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 .
Priority
This application is a 371 of PCT/EP2020/086342 filed 12/16/2020 which claims benefit of provisional application 62/950,167 filed 12/19/2019. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). based on EPO 19217809.3 filed 12/19/2019. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Status of the Claims
Claims 16-29 are pending. Claims 25-28 are withdrawn. Claims 16, 22 and 24 are amended. Claims 30 and 31 are cancelled.
Claims 16-24 and 29 (claim set filed 08/06/2025) are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 08//06/2025 are acknowledged. All of the amendment and arguments have been thoroughly reviewed and considered.
For the purposes of clarity of the record, the reasons for the Examiner's withdrawal and/or maintaining if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner's response to arguments section.
The previous claims 22, 24, 30 and 31 rejection under 35 U.S.C. 112(b) have been withdrawn necessitated by amendment of claims 22 and 24 and cancellation of claims 30 and 31.
The previous claim 22 rejection under 35 U.S.C. 112(a) have been withdrawn necessitated by amendment of claim 22.
The previous claim 31 rejection under 35 U.S.C. 101 have been withdrawn necessitated by cancellation of claim 31.
Maintained/Modified Rejections
The following rejections are maintained and/or modified taking into consideration amendment to claims filed on 08/06/2025.
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 16, 23 and 29 are rejected under 35 U.S.C. 103 as being unpatentable over Bhadra (US 9909148 B2) in view of Hara (Hara et al. Microbial Cell factories, 2014, 13, 173, 1-18).
Regarding claim 16, Bhadra teaches development of microorganism capable to produce fermentation products and method to increase the yield of production of fermentation products (Abstract). The microorganism in Bhadra teaching may be a recombinant host cell (column 4, lines 13-14). Bhadra describes production of different alcohols as chemicals by culturing microorganism in the conditions suitable to produce alcohol and recovering of produced alcohol (column 10, lines 52-59). Bhadra mentions conditions for growing the microorganism (column 56, lines 13-14) and presence of carbon substrates during production (column 48, lines 26-28). Bhadra discloses that cAMP signal transduction pathway is a key regulator of cell growth, proliferation, metabolism and stress responses in yeast (column 7, lines 58-62) and describes that microorganism may be modified to alter expression and/or activity of one or more components of the cAMP signal transduction pathway (column 5, lines 57-60). Bhadra further describes that: “ The levels of cAMP are regulated by its synthesis via adenylate cyclase activity and its degradation via hydrolysis by cyclic nucleotide phosphodiesterases (PDE).” (column 8, lines 13-15). Bhadra teaches modification of the host organism by elimination or reduction of expression of phosphodiesterase gene or elimination or reduction in activity of phosphodiesterase (column 9, lines 11- 14, 16-19) that would prevent cAMP degradation and hence would increase cAMP levels in the host organism. Bhadra mentions that modified microorganism exhibits increased alcohol production measured in several ways, e.g. gram of alcohol produced per hour or per gram of substrate (column 10, lines 18-26).
Fine chemical is defined in the specification as: “Fine chemical according to the invention is a biochemical substance comprising two or more sugar units.” (p. 9, lines 11-12). Bhadra does not explicitly teach production of oligosaccharides.
Hara teaches production of different fine chemicals through microbial fermentation (Abstract), including production of oligosaccharides (p. 14, left column). Hara mentions that microbial system can convert the common carbon source into various fine chemicals and productivity can be improved by genetic engineering of the metabolic pathways (Abstract, p. 2, Figure 1). Hara describes different oligosaccharides, different strategies for production and different host microorganisms and provides examples of specific oligosaccharides production (p. 14, left column).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine Bhadra and Hara teachings and apply production method described by Bhadra, including modification of cultured microorganism to increase level of cAMP to production of oligosaccharides described by Hara. One would have been motivated to do so since Bhadra teaches cAMP to be a key regulator of cell growth, proliferation, metabolism and describes increased production of alcohol at higher cAMP level and Hara teaches genetic modification of microorganisms to increase efficiency of production of oligosaccharides as fine chemicals. A skilled artisan would have reasonably expected success in this combination because Bhadra and Hara teach production of fine chemicals by microbial system. Thus, teachings of Bhadra and Hara render claim 16 obvious.
