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
The application is a 371 application, filed 10/25/2024, of PCT application PCT/US2023/066344, filed 04/28/2023, which claims priority benefits from Provisional No. 63337036, filed 04/29/2022. The effective filing date of this application is 04/29/2022.
Claims status
Amendments filed 10/25/2024 are entered. Claims 1-3, 5-11, 13-16, 18, 20-24 are pending and under examination.
Nucleotide and/or Amino Acid Sequence Disclosures
Summary of Requirements for Patent Applications Filed On Or After July 1, 2022, That Have Sequence Disclosures
37 CFR 1.831(a) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.831(b) must contain a “Sequence Listing XML”, as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.831-1.835. This “Sequence Listing XML” part of the disclosure may be submitted:
1. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter “Legal Framework”) in XML format, together with an incorporation by reference statement of the material in the XML file in a separate paragraph of the specification (an incorporation by reference paragraph) as required by 37 CFR 1.835(a)(2) or 1.835(b)(2) identifying:
a. the name of the XML file
b. the date of creation; and
c. the size of the XML file in bytes; or
2. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation by reference statement of the material in the XML format according to 37 CFR 1.52(e)(8) and 37 CFR 1.835(a)(2) or 1.835(b)(2) in a separate paragraph of the specification identifying:
a. the name of the XML file;
b. the date of creation; and
c. the size of the XML file in bytes.
SPECIFIC DEFICIENCIES AND THE REQUIRED RESPONSE TO THIS NOTICE ARE AS FOLLOWS:
Specific deficiency - The incorporation by reference paragraph required by 37 CFR 1.834(c)(1), 1.835(a)(2), or 1.835(b)(2) is missing, defective or incomplete.
The date of creation of the sequencing file received by the Office is 10/25/2024. The date of creation recited in the Specification is 04/28/2023. The date of creation is the date of creation of the sequence listing, i.e. the electronic version of the sequence data that accompanies the application, and is the day the Applicant submits the sequence data to the Office and the Office receives it (automatically, when electronically submitted).
Required response - Applicant must:
• Provide a substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation by reference paragraph, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
Claim Objections
Claim(s) 5 and 15 is/are objected to because of the following informalities: grammatical and typographical errors. Appropriate corrections are required.
Claim 5 should recite “a” before “mscL gene” to fix a grammatical error.
In claim 15, ‘from” should be deleted before “0.25%” to fix a typographical error, as is consistent with the Specification’s teaching that the adaptive laboratory evolution is done by increasing by 0.25% w/v NaCl increments and not from 0.25% w/v NaCl (e.g. p. 33-34, para. 00102). Also in claim 15, “and” should be added after the semicolon and before limitation (b) to properly indicate both limitations (a) and (b) are required, as is consistent with the rest of the claims.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Subject Matter Eligibility
Claims 1-3, 5-9, 11, 13, and 14 encompass a genus of products wherein some species are subject matter eligible and others are subject matter ineligible. The eligible subject matter are microorganisms that have a naturally occurring counterpart that comprise of both a Large- and Small-conductance mechanosensitive channel gene, so that the product of these claims, which have no large and/or no small conductance genes has no naturally occurring counterpart. The ineligible subject matter is discussed below.
Subject Matter Ineligibility
Claim(s) 1-3, 5-9, 11, 13, and 14 is/are rejected under 35 U.S.C. 101 because the claimed invention is directed to a product of nature without significantly more. The claim(s) recite(s) “an engineered microorganism that is halotolerant or adapted to become halotolerant and comprises a knockout of a large- and/or small-conductance mechanosensitive channel gene”, which is a product-by-process claim wherein the product is a microorganism that is halotolerant and has no large- and/or small-conductance mechanosensitive channel gene. There are naturally occurring counterparts to this product. This judicial exception is not integrated into a practical application. The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception. The analysis is as follows:
Step 1: Is the claim directed to a process, machine, manufacture, or composition of matter?
Claims 1-3, 5-9 directed to an engineered microorganism, are directed to a composition of matter.
Claims 11 and 13-14, directed to a method of producing a protein or chemical using the engineered microorganism of claim 1, are directed to a process.
Step 2A Prong 1: Does the claim recite an abstract idea, law of nature, or natural phenomenon?
