended to remove the parentheses around ARO10, aroF, and tyrA, and thus these specific genes are now required.
New and modified objections and rejections necessitated by amendment
Claim Objections
Claim 7 is objected to because of the following informalities: claim 7 recites “the only
heterologously expressed genes of the transgenic bacterial cell is phenylpyruvate decarboxylase”. This should instead recite “the only heterologously expressed gene of the transgenic bacterial cell is phenylpyruvate decarboxylase”. Appropriate correction is required.
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 1-3, 6-11, 13-14, and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Yang et al., Journal of Agricultural and Food Chemistry; 67(14):3900-8, in view of Koma et al., Applied and Environmental Microbiology; 78(17):6203-16; and as evidenced by Cohrt Bitesize Bio; Millipore Sigma T7 promoter system; Sigma-Aldrich M9 Minimal Salts Medium and NCBI phenylpyruvate decarboxylase aro10 gene, NP_010668.3.
Regarding claim 1, Yang teaches a method of producing tyrosol using transgenic bacteria (Yang “Abstract”). Yang teaches modified strain E. coli BFPG2, which produces tyrosol (p. 3905 Fig. 4).
Yang teaches that E. coli BFPG2 heterologously expresses a phenylpyruvate decarboxylase, ARO10 (p. 3900 para. 4; p. 3902 Table 3).
Yang teaches that E. coli BFPG2 additionally overexpresses a phospho-2-dehydro-3-deoxyheptonate aldolase gene, aroG*; and a prephenate dehydratase gene, tyrA* (p. 3901 first partial para.; p. 3902 Table 3 E. coli BFPG2). Yang teaches that the phospho-2-dehydro-3-deoxyheptonate aldolase AroG is the isoenzyme (i.e., catalyzes same reaction) of AroF (p. 3904 “Initial Pathway Construction”). Yang teaches that the prephenate dehydrogenase activity of TyrA can be inhibited by aromatic amino acids, but mutations of Met53Ile and Ala354Val of TyrA eliminate the inhibitory effect (p. 3906 “Discussion” para. 1). The gene tyrA* encodes the enzyme having these mutations, and tyrA* is overexpressed in E. coli BFPG2.
Yang teaches that E. coli BFPG2 has deletions of, or does not express, pheA (Tables 1 and 3, GenBank ID 947081; chorismate mutase/prephenate dehydratase) and feaB (Tables 1 and 3, GenBank ID 945933; phenylacetaldehyde dehydrogenase).
Yang teaches that this strain is grown in a medium comprising glucose, which is a metabolic precursor of PEP and E4P (p. 3901 “Media and Culture Conditions”). Yang teaches that tyrosol is extracted from the fermentation broth or culture medium (p. 3901-3902 “Media and Culture Conditions”; “HPLC Analysis”).
Regarding claim 2, Yang teaches that the transgenic bacterial cell is of the genus Escherichia, E. coli BL21 (p. 3901 “Strains and Plasmids”).
Regarding claim 3, Yang teaches that the ARO10 gene encoding the phenylpyruvate decarboxylase originates from the yeast S. cerevisiae (p. 3901 Table 1).
Regarding claim 6, the strain E. coli BFPG2 of Yang does not overexpress genes encoding alcohol dehydrogenase, DNA-binding transcriptional regulatory protein (tyrR), and tyrosine aminotransferase (p. 3902 Table 3, showing all genes which are overexpressed in BFPG2).
Regarding claim 7, “heterologously expressed” as defined on p. 1 of the instant specification “means that the gene is derived from a source other than the host species in which it is said to be heterologously expressed”. E. coli BFPG2 as taught by Yang expresses aro10 from S. cerevisiae, and aroA, tyrA*, aroE, and aroG derived from E. coli (Yang p. 3901 Table 1; p. 3902 Table 3). Thus, the only heterologously expressed gene in the cell is phenylpyruvate decarboxylase, aro10.
