DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of Claims
Receipt of Arguments/Remarks filed on 9/24/2025 is acknowledged. Claims 1-9 and 11-19 are pending. Claims 1, 2, 6-9, and 11-13 were amended. New claims 14-19 were added. Claims 11-13 and new claims 14-19 are withdrawn as being directed to non-elected subject matter.
Withdrawn Objections
The amendment filed 9/24/2025 is sufficient to overcome the objection to claims 1, 2, and 6-9. Microorganism names are now italicized.
Maintained Rejections
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 6-8 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., Metabolic engineering; 14(5):521-7 and Willis et al., Proceedings of the National Academy of Sciences; 110(51):20753-8.
Regarding claim 1, Zhang teaches a method for producing the capsular polysaccharide heparosan by culturing metabolically engineered Escherichia coli that has the ability to produce heparosan (Zhang “Abstract”). Zhang teaches the overexpression of genes kfiA, kfiB, kfiC, and kfiD, which are part of the metabolic synthesis pathway for heparosan in E. coli K5 (Zhang Fig. 1; pg. 522).
Regarding claim 2, Zhang teaches that the E. coli strain has a modification to increase the expression of kfiA, kfiB, kfiC, and kfiD genes (Zhang “Abstract”).
Regarding claim 4, Zhang teaches increasing the copy number of kfiA, kfiB, kfiC, and kfiD by introducing these genes into an E. coli strain using medium and high copy plasmids, including pETDuet-1 and pRSFDuet-1 (Zhang pg. 523 Section 2.2).
Regarding claim 6, Zhang teaches that the bacterium is Escherichia coli (Zhang “Abstract”).
Regarding claim 8, Zhang teaches an E. coli strain, BL21 (DE3) expressing kfiA, kfiB, kfiC, and kfiD derived from E. coli strain K5 (Zhang pg. 523 Section 2.1). The kfiA gene from E. coli K5 is 100% identical to instant SEQ ID NO: 34 (see Search results from 6/12/2025, file “us-17-995-352-34.rge” result 2). Similarly, kfiB, kfiC, and kfiD from E. coli K5 are 100% identical to SEQ ID NOs: 35, 36, and 37 (see Search results from 6/12/2025 file “us-17-995-352-35.rge” result 1, file “us-17-995-352-36.rge” result 1, and file “us-17-995-352-37.rge” result 1).
Zhang does not teach that the E. coli strain has a modification for increased expression of the kpsS gene and not the kpsC gene by increasing the copy number or modifying the expression control region of kpsS as recited in instant claims 1 and 3. Zhang does not teach that the kpsS gene has a nucleotide sequence of SEQ ID NO: 33, as recited in instant claim 7.
Regarding claim 1, Willis teaches that KpsS and KpsC are conserved capsular polysaccharide assembly proteins in bacteria (Willis “Abstract”). Willis teaches that capsular polysaccharides contain a (lyso)phosphatidylglycerol moiety, which is attached to the CPS via a poly-Kdo linker, and that KpsS and KpsC synthesize Kdo-linker oligosaccharides in the process of capsular polysaccharide/heparosan biosynthesis (Willis Fig. 4; pg. 20753 col. 2; pg. 20757 col. 1). Willis teaches the construction of single deletion strains and functional truncated derivatives of kpsS and kpsC genes to determine their individual function in capsular polysaccharide synthesis (Willis “Abstract”; pg. 20754-20755 “The KpsC and KpsS Proteins from E. coli and LipA and LipB Proteins from N. meningitidis Are Functionally Exchangeable” and “Construction of Functional Truncated Derivatives of KpsC and KpsS”).
Regarding claim 7, Willis teaches a K1 E. coli strain for genetic manipulation of the kpsS gene (Willis pg. 20757 “Bacterial Strains and Plasmids”). Instant SEQ ID NO: 33 is 95% identical to the kpsS gene sequence in E. coli K1 (GenBank: CP003034.1).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of Zhang and Willis, arriving at a method for producing heparosan by culturing a bacterium with increased expression of the kpsS gene. Both references are directed to genes involved in the production of capsular polysaccharide in E. coli. Zhang teaches that genes in this pathway can be overexpressed for heparosan production. It would have been obvious to a skilled artisan to create a strain with a kpsS overexpression for heparosan production given the role of this gene as a β-Kdo transferase for capsular polysaccharide production.