Claims 1-3, 5-9 is a product-by-process claim, i.e. the engineered microorganism is made by a process comprising of knocking out a Large- and/or Small-conductance mechanosensitive channel gene. According to MPEP section 2106.04(c)(I)(B), regarding the “markedly different characteristics analysis” for product-by-process claims:
“For a product-by-process claim (e.g., a claim to a cloned farm animal produced by a nuclear transfer cloning method), the analysis turns on whether the nature-based product in the claim has markedly different characteristics from its naturally occurring counterpart.”
Therefore, the resultant product of the claims are analyzed. The product of claims 1-3, 5-9 is a microorganism that is halotolerant and does not comprise of a Large- and/or Small-conductance mechanosensitive channel gene.
MPEP section 2106.04(c)(II)(A-C) outlines how to perform the “markedly different characteristics analysis”:
The naturally occurring counterpart product is the naturally occurring microorganism that is halotolerant and does not comprise of a Large- and/or Small-conductance mechanosensitive channel gene. For example, marine bacteria Vibrio alginolyticus and Salinispora tropica both lack the large conductance mechanosensitive channel gene mscL (but do have the mscS gene but do not express it sufficiently to protect themselves against hypoosmotic shock, i.e. down-shock) (Booth, published 2014; p. 19, col. 2, para. 3). Further both bacteria Vibrio alginolyticus (Nakamaru, published 1999; p. 170, col. 1, section: Introduction, para. 2) and Salinispora tropica (Penn, published 2012; p. 8, col. 1, para. 3) are halotolerant, capable of growing in high salt conditions, as they are marine bacteria, and both lyse upon down-shock.
The appropriate characteristics for analysis are the claimed phenotypes, halotolerant; and the phenotype that would naturally flow from the claimed process, lack of conductance mechanosensitive channels.
The characteristics of the claimed product are not markedly different from the naturally occurring counterpart, as they both exhibit the same claimed phenotypes: halotolerance and lack of conductance mechanosensitive channels. Further, Vibrio alginolyticus, for example, can grow in 0.5 M NaCl, which is about 2.9% w/v NaCl (Nakamaru, p. 170, section: 2.1, para. 1) and lack mscL and so would lack the sequences of SEQ ID NO: 1 and SEQ ID NO:2, which is specifically found in E. coli, or any homolog of them. Penn further teaches phylogenetic analysis suggests that the current wildtype Salinispora lost its mscL gene throughout evolution due to marine adaptations, suggesting that the microorganism adapted to growth in high salinity conditions before it lost its mscL gene (Abstract and p. 8, col. 1, section: Comparative genomics based identification of MAGs, para. 2).
When a law of nature or natural phenomenon is claimed as a physical product, the courts have often referred to the judicial exceptions as a “product of nature;” as such, the Office’s eligibility analysis accordingly uses the term “product of nature”.
Therefore, claims 1-3, 5-9 recite a product of nature.
Claims 11, 13, and 14 are process claims directed to a method of producing a recombinant protein or chemical compound from a halotolerant microorganism of claim 1. The halotolerant microorganisms referenced above, Vibrio alginolyticus and Salinispora tropica, both naturally produce proteins or chemical compounds. Therefore, this process is a naturally occurring process. The claims recite a man-made replication of a naturally occurring process.
Therefore, claims 11, 13, and 14 recite a natural phenomenon.
Step 2A Prong 2: If so, does the claim recite additional elements that integrate the judicial exception into a practical application? If the additional elements provide integration into a practical application, then the claim is eligible.
As described for the examples of Vibrio alginolyticus and Salinispora tropica, the additional elements merely describe other naturally occurring properties or processes of these products of nature. For example, although transferring the microorganism to a hypotonic solution to lyse it to isolate proteins or chemical compounds released may be useful, this process is entirely a natural process as they would naturally lyse and release its bioproducts in a hypotonic solution. A practical application requires more than recognition of a use for a natural process.
Step 2B: If the claim is “directed to” a judicial exception, determine whether any additional element, or combination of additional elements, in the claim is sufficient to ensure that the claim as a whole amounts to significantly more than the judicial exception.
