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WO2025240457A1 - Conversion de biomasse en produits chimiques industriels à l'aide de bactéries modifiées - Google Patents

Conversion de biomasse en produits chimiques industriels à l'aide de bactéries modifiées

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Publication number
WO2025240457A1
WO2025240457A1 PCT/US2025/029115 US2025029115W WO2025240457A1 WO 2025240457 A1 WO2025240457 A1 WO 2025240457A1 US 2025029115 W US2025029115 W US 2025029115W WO 2025240457 A1 WO2025240457 A1 WO 2025240457A1
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WO
WIPO (PCT)
Prior art keywords
plant biomass
bacterium
genetically modified
biomass sample
bescii
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/029115
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English (en)
Inventor
Robert M. Kelly
Ryan G. BING
Daniel B. SULIS
Jack P. WANG
Daniel J. WILLARD
Kathryne C. FORD
Michael W. Adams
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Georgia
North Carolina State University
University of Georgia Research Foundation Inc UGARF
Original Assignee
University of Georgia
North Carolina State University
University of Georgia Research Foundation Inc UGARF
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Application filed by University of Georgia, North Carolina State University, University of Georgia Research Foundation Inc UGARF filed Critical University of Georgia
Publication of WO2025240457A1 publication Critical patent/WO2025240457A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
    • C12N15/03—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the genetically modified extremely thermophilic bacterium comprises a heterologous gene encoding a bifunctional alcohol dehydrogenase.
  • the engineered bacterium is a bacterium from the order Caldicellulosiruptorales.
  • the genetically modified bacterium comprises Anaerocellum bescii.
  • the heterologous gene comprises an AdhE gene from Thermoclostridium stercorarium.
  • the AdhE gene from Thermoclostridium stercorarium comprises an Asp492Gly mutation.
  • a gene encoding lactate dehydrogenase expression has been inactivated. In some aspects, a gene encoding lactate dehydrogenase has been inactivated by homologous recombination. In some aspects, the heterologous gene bifunctional AdhE is expressed from a plasmid. In some aspects, the heterologous gene encoding the bifunctional alcohol dehydrogenase is inserted into a genomic locus of the bacterium. In some aspects, the engineered bacterium further includes a promoter operably linked to the heterologous gene encoding the bifunctional alcohol dehydrogenase, wherein the promoter is configured to function at elevated temperatures.
  • the heterologous bifunctional alcohol dehydrogenase gene is inserted downstream of Athe_0949 in the A. bescii genome.
  • described herein are genetically modified A. bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C.
  • described herein is a method for enhancing ethanol production. In some aspects, the method includes: obtaining a biomass sample; contacting the plant biomass sample with a genetically modified bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase under conditions effective to convert the plant biomass to ethanol.
  • the conditions effective include one or more of: (i) maintaining pH between 5.8 and 7, and/or (ii) maintaining an operational temperature of 55-75°C.
  • the genetically modified bacterium comprising A. bescii that has been genetically modified with the insertion of alcohol dehydrogenase gene. Attorney Dkt.10620-156WO1
  • described herein are genetically modified Anaerocellum bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C.
  • a bioreactor system for enhancing ethanol production and selectivity relative to other fermentation byproducts from plant biomass, comprising: a temperature control system configured to maintain a fermentation temperature (e.g., from 55°C to 75°C) for a genetically modified A. bescii strain, the genetically modified A.
  • bescii bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase; a pH control system configured to maintain a pH between 5.8-7.0 (e.g., using sodium bicarbonate and sodium hydroxide); a pressurization system configured to control carbon dioxide head pressure; and an agitation system configured to mitigate cell aggregation.
  • the fermentation byproducts comprise acetate, acetone, lactate, hydrogen, carbon dioxide, acetoin, 2,3-butanediol, pyruvate, butanol, isopropanol, and/or higher chain alcohols.
  • described is a method of producing ethanol from a plant biomass sample.
  • the method can include: obtaining a plant biomass sample; contacting the plant biomass sample with any of the genetically modified thermophilic bacterium described herein; and releasing a fermentable carbohydrate from the plant biomass sample; and fermenting the fermentable carbohydrate to produce ethanol.
  • the plant biomass sample comprises a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose.
  • the poplar tree is a genetically modified poplar tree.
  • the mixing of the plant biomass sample with the genetically modified bacterium occurs at about pH 5.8-7.0. In some aspects, the pH is maintained using NaOH or NaHCO 3 .
  • contacting the plant biomass sample with the genetically modified bacterium occurs at about 55-75 ⁇ C. In some aspects, the method does not comprise a chemical pretreatment of the plant biomass sample.
  • described herein is a method of degrading lignin in a plant biomass sample, comprising: obtaining a plant biomass sample; mixing the plant biomass sample with Attorney Dkt.10620-156WO1 the genetically modified bacterium described herein; and releasing a fermentable carbohydrate from the plant biomass sample.
  • the method further includes: determining a methoxy content number in the plant biomass sample; wherein the methoxy content number is determined using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample.
  • a value of less than or equal to 0.2 is indicative that the plant biomass can be solubilized without chemical pretreatment.
  • the plant biomass sample is from a poplar tree.
  • the poplar tree is a genetically modified poplar tree.
  • mixing the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about pH 5.8-7.0.
  • the pH is maintained using sodium hydroxide.
  • the mixing of the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about 55-75 ⁇ C.
  • the method does not comprise a chemical pretreatment of the plant biomass sample.
  • the fermentable carbohydrate is converted to ethanol.
  • a method of degrading lignocellulose in a plant biomass sample comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; based on the methoxy content number being below a threshold value, contacting the plant biomass sample with a bacterium, wherein the bacterium degrades the lignocellulose; and forming a fermentable carbohydrate from the plant biomass sample.
  • the threshold value is 0.17 or less.
  • the bacterium is a thermophilic bacterium.
  • the bacterium is Anaerocellum (e.g., Anaerocellum bescii), Acetivibrio (e.g., Acetivibrio thermocellus Caldicellulosiruptor, Attorney Dkt.10620-156WO1 Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobius, or Thermoclostridium.
  • the bacterium is a genetically modified strain.
  • the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose.
  • the poplar tree comprises a genetically modified poplar tree.
  • contacting the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about pH 5.0-7.2.
  • the pH is maintained with NaOH.
  • the contacting of the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about 55 ⁇ C-85 ⁇ C.
  • the method does not comprise a chemical pretreatment of the plant biomass sample.
  • the fermentable carbohydrate is converted to ethanol.
  • described herein is a method of determining an amount of a chemical pretreatment of a plant biomass sample, the method comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; and determining, based on the methoxy content number, an amount of a chemical pretreatment needed for the plant biomass sample, wherein a higher methoxy content number indicates more chemical pretreatment and a lower methoxy content number indicates a less or no chemical pretreatment.
  • the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose.
  • the poplar tree comprises a genetically modified poplar tree.
  • Plant biomasses included: sugarcane bagasse, spent coffee beans, Cave-in-Rock switchgrass, wheat straw, Frasier fir, corn stover, hemp fiber, wild-type poplar, and crystalline cellulose (Avicel).
  • Figure 2 Lignin monomers. Monolignols with methoxy group emphasized. Phenol ring carbon numbers of the phenol rings are indicated with small red numbers.
  • Figure 3 A. bescii fermentation of poplar lines. [Panel A] Total fermentation products (acetate, lactate, ethanol) generated by A.
  • Figure 8 Protein alignment of bifunctional alcohol dehydrogenases. AdhE protein sequences from Acetivibrio thermocellus and Thermoclostridium stercorarium subsp.
  • Figure 11 Effect of bicarbonate on RKCB92 ethanol production from cellulose.
  • FIG 14 Transcriptomic analysis of RKCB92 sodium bicarbonate versus hydroxide Avicel bioreactors. RNA sequencing results from late-log phase cells. Heat mapped log 2 fold change of the sodium bicarbonate (NaHCO 3 ) condition compared to sodium hydroxide (NaOH) condition. Log2 counts per million (CPM) are also reported for both conditions. Individual genes are mapped clockwise in order of RKCB92 Gene ID. Specific areas of interest (A-Q) and the 8 most expressed genes with significant fold changes in the bicarbonate condition (1-8) are indicated. Details of these regions and genes are shown in Figure 17. Figure 15 shows a table providing a summary of reported fermentation performance of ethanol producing strains.
  • Figure 16 shows a table providing a summary of RKCB89 and MACB1058 cultures.
  • Figure 17 shows a summary of RKCB92 Fermentations.
  • Figure 18 - Plasmids Maps. Plasmid maps for the five plasmids used in this study.
  • pRGB025 and pRGB026 are E. coli expression vectors for Thermoclostridium stercorarium AdhE with and without the Asp492Gly mutation.
  • pRGB008 and pRGB011 are A. bescii acetate gene (pta, ack) knockout non-replicating vectors.
  • pRGB032 is A. bescii non- replicating vector for integration of T.
  • the term “substantially,” in, for example, the context “substantially identical” or “substantially similar” refers to a method or a system, or a component that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about Attorney Dkt.10620-156WO1 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% by similar to the method, system, or the component it is compared to.
  • amino acid refers to a compound containing both amino (—NH2) and carboxyl (—COOH) groups generally separated by one carbon atom.