Note the recitation “to increase the carbon substrate flexibility of the production … of one or more fine chemicals produced by a host organism” is interpreted as a recitation of intended use. The intended use is given weight to the extent that it imparts a structural limitation and the prior art needs to be capable of performing the intended use. See MPEP 2111.02, section II. In instant case, the prior art renders the instantly claimed method steps obvious and therefore the method described in prior art is capable of achieving the same goals of production, i.e. to increase the carbon flexibility of production.
The recited in the “wherein” clause, i.e. 20% increase of the recited parameter compared to control is a further limitation of the intended use. MPEP 2111.04 states: “ … the court noted that a "‘whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.’" Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003))”. Therefore, the method of production of fine chemicals taught by prior art of Bhadra and Hara is capable to have 20% increase in carbon flexibility of the production compared to control.
Regarding claim 23, Bhadra teaches multiple suitable carbon substrates, including complex and defined substrates: “Suitable carbon substrates may include, but are not limited to, monosaccharides such as fructose or glucose; oligosaccharides such as lactose, maltose, galactose, or sucrose: polysaccharides such as starch or cellulose, or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, corn steep liquor, sugar beet molasses, and barley malt. Other carbon substrates may include ethanol, lactate, succinate and glycerol.” (column 55, lines 5-12). Thus, teachings of Bhadra and Hara render claim 23 obvious.
Regarding claim 29, Hara teaches microbial fermentation of 2’-fucosyllactose which is a human milk oligosaccharide from lactose in a genetically engineered Escherichia coli strain. Hara mentions that: “2′-Fucosyllactose is a functional oligosaccharide present in human milk and protects newborns against infection by enteric pathogens” (p. 14, left column, 3rd paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Hara teaching and apply production method described by Bhadra, including modification of cultured microorganism to increase level of cAMP to production of human milk oligosaccharides such as 2’-fucosyllactose described by Hara. One would have been motivated to do so since Hara teaches that 2′-Fucosyllactose in human milk protects newborns against infection by enteric pathogens. A skilled artisan would have reasonably expected success in this combination because Bhadra and Hara teach production of fine chemicals by microbial system. Thus, teachings of Bhadra and Hara render claim 29 obvious.
Claims 17-22 are rejected under 35 U.S.C. 103 as being unpatentable over Bhadra (US 9909148 B2) in view of Hara (Hara et al. Microbial Cell factories, 2014, 13, 173, 1-18) as applied to claim 16 above, and further in view of Crasnier (Crasnier et al. Mol. Gen. Genet., 1994, 243, 409-416 on record in IDS) as evidenced by UniProt (UniProt, P00936 CYAA_ECOLI [retrieved on 03/28/2025]. Retrieved from the Internet: <https://www.uniprot.org/uniprotkb/P00936/entry>).
The teachings of Bhadra and Hara have been set forth above.
Bhadra and Hara do not teach increase of cAMP level by introduction into the host organism of a mutated adenylate cyclase lacking the regulatory activity and increase in cAMP level in an inducible manner.