For the scope of the claim that encompasses a judicial exception, there are no additional elements sufficient to ensure that the claims as whole amounts to more than the judicial exception, because the elements all occur naturally in certain microorganisms, such as, as nonlimiting examples, Vibrio alginolyticus and Salinispora tropica.
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, 5-10, 18, 21 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.
In claim 1, the phase “adapted to become halotolerant” is not a definite limitation because it is unclear whether the microorganism is or is not yet halotolerant. Is it a microorganism that was non-halotolerant but has been adapted to be halotolerant? Because it is unclear whether claim 1 requires the microorganism to be halotolerant, claim 9 is also indefinite because it is unclear if the microorganism “adapted to grow” on a salt medium of the claimed concentrations can or cannot yet grow in these conditions.
The term “about” in claims 2, 3, 9, and 15 is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Further, “halotolerant” does not provide a limiting lower or upper bound for the NaCl concentrations required for halotolerant. Therefore, the boundaries of the lower and upper limit of NaCl concentrations are indefinite because there is no way to determine what “about” should mean. The dependent claims of claim 1, claims 5-10, 18, 21, do not resolve the indefiniteness of claim 1 and therefore inherit the indefiniteness.
Further in claim 10, it is unclear if the adjective non-natural applies to only the chemical or also to the bioproduct.
A broad range or limitation together with a narrow range or 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 21 recites “greater than 75-90% osmolytic efficiency”. This language makes it unclear whether the ranges are alternatives (e.g. greater than 75% or greater than 90%) or separate limitations (e.g. greater than 75% and greater than 90%). If the latter, the claim(s) are 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.
Claim 24 recites "ΔmscL” and “ΔmscS”. There is insufficient antecedent basis for this limitation in the claim. The terms, ΔmscL and ΔmscS, is understood by the Examiner to be acronyms for the knocked-out genes in claim 1, but they do not explicitly appear in claim 1. Although the Specification provides examples and embodiments of what ΔmscL and ΔmscS is referring to, it does not distinctly define ΔmscL and ΔmscS to always mean the deletion of the mscL and mscS genes, respectively. This rejection for claim 24 may be remediated by inclusion of “ΔmscL” and “ΔmscS” in parenthesis specifically next to their corresponding meanings, so that it is clear which gene corresponds to which acronym; or by amending claim 24 to recite the knockout of the full gene name, as in claim 1.
Claim Rejections - 35 USC § 112
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.
Claim 1-3, 5-11, 13-15, 18, and 20-21 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Written Description
There are two written description issues:
The genus of microorganisms that are not halotolerant and can be adapted to become halotolerant (claims 1-3, 5-11, 13-15, 18, and 20-21)
The genus of non-natural chemicals or bioproducts produced by the microorganism (claim 10).
CLAIMED INVENTION
The claims encompass a genus of microorganisms that are not halotolerant but can be adapted to become halotolerant or a method of generating a halotolerant microorganism from a non-halotolerant microorganism (claims 1-3, 5-11, 13-15, 18, and 20-21).
The claims are also directed to engineered microorganisms that produce the broadly claimed genus of non-natural chemical or bioproduct (claim 10).
WHAT THE SPECIFICATION TEACHES
The specification teaches C. necator strains started in culture medium with 15 g/L, which is 1.5 % w/v, NaCl, and was adapted to culture medium with 3% w/v NaCl, wherein the “evolved strain exhibit[ed] elevated halotolerance” (p. 33-34, paras. 102-103). The specification teaches that adaptive laboratory evolution was used to “improve the halotolerance of C. necator” (p. 12, para. 0042). Therefore, these strains were already halotolerant and were only adapted to have increased halotolerance, by the Specification’s own admission.
The specification teaches that “[b]ecause E. coli BL21 could already grow in elevated NaCl concentration, maintaining around half of its maximum growth rate even in 4% NaCl, further adaptation of the strain was unnecessary” (p. 47, para. 00131), meaning that this was already a halotolerant microorganism.
The specification teaches the production of red fluorescent protein (RFP) by the C. necator and E. coli strains.
THE STATE OF THE ART REGARDING THE ELECTED INVENTION
The state of the art is silent on the complete genus of non-halotolerant microorganisms that can be adapted to be halotolerant or any structure that is predictive of this ability to be adapted to be halotolerant.
