WO2014144293A2 - Development and use of microbes for impurity reduction in biomass hydrolysates and fermentation broths - Google Patents

Development and use of microbes for impurity reduction in biomass hydrolysates and fermentation broths Download PDF

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WO2014144293A2
WO2014144293A2 PCT/US2014/028638 US2014028638W WO2014144293A2 WO 2014144293 A2 WO2014144293 A2 WO 2014144293A2 US 2014028638 W US2014028638 W US 2014028638W WO 2014144293 A2 WO2014144293 A2 WO 2014144293A2
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microbial organism
compound
synthesis
optionally
biomass
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PCT/US2014/028638
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French (fr)
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WO2014144293A3 (en
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Jefferson Clay Lievense
Johannes Pieter Van Dijken
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Genomatica, Inc.
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Publication of WO2014144293A3 publication Critical patent/WO2014144293A3/en

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    • 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
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    • 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
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    • 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
    • C12N1/14—Fungi; Culture media therefor
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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    • C12P21/00—Preparation of peptides or proteins
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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    • 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
    • C12P7/16—Butanols
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    • 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
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • 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

  • This invention generally relates to methods of making compounds using biosynthetic processes.
  • the invention provides co-culture methods for improving the yield, economy or efficiency of a biosynthesis of a compound by a microbial organism.
  • butadiene 1,3 -butadiene, BD, BDE
  • polymers such as synthetic rubbers and ABS resins, and chemicals such as hexamethylenediamine and 1,4-butanediol.
  • Butadiene is typically produced as a by-product of the steam cracking process for conversion of petroleum feedstocks such as naphtha, liquefied petroleum gas, ethane or natural gas to ethylene and other olefins.
  • petroleum feedstocks such as naphtha, liquefied petroleum gas, ethane or natural gas to ethylene and other olefins.
  • the ability to manufacture butadiene from alternative and/or renewable feedstocks represents a major advance in the quest for more sustainable chemical production processes.
  • Butadiene can be produced renewably by fermentation of sugars or other feedstocks to produce diols, such as 1,4-butanediol or 1,3-butanediol, which are separated, purified, and then dehydrated to butadiene in a second step involving metal-based catalysis.
  • diols such as 1,4-butanediol or 1,3-butanediol
  • the invention provides co-culture methods for improving the yield, economy or efficiency of a biosynthesis of a compound by a microbial organism, comprising:
  • the feedstock comprises a carbohydrate-comprising feedstock
  • the carbohydrate-comprising feedstock comprises an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D-xylose, a trehalose, a biomass- derived mixed sugar and/or a biomass-derived mixed sugar stream
  • the nutrient source comprises a carbon source
  • the carbon source is a gas or a syngas, a carbon dioxide gas or a carbon monoxide gas, a methanol, an ethanol, a formate or a glycerol
  • the gas or syngas comprises an H 2 gas, which can be provided alone or as part of a syngas, or a synthesis gas from a burning waste: such as a CO + H 2 , a C0 2 + H 2 , or a CO + C0 2 + H 2 ); or a CO conversion to a C0 2
  • the nutrient source or feedstock comprises a biomass, wherein optionally the biomass comprises: a plant or a microbial biomass; a biomass derived from or comprising bacteria, cyanobacteria, yeast, fungi, or algae;
  • the nutrient source or feedstock comprises a polyhydroxyalkanoate (PHA)-comprising biomass
  • PHA comprises a polyglycolide, a poly-3- hydroxypropionate, a poly-3-hydroxybutyrate, a poly-4-hydroxybutyrate, a poly-5- hydroxyvalerate, or a co-polymer thereof;
  • the compound comprises or is: an alcohol; a diol; a triol; a monohydric, a dihydric or a trihydric alcohol; a lower monohydric, a dihydric or a trihydric alcohol; a butanediol; a germinal or a vicinal diol; or a 1 ,4-Butanediol (BDO);
  • biomass or cell mass of the microorganism, wherein optionally the biomass comprises a cell wall, a protein or a nucleic acid, and/or
  • the compound or a precursor thereof, and optionally the organic impurity generated by synthesis or production of the compound, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4-Hydroxybutyric acid (4HB),
  • the organic impurity generated by synthesis or production of the BDO, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the BDO comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4- Hydroxybutyric acid (4HB),
  • GRAS Generally Recognized As Safe
  • step (c) wherein optionally the at least second microbial organism of claim 1 step (c) is added to the mixing, fermenting, culturing:
  • the culture conditions comprise: aerobic or micro- aerobic conditions, or substantially anaerobic conditions, or use of a liquid culture or a fermentation broth,
  • step (c) when the at least second microbial organism of claim 1 step (c) is added to the mixing, fermenting, culturing after the first microbial organism of (b) begins synthesizing or producing the compound, or is added only after the synthesis or production of the compound is substantially complete,
  • the first microbial organism of (b) is mixed, fermented or cultured under micro-aerobic conditions or substantially anaerobic conditions, and after the second microbial organism of claim 1 step (c) is added, the conditions are changed to increase the amount of oxygen in the mixing, fermenting, culturing conditions.
  • the first microbial organism produces a 1,4-Butanediol (BDO)
  • the at least second microbial organism is capable of metabolizing or converting a BDO synthesis organic impurity or a BDO synthesis toxic or growth-inhibiting byproduct to a BDO.