Regarding claim 3, it would additionally have been obvious to person having ordinary skill in the art to modify the expression of kpsS by increasing the copy number as set forth in claim 3, as this technique is taught by Zhang for expressing kfi genes as discussed above.
The kpsS gene taught by Willis from E. coli K1 is 95% identical to instant SEQ ID NO: 33 from E. coli K5, so a skilled artisan would have found it obvious to express a gene with a nucleotide sequence at least 90% identical to SEQ ID NO: 33 for production of heparosan. Further, as Zhang teaches the expression of kfi genes from E. coli K5 for production of heparosan, it would have been obvious to a person having ordinary skill in the art to express a kpsS gene from E. coli K5, therefore utilizing kpsS with a sequence 100% identical to SEQ ID NO: 33.
A person of ordinary skill in the art would have been motivated to combine these teachings and create a strain with kpsS overexpressed because Willis teaches that KpsS adds the first Kdo linker residue, a critical step in the production of capsular polysaccharides which have a lipid at one end connected via the Kdo linker (Willis pg. 20753 col. 1 and “Significance”). A skilled artisan would be motivated to create a strain with increased expression of the kpsS gene and not the kpsC gene in order to determine the role of each gene individually in the system, similar to the individual deletion strains taught by Willis.
A skilled artisan would have a reasonable expectation of success in making this combination to achieve the predictable outcome of a heparosan-producing E. coli strain with kpsS overexpressed, given the established method of overexpressing genes involved in heparosan biosynthesis in E. coli as taught by Zhang and the involvement of KpsS in capsular polysaccharide production taught by Willis.
Claims 5 and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. and Willis et al. as applied to claims 1-4 and 6-8 above, and further in view of Kalamorz et al., Molecular microbiology; 65(6):1518-33.
Zhang and Willis teach the method according to claim 2, as set forth above.
These references do not teach a deletion of the yhbJ gene as recited in instant claim 5 having the sequence according to SEQ ID NO: 38 as recited in instant claim 9.
Regarding claims 5, Kalamorz teaches that E. coli with yhbJ deleted overproduces glucosamine-6-phosphate (GlcN-6-P) synthase (GlmS), which catalyzes the conversion of fructose-6-phosphate to GlcN-6-P (Kalamorz pg. 1518 “Introduction”).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of Zhang and Willis with the teachings of Kalamorz, utilizing an E. coli strain with yhbJ deleted for the production of heparosan. Zhang teaches genetic modification of genes involved in heparosan biosynthesis. It would have been obvious to a skilled artisan that the yhbJ gene could be modified in this strain in addition to other genes involved in heparosan synthesis.
Regarding claim 9, Zhang teaches that the recombinant strains were made by introducing kfi genes into E. coli BL21 (DE3) (Zhang pg. 524 Section 3.1). SEQ ID NO: 38 is 99% identical to the yhbJ gene of E. coli BL21 (DE3) (GenBank: CP001509.3). Therefore, it would have been obvious for a skilled artisan to delete yhbJ in E. coli BL21 (DE3), having a sequence at least 90% identical to SEQ ID NO: 38.
A person of ordinary skill in the art would have been motivated to incorporate a yhbJ deletion because Kalamorz teaches that deletion of yhbJ results in GlmS overproduction. Zhang teaches that the metabolic synthesis pathway for heparosan involves conversion of fructose-6-phosphate to GlcN-6-P, which would be catalyzed by GlmS as taught by Kalamorz (Zhang Fig. 1). Therefore, a yhbJ deletion strain would be considered advantageous for producing heparosan, as GlmS overproduction would allow for conversion of fructose-6-phosphate to GlcN-6-P, which is a precursor for heparosan biosynthesis.
A skilled artisan would have a reasonable expectation of success in making this combination to achieve the predictable outcome of increased heparosan production in a strain with yhbJ deleted, given the role of this gene in negatively regulating GlmS, which catalyzes the production of a heparosan precursor.