For example, Booth teaches that the non-halotolerant Campylobacter jejuni uniquely lacks MscL, unlike other members of Campylobacter (p. 19, col. 2, para. 3), which might make one suspect it lacking some ability to adapt to osmolarity changes. Glunder teaches that other non-halotolerant Campylobacter species can be adapted to become halotolerant (published 1993; Abstract). However, Campylobacter jejuni, which lacks most conventional osmotic response factors, likely relies on the presence of stress-fit individuals in a heterogenous population (Cameron, published 2012; Abstract), implying that there are Campylobacter jejuni individuals that can and cannot adapt to high salts. Therefore, there is variation even within the same species population.
The state of the teaches that not all non-natural bioproducts or chemicals can be produced by all microorganisms. For example, Tokmakov teaches post-translational modifications (PTMs) are required for proper folding of many proteins, but the low capacity for PTMs by prokaryotes hinder their ability to synthesize many heterologous proteins, particularly human proteins (Abstract). In other words, there is variability in the expression of heterologous proteins by microorganisms. Although there may be methods to try to predict expression success of a polypeptide sequence requiring PTMs, the art is silent on definitive predictors, and experimental data is still needed absent known structure-function correlations.
WHAT WRITTEN DESCRIPTION IS MET BY THE ELECTED INVENTION
Written description is met for the genus of microorganisms that are already known in the art to have been adapted to be halotolerant.
Written description is met for the genus of non-natural products, e.g. heterologous proteins or compounds, that have been shown in the art to producible by specific microorganisms.
WHY THE INVENTION LACKS WRITTEN
The MPEP states that the purpose of the written description requirement is to ensure that the inventor had possession, as of the filing date of the application, of the specific subject matter later claimed. The MPEP lists factors that can be used to determine if sufficient evidence of possession has been furnished in the disclosure of the application. These include “level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention.” The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, disclosure of drawings, or by disclosure of relevant identifying characteristics, for example, structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the Applicants were in possession of the claimed genus.
A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.
There is no reduction to practice or teaching of non-halotolerant microorganism that can be adapted to become halotolerant or teaching of how to identify such microorganism.
The state of the art may provide specific examples of microorganisms that have been experimentally shown to fit these limitations; however, the art is silent on how to identify the entire genus of microorganisms that can be adapted to become halotolerant, absent direct experimental evidence of such ability. Therefore, the art-recognized particular species of this claimed genus of microorganisms is not representative of anything more than itself, because knowing that one species can be adapted to be halotolerant does not help know other species that can be adapted to be halotolerant. As such, the art-recognized particular species alone, absent teachings of any structure-function correlation, are not representative of the entire claimed genus of microorganisms that can be adapted to become halotolerant, and the claimed genus of non-halotolerant microorganisms in the method of generating halotolerant microorganism from a non-halotolerant microorganism.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-3, 5, 10, and 24 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Czech (Czech et al, EctD‑mediated biotransformation of the chemical chaperone ectoine into hydroxyectoine and its mechanosensitive channel‑independent excretion; published 2016).
Regarding claims 1 and 24, Czech teaches an engineered microorganism, i.e. E. coli strains MJF465 and MJF641, that is halotolerant or adapted to become halotolerant, i.e. exposed to sustained high-salinity growth conditions by adding 0.4 M NaCl to its growth medium, LB medium (p. 12, section: Growth media for E. coli strains), and comprises a knockout of a Large- and/or Small-conductance mechanosensitive channel gene, e.g. MscL and MscS (p. 9, col. 1, paras. 2-3), i.e. ΔmscL and ΔmscS, respectively.
The teachings of the references regarding claim 1 are incorporated in its entirety for its dependent claims and discussed further below, as is relevant for each claim.
Regarding claims 2 and 3, Czech teaches the microorganism can grow in 0.4 M NaCl, which is equivalent to 2.34% w/v NaCl (p. 9, col. 1, paras. 2-3).
Regarding claim 5, Czech teaches the large-conductance mechanosensitive channel, MscL channel (p. 9, col. 1, paras. 2-3), which is encoded by the mscL gene.