Regarding claims 17 and 19-22, Crasnier teaches the presence of catalytic and regulatory domains in E. coli adenylate cyclase (Abstract). Crasnier mentions that the junction between the two domains is located on proline 412 (p. 414, right column, last paragraph). Crasnier performed deletions in the C-terminal regulatory part of cya gene of E. coli without disruption of the catalytic part producing cAMP and introduced mutated cya gene into different strains of E coli (as introduction of a transgene) (p. 411, left column, 2nd paragraph). Crasnier describes that truncation and deletion of the regulatory domain resulted in the 10-fold increase in the level of cAMP relative to the wild-type adenylate cyclase in strains deficient in EnzymeIIA (interacting with the regulatory domain) (Abstract). Crasnier concludes that the regulatory domain is inhibitory to the activity of the catalytic domain (Abstract). Please note that cya gene is synonym of claimed CyaA gene of E.coli as evidenced by UniProt (p. 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Crasnier, Bhadra and Hara and introduce into the host organism of Bhadra teaching the mutated adenylate cyclase having truncated or deleted C-terminal regulatory part of adenylate cyclase without disruption of the catalytic part and use CyaA E. coli gene and apply it for production of fine chemicals based on Bhadra and Hara method. One would have been motivated to do so since Crasnier teaches that regulatory domain is inhibitory to adenylate cyclase action and that deletion of the C-terminal domain increases the cAMP level and Bhadra showed increase in the production of fine chemical with the increased level of cAMP. A skilled artisan would have
reasonably expected success in this combination because Bhadra and Hara teach production of fine chemicals by microbial system, Bhadra teaches increase in cAMP for efficient production and Crasnier describes genetic modification to increase in cAMP level in microbial host cells. Thus, teachings of Bhadra, Hara and Crasnier as evidenced by UniProt render claims 17 and 19-22 obvious.
Regarding claim 18, Crasnier teaches introduction of mutated adenylate cyclase gene lacking the regulatory domain into the microbial cell to increase cAMP level as described above. Crasnier discloses that the high level of production of cAMP is toxic to E. coli (p. 413, right column) pointing to necessity to control adenylate cyclase expression and hence the cAMP level.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to introduce into the host organism of Bhadra teaching the mutated adenylate cyclase having deleted C-terminal regulatory part and express it in the inducible manner and apply it for production of fine chemicals based on Bhadra and Hara method. One would have been motivated to do so since Crasnier teaches that high level of cAMP is toxic to E. coli cells and inducible expression of adenylate cyclase will allow to increase cAMP level only for the period of production of fine chemicals preventing toxicity during microbial growth. A skilled artisan would have reasonably expected success in this combination because Bhadra teaches increase in cAMP for efficient production of fine chemicals, Crasnier describes introduction of adenylate cyclase providing increased cAMP level necessary during production and induced expression of introduced gene is routine and conventional. Thus, teachings of Bhadra, Hara and Crasnier render claim 18 obvious.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Bhadra (US 9909148 B2) in view of Hara (Hara et al. Microbial Cell factories, 2014, 13, 173, 1-18) as applied to claim 16 above, and further in view of Deutscher (Deutscher et al. Microbiol. Molec. Biol. Reviews, 2014, 231-256).
The teachings of Bhadra and Hara have been set forth above.
Bhadra and Hara do not teach inactivation or removal of Crr protein.
Deutscher teaches regulation of the bacterial phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) (Abstract). EIIAGlc protein, also called Crr, the component of PTS was found to be central regulator of carbon metabolism (p. 232, left column, 2nd paragraph and p. 233, left column, 2nd paragraph). Deutscher discloses that Crr from E. coli was shown to interact with non-PST proteins (p. 242, left column) and describes that interaction leads to: “inhibition of the non-PTS proteins, which are usually transporters or transporter associated components. Their inhibition prevents the uptake of the corresponding carbon source and consequently the formation of the necessary inducer; this regulation mechanism was therefore called inducer exclusion.” (p. 242, left column). Deutscher demonstrates on Figure 5 that the inducer exclusion is prevented when the EIIAGlc-encoding crr gene is deleted allowing the uptake of the other four carbohydrates, L-arabinose, D-galactose, melibiose, and raffinose (p. 242, Figure 5 legend). Thus, deletion or inactivation of Crr protein results in the uptake of various carbon substrates increasing substrate flexibility.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Deutscher teachings and add inactivation or deletion of Crr protein to the host organism genetically modified to produce fine chemicals based on Bhadra and Hara teachings. One would have been motivated to do so since Deutscher teaches that Crr inhibits transporters or transporter associated components and that deletion or inactivation of Crr leads to uptake of various carbon substrates and hence would increase substrate flexibility. A skilled artisan would have reasonably expected success in this combination because Bhadra and Hara teach production of fine chemicals by microbial system, Bhadra teaches increase in cAMP for efficient production and Deutscher points to genetic modification to increase the substrate flexibility. Thus, teachings of Bhadra, Hara and Deutscher render claim 24 obvious.