  • the central carbon atom may contain a substituent which can be either charged, ionisable, hydrophilic or hydrophobic. Any of 22 basic building blocks of proteins having the formula NH2—CHR— COOH, where R is different for each specific amino acid, and the stereochemistry is in the ‘L’ configuration. Additionally, the term “amino acid” can optionally include those with an unnatural ‘D’ stereochemistry and modified forms of the ‘D’ and ‘L’ amino acids.
  • polypeptide polypeptide
  • peptide or “protein” generally refer to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally-occurring amino acids.
  • protein refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce tertiary and/or quaternary structure.
  • protein excludes small peptides by definition, the small peptides lacking the requisite higher-order structure necessary to be considered a protein. Different modifications of, and/or additions to, the polypeptides constituting the population according to the invention may be performed in order to tailor the polypeptides to the specific use intended.
  • additional amino acids comprised in the same polypeptide chain, or labels and/or therapeutic agents that are chemically conjugated or otherwise bound to the polypeptides constituting the population.
  • additional amino acid residues on the C-terminal end may be preferred. These additional amino acid residues may play a role in the binding of the polypeptide, but may equally well serve other purposes, related for example to one or more of the production, purification, stabilization, coupling or detection of the polypeptide.
  • additional amino acid residues may comprise one or more amino acid residues added for purposes of chemical coupling. An example of this is the addition of a cysteine residue at the very first or very last position in the polypeptide chain, i.e.
  • a cysteine residue to be used for chemical coupling may also be introduced by replacement of another amino acid on the surface of the protein domain, preferably on a portion of the surface that is not involved in target binding.
  • Such additional amino acid residues may also comprise a “tag” for purification or detection of the polypeptide, Attorney Dkt.10620-156WO1 such as a hexahistidyl (His6) tag, or a “myc” tag or a “FLAG” tag for interaction with antibodies specific to the tag.
  • His6 hexahistidyl
  • Myc myc
  • FLAG FLAG
  • additional amino acid residues may also constitute one or more polypeptide domain(s) with any desired function, such as another binding function, or an enzymatic function, or a metal ion chelating function, or a fluorescent function, or mixtures thereof.
  • the “percentage of sequences identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • identical in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same.
  • Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. These definitions also refer to the complement of a test sequence.
  • the identity exists over a region that is at least about 50 nucleotides in length, or more typically over a region that is 100 to 500 or 1000 or more nucleotides in length.
  • the terms “similarity” or “percent similarity” in the context of two or more polypeptide sequences refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined by a conservative amino acid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of Attorney Dkt.10620-156WO1 the following sequence comparison algorithms or by manual alignment and visual inspection.
  • Sequences are “substantially similar” to each other if they are at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% similar to each other.
  • this similarly exists over a region that is at least about 25 amino acids in length (e.g., at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75), or more typically over a region that is at least about 100 to 500 or 1000 or more amino acids in length.
  • typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are commonly used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities or similarities for the test sequences relative to the reference sequence, based on the program parameters.
  • engineered or modified organisms are produced via the introduction of genetic material into a host or parental microorganism of choice thereby modifying or altering the cellular physiology and biochemistry of the microorganism. Through the introduction of genetic material the parental microorganism can acquire new properties, e.g., the ability to produce a new, or greater quantities of, an intracellular metabolite.
  • the genetic material introduced into the parental microorganism contains gene(s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of n-butanol and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. promoter sequences.
  • An engineered or modified microorganism can also include in the alternative or in addition to the introduction of a genetic material into a host or parental microorganism, the disruption, deletion, or knocking out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism.
  • the microorganism acquires new or improved properties (e.g., the ability to produce a new or greater quantities of an intracellular metabolite, improve the flux of a metabolite down a desired pathway, and/or reduce the production of undesirable by-products).
  • the microorganism may be modified to express one or more exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism.
  • a microorganism Attorney Dkt.10620-156WO1 may also be modified to modulate the expression level of an endogenous gene.
  • a genetic modification of a microorganism may be carried out by using techniques known in the art, such as CRISPR-Cas systems described in U.S. Patent No. 11,142,751, which is incorporated by reference herein.
  • condition effective as it relates to the inoculation of a fermentation mixture refers to a set of adjustable process parameters, such as pH, temperature, metabolite environment, and time, which can produce a desired product.
  • the incubation of a fermentation mixture under conditions effective can convert a compound or compounds, such as single carbon compounds, to a target product, such as acetate, at an efficiency.
  • Engineered Bacteria Disclosed herein are engineered bacteria useful for the conversion of a biomass to a carbonaceous product.
  • described herein is a genetically modified extremely thermophilic bacterium, wherein the genetically modified extremely thermophilic bacterium comprises a heterologous gene encoding a bifunctional alcohol dehydrogenase.
  • the term “bifunctional” is intended to include enzymes that catalyze more than one biochemical reaction step.
  • the bifunctional enzyme used herein is an enzyme (adhE) that catalyzes both the alcohol dehydrogenase and acetaldehyde dehydrogenase reactions.
  • Attorney Dkt.10620-156WO1 The genetically modified bacterium described herein may be extremely thermophilic.
  • the engineered bacterium is a bacterium from the order Caldicellulosiruptorales.
  • the bacterium comprises Anaerocellum (e.g., Anaerocellum bescii), Acetivibrio (e.g., Acetivibrio thermocellus Caldicellulosiruptor, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobius, or Thermoclostridium.
  • the bacterium is a genetically modified strain.
  • the genetically modified bacterium comprises A. bescii.
  • the heterologous gene comprises an AdhE gene from a moderately thermophilic bacterium.
  • the term “moderately thermophiles,” also called “facultative thermophiles,” refers to bacterial strains, which are capable of growing at temperatures between 30-65° C, typically having an optimum between 40-60° C.
  • the heterologous gene comprises an AdhE gene from Thermoclostridium stercorarium, e.g., SEQ ID NO:1.
  • the bifunctional alcohol dehydrogenase has 80% or more sequence identity to SEQ ID NO:1.
  • the bifunctional alcohol dehydrogenase has 85% or more sequence identity to SEQ ID NO:1.
  • the bifunctional alcohol dehydrogenase has 90% or more sequence identity to SEQ ID NO:1.
  • the bifunctional alcohol dehydrogenase has 95% or more sequence identity to SEQ ID NO:1. In some aspects, the bifunctional alcohol dehydrogenase has 97% or more sequence identity to SEQ ID NO:1. In some aspects, the bifunctional alcohol dehydrogenase has 99% or more sequence identity to SEQ ID NO:1. In some aspects, the AdhE gene from Thermoclostridium stercorarium comprises an Asp492Gly mutation. In some aspects, the bifunctional alcohol dehydrogenase has 80% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase has 85% or more sequence identity to SEQ ID NO:2.
  • the bifunctional alcohol dehydrogenase has 90% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase has 95% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase has 97% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase has 99% or more sequence identity to SEQ ID NO:2. In some aspects, the bifunctional alcohol dehydrogenase comprises SEQ ID NO:2. In some aspects, a gene encoding lactate dehydrogenase expression has been inactivated.
  • a gene encoding lactate dehydrogenase has been inactivated by Attorney Dkt.10620-156WO1 homologous recombination.
  • the heterologous gene bifunctional AdhE is expressed from a plasmid.
  • the heterologous gene encoding the bifunctional alcohol dehydrogenase is inserted into a genomic locus of the bacterium.
  • the engineered bacterium further includes a promoter operably linked to the heterologous gene encoding the bifunctional alcohol dehydrogenase, wherein the promoter is configured to function at elevated temperatures.
  • the heterologous bifunctional alcohol dehydrogenase gene is inserted downstream of Athe_0949 in the A.
  • bescii genome In some aspects, described herein are genetically modified A. bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C, e.g., from 55°C to 70°C, from 55°C to 65°C, from 55°C to 60°C, from 60°C to 75°C, from 65°C to 75°C, from 70°C to 75°C, from 60°C to 70°C, or about 65°C. In some aspects, described herein is a method for enhancing ethanol production.
  • the method includes: obtaining a biomass sample; contacting the plant biomass sample with a genetically modified bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase under conditions effective to convert the plant biomass to ethanol.
  • the conditions effective include one or more of: (i) maintaining pH between 5.8 and 7.0, and/or (ii) maintaining an operational temperature of 55-75°C.
  • the genetically modified bacterium comprising A. bescii that has been genetically modified with the insertion of alcohol dehydrogenase gene.
  • described herein are genetically modified A.
  • bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C, e.g., from 55°C to 70°C, from 55°C to 65°C, from 55°C to 60°C, from 60°C to 75°C, from 65°C to 75°C, from 70°C to 75°C, from 60°C to 70°C, or about 65°C.
  • 55°C to 75°C e.g., from 55°C to 70°C, from 55°C to 65°C, from 55°C to 60°C, from 60°C to 75°C, from 65°C to 75°C, from 70°C to 75°C, from 60°C to 70°C, or about 65°C.
  • bescii strains configured to selectively form ethanol over acetate under anaerobic conditions at a temperature range of 55°C to 75°C, e.g., from 55°C to 70°C, from 55°C to 65°C, from 55°C to 60°C, from 60°C to 75°C, from 65°C to 75°C, from 70°C to 75°C, from 60°C to 70°C, or about 65°C.