  • (b) and the at least second microbial organism of (c), are in syntrophy, or artificial syntrophy, and optionally the first microbial organism of (b) is auxotrophic for a composition made only by the at least second microbial organism of (c), and/or
  • the at least second microbial organism of (c) is auxotrophic for a composition made only by the first microbial organism of (b),
  • composition is an amino acid
  • the compound is made by metabolizing or converting an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D-xylose, a biomass-derived mixed sugar and/or a biomass-derived mixed sugar stream to the compound;
  • the microorganism is a bacteria, a Saccharomycetales , or a yeast, or a member of the genus Saccharomyces, Escherichia, Pseudomonas or Acinetobacter,
  • yeast Saccharomyces cerevisiae, Pseudomonas putida, Escherichia coli or Acinetobacter calcoaceticus ,
  • the first microbial organism of (b) is a bacteria and the second microbial organism is a yeast;
  • step (c) the at least second microbial organism of claim 1 step (c) itself cannot or substantially cannot grow on or metabolize: the nutrient, feedstock or biomass, or a sugar or a carbohydrate or any biomass-derived mixed sugar, or at least one sugar, carbohydrate or biomass-derived mixed sugar,
  • the at least second microbial organism of claim 1 step (c) cannot or substantially cannot grow on or metabolize an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D-xylose, and/or a biomass-derived mixed sugar,
  • PQQ pyrroloquinoline-quinone
  • alcohol dehydrogenase or
  • step (f) if the at least second microbial organism of claim 1 step (c) is a Saccharomyces or a Saccharomyces cerevisiae, then a hexokinase or hexokinases and a glucokinase, and a galactokinase if galactose is in the feedstock, is deleted;
  • step (g) if the at least second microbial organism of claim 1 step (c) is a Pseudomonas or a Pseudomonas putida , or a P. putida SI 2, which comprise a glucokinase and at least two PQQ-glucose dehydrogenases, then the glucokinase and at least two PQQ-glucose dehydrogenases must be deleted, inactivated or disabled or substantially inactivated or disabled, or the operon for PQQ biosynthesis is deleted, inactivated or disabled or substantially inactivated or disabled,
  • a fructose-containing feedstock a fructose PTS or a fructose-
  • 1-P kinase must be deleted, inactivated or disabled or substantially inactivated or disabled; or (h) the first microbial organism of (b) and/or the at least second microbial organism of
  • (c) are genetically engineered or modified to comprise or contain and express a heterologous protein or enzyme.
  • the co-culture methods further comprise measuring or monitoring conductivity of the liquid culture or fermentation broth during the culture or fermentation to determine the amount or level or rate of consumption of all organic acids or an organic acid.
  • the ratio of the amount of time the first microbial organism of claim 1 step (b) is mixed, fermented or cultured to synthesize or produce the compound (without the second microbial organism of claim 1 step (c)) (the so- called "ferment period") to the amount of time the first microbial organism of claim 1 step (b) is co-cultured with the at least second microbial organism of claim 1 step (c) (the so-called "burn-off period") is 2: 1, 3 : 1, 4: 1, 5: 1, or 6: 1, or between about 2: 1 and 6: 1 (the so-called "burn-off period ratio"),
  • ferment period:burn-off period is about 30 hours:about 6 hours, or is about 25 to 30 hours (ferment period):about 2 to 10 hours (burn-off period).
  • the organic impurity generated by synthesis or production of the BDO, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the BDO comprises: an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a
  • the organic impurity generated by synthesis or production of the adipate, 6-aminocaproic acid or caprolactam, or the toxic, growth- inhibiting or synthesis-inhibiting byproduct generated by synthesis of the adipate, a 6- aminocaproic acid or a caprolactam comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ), an adipate semialdehyde, an alpha- ketoadipate, an alpha-ketohydroxyadipate, and/or a hexa-2-enedionate.
  • the organic impurity generated by synthesis or production of the hexametheylenediamine or levulinic acid, or the toxic, growth-inhibiting or synthesis- inhibiting byproduct generated by synthesis of the hexametheylenediamine or levulinic acid comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a
  • pyrroloquinoline-quinone PQQ
  • an adipate an adipate semialdehyde, a 3-oxoadipate, a 5- carboxy-2-pettenoate, a 5-carboxy-2-pentenoate, an 6-aminocaproate semialdehyde, and/or a caprolactam.
  • the invention provides a composition or method according to any embodiment of the invention, substantially as hereinbefore described, or described herein, with reference to any one of the examples.
  • the details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
  • Figure 1 illustrates exemplary metabolic pathways enabling the extraction of reducing equivalents from methanol.
  • the enzymatic transformations shown are carried out by the following enzymes: 1A) a methanol methyltransferase, IB) a methylenetetrahydro folate reductase, 1C) a methylenetetrahydro folate dehydrogenase, ID) a methenyltetrahydrofolate cyclohydrolase, IE) a formyltetrahydrofolate deformylase, IF) a formyltetrahydrofolate synthetase, 1G) a formate hydrogen lyase, 1H) a hydrogenase, II) a formate dehydrogenase, 1J) a methanol dehydrogenase, IK) a formaldehyde activating enzyme, 1L) a formaldehyde dehydrogenase, 1M) a S-
  • FIG. 2 illustrates exemplary pathways for BDO production, which can be used to increase BDO yields from carbohydrates when reducing equivalents produced by a methanol metabolic pathway provided herein are available.
  • BDO production is carried out by the following enzymes: 2A) a succinyl-CoA transferase or a succinyl-CoA synthetase, 2B) a succinyl-CoA reductase (aldehyde forming), 2C) a 4-hydroxybutyrate dehydrogenase, 2D) a 4-hydroxybutyrate kinase, 2E) a phosphotrans-4-hydroxybutyrylase, 2F) a 4-hydroxybutyryl- CoA reductase (aldehyde forming), 2G) a 1,4-butanediol dehydrogenase, 2H) a succinate reductase, 21) a succinyl-CoA reductase (alcohol forming), 2J
  • Figure 3 illustrates an exemplary formaldehyde assimilation pathway.
  • the enzymatic transformations are carried out by the following enzymes: 3A) a hexulose-6-phosphate synthase, and 3B) a 6-phospho-3-hexuloisomerase.
  • Figure 4 illustrates an exemplary formaldehyde assimilation pathway.
  • the enzymatic transformations are carried out by the following enzymes: 4A) a dihydroxyacetone synthase, and 4B) a dihydroxyacetone kinase.
  • the invention provides methods for improving the yield, economy or efficiency of a biosynthesis of a compound by a microbial organism.
  • the methods comprise use of co-cultures of microorganisms, wherein a first microbial organism capable of synthesizing, producing or making the compound by enzymatic conversion or biosynthesis of the nutrient source, carbon source or carbohydrate to the compound is co-cultured with at least a second microbial organism capable of metabolizing or converting: an organic impurity generated by synthesis or production of the compound; or, a toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound (made by the first microbial organism) to: a non-toxic
  • composition a non-growth-inhibiting composition, a non-synthesis-inhibiting composition, a carbon dioxide, a biomass or cell mass of the microorganism, wherein optionally the biomass comprises a cell wall, a protein or a nucleic acid, and/or the compound made by the first microbial organism, or a precursor of the compound of interest, or any combination of these.
  • the second organism is added to the first organism culture before, at the same time, during production of the compound of interest, or even after all or substantially all compound of interest has been produced (e.g., the culture has reached its maximum yield of compound of interest).
