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 .
DETAILED ACTION
Claims 1-17 are pending and under consideration in this action.
Priority
The instant claims are entitled to an effective filing date of 06/21/2021.
Claim Objections
Claim 12-13 are objected to because of the following informalities:
Claims 12-13 recite “a bacteria species” in lines 1-2, which should be replaced with “a bacterium species”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
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 1-17 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for: a method for regulating a microbial consortium, comprising: providing a plurality of microorganisms, including a first microorganism of Escherichia coli, Saccharomyces cerevisiae, or Pseudomonas putida that includes an optogenetic gene expression system to control its growth rate using a specific wavelength of light or using darkness, and co-culturing the plurality of microorganisms in a light-controlled fermentation by adjusting light conditions to control the microbial consortium composition and production of a chemical of interest; does not reasonably provide enablement for: a method for regulating a microbial consortium, comprising: providing a plurality of microorganisms, including a first microorganism including an optogenetic gene expression system to control the first organism’s growth rate using a specific wavelength of light or using darkness, and co-culturing the plurality of microorganisms in a light-controlled fermentation by adjusting light conditions to control the microbial consortium composition and production of a chemical of interest. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The factors to be considered in determining whether a disclosure would require undue experimentation include:
A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims and the nature of the invention:
Claim 1 entails providing any plurality of microorganisms, including a first microorganism that includes an optogenetic gene expression system to control its growth rate using a specific wavelength of light or using darkness, and co-culturing the plurality of microorganisms in a light-controlled fermentation by adjusting the light conditions to control the microbial consortium composition and the production of a chemical of interest. Therefore, the claim encompasses any combination of microorganisms, as long as one microorganism includes the required optogenetic gene expression system.
Dependent claims 1-17 do not limit the first microorganism to any particular strain. Claim 11 requires the plurality of microorganisms to comprise one or more strains of E. coli, one or more strains of S. cerevisiae, one or more strains of P. putida, or a combination thereof. However, claim 11 does not require the first microorganism to be E. coli, S. cerevisiae, or P. putida. Therefore, in accordance with claim 11 the first microorganism can be: E. coli, S. cerevisiae, P. putida, or any microorganism that can be co-culture with a plurality of microorganisms that comprises E. coli, S. cerevisiae, and/or P. putida. According to the instant specification, maintaining population stability remains an obstacle in engineering microbial consortia. See [0005].
The state of the prior art and the level of predictability in the art:
With respect to the state of the art of co-culturing, Aditya, (bioRxiv 2021.06.09. 447744; version posted June 9, 2021) teaches optogenetically controlling the growth rate and composition of a consortium. See the sentences spanning p. 8-9. Aditya discloses that co-culturing approaches rely on various inter-species interactions to ensure the co-existence of different species. Despite considerable advances in engineering microbial consortia and understanding of community interactions, dynamic control of consortium composition remains a key challenge in the field. Additionally, scaling the consortium to include more than two species requires non-trivial considerations that may not lead to stable co-existence. See p. 2 second paragraph. Furthermore, Aditya discloses that the dynamic control of a consortium composition remains relatively unexplored. See p. 8 second sentence. Lalwani (ACS Synth Biol. 2021 Aug 20;10(8):2015-2029), a past filing date, adds that stabilizing and optimizing microbial subpopulations for maximal chemical production remains a major obstacle in the field. See the abstract.
The amount of direction provided by the inventor and the existence of working examples:
The instant specification provides three working examples of controlling E. coli and S. cerevisiae consortia with optogenetics. See [0068]. In example 2, the specification teaches regulating the growth of E. coli with OptoTA. See [0069]. OptoTa expresses mazF toxin using pDusk and mazE anti-toxin using pDawn to control bacterial growth with light. See [0061]. For the co-culture, the specification teaches incubating E. coli and S. cerevisiae under blue light. See [0075]. In example 3, the specification teaches using an E. coli and S. cerevisiae consortium for the production of isobutyl acetate. See [0082]. E. coli is engineered to produce isobutanol, and S. cerevisiae combines isobutanol with acetyl-coA to produce isobutyl acetate. The E. coli is transformed with plasmids containing the OptoTa. See [0084]. In example 4, the specification teaches using an E. coli and S. cerevisiae consortium for the production of naringenin. See [0094]. Light responsive E. coli is engineered to produce tyrosine. See [0096]. The S. cerevisiae strain produces naringenin from imported tyrosine. See [0099]. Thus, the specification provides working examples of E. coli and S. cerevisiae consortia.