  • a bioreactor system for enhancing ethanol production and selectivity relative to other fermentation byproducts from plant biomass, comprising: a temperature control system configured to maintain a fermentation temperature (e.g., from Attorney Dkt.10620-156WO1 55°C to 75°C) for a genetically modified A. bescii strain, the genetically modified A. bescii bacterium comprising a heterologous gene encoding a bifunctional alcohol dehydrogenase; a pH control system configured to maintain a pH between 5.8-7.0 (e.g., using sodium bicarbonate and sodium hydroxide); a pressurization system configured to control carbon dioxide head pressure; and an agitation system configured to mitigate cell aggregation.
  • a temperature control system configured to maintain a fermentation temperature (e.g., from Attorney Dkt.10620-156WO1 55°C to 75°C) for a genetically modified A. bescii strain, the genetically modified A. bescii bacterium comprising a
  • the fermentation byproducts comprise acetate, lactate, hydrogen, carbon dioxide, acetoin, 2,3-butanediol, pyruvate, butanol, isopropanol, and/or higher chain alcohols.
  • bioreactor systems may typically include one or more vessels, towers, or piping arrangements, such as a continuous stirred tank reactor (CSTR), immobilized cell reactor (ICR), trickle bed reactor (TBR), bubble column, gas lift fermenter, static mixer, or other vessel or other device suitable for gas-liquid contact.
  • one or more of the bioreactors can include a growth reactor which can be used to seed a fermentation reactor.
  • Bioreactors can range in size from a few liters to several cubic meters (i.e. several 1000 liters) or larger and can be formed using a number of different materials, e.g. stainless steel or glass. Based on the mode of operation, a bioreactor may be classified as batch, fed-batch or continuous.
  • the bioreactor is typically equipped with one or more inlets for supplying culture medium to the cells, and with one or more outlets for harvesting product or emptying the bioreactor. Additionally, the bioreactor may be equipped with at least one outlet constructed in such a way that a separation device can be attached to the bioreactor.
  • the bioreactor's environmental conditions such as gas (i.e., air, oxygen, nitrogen, carbon dioxide) flow rates, temperature, pH and dissolved oxygen levels, and agitation speed/circulation rate can be closely monitored and controlled.
  • gas i.e., air, oxygen, nitrogen, carbon dioxide
  • temperature, pH and dissolved oxygen levels, and agitation speed/circulation rate can be closely monitored and controlled.
  • the fermentation mixture(s) are maintained in an aqueous culture medium including nutrients, vitamins, and/or minerals sufficient to permit growth of the microorganism(s). Suitable media are well known in the art as instructed by this disclosure.
  • described is a method of producing ethanol from a plant biomass sample.
  • the method can include: obtaining a plant biomass sample; contacting the plant biomass sample with any of the genetically modified thermophilic bacterium described herein; and releasing a fermentable carbohydrate from the plant biomass sample; and fermenting the fermentable carbohydrate to produce ethanol.
  • the plant biomass sample comprises a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose.
  • the poplar tree is a genetically modified poplar tree.
  • the plant biomass comprises a plant biomass that has been genetically modified to have a lower methoxy content compared to a wild-type plant biomass.
  • the mixing of the plant biomass sample with the genetically modified bacterium occurs at about pH 5.8-7.0. In some aspects, the pH is maintained using NaOH or NaHCO 3 . In some aspects, contacting the plant biomass sample with the genetically modified bacterium occurs at about 55-75 ⁇ C. In some aspects, the method does not comprise a chemical pretreatment of the plant biomass sample.
  • described herein is a method of degrading lignin in a plant biomass sample, comprising: obtaining a plant biomass sample; mixing the plant biomass sample with the genetically modified bacterium described herein; and releasing a fermentable carbohydrate from the plant biomass sample.
  • the method further includes: determining a methoxy content number in the plant biomass sample; wherein the methoxy content number is determined using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample.
  • a value of less than or equal to 0.2 is indicative that the plant biomass can be solubilized without chemical pretreatment.
  • the plant biomass sample is from a poplar tree.
  • the poplar tree is a genetically modified poplar tree.
  • mixing the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about pH 5.8-7.0.
  • the pH is maintained using sodium hydroxide.
  • the mixing of the plant biomass sample comprising a low methoxy lignin content with the bacterium is done at about 55-75 ⁇ C.
  • the method does not comprise a chemical pretreatment of the plant biomass sample.
  • the fermentable carbohydrate is converted to ethanol, e.g., at a high titer.
  • the term “titer” refers to the quantity of targeted product (e.g., ethanol) produced per unit volume of host cell culture.
  • a titer of the ethanol is 25 mM or more, such as 30 mM or more, 35 mM or more, 40 mM or more, 45 mM or more, 50 mM or more, 55 mM or more, 60 mM or more, 65 mM or more, 70 mM or more, 75 mM or more, Attorney Dkt.10620-156WO1 80 mM or more, 85 mM or more, 90 mM or more, 95 mM or more, 100 mM or more, 105 mM or more, 110 mM or more, 115 mM or more, 120 mM or more, 125 mM or more, 130 mM or more, 135 mM or more, 140 mM or more, 145 mM or more, or 150 mM or more.
  • a titer of ethanol is produced is from 25 mM to 150 mM, such as from 35 mM to 150 mM, from 50 mM to 150 mM, from 65 mM to 150 mM, from 75 mM to 150 mM, from 95 mM to 150 mM, from 25 mM to 135 mM, from 45 mM to 135 mM, from 65 mM to 135 mM, from 75 mM to 135 mM, from 85 mM to 135 mM, from 95 mM to 135 mM, from 100 mM to 135 mM, or about 135 mM.
  • a method of degrading lignocellulose in a plant biomass sample comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; based on the methoxy content number being below a threshold value, contacting the plant biomass sample with a bacterium, wherein the bacterium degrades the lignocellulose; and forming a fermentable carbohydrate from the plant biomass sample.
  • the threshold value is 0.17 or less.
  • the bacterium is an extremely thermophilic bacterium.
  • the bacterium is Anaerocellum (e.g., A. bescii), Acetivibrio e.g., Acetivibrio thermocellus Caldicellulosiruptor, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobius, or Thermoclostridium.
  • the bacterium is a genetically modified strain.
  • the biomass includes a lignocellulose hydrolysate.
  • lignocellulose hydrolysate refers to hydrolysis products of lignocellulose or lignocellulosic material comprising cellulose and/or hemicellulose, oligosaccharides, mono- and/or disaccharides, acetic acid, formic acid, other organic acids, furfural, hydroxymethyl furfural, levulinic acid, phenolic compounds, other hydrolysis and/or degradation products formed from lignin, cellulose, hemicellulose and/or other components of lignocellulose, nitrogen compounds originating from proteins, metals and/or non- hydrolyzed or partly hydrolyzed fragments of lignocellulose.
  • lignocellulose hydrolysates are obtained from a lignocellulosic biomass such as paper, paper Attorney Dkt.10620-156WO1 products, wood, wood-related materials, particle board, grasses, rice hulls, bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, coconut hair, algae, seaweed, altered celluloses, e.g., cellulose acetate, regenerated cellulose, and the like, or combinations thereof.
  • the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose.
  • the poplar tree comprises a genetically modified poplar tree.
  • contacting the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about pH 5.0-7.2.
  • the pH is maintained with NaOH.
  • the contacting of the plant biomass sample comprising a low methoxy lignin content with the bacterium occurs at about 55 ⁇ C-85 ⁇ C, e.g., from 55°C to 75°C, from 55°C to 70°C, from 55°C to 65°C, from 55°C to 60°C, from 60°C to 75°C, from 65°C to 75°C, from 70°C to 75°C, from 60°C to 70°C, or about 65°C.
  • the method does not comprise a chemical pretreatment of the plant biomass sample.
  • the fermentable carbohydrate is converted to ethanol.
  • a method of determining an amount of a chemical pretreatment of a plant biomass sample comprising: determining a methoxy content number of a plant biomass sample; wherein the methoxy content number is calculated using the formula: , wherein G represents an amount of coniferyl alcohol subunits in the plant biomass sample, and S represents an amount of sinapyl alcohol subunits in the plant biomass sample; and determining, based on the methoxy content number, an amount of a chemical pretreatment needed for the plant biomass sample, wherein a higher methoxy content number indicates more chemical pretreatment and a lower methoxy content number indicates a less or no chemical pretreatment.
  • the plant biomass sample is obtained or derived from a poplar tree, sugarcane bagasse, coffee beans, switchgrass, wheat straw, Frasier fir, corn stover, hemp, or crystalline cellulose.
  • the poplar tree comprises a genetically modified poplar tree.
  • Lignocellulose is composed of polysaccharides cross-linked with the phenolic polymer lignin such that processes utilizing lignocellulose need to liberate carbohydrates from lignin.
  • thermophilic bacteria i.e. genera Caldicellulosiruptor, Anaerocellum, Acetivibrio
  • Anaerocellum natively solubilize and utilize a wide range of plant polysaccharides employing large sets of extracellular enzymes and have been metabolically engineered to make fuels and chemicals.
  • Anaerocellum f.
  • Caldicellulosiruptor bescii, belonging to the extremely thermophilic Caldicellulosiruptorales (T opt > 70°C), has demonstrated the capacity to degrade a wide range of plant biomasses, resist contamination, and to produce industrially relevant products.