Regarding claim 10, Czech further teaches the engineered microorganism is further engineered to produce a non-natural chemical or bioproduct. In this case, the E. coli strain is engineered to express the protein encoded by the ectD gene, ectoine hydroxylase, from Pseudomonas stutzeri A1501 (Abstract), which is a non-natural bioproduct for the host strain.
Claim(s) 6 and 8 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Czech (Czech et al, EctD‑mediated biotransformation of the chemical chaperone ectoine into hydroxyectoine and its mechanosensitive channel‑independent excretion; published 2016), as evidenced by Walton (Walton et al, US 9046525 B2, published 2015) and Schwaneberg (US 20100189777 A1).
Regarding claims 6 and 8, Czech teaches the large-conductance mechanosensitive channel, MscL channel (p. 9, col. 1, paras. 2-3), which is encoded by the mscL gene in E. coli.
As evidenced by Walton, the E. coli mscL gene is the amino acid sequence of SEQ ID NO: 3 (Figure 4B and col. 4, lines 29-30), which has a 100% sequence identity match to instant SEQ ID NO: 2. Schwaneberg, in turn, teaches the amino acid sequence of SEQ ID NO: 68 also matches 100% to instant SEQ ID NO: 2, and further teaches that the corresponding nucleic acid sequence encoding this polypeptide is SEQ ID NO: 32 (p. 2-3, para. 0025), which has a 100% sequence identity to sequence instant SEQ ID NO: 1. Therefore, the mscL gene in E. coli taught by Czech is encoded by the instantly claimed SEQ ID NO: 1 and produces the polypeptide of instantly claimed SEQ ID NO: 2, as evidenced by Walton identifying the amino acid sequence of E. coli mscL gene and Schwaneberg teaching the corresponding DNA sequence.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Czech (published 2016), as applied to claim 5, in the 35 U.S.C. 102 above, and further in view of GenBank: CP038353.1 (published 05/26/2020).
Claim 7 depends on claim 5 and relates to claim 6. The teachings of the references regarding claim 5 and 6 are discussed in the 5 U.S.C. 102 rejection above, and are incorporated in its entirety for claim 7 and discussed further below, as is relevant for each claim.
Regarding claim 7, Czech does not explicitly teach mscL gene homologs to SEQ ID NO: 1, which is the common E. coli mscL gene.
However, GenBank: CP038353.1 teaches that there are sometimes strain specific variations in the mscL gene, and teaches the mscL gene in E. coli O157:H7 strain, which is a homolog to the mscL gene of instant SEQ ID NO: 1, has a sequence that is 98.1% identical, which is at least 80% identical, to instant SEQ ID NO: 1.
PNG
media_image1.png
400
812
media_image1.png
Greyscale
PNG
media_image2.png
568
764
media_image2.png
Greyscale
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that strain-specific sequence variations of mscL gene homologs can occur, as shown by the variation even within E. coli.
One skilled in the art, before the effective filing date of the instant application, would be motivated to delete the mscL gene sequence specific to the host strain to achieve the desired knock-out effect.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success that deleting gene homologs would lead to similar effects since they are, by definition, genes that encode the same polypeptide but in different organisms, and it would be reasonable to expect the polypeptide to function the same.
Claim(s) 9, 11, 13, and 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Czech (published 2016), as applied to claim 1 in the 35 U.S.C. 102 above, and further in view of in view of Chure (Chure et al, Connecting the Dots between Mechanosensitive Channel Abundance, Osmotic Shock, and Survival at Single-Cell Resolution, published 2018) and Levina (Levina et al, Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity, published 1999) and Leduc (Leduc et al, Autolysis of Escherichia coli, published 1980) and Margesin (Margesin et al, Potential of halotolerant and halophilic micoorganisms for biotechnology, published 2001).
Regarding claim 9, Czech teaches the MJF465 and MJF641 strains, which have the large-conductance mechanosensitive channel gene knocked out, are then exposed to high salinity growth conditions (p. 9, col. 1, para. 3). Regarding claim 11, Czech further teaches a method of producing a desired recombinant protein, e.g. ectoine hydroxylase (EctD), comprising transforming an engineered microorganism with a vector encoding the desired recombinant protein or polypeptides that synthesize the desired chemical compound, e.g. 5-hydroxyectoine (Abstract).