  • the at least second microbial organism is added to the mixing, fermenting, culturing after the first microbial organism (which makes the compound of interest) begins synthesizing or producing the compound, or is added only after the synthesis or production of the compound is substantially complete.
  • the first microbial organism is mixed, fermented or cultured under micro-aerobic conditions or substantially anaerobic conditions, and after the second microbial organism is added, the conditions are changed to increase the amount of oxygen in the mixing, fermenting, culturing conditions.
  • the first organism and the at least a second microbial organism are in separate compartments, or cultures, and common media is circulated between the two compartments such that the at least a second microbial organisms can e.g., metabolize or convert an organic impurity generated by synthesis or production of the compound; or, convert a toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound, to: a non-toxic composition, a non-growth- inhibiting composition, a non-synthesis-inhibiting composition, a carbon dioxide, a biomass or cell mass of the microorganism, wherein optionally the biomass comprises a cell wall, a protein or a nucleic acid, and/or the compound of interest or a precursor thereof.
  • this separate module or compartment setup makes it easier to harvest a compound of interest, e.g., a butadiene gas.
  • the culture feedstock comprises a carbohydrate-comprising feedstock, e.g., an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D- xylose, a trehalose, a biomass-derived mixed sugar and/or a biomass-derived mixed sugar stream, and the like.
  • the feedstock can further contain methanol, optionally to produce formaldehyde and/or reducing equivalents
  • the nutrient source comprises a carbon source, e.g., such as a syngas, a carbon dioxide gas or a carbon monoxide gas, a methanol, an ethanol, a formate or a glycerol, and the like.
  • a carbon source e.g., such as a syngas, a carbon dioxide gas or a carbon monoxide gas, a methanol, an ethanol, a formate or a glycerol, and the like.
  • the nutrient source comprises a carbon source such as a gas or a syngas, a carbon dioxide gas or a carbon monoxide gas, a methanol, an ethanol, a formate or a glycerol
  • the gas or syngas comprises an H 2 gas, which can be provided alone or as part of a syngas, or a synthesis gas from a burning waste: such as a CO + H 2 , a C0 2 + H 2 , or a CO + C0 2 + H 2 ); or a CO conversion to a C0 2 .
  • the feedstock contains glycerol and methanol, especially as crude glycerol that contains methanol or as crude glycerol with the addition of methanol.
  • the nutrient source or feedstock comprises a biomass, e.g., such as a plant or a microbial biomass; an agricultural crop or a byproduct of crop production, e.g., bagasse (fibrous matter that remains after sugarcane or sorghum stalks are crushed to extract their juice), stover or corn stover (stalk, the leaf, husk and cob remaining in the field following the harvest of cereal grain) and the like.
  • the nutrient source or feedstock comprises a biomass derived from or comprising any microbial organism, e.g., such as a bacteria, cyanobacteria, yeast, fungi, or algae and the like.
  • the feedstock can further contain methanol, optionally to produce formaldehyde and/or reducing equivalents.
  • the nutrient source or feedstock comprises a polyhydroxyalkanoate (PHA)-comprising biomass, e.g., such as a polyglycolide, a poly-Shy droxypropionate, a poly-3-hydroxybutyrate, a poly-4-hydroxybutyrate, a poly-5- hydroxyvalerate, or a co-polymer thereof.
  • PHA polyhydroxyalkanoate
  • the invention uses at least a first microbial organism capable of synthesizing, producing or making a compound of interest by metabolic or enzymatic conversion or biosynthesis of the nutrient source, carbon source or carbohydrate to the compound, and at ast a second microbial organism, as described herein.
  • the first and the second microbial organisms can be the same or different, and in alternative embodiment, the microorganism can be a bacterium, a Saccharomycetales , or a yeast, or a member of the genus Saccharomyces, Escherichia, Pseudomonas or Acinetobacter, or the bacteria or a yeast is a: Saccharomyces cerevisiae, Pseudomonas putida, Escherichia coii or Acinetobacter calcoaceticus .
  • different microorganisms can be particularly paired for production of a particular purpose, e.g., when the first microbial organism of is a bacteria and the second microbial organism is a yeast.
  • any organism that exists as a microscopic cell that is included within the domains of Archaea, bacteria or eukarya can be used to practice the invention.
  • any prokaryotic or eukaryotic cells or organisms having a microscopic size can be used, e.g., including bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi.
  • any cell cultures of any species that can be cultured for the production of a biochemical or a compounds can be used to practice the invention.
  • At least one microbial organism that makes at least one compound of interest is co-cultured with at least a second microbial organism, wherein the second microbial organism allows for e.g., a greater or a more efficient yield of compound, e.g., the at least a second microbial organism capable of metabolizing or converting: an organic impurity generated by synthesis or production of the compound; or, a toxic, growth- inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound to another composition, as described herein. Any culture system or apparatus known in the art can be used.
  • microreactors, biological reactors, petri dishes, tubes, vats, cultivation chambers, fermenters, microfluidic devices and the like are used to practice the methods of the invention.
  • a cultivation chamber as described in USPN 7,897,390 can be used; or, a disposable bioreactor as described in USPN 6,432,698, can be used; or an inflated plastic bag as described in USPN 6, 190,913, can be used; or a microreactors or biological reactors as described in USPN 7,485,454, can be used; or a culture reaction chamber as described in USPN 7,052,880, can be used; or microscale bioreactors or microfermentors as described in US Pat App Pub No. 20060199260, can be used; or culture methods and devices as described in USPN 6,703,217, can be used.
  • the methods of the invention incorporate use of compositions and apparatus that can efficiently detect the compound of interest, e.g., a butadiene or a butadiene gas, produced as a product of a biosynthetic process, e.g., as a product of the first microbial organism biosynthetic process.
  • this allows data on when to add nutrients and/or the at least second microorganism, e.g., when adding the at least second microorganism after the first microorganism, or when the at least second microorganism is added only after the synthesis or production of the compound is substantially complete, and the like.
  • the methods of the invention incorporate use of aerobic or micro- aerobic conditions, or substantially anaerobic conditions, or use of a micro- aerobic or substantially anaerobic liquid culture or a fermentation broth.
  • the first microbial organism is mixed, fermented or cultured under micro-aerobic conditions or substantially anaerobic conditions, and after the second microbial organism is added, the conditions are changed to increase the amount of oxygen in the mixing, fermenting, culturing conditions.