  • T opt > 70°C extremely thermophilic Caldicellulosiruptorales
  • lignin remains a barrier to highly efficient biomass degradation by A. bescii, which is reflected in the disparate levels of carbohydrate solubilization of low and high lignin plant biomasses (such as soybean hulls as compared to poplar wood).
  • the lignin barrier extends to other microbes, such as Acetivibrio thermocellus as well.
  • CRISPR edited poplar trees can have superior wood properties for fiber pulping, producing trees with lower lignin, higher carbohydrate to lignin ratio, and increased S/G ratio all while preserving the overall fitness of the trees.
  • the issue considered here is whether plant biomasses, including poplar, best for fiber pulping are also amenable to microbial conversion and what features are most significant for solubilization to fermentable sugars.
  • composition and fermentation data for selected plant biomasses Attorney Dkt.10620-156WO1 PB: p-hydroxybenzoic acid; H: H-subunits; G: G-subunits; S: S-subunits; Other Lignin Acids; %: percentage volume of total lignin.*Other reports may suggest up to 100% mass solubilization and >30 mM products, these numbers are used for culture condition consistency Lignin is a highly complex polymer, containing a wide variety of subunits and chemical linkages, making direct quantification of their structures difficult. However, the majority of the subunits are derived from monolignol precursors: p-coumaryl alcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S).
  • H p-coumaryl alcohol
  • G coniferyl alcohol
  • S sinapyl alcohol
  • Equation 1 weights monolignols (as percent of monolignols, H + G + S from 2D-NMR data) by their number of methoxy substitutions, then multiplies this by total lignin content (mass fraction of total plant biomass).
  • Linear regression of A. bescii fermentation products and mass solubilization resulted in R 2 values of 0.66 and 0.91, respectively ( Figure 1, panels C, D).
  • mass solubilization is the primary measurement of microbial ability to solubilize polysaccharides contained in the lignocellulose, and fermentation products measure both solubilization and subsequent conversion.
  • A. bescii Due to the small number of data points (ten) in Figure 1 and the limited availability of lignin composition for plant biomasses, further validation of the methoxy content correlation was addressed with A. bescii fermentation of genetically modified poplar lines with highly variable lignin content and composition.
  • Two strains of A. bescii wild-type strain DSM6725 and recombinant strain MACB1058 were used to screen wild-type poplar and 133 genetically modified poplar lines (including 18 lines from previous studies ) for conversion of plant carbohydrates into primary fermentation products (acetate, ethanol, lactate) as a function of tree fitness (Figure 3, panel A).
  • MACB1058 is an A.
  • RNAi-modified Line i20-3-1 could also be fermented to a high degree (21 mM), albeit below the levels observed for Lines 54 and 80, but with no significant impact on tree fitness; Line i20-3-1 actually grew better than wild-type.
  • CRISPR-edited Lines H-4-1 and E-3-1 had similar lignin levels ( ⁇ 15% compared to ⁇ 22% for WT), but H-4-1 generated 22.1 mM fermentation products compared to 13.0 mM for E- 3-1. H-4-1 had no significant fitness issues, in contrast to E-3-1 which did ( Figure 3, panel A).
  • Line 80 had fermentable mass equivalent to 20.5% of a wild-type tree, even though at 5 g/L mass Attorney Dkt.10620-156WO1 loading more fermentation products were generated (i.e., lower microbial recalcitrance, but poor growth).
  • the most improved lines for microbial conversion (H-4-1, i20-3-1) are represented in the upper right of Figure 3, panel B. While total fermentation products are inversely proportional to poplar total lignin content, these data had a linear correlation coefficient (R 2 ) of 0.5 ( Figure 3, panel C), in line with data shown in Figure 1, panel A for non-genetically edited plant biomasses. High S/G ratio and low H is desirable for pulp and paper feedstocks.
  • Wild-type poplar was more recalcitrant to fermentation than the genetically modified lines that had lower lignin and significant fitness issues (Lines 54, 80 and E-3-1).
  • the other reduced lignin lines (i20-3-1 and H-4-1) were solubilized and fermented to a greater extent than wild-type poplar, but with no fitness issues.
  • Lines i20-3-1 and H-4-1 had 2.7 and 3 times, respectively, more fermentable mass in a 6-month-old tree than wild-type poplar (Figure 4, panel B, relative growth multiplied by mass solubilization), and the carbohydrate content was 2-2.5 times more accessible (Figure 4, panel B, total fermentation products).
  • Experimental scale, degree of mixing, and pH control were not significant factors.
  • Line H-4-1 was compared at 50 mL serum bottle scale with no pH control to 1L bioreactor scale with pH control; no substantial differences in solubilized carbohydrate content was noted (Figure 4, panel C, panel D).
  • Poplar lignin content and composition impacts microbial solubilization and conversion. Even though CRISPR-edited Lines E-3-1 and H-4-1 had similar reduced lignin content (15% and 15.5%, respectively), the microbial total mass solubilization (35.3% vs. Attorney Dkt.10620-156WO1 63.4%, respectively) and fermentation products (13.0 mM vs.22.1 mM, respectively) differed significantly (two-way t-test, ⁇ 99.5% confidence). This result parallels the data seen in Figure 1, panels A, B for non-genetically modified plants with lignin ⁇ 20-22%. This difference was examined in greater detail.
  • H-4-1 had better growth characteristics than E-3-1 as indicated by stem volume as percent of wild-type (106% vs.42%, respectively). Although total lignin content was comparable for H-4-1 and E-3-1, lignin composition differed significantly between the two lines (Table 2).
  • Line 80 is 30% lignin aldehydes Attorney Dkt.10620-156WO1 (G and S unit monolignol precursors). Weighting the methoxy content (Equation 1) by the alcohol content of lignin (accounting for acids and aldehydes) improves the fit for Line 80 and grasses containing ferulic and coumaric acid (sugarcane, wheat straw), but worsens the fits for other high aldehyde lines (E and F CRISPR-edit poplar lines targeting PtrPAL genes).
  • thermocellus ( Figure 6, panel C). This is due to contamination from indigenous microbial life that grows only under 75°C, as seen for the autoclaved versus not autoclaved sugarcane bagasse and Avicel (reported to have no contaminants). As Ac. thermocellus is unable to consume pentoses (even though it solubilizes them from hemicellulose), and does not grow above the thermophilic threshold to resist contamination, fermentation products produced from non- pretreated plant biomass are inconsistent; this emphasizes the need to use sterilizing pretreatments with moderately thermophilic Ac. thermocellus. However, as Ac. thermocellus is still the primary lignocellulose degrader, the mass solubilization (Figure 6, panel D) has a better linear fit with methoxy content.
  • H-4-1 is predominantly G lignin
  • a shift to higher H and even lower S lignin could generate poplar with even lower methoxy content.
  • the relationship between low lignin and methoxy content relates to plant biomass recalcitrance is not completely clear. Bonds associated with methoxy groups may result in a higher degree of carbohydrate-lignin cross-links that are less enzymatically available.
  • Attorney Dkt.10620-156WO1 Higher S/G ratio has been associated with less condensed lignin polymer harboring lower levels of carbon-carbon linkages between subunits of lignin, which may reduce interference during chemical or enzymatic deconstruction.
  • thermocellus lack carbohydrate active enzyme(s) needed to deconstruct specific carbohydrates, within corn fiber and soybean hulls, respectively. Additionally, deviation of Line 80 poplar from the fit in Figure 6, panels A, B is likely due to the incorporation of specific monolignol intermediates (most likely aldehydes) into the final lignin polymers. How hydroxycinnamaldehyde units in lignin contribute to the overall methoxy content correlation remains unclear from this data. Further evaluation of these aldehyde units in lignin may be needed. Despite this, most lignocellulosic substrates are primarily cellulose, xylan, and lignols. For these substrates, the methoxy content correlation is highly predictive.
  • methoxy content represents the primary barrier to microbial solubilization and conversion of cellulose- and xylan-rich lignocellulose
  • the recalcitrance of plant biomasses can now be predicted. This establishes specific lignin compositional goals for feedstock engineering for microbial biorefineries. While low methoxy content substrates do not Attorney Dkt.10620-156WO1 require chemical pre- or post-treatments, opportunity exists to reduce recalcitrance in higher methoxy substrates through chemical means that leverage the divergent lignin composition preferences for chemical and microbial solubilization. Materials and Methods Bacterial strains and culture conditions. Anaerocellum (f.
  • Caldicellulosiruptor bescii wild-type strain (DSM6725) was obtained from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH and maintained, as previously described.
  • A. bescii strain MACB1058 was developed as reported previously. Fermentations with wild-type A. bescii were conducted at 75°C, while MACB1058 was fermented at 65°C to allow ethanol production. All fermentations were conducted at 5 g/L substrate loading in modified D671 medium (without MOPS) at 50 mL culture volume in 125 mL sealed serum bottles in New Brunswick Innova 42 incubator shakers at 150 rpm as previously described. Poplar Source.
  • Poplar biomass was washed with 65°C water (30 g/L) 3 times, then dried at the same temperature in an oven. Hemp fiber was processed in a similar manner as previously described. Bacterial solubilization of plant biomass.