Specifically regarding claim 9, Czech does not explicitly teach the microorganism is adapted to grow in high salt conditions of 1.5% to about 3.25% (w/v) NaCl prior to knocking out the large-conductance mechanosensitive channel gene.
Specifically regarding claim 11, similarly, Czech does not explicitly teach that the engineered microorganism is already halotolerant prior to the transformation with the vector encoding the desired recombinant product.
However, Chure teaches that E. coli strains engineered to have reduced expression of MscL and lacking all mechanosensitive channel genes showed decreased survival rates with decreasing MscL expression (p. 11, section: Materials and Methods, para. 1). Chure further teaches that incorporation of a 500 mM NaCl osmotic downshock into the selection process favored the survival of MscL-expressing strains (p. 11, section: Materials and Methods, para. 1), supporting the logical conclusion that knocking out the mscL gene in a non-halotolerant microorganism puts them at risk of lysis during osmolarity changes.
Additionally, Levina teaches that E. coli mutants lacking both MscS and MscL channels have limited solute release during a 0.2 M NaCl (equivalent to 1.17 % w/v NaCl) downshock, whereas E. coli mutants lacking MscL exhibit extensive cell lysis during a 0.5 M NaCl (equivalent to 2.9% w/v NaCl) downshock, showing that the magnitude of the downshock affects the extent of cell lysis.
Additionally, Leduc teaches osmotic shock as a method for triggering cell lysis (Abstract).
Additionally, Margesin teaches that halotolerant microorganisms offer a multitude of actual or potential application for biotechnology, such as the production of food supplements, and the production of enzymes that are more stable in high salt concentrations (Abstract).
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the microorganism should be halotolerant before the knockout of the large- and/or small-conductance mechanosensitive channel gene because, as taught by Chure, doing it in the opposite order would kill off those without the mechanosensitive gene so both traits could not be selected for. As taught by Levina, these two traits together, halotolerance and lack of mechanosensitive channel genes, enable a cell to be lysed by a simple, but large, downshock, which is a desirable ability, as Leduc teaches the desire to promote E. coli cell lysis by osmotic shock, and it would be obvious to one skilled in the art, before the effective filing date of the instant application, that this could be utilized in many biotechnology purposes that require cell lysis. Although, Leduc teaches a combination of downshock and upshock in quick succession, this is understood to be necessary because these E. coli cells started at regular salinity, and thus to achieve the required magnitude of osmotic shock taught by Levina, the osmolarity had to be dropped to 0 M Na+ before going up to 0.5 M Na+. It would be obvious that if the cell started at a high osmolarity, the magnitude of the downshock would be sufficient to lyse the cell. Therefore, the first advantage conferred by starting with a halotolerant microorganism is the ability to lyse with only one osmotic shock – a downshock from its high salinity culture. Specifically, it would be obvious to one skilled in the art, before the effective filing date of the instant application, that to downshock to 0 M Na+, starting at 0.5 M Na+ (equivalent to 2.9% w/v NaCl) conditions would yield extensive cell lysis, based on Levina teaching this result. Furthermore, it would have been obvious to one skilled in the art, before the effective filing date of the instant application, that a second advantage to starting with a halotolerant organism are the unique advantages to producing recombinant products from halotolerant organisms, in light of the teachings by Margesin.
One skilled in the art, before the effective filing date of the instant application, would be motivated to give a microorganism these two traits, specifically adapted to the salt conditions claimed, and in the order claimed because the order is important to the selection of both traits and both traits confer the advantages discussed above. Specifically, one would be motivated to give a microorganism, such as E. coli, these two traits because it is widely used to produce recombinant products, and these traits would enable easier cell lysis and potentially allow E. coli to produce new products that are more stable at higher salinities than E. coli is usually grown in.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success because it is mimicking the natural lysis of halotolerant microorganisms, and E. coli, through these references, has already been shown be capable of being made halotolerant.
Claim 13 depends on claim 11. Claim 14 depends on claim 13. The teachings of the references regarding claim 11 and 13 are incorporated in its entirety for their dependent claims and discussed further below, as is relevant for each claim.
Regarding claim 13, Czech further teaches the engineered microorganism is cultured to produce the desired recombinant product, i.e. 5-hydroxyectoine (p. 9, col. 1, para. 3).