  • the methods of the invention incorporate use of "substantially anaerobic" culture or growth conditions, e.g., where an amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media; including use of sealed chambers of liquid or solid medium.
  • the methods of the invention incorporate use of "growth- coupled" production of a compound of interest, e.g., a biochemical product, e.g., the biosynthesis of the compound of interest is produced during the growth phase of a first microorganism.
  • the growth-coupled production can be obligatory, meaning that the biosynthesis of the compound of interest is an obligatory product produced during the growth phase of a first microorganism.
  • the term "growth-coupled" when used in reference to the consumption of a feedstock or nutrient means that the referenced feedstock or nutrient is consumed during the growth phase of a microorganism.
  • the compound of interest can be any compound produced by a non-naturally- occurring micro-organism comprising enzymes of a metabolic pathway to produce that compound of interest. More generally, the compound-of-interest (COT) synthesis organic impurity or a compound-of-interest (COI) synthesis toxic or growth-inhibiting byproduct will be a non-macromolecular organic compound that is an intermediate in the metabolic pathway to the compound of interest, preferably where the pathway is specific to the production of the compound of interest, and that can diffuse or be transported through the first mieobe's cell membrane or cell wall to the fermentation broth.
  • the impurity or byproduct compound can be highly oxidized such as an organic acid or reduced such as an alcohol, e.g.
  • 4ITB is such a compound of interest while CoA -conjugates such as 4HB-CoA are not.
  • CoA -conjugates such as 4HB-CoA are not.
  • crotooate, crotyl-alcohol and/or crotonyl-CoA would be product specific pathway synthesis intermediates, but crotonate and crotyl-alcohol would be by-products subject to the present invention whereas crotonyl-CoA would not.
  • the COT synthesis organic impurity or a COI synthesis toxic or growth-inhibiting byproduct can include and comprise, as non-limiting examples, an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4-Hydroxybutyric acid (4HB), acetate, succinate, alanine, alp a-ketoglutatrate, fumarate, crotonate, crotyl alcohol, and gamma-ammobutyric acid (GAB A).
  • an organic acid an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4-Hydroxybutyric acid (4HB)
  • compounds of interest made by a co-culture of the invention comprise: an alcohol; a diol; a triol; a monohydric, a dihydric or a trihydric alcohol; a lower monohydric, a dihydric or a trihydric alcohol; a butanediol; a germinal or a vicinal diol; or a 1,4-Butanediol (BDO), and the like.
  • compounds of interest made by a co-culture of the invention comprise: 1,3 -Propanediol (1,3-PDO) and other glycols including ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol, dipropylene glycol, tripropylene glycol, neopentyl glycol and bisphenol A.
  • 1,3-Propanediol 1,3-Propanediol
  • other glycols including ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol, dipropylene glycol, tripropylene glycol, neopentyl glycol and bisphenol A.
  • compounds of interest made by a co-culture of the invention comprise: alkanes, butadiene and alkenes, alkynes, an adipate, a 6-aminocaproic acid, a caprolactam, a hexane - 1,6 diamine (HMD A), caprolactam, an organic acid, a succinic acid, and the like, e.g., as described in U.S. Pat. App. Pub. No. 20120309062.
  • compounds of interest made by a co-culture of the invention comprise: a hexametheylenediamine or levulinic acid, e.g., as described in U.S. Pat. App. Pub. No. 20120282661.
  • compounds of interest made by a co-culture of the invention comprise: a 2-hydroxyisobutyric acid, a 3-hydroxyisobutyric acid or a methacrylic acid, e.g., as described in U.S. Pat. App. Pub. No. 20120276605.
  • compounds of interest made by a co-culture of the invention comprise: butadiene, crotyl alcohol, 1,3-butanediol, and the additional compounds and their metabolic pathways as described in U.S. Patent US8268607, WIPO App. Pubs. WO2013036764A1, WO2013028519A1, WO2013012975A1, and WO2012177710A1.
  • the first microbial organism produces a 1 ,4-Butanediol (BDO)
  • the at least second microbial organism is capable of metabolizing or converting a BDO synthesis organic impurity or a BDO synthesis toxic or growth-inhibiting byproduct to a BDO.
  • the organic impurity generated by synthesis or production of the BDO, or the toxic, growth- inhibiting or synthesis-inhibiting byproduct generated by synthesis of the BDO comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4-
  • Hydroxybutyric acid (4HB),more preferably acetate, 4HB and/or glutamate, most preferably glutamate and/or 4HB.
  • the organic acid cars include one or more acetate, pyruvate, succinate, alanine, glutamate, alpha-ketoglutatrate, fumarate, and gamma-aminoburyric acid (GABA)
  • GABA gamma-aminoburyric acid
  • the method incorporates use of compositions and apparatus that can efficiently detect one or more COI synthesis organic impurity or COI synthesis toxic or growth-inhibiting byproduct produced as a product of a biosynthetic process, e.g., as a product of the first microbial organism biosynthetic process.
  • this allows data on when to add nutrients and/or the at least second microorganism, e.g., when adding the at least second microorganism after the first microorganism, or when the at least second microorganism is added only after the synthesis or production of the compound is substantially complete, and the like.
  • the first microbial organism comprises a methanol metabolic pathway, e.g., where the microorganism has at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol.
  • the methanol metabolic pathway comprises one or more enzymes selected from the group consisting of a methanol methyltransferase; a methylenetetrahydrofolate reductase; a methylenetetrahydrofolate dehydrogenase; a methenyltetrahydrofolate cyclohydrolase; a formyltetrahydrofolate deformylase; a formyltetrahydrofolate synthetase; a formate hydrogen lyase; a hydrogenase; a formate dehydrogenase; a methanol dehydrogenase; a formaldehyde activating enzyme; a formaldehyde dehydrogenase; a S-(hydroxymethyl)glutathione synthase; a glutathione-dependent formaldehyde dehydrogenase; and an S-formylglutathione hydrolase.
  • Such organisms advantageously allow for the
  • the first microbial organism comprises a BDO synthetic pathway (a product specific pathway); e.g., where the first microorganism comprises at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO.
  • BDO synthetic pathway a product specific pathway
  • the BDO pathway enzyme is: a succinyl- CoA transferase or a succinyl-CoA synthetase (or succinyl-CoA ligase); a succinyl-CoA reductase (aldehyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde forming); a 1 ,4-butanediol dehydrogenase; a succinate reductase; a succinyl-CoA reductase (alcohol forming); a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyrate reductase; a 4-hydroxybutyryl-phosphate reductase;
  • the first microbial organism comprises a methanol metabolic pathway, either alone or in combination with a BDO pathway, as provided herein, and can further comprise a formaldehyde assimilation pathway that utilizes formaldehyde, e.g., obtained from the oxidation of methanol, in the formation of intermediates of certain central metabolic pathways that can be used, for example, in the formation of biomass.