  • A. bescii strains were adapted to plant biomass (poplar lines or hemp fiber), as previously described, with 5 g/L mass loading of substrate. Briefly, A. bescii freezer stocks were revived on medium containing 1 g/L cellobiose, 2 g/L Avicel (PH-101), and 2 g/L beechwood xylan and allowed to grow for 48 h.
  • Cultures were passaged to medium containing 5 g/L milled and washed substrate, 0.1 g/L cellobiose, 0.2 g/L Avicel, and 0.2 g/L beechwood xylan, and allowed to grow for 48 h. Cultures were then passaged to medium containing 5 g/L substrate at and allowed to grow for ⁇ 3 days or until cell density reached ⁇ 5x10 8 cells/mL. Biological replicate cultures were Attorney Dkt.10620-156WO1 started with 5 g/L substrate (exact amount recorded) at 1x10 7 cells/mL and allowed to ferment for 7 d. Cell densities were determined by epifluorescence microscopy, as previously described.
  • Line H-4-1 poplar was fermented at 5 g/L loading at 1 L culture volume in 3 L glass bioreactors (Chem Glass CLS-1380-01) at 75°C.
  • a double impeller structure was used with an uplift marine style impeller (Chem Glass CLS-1380-07) at the bottom of the shaft and a Rushton style impeller (Chem Glass CLS-1380-08) located 2 cm below the liquid surface.
  • Reactors were agitated at 150 rpm, sparged at 25 SCCM with N 2 /CO 2 (80/20 v/v) mix, and heat supplied via an electrical heating mantel. pH 6.5 was maintained with 1 M sodium hydroxide.
  • An Applikon Bio Console was used to control the bioreactors.
  • Inocula for the bioreactors were prepared in the same manner, as described above, except that the final culture passage was inoculated into a single bioreactor. Bioreactor experiments were conducted in duplicate, each with separately adapted cultures. Fermentations were allowed to progress for 7 d, and processed and analyzed identical to the 50 ml solubilization cultures. Quantitative saccharification. Plant biomass compositions were determined using the Klason lignin method with sugar HPLC analysis conducted with a Shodex SP0810 column, as previously described. Microscopy of poplar particles. For scanning electron microscopy (SEM), dry poplar particles before and after fermentation were sputter-coated with Au/Pd for 60 seconds.
  • SEM scanning electron microscopy
  • a HITACHI SU3900 SEM instrument was used to obtain SEM images. To visualize the distribution of lignin and cellulose in samples, several cross-sections of the poplar particles were prepared. Sample preparation and embedding was carried out per LR White manufacturer recommendations (Electron Microscopy Sciences, USA). Poplar particles were chemically fixed using a solution of 4% paraformaldehyde and 0.05% Attorney Dkt.10620-156WO1 glutaraldehyde.
  • Dehydration and embedding were carried out sequentially as follows: 50% and 70% ethanol in water for 15 min, 80% ethanol in water for 10 min, 2:1 LR white resin to 70% ethanol, and 100% LR white resin for 1 h, 100% LR white resin overnight, and 100% LR white resin for 30 min.
  • the North Carolina State University Analytical Instrumentation Facility (NCSU AIF) polymerized the resin, cut ⁇ 250 ⁇ m thick cross sections with an ultramicrotome, and placed the cross sections on microscope slides.
  • Calcofluor White was excited with 365 nm LED (0.3 %) to visualize cellulose; images were collected using the DAPI filter with 100 ms exposure, gain setting of 4.4 / 5.0 / 5.0 / 5.0. Lignin autofluorescence was excited with 555 nm LED (100.0%); images were collected using the Texas Red filter with 500 ms exposure and gain setting of 5.0. Image histograms were set for blue (0 to 750) and red (150 to 500). Figure 5 images are the composite images of blue and red channels. Confocal images used the CS263X water immersion objective with a glass coverslip. For confocal, Calcofluor White was excited with 405 nm laser and blue channel images captured with the DAPI filter.
  • Lignin autofluorescence was excited with 561 nm laser and red channel images captured with the Texas Red filter. Confocal settings used 600 Hz bidirectional scan speed and line averaging with 2 repetitions. Image histograms were set for blue (0 to 170) and red (0 to 30). Figure 5 shows composite images of blue and red channels. All image processing used Leica LAS X Office (1.4.4.26810) software. 2D-NMR. Poplar lignin composition was determined with 2-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), as previously described. Extractive-free wood samples were ground to 40-60 mesh as woodmeal using Willey mill and dried over P 2 O 5 .
  • 2D-NMR 2-dimensional nuclear magnetic resonance spectroscopy
  • the woodmeal ( ⁇ 2 g) was further milled at 600 rpm using 17 ZrO 2 balls for a duration of 6 h using a Pulverisette 7 Planetary ball mill. Each milling cycle comprised a 15-minute milling period followed by a 30-minute pause. Subsequent to the ball milling process, the samples were stored under vacuum conditions with P 2 O 5 prior to their utilization.
  • ⁇ 40 mg of each ball milled sample was directly introduced into a 5 mm NMR tube with addition of 500 ⁇ L of premixed DMSO-d6/Pyriding-d5 (4:1).
  • the NMR sample was sonicated for a duration ranging from 1 to 6 h in an ultrasonic bath until achieving a uniform gel consistency.
  • the 2D HSQC spectra was recorded in a 700 MHz Bruker Avance NEO magnet equipped with a 5 mm TCI helium-cooled probe.
  • the pulse program hsqcetgpsisp.2 was used to acquire the spectra with 2,048 points in F2 and 512 points in F1 for acquisition times of 125 ms and 6.6 ms, D1 delay of 1 s and 32 scans.
  • the spectra were processed using Topspin 4.1.1.
  • the relative abundances of interunit linkages were measured by integrating the C ⁇ /H ⁇ contours and expressed on the basis of the sum of ( ⁇ -O-4’) + ( ⁇ -5’) + ( ⁇ - ⁇ ’) + ( ⁇ -1’) levels 2D-NMR lignin. Additional experimental results are illustrated in Bing, Ryan G., et al., “Beyond low lignin: Identifying the primary barrier to plant biomass conversion by fermentative bacteria.” Science Advances 10.42 (2024): eadq4941, which is herein expressly incorporated by reference in its entirety.
  • Example 2 Engineering ethanologenicity into the extremely thermophilic bacterium Anaerocellum
  • the anaerobic bacterium Anaerocellum f. Caldicellulosiruptor
  • bescii natively ferments the carbohydrate content of plant biomass (including microcrystalline cellulose) into predominantly acetate, H 2 , and CO 2 , and smaller amounts of lactate, alanine and valine.
  • This extreme thermophile (growth Topt 78°C) is not natively ethanologenic, it has been previously metabolically engineered with this property, albeit initially yielding low solvent titers ( ⁇ 15 mM).
  • the current application shows significant progress on improving ethanologenicity in A.
  • the moderately thermophilic bacterium Acetivibrio thermocellus (f. Clostridum thermocellum) (T opt 60°C) is an option here with the additional advantage that it also natively produces ethanol; in fact, strains of A. thermocellus have been developed that can generate high ethanol titers.
  • a concern for metabolically engineered moderate thermophiles is that native biomasses contain indigenous moderate thermophiles that could interfere with engineered microorganisms by concomitantly converting plant biomass carbohydrates into undesirable fermentation byproducts; industrially, this forces the use of sterilizing pretreatments to avoid out-competition of indigenous microbes.
  • thermophilic threshold was determined such that temperatures approach 75°C this issue can be mitigated by precluding growth and metabolism of less thermophilic fermentative microorganisms.
  • extremely thermophilic bacteria belonging to the order Caldicellulosiruptorales which grow optimally at or above 75°C, have been considered. These bacteria were initially of interest for their capacity to produce molecular hydrogen from carbohydrates at yields that approach the Thauer limit.
  • Caldicellulosiruptorales strains and enzymes have been engineered to produce industrial chemicals, such as lactate, ethanol, acetone, 2,3-butanediol, and acetoin, albeit at low titers.
  • bescii is the most studied of the Caldicellulosiruptorales, capable of fermenting microcrystalline cellulose and other biomass polysaccharides, primarily generating hydrogen, acetate, and CO 2 , with smaller amounts of lactate and amino acids.
  • A. bescii is capable of solubilizing native and transgenic biomasses, allowing them to remain compatible with metabolic engineering efforts to improve biomass feedstock species.
  • A. bescii natively produces neither ethanol nor any other carbon-based industrially relevant chemical in significant amounts.
  • meaningful Attorney Dkt.10620-156WO1 progress has been made towards establishing A. bescii as a metabolic engineering platform ( Figure 15).
  • bescii strains engineered to produce acetone and molecular hydrogen from plant biomass showed economic viability if certain biomass solubilization and metabolic engineering targets could be met. This motivates further improvements in titer, selectivity, and productivity of industrial chemicals to fully leverage A. bescii’s capabilities for lignocellulose solubilization and conversion.
  • the A. bescii genetic toolkit relies on uracil auxotrophy, initially generated as a random deletion in the pyrimidine biosynthesis locus (strain JWCB005, ⁇ pyrFA). Later introduction of a thermostable kanamycin resistance gene allowed for targeted knock out of the pyrE gene (strain MACB1018).