Regarding claim 14, Czech further teaches isolating the desired product released from the engineered microorganism.
Czech does explicitly teach transferring the engineered microorganism to the hypotonic solution to lyse the microorganism.
However, as previously presented in the teachings by Chure, Levina, Leduc, and Margesin and rationale discussion for claim 11, it would have been obvious to one skilled in the art, before the effective filing date of the instant application, to use osmotic downshock with a hypotonic solution to lyse the halotolerant microorganisms.
One skilled in the art, before the effective filing date of the instant application, would be motivated to utilize this simple method of cell lysis to retrieve the desired recombinant product made by the microorganism.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success because it is mimicking the natural lysis of halotolerant microorganisms, and E. coli, through these references, has already been shown be capable of being made halotolerant.
Claim(s) 15, 16, 18, 20, 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Czech (published 2016), in view of Chure (published 2018) and Levina (published 1999) and Leduc (published 1980) and Margesin (published 2001) and Wu (Wu et al, Adaptation of Escherichia coli to Elevated Sodium Concentrations Increases Cation Tolerance and Enables Greater Lactic Acid Production, published 2014).
Regarding claim 15, the teachings of Czech in view of Chure and Levina and Leduc and Margesin are taught above for claims 9 and 11 to arrive at the product of the claimed method, i.e. the halotolerant microorganism that grows within the claimed salt concentration with its large and/or small conductance mechanosensitive channel gene knocked out, and incorporated here.
Czech in view of Chure and Levina and Leduc and Margesin do not explicitly teach the steps of generating a halotolerant microorganism from a non-halotolerant microorganism by passaging the non-halotolerant microorganism on media that increases in salt concentration by 0.25% w/v NaCl.
However, Wu teaches a adaptive laboratory evolution method of generating halotolerant, i.e. elevated sodium (Na+)-tolerant, E. coli strains, from a non-halotolerant microorganism comprising passing the non-halotolerant microorganism in media that increases in salt concentration, i.e. serial transfer in medium containing progressively greater Na+ concentrations, to obtain a laboratory evolved halotolerant strain (Abstract and p. 2881, Figure 1). Wu teaches incrementing from 0.1M to 0.2M (about a 0.59% w/v change) to 0.4M (about a 1.17% w/v change) NaCl in the beginning passages, and then progressively smaller increments at higher concentrations when the viability of the cell starts to drop (Figure 1). Wu further teaches that after 0.4 M NaCl, the evolved strains started to grow better than the wildtype strain (p. 2882, col. 1, para. 1), indicating the adaptive evolution is having an effect on growth under higher saline conditions, i.e. salinity tolerance.
Wu does not explicitly teach incrementing specifically by 0.25% w/v NaCl.
However, as previously presented for claim 9 and 11, Levina teaches that the magnitude of osmolarity change impacts the cells survival and lysis, i.e. a larger change at once leads to more cell lysis than smaller change.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the magnitude of the NaCl concentration change is a result effective variable that affects the cell viability during the adaptive laboratory evolution, and would be specific to each microorganism based on its baseline tolerance for salinity. Based on the teachings of Wu that a 0.59% or 1.17% w/v NaCl change was possible for E. coli but that lower magnitude changes were needed as the E. coli was pushing the limits of its salinity tolerance, it would be obvious to one skilled in the art, for a method to be generalizable to more microorganisms, some with a lower baseline salinity tolerance than E. coli, to start with a lower gradient change. Especially in light of the Wu teaching in Figure 1 that the E. coli growth and viability started to stabilize more when they switched to lower increment changes in NaCl concentrations, such as between 0.5M NaCl and under 0.9 M NaCl.
One skilled in the art, before the effective filing date of the instant application, would be motivated to choose a lower increment changes in NaCl during the adaptive laboratory evolution for the advantage of maintaining cell viability and growth.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success based on the teaching that higher increment changes are possible but lower increment changes better maintain cell growth and viability.
The teachings of the references regarding claim 15 are incorporated in its entirety for its dependent claims and discussed further below, as is relevant for each claim.
Regarding claim 16, as previously presented, the teachings of Czech in view of Chure and Levina and Leduc and Wu all used E. coli as the exemplary non-halotolerant microorganism.