  • the formaldehyde assimilation pathway comprises a hexulose-6- phosphate synthase, 6-phospho-3-hexuloisomerase, dihydroxyacetone synthase or dihydroxyacetone kinase.
  • the first microbial organism comprises a methanol metabolic pathway, e.g., comprising at least one exogenous nucleic acid encoding a methanol dehydrogenase expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or expressed in a sufficient amount to convert methanol to formaldehyde.
  • the microbial organism further comprises a formaldehyde assimilation pathway.
  • the first microbial organism further comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of glycolysis.
  • the formaldehyde assimilation pathway enzyme is a hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, dihydroxyacetone synthase and/or dihydroxyacetone kinase.
  • the first microbial organism comprises one or more gene disruptions, occurring in one or more endogenous genes encoding protein(s) or enzyme(s) involved in native production of ethanol, glycerol, acetate, lactate, formate, CO2, and/or amino acids by said microbial organism, wherein said one or more gene disruptions confer increased production of BDO in said microbial organism.
  • one or more endogenous enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2 and/or amino acids by the microbial organism has attenuated enzyme activity or expression levels.
  • the organism any number of gene disruptions, e.g., from one to about twenty-five gene disruptions, or more.

Abstract

In alternative embodiments, the invention provides co-culture methods for improving the yield, economy or efficiency of a biosynthesis of a compound by a. microbial organism. In alternative embodiments, the invention provides co-culture methods for improving the yield, economy or efficiency of a biosynthesis of: an alcohol; a diol; a triol; a monohydric, a dihydric or a trihydric alcohol; a lower monohydric, a dihydric or a trihydric alcohol; a butanediol; a germinal or a vicinal diol; or a 1,4-Butanediol (BDO). In alternative embodiments, a co-culture of the invention uses a nutrient source comprising a biomass or a feedstock.

Description

DEVELOPMENT AND USE OF MICROBES FOR IMPURITY REDUCTION IN BIOMASS HYDROLYSATES AND FERMENTATION BROTHS
TECHNICAL FIELD
This invention generally relates to methods of making compounds using biosynthetic processes. In alternative embodiments, the invention provides co-culture methods for improving the yield, economy or efficiency of a biosynthesis of a compound by a microbial organism.
BACKGROUND
Over 25 billion pounds of butadiene (1,3 -butadiene, BD, BDE) are produced annually and are applied in the manufacture of polymers such as synthetic rubbers and ABS resins, and chemicals such as hexamethylenediamine and 1,4-butanediol. Butadiene is typically produced as a by-product of the steam cracking process for conversion of petroleum feedstocks such as naphtha, liquefied petroleum gas, ethane or natural gas to ethylene and other olefins. The ability to manufacture butadiene from alternative and/or renewable feedstocks represents a major advance in the quest for more sustainable chemical production processes. Butadiene can be produced renewably by fermentation of sugars or other feedstocks to produce diols, such as 1,4-butanediol or 1,3-butanediol, which are separated, purified, and then dehydrated to butadiene in a second step involving metal-based catalysis.
Improved microbial organisms and methods for effectively producing butadiene from cheap renewable feedstocks such as molasses, sugar cane juice, and sugars derived from biomass sources, including agricultural and wood waste, as well as CI feedstocks such as syngas and carbon dioxide, are needed.
SUMMARY
In alternative embodiments, the invention provides co-culture methods for improving the yield, economy or efficiency of a biosynthesis of a compound by a microbial organism, comprising:
(a) providing a co-culture nutrient source, a biomass, or a feedstock,
wherein optionally the feedstock comprises a carbohydrate-comprising feedstock, and optionally the carbohydrate-comprising feedstock comprises an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D-xylose, a trehalose, a biomass- derived mixed sugar and/or a biomass-derived mixed sugar stream,
and optionally the nutrient source comprises a carbon source, and optionally the carbon source is a gas or a syngas, a carbon dioxide gas or a carbon monoxide gas, a methanol, an ethanol, a formate or a glycerol, and optionally the gas or syngas comprises an H2 gas, which can be provided alone or as part of a syngas, or a synthesis gas from a burning waste: such as a CO + H2, a C02 + H2, or a CO + C02 + H2); or a CO conversion to a C02, and optionally the nutrient source or feedstock comprises a biomass, wherein optionally the biomass comprises: a plant or a microbial biomass; a biomass derived from or comprising bacteria, cyanobacteria, yeast, fungi, or algae;
and optionally the nutrient source or feedstock comprises a polyhydroxyalkanoate (PHA)-comprising biomass, and optionally the PHA comprises a polyglycolide, a poly-3- hydroxypropionate, a poly-3-hydroxybutyrate, a poly-4-hydroxybutyrate, a poly-5- hydroxyvalerate, or a co-polymer thereof;
(b) providing a first microbial organism capable of synthesizing, producing or making the compound by metabolic or enzymatic conversion or biosynthesis of the nutrient source, carbon source or carbohydrate to the compound,
wherein optionally the compound comprises or is: an alcohol; a diol; a triol; a monohydric, a dihydric or a trihydric alcohol; a lower monohydric, a dihydric or a trihydric alcohol; a butanediol; a germinal or a vicinal diol; or a 1 ,4-Butanediol (BDO);
(c) providing at least a second microbial organism capable of metabolizing or converting: an organic impurity generated by synthesis or production of the compound; or, a toxic, growth- inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound, to:
a non-toxic composition,
a non-growth-inhibiting composition,
a non-synthesis-inhibiting composition,
a carbon dioxide,
a biomass or cell mass of the microorganism, wherein optionally the biomass comprises a cell wall, a protein or a nucleic acid, and/or
the compound or a precursor thereof, and optionally the organic impurity generated by synthesis or production of the compound, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound, comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4-Hydroxybutyric acid (4HB),
and optionally, when the compound is BDO, the organic impurity generated by synthesis or production of the BDO, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the BDO, comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4- Hydroxybutyric acid (4HB),
and optionally the at least second microbial organism:
is an obligate aerobe,
is a Generally Recognized As Safe (GRAS) organism,
has an affinity for oxygen equal to or greater than the compound-synthesizing first microorganism,
cannot grow on, metabolize or oxidize, or substantially grow on, metabolize or oxidize the compound,
cannot excrete or produce, or substantially cannot excrete or produce, a substance or composition that negatively affects growth or compound product formation by the co-culture,
wherein optionally: cannot excrete or produce, or substantially cannot excrete or produce: an organic acid, an acetate, a glutamate, a pyrroloquinoline-quinone ( PQQ) and/or a 4-Hydroxybutyric acid (4HB); and
(d) mixing, fermenting, culturing or incubating the carbohydrate-comprising feedstock, the first microbial organism of (b) and the at least second microbial organism of (c) under conditions such that the compound is synthesized or produced by the first microbial organism of (b),
wherein optionally the at least second microbial organism of claim 1 step (c) is added to the mixing, fermenting, culturing:
before, during, and/or after the synthesis or production of the compound by the first microbial organism of claim 1 step (b), or
after substantially most of the synthesis or production of the compound by the first microbial organism of claim 1 step (b), or
when production of the compound is substantially complete, and optionally the culture conditions comprise: aerobic or micro- aerobic conditions, or substantially anaerobic conditions, or use of a liquid culture or a fermentation broth,
and optionally, when the at least second microbial organism of claim 1 step (c) is added to the mixing, fermenting, culturing after the first microbial organism of (b) begins synthesizing or producing the compound, or is added only after the synthesis or production of the compound is substantially complete,
and optionally the first microbial organism of (b) is mixed, fermented or cultured under micro-aerobic conditions or substantially anaerobic conditions, and after the second microbial organism of claim 1 step (c) is added, the conditions are changed to increase the amount of oxygen in the mixing, fermenting, culturing conditions.