Response to Arguments
Applicant's arguments filed 9/24/2025 have been fully considered but they are not persuasive.
Rejections under 35 U.S.C. § 103
Applicant argues that a person of ordinary skill in the art would not have arrived at the claimed invention based on the disclosures of Zhang et al., Willis et al., and Kalamorz et al. Applicant argues that Zhang does not teach a strain which has a genetic modification increasing expression of a kpsS gene as compared to expression of the kpsS gene of an unmodified strain, and (b) which does not have a genetic modification increasing expression of a kpsC gene as compared to expression of the kpsC gene of an unmodified strain, while retaining (c) heparosan-producing ability in a medium. Applicant argues that Willis et al. does not teach or suggest that one of kpsS and kpsC affects heparosan production more than the other gene and does not suggest that heparosan production could be increased through the mutation of one gene without the other gene, a person of ordinary skill in the art would not have modified the E. coli that has been metabolically engineered to overexpress the kfiA, kfiB, kfiC, and kfiD genes as disclosed in Zhang et al. so as to overexpress the kpsS gene and not overexpress the kpsC gene. Similarly, Kalamorz et al. does not provide any reason for a person of ordinary skill in the art to do so.
In response to this argument, it is noted that while it is true that Willis does not teach that one of kpsS and kpsC affects heparosan production more than the other, this is not necessary to lead a skilled artisan to create a strain with a kpsS modification and not a kpsC modification. As taught by Willis, it is a common strategy in the field of molecular biology to create strains with modifications of individual genes in a relevant pathway, in addition to a strain in which multiple genes are modified from a pathway (see Willis “Construction of Functional Truncated Derivatives of KpsC and KpsS”). Willis further teaches that each of these genes has a distinct role in capsular polysaccharide production (Willis Abstract). Thus, a skilled artisan would have found it obvious to create a strain with a modification, such as overexpression, of kpsS and not kpsC, as each of these genes is important for capsular polysaccharide production but performs a different function. A skilled artisan would have been motivated to do so with a reasonable expectation of success given the successful overexpression of other genes involved in heparosan biosynthesis, and would have found it obvious to culture such a strain in a method of producing heparosan as taught by Zhang. Therefore, it is considered that a skilled artisan would have found it obvious to modify one of these genes and not the other in addition to the other capsular polysaccharide synthesis genes as taught by Zhang.
Applicant argues that unobviousness of the present invention as defined by claims 1-9 is further evidenced by the beneficial effect of the present invention. Applicant argues that the amount of heparosan accumulated in the culture medium of a bacterium of the genus Escherichia, which has a heparosan-producing ability, is significantly increased by increasing expression of only the kpsS gene while not increasing expression of the kpsC gene of the bacterium. Applicant cites the experimental data set forth in Table 3 of the present application, which demonstrates the amount of heparosan accumulated by a strain which has only the genetic modification increasing expression of the kpsS gene was increased by 3.3 times as compared with that in the unmodified parent strain. This is in contrast to strains with genetic modifications increasing expression of kpsS and kpsC genes; or genetic modifications increasing expression of kpsF, kpsE, kpsD, kpsU, kpsS, and kpsC genes, wherein the amount of heparosan accumulated was increased by 1.7 times and 1.8 times, respectively, as compared with the unmodified parent strain. Applicant argues that such a beneficial effect is not disclosed or suggested in Zhang et al., Willis et al., and Kalamorz et al. and could not have been reasonably expected by a person of ordinary skill in the art prior to the present invention.
In response to this argument, as discussed above, a skilled artisan would have found it obvious and would have been motivated, with a reasonable expectation of success, to culture a strain with an overexpressed kpsS gene and not a kpsC gene in a method of producing heparosan. The beneficial effects stated by Applicant are a result of culturing a strain having such a genetic modification. Therefore, these results would be expected to occur in the method rendered obvious by Zhang and Willis as discussed above.
Conclusion
Claims 1-9 are rejected. No claims are allowed.
THIS ACTION IS MADE FINAL. 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.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Sharmila Landau can be reached at (571)272-0614. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653
Prosecution Insights