Regarding claim 18, as previously presented, Czech teaches the large conductance mechanosensitive channel gene is mscL and the small conductance mechanosensitive channel gene is mscS, and both are knocked out (Abstract).
Regarding claim 20, the teachings to arrive at the method of producing this halotolerant microorganism is discussed for claim 15; therefore, the teachings also teach the resultant halotolerant microorganisms obtained by the method of claim 15.
Regarding claim 21, Chure teaches that a cell that has fewer than approximately 100 MscL channels per cell, and no MscS channels, never survived an osmotic downshock in their single-cell experiments.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that a microorganism that completely lacks any MscL channels, due to complete knockout of the gene, would not survive osmotic downshock, effectively achieving 100% osmolytic efficiency upon osmotic downshock.
One skilled in the art, before the effective filing date of the instant application, would be motivated to efficiently lyse all cells, such as in applications that require recovering bioproducts made by the cell.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success due to the direct teachings by Chure regarding the effect that a lack of MscL and MscS channels have on the lysis of cells upon osmotic downshock.
Claim(s) 22 and 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Czech (published 2016) in view of Chure (published 2018) and Levina (published 1999) and Leduc (published 1980) and Margesin (published 2001) and Wu (published 2014) and Sedlaceka (Sedlaceka et al, PHA granules help bacterial cells to preserve cell integrity when exposed to sudden osmotic imbalances, published 2019) and Passanha (Passanha et al, The use of NaCl addition for the improvement of polyhydroxyalkanoate production by Cupriavidus necator, published 2014) and Berezina (Berezina et al, Novel approach for productivity enhancement of polyhydroxyalkanoates (PHA) production by Cupriavidus necator DSM 545, published 2013).
Regarding claims 22 and 23, the teachings of Czech in view of Chure and Levina and Leduc and Margesin and Wu to arrive at halotolerant microorganisms, such as E. coli, that grows on 3% NaCl LB broth and lacks expression of a large conductance mechanosensitive channel protein are previously presented and incorporated for the claims below.
Czech in view of Chure and Levina and Leduc and Wu do not explicitly teach the microorganism is C. necator.
However, Sedlaceka teaches the non-halophilic bacterium Cupriavidus necator, which naturally produce polyhydroxyalkanoates (PHA), is able to withstand osmotic up-shock induced by 200 g/L NaCl (Abstract; p. 130, section: Microorganisms and section: Results, para. 1).
Additionally, Passanha teaches that increasing NaCl concentration increases production of PHA, up to a certain concentration (Abstract), and the utility of PHA as a desirable plastic source.
Additionally, Berezina teaches that Cupriavidus necator can grow on LB, and the most efficient biomass and PHA production were found when culturing C. necator in two tested media, one of which is LB.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that (a) Cupriavidus necator is a non-halotolerant bacterium useful for biotechnology applications that favors culture at high salt concentrations, and that Cupriavidus necator can likely be made halotolerant due to its demonstrated high tolerance for salinity; and (b) that Cupriavidus necator can grow on the claimed LB media with 3% NaCl, and (c) to further knockout its large mechanosensitive conductance channel gene for the same reasons explained above, pertaining to enabling its ability to be used in high salt production systems and simple lysis by downshocking in water.
One skilled in the art, before the effective filing date of the instant application, would be motivated to improve on the ability of Cupriavidus necator to produce bioproducts, such as PHA, that are more efficiently produced at higher salinity cultures.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success because the of the demonstrated teachings that Cupriavidus necator can be tolerant to high salt concentrations and can grow and produce bioproducts, such as PHA, on LB. It would also be reasonably expected that the knockout of the large mechanosensitive conductance channel gene homolog in C. necator would also confer the same effects as those demonstrated in E. coli, as the large mechanosensitive conductance channel serves the same purpose in all the microorganisms they are present in.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BONIRATH CHHAY whose telephone number is (571)272-0682. The examiner can normally be reached Mon-Thu 8AM-5PM EST.
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, Bao-Thuy Nguyen can be reached at (571) 272-0824. 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.
/BONIRATH CHHAY/Examiner, Art Unit 1645
August 22, 2026
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 August 24, 2026
Prosecution Insights