Response to Arguments
Applicant's arguments filed 08/06/2025 have been fully considered but they are not persuasive.
Applicant argues (addressing p. 7 of the Remarks) that amended claim 16 requires “at least 20% increase as to carbon flexibility, space time yield, or carbon conversion efficiency. As a result, the Examiner's dismissal of these improvements on page 10 as being intended use is now rendered incorrect, as it is a required outcome of the process of claim 16.” Applicant further argues that: “… the Examiner asserts that the prior art "is capable" of having such a value. As an initial matter, there is nothing in the prior art that would have suggested this capability. Second, "is capable" is insufficient to support inherency of a claim limitation, as inherency requires that the property is necessarily present. In this case the properties are not necessarily present, and therefore not inherent.” These arguments are not persuasive because:
The combination of prior art of Bhadra and Hara makes the instant method steps obvious as described in the rejection above. The increase of carbon substrate flexibility (which is the elected species) is interpreted as intended use of the instant method and the 20% increase of carbon substrate flexibility is interpreted as further limitation of the intended use. The combination of Bhadra and Hara will necessarily have the property to increase the carbon substrate flexibility as evidenced by the following prior art: (i) Shimada (Shimada et al. PLOS One, 2011, 6, e20081, 1-11) teaches the roles of cAMP receptor protein (CRP) in regulation of transport and metabolism of carbon sources (Title). Shimada describes that CRP which is a transcriptional activator, plays a key role in activation of the genes for utilization of carbon sources and CRP requires cAMP for conversion to the activated form (p. 1, left column, 2nd paragraph). Shimada discloses that 378 promoters are the regulatory targets of cAMP-CRP and that “CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration” (Abstract). More specifically, Shimada mentions that almost all genes for transport of carbon sources including substrate-specific transport systems PTS, ABC and MFS, are identified to be under the direct control of cAMP-CRP (p. 7, right column, 5th paragraph). Shimada teaching indicates that increase in cAMP will increase the transport of various carbon substrates and their metabolism and hence will increase substrate flexibility during fine chemical synthesis; (ii) similarly Franchini (Franchini et al. PLOS One, 2015, 10, e01333793, 1-24) teaches that the inability to synthesize cAMP has strong negative effect on the expression of alternative carbon source uptake and degradation systems and that cAMP is crucial for catabolic flexibility during slow, carbon-limited growth (Abstract). Franchini discloses that: “the expression of a broad range of high-affinity uptake systems and of enzymes involved in the central metabolism (citric acid cycle and of glycolysis) were severely repressed in the absence of cAMP.” (p. 18, 4th paragraph); and (iii) Pastan (Pastan and Perlman, Science, 1970, 169, 339-344) teaches that the E. coli mutant strain deficient in adenyl cyclase and producing undetectable level of cAMP was unable to grow on lactose, maltose, arabinose, mannitol or glycerol and grew slowly on glucose, fructose and galactose. The addition of cAMP permitted normal utilization of all of these carbon sources (p. 340, right column, 1st paragraph, p. 341, Table 3) and hence increased substrate flexibility.
Thus, method steps of increasing cAMP levels during culturing of the host microorganism in the presence of substrates and under conditions for production of fine chemicals as taught by combination of prior art of Bhadra and Hara will necessarily provide increase in carbon substrate flexibility. Therefore, the 35 U.S.C. 103 rejection is maintained and modified necessitated by amendment of the claims.
Conclusion
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 LIOUBOV G KOROTCHKINA whose telephone number is (571)270-0911. The examiner can normally be reached Monday-Friday: 8:00-5:30.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Sharmila G 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.
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/L.G.K./ Examiner, Art Unit 1653
/SHARMILA G LANDAU/ Supervisory Patent Examiner, Art Unit 1653