Regarding claim 8, Yang teaches that the overexpressed genes are introduced into the transgenic cell via plasmid vectors, including pETDuet-1 plasmids, which have a copy number 40, or pCDFDuet, which has a copy number of 20 (p. 3901 “Strains and Plasmids”; p. 3904 “Effects of the Coordinated Expression of the Key Genes from Modules 2 and 3 on Tyrosol Production”; p. 3905 “Effects of the Expression Levels of the Key Genes aroA and tyrA* from Module 2 on Tyrosol Production”). Medium copy number plasmids are considered to have average copy numbers of 20-100 (see Cohrt p. 2). Thus, pETDuet-1 and pCDFDuet are considered medium copy number plasmids. Yang teaches various combinations of genes expressed on plasmids having different copy numbers to optimize tyrosol production (Yang Tables 2 and 3; Fig. 4). It would have been obvious for a skilled artisan to express all of phenylpyruvate decarboxylase, phospho-2-dehydro-3-deoxyheptonate aldolase, and prephenate dehydrogenase on medium copy plasmids, as Yang teaches the use of medium copy plasmids and teaches that the type of plasmid used to express each gene and plasmid copy number is optimized depending on the desired experimental outcome.
Regarding claims 9 and 16, Yang teaches that the genes encoding phenylpyruvate decarboxylase, phospho-2-dehydro-3-deoxyheptonate aldolase, and prephenate dehydrogenase are under the control of a T7 promoter (p. 3901 “Strains and Plasmids”). The T7 promoter has a sequence according to instant SEQ ID NO: 31 (see Millipore Sigma reference).
Regarding claim 10, Yang teaches that the expression of heterologous and/or overexpressed genes is induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG) (p. 3902 “Media and Culture Conditions”).
Regarding claim 11, Yang teaches that the medium comprises 1% w/v of glucose, which is equal to 1g/100mL, or 10 g/L (p. 3901 “Media and Culture Conditions”).
Regarding claim 13, Yang teaches M9Y minimal medium containing 1x M9 minimal salts, 5 mM MgSO4, 0.1 mM CaCl2, 1% w/v glucose, and 0.025% w/v yeast extract, with kanamycin (antibiotic) final concentration of 50 μg/mL (p. 3901 “Media and Culture Conditions”).
0.1 mM CaCl2, which has a molar mass of 110.98 g/mol, is equivalent to 0.011 g/L CaCl2.
5x M9 minimal salts from Sigma-Aldrich (see Sigma-Aldrich reference, p. 3) contains:
33.9 g/L Na2HPO4-
15 g/L KH2PO4
2.5 g/L NaCl
5 g/L NH4Cl
Thus, a 1x solution of M9 minimal salts, as used by Yang, comprises:
6.78 g/L Na2HPO4-
3 g/L KH2PO4
0.5 g/L NaCl
1 g/L NH4Cl
All of these values are within the claimed concentration ranges. As such, the medium
taught by Yang anticipates the claimed medium.
Regarding claim 14, given the use of “and/or” it is considered that only one of options a, b, c, or d is required. Yang teaches (a) that the phenylpyruvate decarboxylase aro10 gene has GenBank ID 851987 (Yang Table 1). The associated protein sequence is NCBI Reference Sequence: NP_010668.3 (see attached NCBI reference). This sequence is 100% identical to instant SEQ ID NO: 1 and is thus considered to have the same catalytic activity (see Sequence Alignment in OA appendix).
Yang does not teach that the transgenic bacterial cell overexpresses the phospho-2-dehydro-3-deoxyheptonate aldolase aroF (claim 1).
Regarding claim 1, Koma teaches a method of producing aromatic compounds, including 2-(4-hydroxyphenyl)ethanol (4HPE), also known as tyrosol, using transgenic E. coli cells (Koma Abstract; p. 6204 para. 2). Koma teaches a transgenic E. coli strain PAR-104 used to produce 4HPE, or tyrosol (Koma Table 5; p. 6211 first partial para.). Strain PAR-104 expresses feedback-resistant (fbr) aroF and tyrA, and does not express pheA or feaB (Koma Table 1; Table 5; p. 6210 para. 2; p. 6211 first partial para.).