The quantity of experimentation needed to make or use the invention:
In view of the nature of the invention, the breadth of the claims, the guidance and
working examples in the specification, and the level of predictability within the art, as
evidenced above, one skilled in the art could not use the disclosed method to regulate a microbial consortium, without undue experimentation. Prior to the effective filing date of the instantly claimed invention, it was well known that co-culturing relies on various inter-species interactions to ensure the co-existence of different species, as evidenced by Aditya. To date, stabilizing microbial subpopulations for maximum chemical production remains an obstacle, as evidenced by Lalwani. The instant specification provides three working examples in which a consortium of E. coli and S. cerevisiae are co-cultured. Consequently, there is no indication that the claimed method can be extended to any microbial consortium comprising of any plurality of microorganisms, which is commensurate with the scope of the claims.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-17 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A broad limitation together with a narrow limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 1 recites the broad recitation “the first organism’s” in line 3, and the claim also recites “a first microorganism” which is the narrower statement of the limitation. The claim is considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. In instant claim 1, it is unclear whether the first organism is limited to a first microorganism. Claim 5 depends from claim 1 and further recites the broad limitation “organism” in lines 1 and 3, and “microorganisms” in lines 1 and 3, which is a narrower limitation. As such, it is unclear whether the organisms recited in claims 1, and 5 are limited to microorganisms.
Claims 2-4, 6-17 depend from claim 1 and are rejected for the reason set forth above.
Claim 7 recites “the genetic circuit that uses an optogenetic gene expression system to control an organism’s growth rate” in lines 2-3, which renders the claim indefinite because in one interpretation, the genetic circuit of claim 7 is the “gene expression system to control the first organism’s growth rate” (claim 1 line 3); and under an alternative interpretation, the genetic circuit of claim 7 is a separate genetic circuit that uses optogenetic gene expression to control any organism’s growth rate.
Claims 7-9 recite “the microorganism” in line 2, which renders the claims indefinite because in one interpretation, the microorganism is the first microorganism required in claim 1, and under an alternative interpretation, claims 7-9 require transforming any of the plurality of microorganisms, such that different microorganisms may be transformed with different plasmids.
Claim 10 depends from claim 9 and is rejected for the reasons set forth above,
Claim Rejections - 35 USC § 103
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.
Claims 1-17 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Eng Life Sci. 2017 Jun 1;17(9):1021-1029) in view of Avalos (US 2019/0119331).
Regarding claim 1, Zhang teaches co-culturing engineered E. coli and S. cerevisiae for the production of naringenin. See p. 1022 last passage. Zhang teaches selecting a strong promoter, TEF1p, as part of constructing a naringenin biosynthetic pathway in S. cerevisiae. See p. 1025 section 3.1 first paragraph. Zhang suggests that co-cultures are advantageous compared with traditional mono-cultures. See p. 1025 section 3.1 last sentence of the section.
Zhang does not teach a first microorganism including an optogenetic gene expression system to control the first organism’s growth rate using a specific wavelength of light or using darkness.
Zhang does not teach a light-controlled fermentation by adjusting light conditions to control the microbial consortium composition and production of a chemical of interest.
Avalos teaches a method for controlling the expression of genes in yeast for the purpose of producing a desired, valuable end product. See [0136]. Avalos teaches constructing an optical gene expression system (OptoEXP) in S. cerevisiae. See [0061]. OptoEXP includes PTEF1 (TEF1 promoter) and Pc120. See [0064]. PC120 is a promoter that is activated upon light stimulation. See [0062]. Avalos teaches YEZ61, which includes PC120. See [0093]. Avalos discloses that the growth rate of YEZ61 is close to zero in complete darkness, but increases with increasing light dose. See [0097]. Avalos teaches growing YEZ61 cells under blue light. See [0098]. Avalos suggests adjusting the light schedule of a fermentation to increase the overproduction of a desired chemical. See [0143].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Avalos’s optogenetic gene expression system (OptoEXP) for Zhang’s promoter (TEF1p) expression system, and to further adjust the light conditions of the co-culture based on Avalos’s suggestion. One of ordinary skill in the art would have been motivated to use Avalos’s optogenetic gene expression system, because Avalos suggests that the system can be used to control gene expression in yeast for producing a desired product. There would have been a reasonable expectation of success because Zhang demonstrates expressing genes in S. cerevisiae under a TEF1 promoter, and Avalos teaches expressing genes in S. cerevisiae under OptoEXP, which includes a TEF1 promoter. One of ordinary skill in the art would have been further motivated to adjust the light conditions, because Avalos suggests that the fermentation light schedule can be adjusted to increase chemical production. There would have been a reasonable expectation of success because Avalos demonstrates culturing cells under blue light to control the growth rate.
Regarding claim 2, Avalos teaches varying the schedule of illumination during the production phase of fermentation to achieve high yields of desired chemical production. See [0034].
Regarding claim 3, Avalos teaches testing different light schedules, consisting of 30 minutes of light at exposure at a duty cycle of 15 s (i.e. light) / 65 s (i.e. dark), every 5 h, 10 h, or 20 h. See [0114].
Regarding claim 4, Avalos teaches measuring or detecting visual results for a variety of reasons including providing feedback to a controller or automating at least some portion of the fermentation process. The controller automates or adjusts the light schedules of the fermentation phase. See [0143]. Avalos teaches an element that can be visually detected such as green fluorescent protein (GFP). See [0046].