  • MACB1018 and its derived strains have improved genomic stability over strains belonging to the JWCB005 lineage. Both lineages contain strains with disruption or deletion of the gene encoding lactate dehydrogenase (ldh) that diverts carbon flux towards acetate and electron flux towards H 2 . Subsequent genomic insertion of the gene encoding a bifunctional acetaldehyde-CoA /alcohol dehydrogenase (AdhE) from the moderate thermophile A. thermocellus in ldh disrupted strains, enabled and enhanced ethanol production.
  • Figure 15 summarizes reported fermentation performance of ethanol producing strains.
  • Figure 7 displays the central metabolism for A.
  • A. bescii strain JWCB032 (derivative of JWCB005) produced ethanol from simple sugars and plant biomass at measurable but still low titers (15 mM) at 65°C.
  • Swapping out the AdhE from A. thermocellus for the AdhE or AdhB from Thermoanaerobacter pseudoethanolicus 39E allowed ethanol production at temperatures closer to the thermophilic contamination threshold of 75°C, but resulted in lower titers ( ⁇ 2 mM) and lower ethanol selectivity (higher amounts of acetate) (JWCB049, JWCB054).
  • Increased titers were achieved in bioreactors through use of the more genetically stable parent strain MACB1034 ( ⁇ pyrE, ⁇ ldh, derivative of MACB1018) expressing the A. thermocellus AdhE from a different locus (MACB1058) to produced up to 61 mM ethanol and 45 mM acetate at 60°C. Further insertion of genes encoding the Rnf complex from Thermoanaerobacter sp. X14 to improve redox balancing (MACB1062) resulted in up to 76 mM ethanol and 52 mM acetate at 60°C.
  • This engineered strain produced acetone (9 mM) and ethanol (3 mM) while consuming acetate from exogenous sources.
  • Improved ethanologenicity in A. bescii in terms of titer, productivity, and selectivity is demonstrated in the current application. This is achieved by redirecting carbon away from acetate through modulation of growth rate via optimized bioreactor operation strategies and strains engineered for improved redox control and thermophilicity. Disruption of acetate formation in A. bescii improves ethanol: acetate selectivity.
  • RKCB89 was determined to be a permanent 3’ (downstream of ack) first crossover. This strain had major genomic mutations, formed aggregates, and grew poorly above 70°C, likely related to the thermostability of the inserted AdhE from A. thermocellus and the reduced ability to produce acetate for bionenergetic benefit. Further analysis of RKCB89 was done to explore the impact of media composition (e.g., varying amounts of uracil, NH4, cellobiose, trace elements, vitamins, phosphate, cysteine), degree of mixing, pH, and temperature ( Figure 16).
  • media composition e.g., varying amounts of uracil, NH4, cellobiose, trace elements, vitamins, phosphate, cysteine
  • RKCB89 is a permanent first crossover with genomic mutations, further genetic edits are not possible (i.e., it is kanamycin and 5-FOA resistant).
  • the aim was for improved thermophilicity and better ethanol:acetate selectivity along with high ethanol titers.
  • Thermophilic Thermoclostridium stercorarium subsp. strain RKWS1 (Tster) (Topt 68°C) that was isolated from wheat straw provided a path to improved thermophilicity. Since this bacterium grows optimally at about 10°C above A. thermocellus, the AdhE encoded in its genome was likely more thermostable than A. thermocellus AdhE.
  • thermophilic AdhE contains 36 extra residues on the C-terminus compared to A. thermocellus AdhE. Reports on thermophilic AdhE’s revealed differences in cofactor requirements (NADH/NAPDH); Asp to Gly mutations in A. thermocellus and Thermoanaerobacterium saccharolyticum allowed both cofactor formats (NADH and NAPDH) to be used by the enzyme for the alcohol dehydrogenase step.
  • RKWS1 AdhE Asp492Gly differs from A. thermocellus and T.
  • the final OD680 was similar across all conditions despite differing growth rates at 65°C (t d ⁇ 2.7 h), 70°C (t d ⁇ 2.1 h) and 75°C (t d ⁇ 1.9 h), which reflects the optimum temperature of A. Additionally, fermentation product distributions were significantly affected, with ethanol titer and selectivity inversely correlating with temperature (ethanol:acetate (mM:mM) 65°C - 54:17, 70°C - 24:26, 75°C - 22:26). The optimal temperature of Tster AdhE (Asp492Gly) conversion of acetaldehyde to ethanol was determined to be between 70-75°C, with slight differences for NADH and NADPH usage ( Figure 18).
  • the floccing phenotype of both RKCB89 and RKCB92 was dependent on temperature and correlated with ethanol selectivity: for RKCB92, no flocking at ⁇ 78°C (no ethanol produced) and peak floccing at 65-68°C (highest ethanol selectivity). With sufficient agitation, this phenotype manifests as formation of 1-3 large white clumps amongst otherwise planktonic cells. Flocculation has been observed as a response to external stressors in Anaerocellum and Caldicellulosiruptor species. The potential of ethanol damage was examined by inducing this phenotype in the parent strain (MACB1034), but exogenous ethanol failed to induce floccing.
  • floccing is observed to correlate with ethanol production in mesophilic organisms such as Attorney Dkt.10620-156WO1 Zymomonas sp., though the mechanism is not known. This phenotype presented in A. bescii may be due to a similar fermentation-related process, though other unexplored stressors cannot be ruled out. In context of industrial ethanol fermentation, floccing can be advantageous, as it helps reduce down-stream process intensity required to separate cells from fermentation broth. The impact of fermentation conditions on ethanol titer and selectivity was investigated with the RKCB92 strain (Figure 17).
  • bicarbonate simultaneously maintains pH and replenishes carbonic acid and CO 2 as they leave the system. This maintains a higher concentration of carbonic acid in solution and keeps the pH near the bicarbonate carbonic acid pK.
  • the use of bicarbonate avoids the need to pressure the bioreactor to achieve this and enhances ethanol production.
  • Carbon consumption based on the amount of Avicel utilized and fermentation products detected when input into the metabolic model for A. bescii showed 72% and 74% Attorney Dkt.10620-156WO1 carbon closures, for the NaHCO3 and NaOH cases, respectively. Note that significant soluble cellooligosaccharides from Avicel hydrolysis were present but not quantified in the terminal cultures.
  • the carbonic acid effect was further examined by comparing genomic and transcriptomic differences across the engineered A. bescii strains. Genomes of ethanol-producing A. bescii strains. As the genetic heritage of engineered A. bescii has previously had severe impact on bioproduction (i.e., JWCB005 vs MACB1018 lineages), the genomes of ethanologenic A. bescii strains of the MACB1018 lineage were reexamined, in the context of their parent strains, to determine whether any unexpected modifications occurred.
  • Region 12 shows the intended pyrE deletion in Attorney Dkt.10620-156WO1 MACB1018, MACB1058, and RKCB92.
  • RKCB89 regained pyrE in the acetate locus (Athe_1493-1494) via integration of a non-replicating vector (permeant first crossover, Region 14) and deleted a large region of the pyrimidine biosynthesis locus (Region 11). This apparently allowed the ack knockdown strain RKCB89 to tolerate 5-FOA while retaining pyrE and resist chloroacetate from the 3’ vector integration in the acetate locus.
  • lactate dehydrogenase (Athe_1918) was deleted, as expected in MACB1034, MACB1058, MACB1062, RKCB89, RKCB92.
  • A. bescii natively harbors two plasmids (pAthe01 and pAthe02).
  • Nanopore long reads provided new insight into their structures, where both plasmids, especially the larger pAthe01, appear to exist in a multimeric state where long reads filtered to a 20kB cutoff were still able to produce circular contigs representing ⁇ 4-mers of pAthe01. Monomers and dimer contigs for pAthe02 were generated in some assemblies.
  • A. bescii The final assemblies provided here list the plasmids as monomers, but further investigation is needed to determine which multimers are the most dominant in A. bescii. Additionally, in RKCB92, pAthe01 was lost, while retaining pAthe02. Note that the A. bescii genome encodes genes related to spore formation (including spore coat, Stage II, III, IV, V proteins, as well as spore associated transcriptional regulators and sigma factors). A previous report gave A. bescii an 85% likelihood of being capable of spore formation, suggesting the right conditions to induce endospore formation had just not yet been found. These genes could contribute to the spore-like formations observed during bioreactor experiments with NaHCO3 ( Figure 12).
  • FIG. 14 summarize transcriptional data comparing late exponential phase (corresponding to the highest ethanol selectivity) of RKCB92 for the NaOH and NaHCO3 Avicel bioreactor cases (both at 1E9 cells/mL). A total of 455 of 2,587 coding sequences in the genome were significantly differentially transcribed.
  • the bulk of down-regulated genes in the NaHCO 3 case are growth-associated, including the entire Glucan Degradation Locus (GDL) ( Figure 14 - Region J), by 5- to 13-fold.
  • the NaHCO 3 case has a large number of upregulated iron, sulfur, and molybdopterin associated genes (Regions B, D, F, H, M, N, and Q).
  • Region D contains 21 up-regulated genes, up to 212-fold (sulfurtransferase), related to sulfur assimilation. These include a [4Fe-4S]-containing ferredoxin-like protein and sulfonate, molybdopterin, sulfite, uroporphyrinogen, and porphobilinogen related proteins. Additionally, the cysteine synthase, up-regulated 14-fold, was the 8 th most highly expressed Attorney Dkt.10620-156WO1 gene (based on transcript counts) in the NaHCO3 case (224 th highest in NaOH).