In alternative embodiments of the co-culture methods, the first microbial organism produces a 1,4-Butanediol (BDO), and the at least second microbial organism is capable of metabolizing or converting a BDO synthesis organic impurity or a BDO synthesis toxic or growth-inhibiting byproduct to a BDO.
In alternative embodiments of the eo-cuUure methods, the first microbial organism of
(b) and the at least second microbial organism of (c), are in syntrophy, or artificial syntrophy, and optionally the first microbial organism of (b) is auxotrophic for a composition made only by the at least second microbial organism of (c), and/or
the at least second microbial organism of (c) is auxotrophic for a composition made only by the first microbial organism of (b),
wherein optionally the composition is an amino acid.
In alternative embodiments of the eo-cuUure methods:
(a) the compound is made by metabolizing or converting an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D-xylose, a biomass-derived mixed sugar and/or a biomass-derived mixed sugar stream to the compound;
(b) the microorganism is a bacteria, a Saccharomycetales , or a yeast, or a member of the genus Saccharomyces, Escherichia, Pseudomonas or Acinetobacter,
or the bacteria or a yeast is a: Saccharomyces cerevisiae, Pseudomonas putida, Escherichia coli or Acinetobacter calcoaceticus ,
and optionally the first microbial organism of (b) is a bacteria and the second microbial organism is a yeast;
(c) the at least second microbial organism of claim 1 step (c) itself cannot or substantially cannot grow on or metabolize: the nutrient, feedstock or biomass, or a sugar or a carbohydrate or any biomass-derived mixed sugar, or at least one sugar, carbohydrate or biomass-derived mixed sugar,
(d) the at least second microbial organism of claim 1 step (c) cannot or substantially cannot grow on or metabolize an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D-xylose, and/or a biomass-derived mixed sugar,
(e) the at least second microbial organism of claim 1 step (c) that cannot or substantially cannot grow on or metabolize a sugar or a carbohydrate or any biomass-derived mixed sugar:
lacks an enzyme, enzymes or operon involved in synthesizing a pyrroloquinoline- quinone ( PQQ) or an alcohol dehydrogenase, or
comprises a disabled or a non-functioning enzyme, enzymes or operon involved in synthesizing a pyrroloquinoline-quinone ( PQQ) or an alcohol dehydrogenase, or
lacks or comprises a disabled or non-functioning PQQ operon;
(f) if the at least second microbial organism of claim 1 step (c) is a Saccharomyces or a Saccharomyces cerevisiae, then a hexokinase or hexokinases and a glucokinase, and a galactokinase if galactose is in the feedstock, is deleted;
(g) if the at least second microbial organism of claim 1 step (c) is a Pseudomonas or a Pseudomonas putida , or a P. putida SI 2, which comprise a glucokinase and at least two PQQ-glucose dehydrogenases, then the glucokinase and at least two PQQ-glucose dehydrogenases must be deleted, inactivated or disabled or substantially inactivated or disabled, or the operon for PQQ biosynthesis is deleted, inactivated or disabled or substantially inactivated or disabled,
and optionally if a fructose-containing feedstock is used a fructose PTS or a fructose-
1-P kinase must be deleted, inactivated or disabled or substantially inactivated or disabled; or (h) the first microbial organism of (b) and/or the at least second microbial organism of
(c) are genetically engineered or modified to comprise or contain and express a heterologous protein or enzyme.
In alternative embodiments the co-culture methods further comprise measuring or monitoring conductivity of the liquid culture or fermentation broth during the culture or fermentation to determine the amount or level or rate of consumption of all organic acids or an organic acid.
In alternative embodiments, when the at least second microbial organism of a method of the invention (claim 1 step (c)) is added to the mixing, fermenting, culturing during and/or after the production of the compound by the first microbial organism of claim 1 step (b) has begun or is complete or substantially complete, the ratio of the amount of time the first microbial organism of claim 1 step (b) is mixed, fermented or cultured to synthesize or produce the compound (without the second microbial organism of claim 1 step (c)) (the so- called "ferment period") to the amount of time the first microbial organism of claim 1 step (b) is co-cultured with the at least second microbial organism of claim 1 step (c) (the so-called "burn-off period") is 2: 1, 3 : 1, 4: 1, 5: 1, or 6: 1, or between about 2: 1 and 6: 1 (the so-called "burn-off period ratio"),
wherein optionally the ferment period:burn-off period is about 30 hours:about 6 hours, or is about 25 to 30 hours (ferment period):about 2 to 10 hours (burn-off period).
In alternative embodiments of the methods of the invention, when the compound is BDO, the organic impurity generated by synthesis or production of the BDO, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the BDO, comprises: an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a
pyrroloquinoline-quinone (PQQ) and/or a 4-Hydroxybutyric acid (4HB).