Regarding claim 5, Zhang teaches that E. coli can excrete acetate, while S. cerevisiae can utilize acetate (i.e. an intermediate) as a carbon source. See the sentence spanning p. 1021-1022. In figure 1, Zhang teaches an artificial S. cerevisiae biosynthetic pathway that converts acetate to naringenin.
Regarding claim 6, Zhang teaches a naringenin final product. Naringenin is a flavonoid. See p. 1021 right column first two sentences.
Regarding claim 7, Avalos teaches transforming YEZ61 yeast with an EZ_L143 (i.e. first plasmid) plasmid containing the P--C120 promoter. See [0093]. PC120 is a promoter that is activated upon light stimulation. See [0062]. Avalos discloses that the YEZ61 growth rate is close to zero in complete darkness. See [0097].
Regarding claim 8, Zhang teaches modifying E. coli to improve the precursor amino acid L-tyrosine (i.e. intermediate metabolite) supply to the whole co-culture system. The modified E. coli is BKT5. See p. 1025 section 3.2. The BKT5 strain includes the pYBT5 plasmid (i.e. second plasmid), which includes aroGfbr, tyrAfbr, aroE, ppsA, and tktA . See table 1. As shown in figure 1, aroGfbr, tyrAfbr, aroE, ppsA,and tktA, form a biosynthetic pathway for L-tyrosine production.
Regarding claim 9, Zhang teaches transforming gene modules related to the naringenin pathway into yeast. See p. 1023 right column first full paragraph. Zhang teaches a pZW01 plasmid (i.e. third plasmid), which is the pRS425K harboring cassette TEF1p-4CL-CYC1t. See table 1. The pRS425K vector includes chalcone isomerase (CHI), chalcone synthase (CHS), 4-coumarate-CoA ligase (4CL) and tyrosine ammonia lyase (TAL) modules. See p. 1023 left column last passage. As shown in figure 1, the encoded enzymes make up a biosynthetic pathway that converts L-tyrosine (i.e. intermediate metabolite) to naringenin (i.e. chemical of interest).
Regarding claim 10, Zhang teaches modifying E. coli to improve the precursor amino acid L-tyrosine (i.e. aromatic amino acid) supply to the whole co-culture system. See p. 1025 section 3.2.
Regarding claim 11, Zhang teaches co-culturing E. coli and S. cerevisiae. See p. 1022 last passage.
Regarding claim 12, Zhang teaches optimizing the inoculation ratio of the two E. coli and S. cerevisiae microorganisms. See p. 1023 left column first passage. Zhang teaches setting the inoculum ratio of the co-culture to over inoculation of S. cerevisiae, using the four ratios: 70:1, 50:1, 30:1 and 10:1 (i.e. yeast:bacteria). See p. 1026 section 3.4 first paragraph. Therefore, Zhang teaches bacteria-to-yeast ratios of 1:70, 1:50, 1:30 and 1:10, all of which are ≤1 as instantly required.
Regarding claim 13, Zhang teaches setting the inoculum ratio of the co-culture to over inoculation of S. cerevisiae, using the four ratios: 70:1, 50:1, 30:1 and 10:1 (i.e. yeast:bacteria). See p. 1026 section 3.4 first paragraph. Zhang suggests that low inoculation of E. coli limits the population of yeast by acetate concentration. See p. 1027 left column first passage.
Avalos and Zhang do not teach a bacteria-to-yeast ratio that is > 1.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to adjust the bacteria-to-yeast ratio of the co-culture based on Zhang’s suggestion. One of ordinary skill in the art would have been motivated to do so because Zhang suggests that low E. coli inoculation can limit the yeast population as E. coli produces acetate which is used by the yeast population (see fig. 1). There would have been a reasonable expectation of success because Zhang provides a starting ratio from which one of ordinary skill in the art could optimize. MPEP 2144.05(II)(A) indicates that differences in concentration generally amount to “routine optimization” and will not support patentability unless there is evidence indicating the claimed feature is critical. “Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 14, Zhang teaches that the inoculation of E. coli involved into the co-culture is set as OD600=0.1 (i.e. starting OD600), which is ≤5 as instantly required. See p. 1026 left column section 3.3 first paragraph.
Regarding claim 15, Zhang teaches that the inoculation of E. coli involved into the co-culture is set as OD600=0.1 (i.e. starting OD600), which is >1x10-3 as instantly required. See p. 1026 left column section 3.3 first paragraph.
Regarding claim 16, Zhang teaches the OD600 of the yeast initial inoculation. See p. 1026 left column last full paragraph. Zhang suggests that the initial yeast inoculation of OD600=5, which is ≥0.1 as instantly required, produces the highest naringenin production. See p. 1026 the sentences bridging the left and right columns.
Regarding claim 17, Zhang suggests that an initial yeast inoculation of OD600=5, which is ≤10 as instantly required, produces the highest naringenin production. See p. 1026 the sentences bridging the left and right columns.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KIMBERLY C BREEN whose telephone number is (571)272-0980. The examiner can normally be reached M-Th 7:30-4:30, F 8:30-1:30 (EDT/EST).
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657
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