  • Flagella and chemotaxis related proteins were downregulated in the NaHCO 3 case (-2.3 to -4 fold, Region L), which may reflect the higher degree of aggregation seen in the NaOH case.
  • the acetate formation genes (pta, ack) were not responsive.
  • the Surface Layer Protein (Slp) (highest transcript in both cases) was down-regulated slightly (1.4-fold) in the NaHCO 3 case, while the AdhE was slightly up-regulated 1.7-fold; these were the 3 rd and 7 th highest transcript in NaHCO3 and NaOH, respectively.
  • Carry-over transcription from Athe_0949 may explain the increased adhE transcript in the NaHCO 3 case compared to the NaOH.
  • the second most down-regulated gene in the NaHCO3 condition compared to NaOH encodes a predicted carbonic anhydrase. These enzymes bidirectionally convert CO 2 to carbonic acid and have been known to have regulatory roles. This difference in expression demonstrates the severe impact of NaHCO3 on metabolic regulation within the cell and the inferred associated increased carbonic acid level. Cells need less carbonic anhydrase because there is already abundant carbonic acid intracellularly. This further supports the hypothesis that increased carbonic acid is the driver for the observed changes.
  • the differential regulation surrounding [4Fe-4S]-containing enzymes and molybdopterin-associated proteins point to ferredoxin electron balancing.
  • the glycolytic enzyme glyceraldehyde-3- Attorney Dkt.10620-156WO1 phosphate ferredoxin oxidoreductase contains [4Fe-4S] clusters as well as molybdopterin that binds a tungsten atom.
  • the stress response genes and ferredoxin-related genes are indicative of redox stress.
  • the reoccurring aggregation phenotype seen in ethanol strains is most evident when ethanol selectivity is highest. This phenotype appears to be linked to the induced redox stress.
  • a second transcriptome from stationary phase ( ⁇ 4E9 cell/ml) largely aligned with results from the exponential phase, with the exception that the sulfur assimilation genes (Region D) were not differentially regulated.
  • the acetate formation gene, pta was slightly up-regulated in the NaHCO 3 case (1.9-fold), while the ack gene remained unaffected.
  • the AdhE was up-regulated in the NaHCO3 case (1.8-fold, 3 rd and 5 th highest transcript in NaHCO3 and NaOH, respectively).
  • the surface layer protein was again down-regulated in the NaHCO 3 case (-1.8-fold, 1 st and 2 nd 5 th highest transcript in NaHCO 3 and NaOH, respectively).
  • the differential transcriptional response between NaHCO3 and NaOH bioreactors could be directly related to ethanol production as there is slight up-regulation of AdhE (in both growth phases).
  • the up-regulation of the pta gene in NaHCO 3 stationary phase may explain the reduced ethanol selectivity in mid- to late-stationary (but still better than the NaOH case). While these transcriptional fold-changes are relatively low, the AdhE is consistently among the highest transcripts in the genome. As such, a small increase in transcription could nonetheless generate substantial amounts of protein, thereby outcompeting the acetate formation genes. Conversely, there is consistently 10-20 times more adhE transcript than pta and ack in all cases. As such, it is unlikely that the Pta and Ack are out competing the AdhE.
  • RKCB92 under high NaHCO3 conditions is a candidate for laboratory evolution to increase cell growth and extend metabolic activity. What drives A. bescii into stationary phase, eventually ceasing ethanol generation, is not clear. While phosphate, Attorney Dkt.10620-156WO1 uracil, ammonium, carbohydrate, iron, sulfur (presumably), and vitamins were ruled out as limiting nutrients, insufficient supplies of other nutrients cannot be excluded. Beyond the ethanologenicity of A. bescii, this work informs future engineering efforts to generate other non-native products. Improved culture conditions have increased cell densities from ⁇ 5E8 to 8E9 cells/ml.
  • a fed-batch system for growth on soluble sugars avoids possible osmotic stress.
  • Improved strains and fermentation conditions established here form the basis for further development of A. bescii as a platform for industrial chemical production from lignocellulosic plant biomass, thereby exploiting thermophilicity as a bioprocessing advantage.
  • thermophilicity as a bioprocessing advantage.
  • the focus here was on converting plant biomass to ethanol and minimizing acetate production, future metabolic engineering efforts with A. bescii should also consider maximizing acetate production and minimizing CO2 formation as another route to valorizing thermophilic fermentation physiology.
  • Materials and Methods Bacterial strains and culture conditions. Anaerocellum (f.
  • Caldicellulosiruptor bescii wild-type strain (DSM6725) was obtained from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH and maintained as previously described. A. bescii strains MACB1018, MACB1034, and MACB1058 were developed, as reported previously. Modified defined DSMZ 516 medium was used for all cultures, as previously described. Several additional modifications were made as indicated. These include addition of buffering agents, MOPS (3-(N-morpholino)propanesulfonic acid) and/or MES (2-(N- morpholino)ethanesulfonic acid), at combined concentrations from 20 to 60 mM.
  • MOPS 3-(N-morpholino)propanesulfonic acid
  • MES 2-(N- morpholino)ethanesulfonic acid
  • Increased ammonium chloride was routinely used at 36 mM, occasionally 48 mM, or by fed batch by addition of 3.6 M ammonium chloride in 12 mM amounts as needed in bioreactors. Vitamins and phosphate loadings were routinely doubled. Uracil was added up to 1 mM, but routinely at 0.3 mM. Uracil was prepared as a 0.25 M stock with enough sodium hydroxide Attorney Dkt.10620-156WO1 to maintain pH between 10-10.5. Carbohydrate sources were varied. Cellobiose was routinely used and sterilized by filtration of final medium.
  • Insoluble substrates Avicel (microcrystalline cellulose) and beechwood xylan, were sterilized by a 60 min 121°C autoclave cycle, then directly added to sterile serum bottles or bioreactors. Initial pH was 7.2 at room temperature, unless indicated otherwise. N 2 /CO 2 (80/20 v/v) gas mix was used to degas or sparge cultures. All bottle cultures were conducted as 50 ml cultures in 125 ml sealed serum bottles (unless noted otherwise) with 5 psig initial head pressure. Head pressure was intermittently monitored at temperature for active cultures with a pressure gauge fitted to a syringe needle.
  • Vented bottles, 1 L and 1.5 L bioreactors used luer-lok check valves on gas outlets (Masterflex 30505-92) resulting in ⁇ 1 psig head pressure.
  • Culture inocula were passaged twice from revived freezer stocks on medium identical to the experimental case in serum bottles. Inocula (3%) were routinely used from cultures in late exponential phase (OD 680 0.4-0.6 or 5E8 - 1E9 cells/ml). Experimental conditions were all started at ⁇ 5E6 cells/ml.
  • culture transfers were appropriately scaled to obtain enough volume for inoculation. Sealed 1 L (300 mL culture) and 2 L (600 mL culture) screw-top Erlenmeyer flasks were used for larger cultures.
  • A. bescii chloroacetate toxicity was determined for use as a selective agent. Wildtype A. bescii was grown for 45 h in serum bottles at 75°C, with variable concentration of sodium chloroacetate: 0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 mM. Minimal toxicity was observed up to 1.6 mM, 3.2 mM had ⁇ 60% growth compared to 0 mM, and no growth was observed at either 6.4 or 12.8 mM sodium chloroacetate. As such, 6.4 mM was determined to be above wildtype tolerance to chloroacetate. Strain construction and bacterial genetics.
  • pRGB0115’ flanking region contains the entire pta without promotor (starts at the start codon).
  • Two E. coli expression vectors (pRGB025 and pRGB026) were constructed using the pRSF Attorney Dkt.10620-156WO1 backbone; sequence confirmed plasmid was also transformed into Rosetta (DE3) pLysS E. coli, as previously described.
  • the sequence encoding the bifunctional alcohol dehydrogenase from Thermoanaerobacter stercorarium RKWS1 was PCR amplified from genomic DNA. This was assembled into the pRSF backbone to create pRGB025, containing a 6x histidine N-terminal tag.
  • a KLD reaction (kinase, ligase, DPN1) was used to alter 2 base pairs to induce the Asp492Gly mutation (GAC to GGT) to create pRGB026.
  • the mutated gene was PCR amplified and then inserted into the E. coli pSC101 origin A. bescii targeting non-replicating vector with flanking regions for the Athe_0949 locus, to generate pRGB032.
  • a synthetic DNA fragment including the 307 bp of the AdhE and the Calkro_0402 terminator was used to aid in Gibson assembly of pRGB032.
  • A. bescii genetics were carried out, as previously described, with a few modifications. Briefly, A.
  • bescii cells were grown to an OD 680 of 0.06-0.07, pelleted and washed with sucrose, then electroporated at 1.8 kV, 400 ⁇ , 25 ⁇ F. Selection was carried out with 50 ⁇ g/mL kanamycin. Cultures were plate purified 6 times on solid kanamycin containing medium to isolate pure 1 st crossover cultures. Freshly grown 1 st crossover cultures were then passaged (3% inoculum) to non-selective medium with 80 ⁇ M uracil for 4 h, then plated on solid medium with 8 mM 5-fluoroorotic acid (5-FOA) and 80 ⁇ M uracil. Colonies were screened by PCR for 2 nd crossovers.
  • 5-FOA 5-fluoroorotic acid
  • Plasmid pRGB032 generated A. bescii strain RKCB92.