It would have been obvious for a skilled artisan to modify the cell taught by Yang, and overexpress the phospho-2-dehydro-3-deoxyheptonate aldolase aroF. Both Yang and Koma are directed to transgenic E. coli cells for the production of tyrosol. Both references teach strains which express tyrA, and do not express pheA or feaB, for this purpose. Yang teaches that the strain overexpresses aroG, which is the isoenzyme of aroF. Thus, it would have been obvious to a skilled artisan that aroF could be expressed in the cell as taught by Yang in place of aroG, because aroF and aroG catalyze the same reaction and are both expressed in a tyrosol-producing cell. This would be considered a simple substitution of one known element for another, with a reasonable expectation of predictable results, as both aroG and aroF catalyze the same reaction (are isoenzymes) and have both been overexpressed in E. coli cells for the production of tyrosol (see Yang p. 3904 “Initial Pathway Construction”; Koma Fig. 1 showing aroF and aroG catalyzing the conversion of PEP and E4P to DAHP).
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Yang and Koma as applied to claims 1-3, 6-11, and 13-14 above, in view of Menzella, Microbial cell factories; 10(1):15.
Yang teaches the method according to claim 1, as set forth above. Yang does not teach that phenylpyruvate decarboxylase ARO10 is codon-optimized for expression in the transgenic bacterial cell as set forth in claim 12.
Regarding claim 12, Menzella teaches that for expressing enzymes in host cells, such as the preferred host cell, E. coli, species-specific variations in codon usage must be considered (Menzella “Background”). Menzella teaches that variations in codon usage between species are one of the major causes affecting recombinant protein expression levels, with a significant impact on the economy of industrial enzyme production processes (Menzella “Abstract”). Menzella teaches common strategies for codon optimization to allow for optimal expression in E. coli (Menzella “Abstract”).
It would have been obvious to a skilled artisan, before the effective filing date, to modify the method of Yang and codon-optimize the aro10 gene from S. cerevisiae, for expression in E. coli. This is well-established technique, as taught by Menzella, and a skilled artisan would be aware of the differences in codon usage between species and the need for codon optimization of DNA sequences for efficient protein expression. It would have been obvious for an ordinary artisan to use a well-known codon-optimization technique for expression of a gene in the transgenic cell of Yang.
A person of ordinary skill in the art would have been motivated to modify the teachings of Yang and codon-optimize the aro10 gene from S. cerevisiae because, as taught by Menzella, differences in codon usage between species are a major issue in recombinant protein expression that can lead to poor expression of protein/enzymes in industrial settings. Further, it is desirable to express proteins in E. coli as a host (Menzella “Background”). As Yang teaches expression of S. cerevisiae aro10 in an E. coli host, an ordinary artisan would have been motivated to improve the expression of this gene derived from a different species using a common technique of codon optimization.
A skilled artisan would have a reasonable expectation of success in codon-optimizing the transgene of Yang. As codon optimization is a well-established and known technique for adapting DNA sequences to be expressed in various host species, particularly in E. coli, as taught by Menzella, a skilled artisan could expect to successfully codon optimize the transgene sequence of Yang for expression in E. coli.
Response to Arguments
In light of amendments to the claims, the rejection of claims 1-3, 6-11, and 13-14 under 35 U.S.C. § 102; and the rejection of claim 12 under 35 U.S.C. § 103 in view of Yang and Menzella have been withdrawn. However, upon further consideration, new grounds of rejection of claims 1-3, 6-11, and 13-14 are made under 35 U.S.C. § 103 in view of Yang and Koma as set forth above. Given these new grounds of rejection, the arguments presented regarding claims rejected under 35 U.S.C. § 102 or under 35 U.S.C. § 103 in view of Yang and Menzella are moot.
Regarding the declaration under 37 CFR 1.132, the arguments and evidence presented in the declaration are directed to the unexpected result of tyrosol production in a strain overexpressing aroF, and that Yang does not teach overexpression of aroF. However, the current rejection is in view of Yang and Koma, and Koma teaches a strain overexpressing aroF which produces tyrosol. Therefore, the arguments presented in the declaration are moot in light of the new rejection necessitated by amendment.
Conclusion
Claims 1-3, 6-14, and 16 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.
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/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653
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