In alternative embodiments of the methods of the invention, when the compound is an adipate, a 6-aminocaproic acid or a caprolactam, the organic impurity generated by synthesis or production of the adipate, 6-aminocaproic acid or caprolactam, or the toxic, growth- inhibiting or synthesis-inhibiting byproduct generated by synthesis of the adipate, a 6- aminocaproic acid or a caprolactam, comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ), an adipate semialdehyde, an alpha- ketoadipate, an alpha-ketohydroxyadipate, and/or a hexa-2-enedionate.
In alternative embodiments of the methods of the invention, when the compound is a hexametheylenediamine, the organic impurity generated by synthesis or production of the hexametheylenediamine or levulinic acid, or the toxic, growth-inhibiting or synthesis- inhibiting byproduct generated by synthesis of the hexametheylenediamine or levulinic acid, comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a
pyrroloquinoline-quinone (PQQ), an adipate, an adipate semialdehyde, a 3-oxoadipate, a 5- carboxy-2-pettenoate, a 5-carboxy-2-pentenoate, an 6-aminocaproate semialdehyde, and/or a caprolactam.
In alternative embodiments, the invention provides a composition or method according to any embodiment of the invention, substantially as hereinbefore described, or described herein, with reference to any one of the examples. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figures are described and discussed herein.
Figure 1 illustrates exemplary metabolic pathways enabling the extraction of reducing equivalents from methanol. The enzymatic transformations shown are carried out by the following enzymes: 1A) a methanol methyltransferase, IB) a methylenetetrahydro folate reductase, 1C) a methylenetetrahydro folate dehydrogenase, ID) a methenyltetrahydrofolate cyclohydrolase, IE) a formyltetrahydrofolate deformylase, IF) a formyltetrahydrofolate synthetase, 1G) a formate hydrogen lyase, 1H) a hydrogenase, II) a formate dehydrogenase, 1J) a methanol dehydrogenase, IK) a formaldehyde activating enzyme, 1L) a formaldehyde dehydrogenase, 1M) a S-(hydroxymethyl)glutathione synthase, IN) a glutathione-dependent formaldehyde dehydrogenase, and 10) a S-formylglutathione hydrolase. In certain embodiments, steps K and/or M are spontaneous.
Figure 2 illustrates exemplary pathways for BDO production, which can be used to increase BDO yields from carbohydrates when reducing equivalents produced by a methanol metabolic pathway provided herein are available. BDO production is carried out by the following enzymes: 2A) a succinyl-CoA transferase or a succinyl-CoA synthetase, 2B) a succinyl-CoA reductase (aldehyde forming), 2C) a 4-hydroxybutyrate dehydrogenase, 2D) a 4-hydroxybutyrate kinase, 2E) a phosphotrans-4-hydroxybutyrylase, 2F) a 4-hydroxybutyryl- CoA reductase (aldehyde forming), 2G) a 1,4-butanediol dehydrogenase, 2H) a succinate reductase, 21) a succinyl-CoA reductase (alcohol forming), 2J) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, 2K) a 4-hydroxybutyrate reductase, 2L) a 4- hydroxybutyryl-phosphate reductase, and 2M) a 4-hydroxybutyryl-CoA reductase (alcohol forming). Figure 3 illustrates an exemplary formaldehyde assimilation pathway. The enzymatic transformations are carried out by the following enzymes: 3A) a hexulose-6-phosphate synthase, and 3B) a 6-phospho-3-hexuloisomerase.
Figure 4 illustrates an exemplary formaldehyde assimilation pathway. The enzymatic transformations are carried out by the following enzymes: 4A) a dihydroxyacetone synthase, and 4B) a dihydroxyacetone kinase.
Like reference symbols in the various drawings indicate like elements, unless otherwise stated.
Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.
DETAILED DESCRIPTION
In alternative embodiments, the invention provides methods for improving the yield, economy or efficiency of a biosynthesis of a compound by a microbial organism. In alternative embodiment, the methods comprise use of co-cultures of microorganisms, wherein a first microbial organism capable of synthesizing, producing or making the compound by enzymatic conversion or biosynthesis of the nutrient source, carbon source or carbohydrate to the compound is co-cultured with at least a second microbial organism capable of metabolizing or converting: an organic impurity generated by synthesis or production of the compound; or, a toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound (made by the first microbial organism) to: a non-toxic
composition, a non-growth-inhibiting composition, a non-synthesis-inhibiting composition, a carbon dioxide, a biomass or cell mass of the microorganism, wherein optionally the biomass comprises a cell wall, a protein or a nucleic acid, and/or the compound made by the first microbial organism, or a precursor of the compound of interest, or any combination of these.
In alternative embodiments, the second organism is added to the first organism culture before, at the same time, during production of the compound of interest, or even after all or substantially all compound of interest has been produced (e.g., the culture has reached its maximum yield of compound of interest). For example, in alternative embodiments, the at least second microbial organism is added to the mixing, fermenting, culturing after the first microbial organism (which makes the compound of interest) begins synthesizing or producing the compound, or is added only after the synthesis or production of the compound is substantially complete. In alternative embodiments, the first microbial organism is mixed, fermented or cultured under micro-aerobic conditions or substantially anaerobic conditions, and after the second microbial organism is added, the conditions are changed to increase the amount of oxygen in the mixing, fermenting, culturing conditions.
In alternative embodiments, the first organism and the at least a second microbial organism are in separate compartments, or cultures, and common media is circulated between the two compartments such that the at least a second microbial organisms can e.g., metabolize or convert an organic impurity generated by synthesis or production of the compound; or, convert a toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound, to: a non-toxic composition, a non-growth- inhibiting composition, a non-synthesis-inhibiting composition, a carbon dioxide, a biomass or cell mass of the microorganism, wherein optionally the biomass comprises a cell wall, a protein or a nucleic acid, and/or the compound of interest or a precursor thereof. In alternative embodiments, this separate module or compartment setup makes it easier to harvest a compound of interest, e.g., a butadiene gas.