  • acetate knockout plasmids pRGB008 and pRGB011
  • 6.4 mM chloroacetate was also added to liquid and solid 5-FOA medium.
  • Over 1,000 colonies were screened both by colony PCR, as well as HPLC analysis, to detect acetate formation.
  • A. bescii strain RKCB89 resulted from pRGB0113’ crossover insertion into MACB1058.
  • RKCB89 was the end strain after passage on liquid medium containing 6.4 mM chloroacetate, 8 mM 5-FOA, followed by plating on solid medium of the same composition.
  • Bioreactor conditions All ethanol producing bioreactors were allowed to progress until ethanol titers peaked. All bioreactors used a 4°C condenser on gas outlets. All other ports were sealed and pressure leak tested.
  • Bioreactor cultures (1 L and 1.5 L working volumes) were conducted in 3 L glass vessels with stainless steel head plates (Chem Glass CLS-1380-01) and double Rushton impellers (Chem Glass CLS-1380-08) located at the bottom of the shaft - 2 cm below liquid surface. Reactors were controlled with an Applikon Bio Console.
  • Control of pH was effected by addition of 2M NaOH or ⁇ 9% saturated (at 22°C) NaHCO3.
  • Hamilton Attorney Dkt.10620-156WO1 EasyFerm PHI K8 sensors (ChemGlass CLS-1435-P70), stable to 140°C, were used and no stability issues were noted.
  • Bioreactor samples were routinely re-checked with a standalone pH probe to verify accuracy. Gas sparge rate of 25 SCCM of N 2 /CO 2 (80/20 v/v) was used. Bioreactors were sterilized by autoclaving for 60 min at 121°C with the pH probe installed.
  • Avicel was then separated from cell mass via centrifugation at 200 x g for 2 min, followed by decanting of planktonic cells. Three rounds of 200 x g spins were done to collect Avicel from cells. Subsequently, cell mass was pelleted at 6,000 x g for 15 min. Avicel was washed 2x with water, vortexed to resuspend, and pelleted at 200 x g. Water supernatant was then pelleted at 6,000 x g to collect any residual cell mass that was recovered from Avicel. Avicel and cell pellets were then dried at 110°C for 24 h to determine dry mass. Recombinant expression of Thermoclostridium stercorarium RKWS1 AdhEs. E.
  • coli Rosetta (DE3)pLysS strains harboring pRGB025 and pRGB026 were grown in ZYM- 5052 auto-induction medium, as previously described.
  • Cells were pelleted, resuspended in IMAC buffer A (500 mM NaCl; 20 mM sodium phosphate; 20 mM imidazole, pH 8.0) at 4 mL/g-cell-wet-weight with 0.001 g lysozyme/mL. Solutions were lysed using a French Pressure Vessel, heat treated at 55°C for 30 min, then centrifuged and filtered to clarify.
  • AdhE Alcohol dehydrogenase specific activity of Thermoclostridium stercorarium RKWS1 AdhE enzymes, with and without the Asp492Gly mutation, were determined.
  • Purified recombinantly expressed AdhE’s were assayed under the following conditions in a Coy Anaerobic Chamber (95% N2, 5% H2 gas phase): final 200 ⁇ L reactions contained 100 mM Tris-HCl (pH 7.5), 5 ⁇ M FeSO4, 1 mM dithiothreitol (DTT), 0.25 mM NADH or NADPH, 20 mM acetaldehyde, and 10 ⁇ g/ml purified AdhE.
  • a Waters 2414 refractive index detector and Waters 2998 photodiode array detector were used to quantify products, as previously described. Ammonium was measured using reagents from API Freshwater/Saltwater Ammonia Test Kit (salicylate-based ammonia test). “Bottle 1” solution (11 ⁇ L) was added to 120 ⁇ L diluted sample or standard followed by 11 ⁇ L of “Bottle 2” solution. Samples were vortexed for 10 s, then allowed to incubate at room temperature for 10 min. Absorbance was read at 690 nm. Water was used as a blank. Culture samples were diluted 100-fold. Detection was reliable from sample concentrations of 0.5 to 24 mM NH4.
  • Quantification above 24 mM used 300x dilutions. Quantification of amino acids was performed using a Waters AccQ Tag Amino Acids C18 Column (3.9 mm x 150 mM, WAT052885) on the Arc HPLC with a Waters 2475 Fluorescence Detector, per manufacture instructions. Derivatization of samples via the Waters AccQ-Fluor Reagent Kit (WAT052880) was done per manufacture instructions. A. bescii culture samples were diluted 1:100 in water and 10 ⁇ L input as the sample to the kit.
  • RNA Protection Reagent NEB Monarch Total RNA Miniprep Kit, New England Biolabs, Inc.
  • Frozen cells were stored until processed (2 weeks); cells were pelleted from protection reagent, and then processed the same as log phase cells as follows. Cells (immediately for log phase cells), were resuspended in 240 ⁇ L PBS, 60 ⁇ L of 25 mg/mL lysozyme was added, vortexed briefly, then 300 ⁇ L of Tissue Lysis Buffer (from NEB Monarch Genomic DNA Purification Kit) and vortexed.
  • RNA Lysis Buffer was added, then proceeded as per manufacture instruction for Monarch Total RNA Miniprep Kit, including the on-column DNAse I digest.
  • Quibit Broad Range RNA Kit was used to quantify RNA. Ribosomal RNA depletion, dscDNA synthesis, and sequencing. Total RNA samples were depleted of ribosomal RNA by RNAse H digest. A mixture of ssDNA probes targeting A.
  • bescii ribosomal RNA 120 probes at 2 ⁇ M, Table 3 each, were hybridized Attorney Dkt.10620-156WO1 with 10 ⁇ g of total RNA at 95°C for 5min with 40U of murine RNAse Inhibitor (NEB); 10 mM tris-HCl pH 8.0, 100 mM NaCl, and 1 mM EDTA. Thermostable RNAse H and buffer were added (31.25U, NEB) then incubated at 50°C for 30 min. TurboDNAse and buffer were added (7.36U, Invitrogen) then incubated at 37°C for 30 min.
  • NEB murine RNAse Inhibitor
  • RNAClean XP Bead cleanup was used to purify ribodepleted RNA per manufacture instructions with 16 ⁇ L elution. Modified protocols from Oxford Nanopore Technologies (ONT) were used in the following steps. RNAClean XP and AMPure XP beads were used per manufacture and ONT instructions where required. Ribodepleted RNA was polyadenylated with E. coli Poly(A) Polymerase (NEB).
  • Reverse Transcription and second strand synthesis were carried out per ONT instructions except custom primers were used: 2 ⁇ M Oligo(dt)VN primer, 10 ⁇ M Template Switching Oligo (TSO-1), and 10 ⁇ M second strand synthesis primer (S3P) (Table 3), resulting in double stranded cDNA (dscDNA).
  • ONT Native Barcoding Kit 24 V14 and 10.4.1 flowcells on a MinION Mk1B with high-accuracy model, 400 bps base- calling (MinKNOW v23.11.5) were used per manufacture instructions to sequence dscDNA starting at the End-prep step, multiplexing 6 samples at a time. Transcriptomics analysis.
  • MinKNOW trimmed reads were filtered to Q9 and 200bp with Nanofilt v2.8.0. Reads were aligned to RKCB92 genome with BowTie2 local alignment and then counted with HTSeq. Differential expression levels were generated from read counts using a generalized linear model in EdgeR with RStudio. Metabolic modeling. Metabolic modeling was performed using the A. bescii genome scale metabolic model. Model simulations were performed with the PSAMM software v1.2.1, using IBM ILOG CPLEX Optimizer version 22.1.0 and Python version 3.9.15. The model was modified to enable the accumulation of amino acids (i.e by adding sink reactions for Gly, Glu, Ile, Leu, Pro, Met) following experimental observations.
  • Flux constraints were calculated for cellobiose consumption and for amino acid, product, and protein yields by taking the difference between the highest and lowest measured concentrations during RKCB92 bioreactor experiments (Table 4).
  • the modeled maximum Avicel consumption was calculated using fba while fixing the constraints of production and protein yields.
  • Carbon closure was calculated by dividing the maximum Avicel consumption in the model by the experimentally measured Avicel consumption.
  • the minimum required Cysteine consumption was calculated using fva while fixing the constraints of production and protein yields.
  • Table 3 Primers and Synthetic Gene Sequences Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1 Attorney Dkt.10620-156WO1
  • 55. R. G. Bing, M. J. Carey, T. Laemthong, D. J. Willard, J. R. Crosby, D. B. Sulis, J. P. Wang, M. W. W. Adams, R. M. Kelly, Fermentative conversion of unpretreated plant biomass: A thermophilic threshold for indigenous microbial growth. Bioresource Technology 367, 128275 (2023).

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Abstract

L'invention concerne des bactéries extrêmement thermophiles modifiées comprenant un gène hétérologue codant pour une alcool déshydrogénase bifonctionnelle. L'invention concerne également des systèmes et des procédés pour améliorer la récupération sélective de produits souhaitables à partir de biomasse végétale.
PCT/US2025/029115 2024-05-13 2025-05-13 Conversion de biomasse en produits chimiques industriels à l'aide de bactéries modifiées Pending WO2025240457A1 (fr)

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