Nutrients, Feedstock or Biomass
In alternative embodiments, to make the compound of interest by the first microbial organism, a nutrient source, a biomass, or a feedstock, and the like, is added to the co-culture. In alternative embodiments, the culture feedstock comprises a carbohydrate-comprising feedstock, e.g., an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D- xylose, a trehalose, a biomass-derived mixed sugar and/or a biomass-derived mixed sugar stream, and the like. The feedstock can further contain methanol, optionally to produce formaldehyde and/or reducing equivalents
In alternative embodiments, the nutrient source comprises a carbon source, e.g., such as a syngas, a carbon dioxide gas or a carbon monoxide gas, a methanol, an ethanol, a formate or a glycerol, and the like. In alternative embodiments, the nutrient source comprises a carbon source such as a gas or a syngas, a carbon dioxide gas or a carbon monoxide gas, a methanol, an ethanol, a formate or a glycerol, and optionally the gas or syngas comprises an H2 gas, which can be provided alone or as part of a syngas, or a synthesis gas from a burning waste: such as a CO + H2, a C02 + H2, or a CO + C02 + H2); or a CO conversion to a C02. In one embodiment, the feedstock contains glycerol and methanol, especially as crude glycerol that contains methanol or as crude glycerol with the addition of methanol.
In alternative embodiments, the nutrient source or feedstock comprises a biomass, e.g., such as a plant or a microbial biomass; an agricultural crop or a byproduct of crop production, e.g., bagasse (fibrous matter that remains after sugarcane or sorghum stalks are crushed to extract their juice), stover or corn stover (stalk, the leaf, husk and cob remaining in the field following the harvest of cereal grain) and the like. In alternative embodiments, the nutrient source or feedstock comprises a biomass derived from or comprising any microbial organism, e.g., such as a bacteria, cyanobacteria, yeast, fungi, or algae and the like. The feedstock can further contain methanol, optionally to produce formaldehyde and/or reducing equivalents.
In alternative embodiments, the nutrient source or feedstock comprises a polyhydroxyalkanoate (PHA)-comprising biomass, e.g., such as a polyglycolide, a poly-Shy droxypropionate, a poly-3-hydroxybutyrate, a poly-4-hydroxybutyrate, a poly-5- hydroxyvalerate, or a co-polymer thereof.
Microbial Organisms
In alternative embodiments, the invention uses at least a first microbial organism capable of synthesizing, producing or making a compound of interest by metabolic or enzymatic conversion or biosynthesis of the nutrient source, carbon source or carbohydrate to the compound, and at ast a second microbial organism, as described herein. The first and the second microbial organisms can be the same or different, and in alternative embodiment, the microorganism can be a bacterium, a Saccharomycetales , or a yeast, or a member of the genus Saccharomyces, Escherichia, Pseudomonas or Acinetobacter, or the bacteria or a yeast is a: Saccharomyces cerevisiae, Pseudomonas putida, Escherichia coii or Acinetobacter calcoaceticus .
In alternative embodiments, for a particular purpose, different microorganisms can be particularly paired for production of a particular purpose, e.g., when the first microbial organism of is a bacteria and the second microbial organism is a yeast.
In alternative embodiments, any organism that exists as a microscopic cell that is included within the domains of Archaea, bacteria or eukarya can be used to practice the invention. In alternative embodiments, any prokaryotic or eukaryotic cells or organisms having a microscopic size can be used, e.g., including bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. In alternative embodiments, any cell cultures of any species that can be cultured for the production of a biochemical or a compounds can be used to practice the invention.
Co-Culture Systems
In alternative embodiments, at least one microbial organism that makes at least one compound of interest is co-cultured with at least a second microbial organism, wherein the second microbial organism allows for e.g., a greater or a more efficient yield of compound, e.g., the at least a second microbial organism capable of metabolizing or converting: an organic impurity generated by synthesis or production of the compound; or, a toxic, growth- inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound to another composition, as described herein. Any culture system or apparatus known in the art can be used. For example, in alternative embodiments, microreactors, biological reactors, petri dishes, tubes, vats, cultivation chambers, fermenters, microfluidic devices and the like are used to practice the methods of the invention. For example, a cultivation chamber as described in USPN 7,897,390, can be used; or, a disposable bioreactor as described in USPN 6,432,698, can be used; or an inflated plastic bag as described in USPN 6, 190,913, can be used; or a microreactors or biological reactors as described in USPN 7,485,454, can be used; or a culture reaction chamber as described in USPN 7,052,880, can be used; or microscale bioreactors or microfermentors as described in US Pat App Pub No. 20060199260, can be used; or culture methods and devices as described in USPN 6,703,217, can be used.
In alternative embodiments, the methods of the invention incorporate use of compositions and apparatus that can efficiently detect the compound of interest, e.g., a butadiene or a butadiene gas, produced as a product of a biosynthetic process, e.g., as a product of the first microbial organism biosynthetic process. In alternative embodiments, this allows data on when to add nutrients and/or the at least second microorganism, e.g., when adding the at least second microorganism after the first microorganism, or when the at least second microorganism is added only after the synthesis or production of the compound is substantially complete, and the like.
In alternative embodiments, the methods of the invention incorporate use of aerobic or micro- aerobic conditions, or substantially anaerobic conditions, or use of a micro- aerobic or substantially anaerobic liquid culture or a fermentation broth. In alternative embodiments, the first microbial organism is mixed, fermented or cultured under micro-aerobic conditions or substantially anaerobic conditions, and after the second microbial organism is added, the conditions are changed to increase the amount of oxygen in the mixing, fermenting, culturing conditions. In alternative embodiments, the methods of the invention incorporate use of "substantially anaerobic" culture or growth conditions, e.g., where an amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media; including use of sealed chambers of liquid or solid medium.
In alternative embodiments, the methods of the invention incorporate use of "growth- coupled" production of a compound of interest, e.g., a biochemical product, e.g., the biosynthesis of the compound of interest is produced during the growth phase of a first microorganism. In a particular embodiment, the growth-coupled production can be obligatory, meaning that the biosynthesis of the compound of interest is an obligatory product produced during the growth phase of a first microorganism. In alternative embodiments, the term "growth-coupled" when used in reference to the consumption of a feedstock or nutrient (e.g., a biochemical) means that the referenced feedstock or nutrient is consumed during the growth phase of a microorganism.
Compounds of Interest (COI)
The compound of interest can be any compound produced by a non-naturally- occurring micro-organism comprising enzymes of a metabolic pathway to produce that compound of interest. More generally, the compound-of-interest (COT) synthesis organic impurity or a compound-of-interest (COI) synthesis toxic or growth-inhibiting byproduct will be a non-macromolecular organic compound that is an intermediate in the metabolic pathway to the compound of interest, preferably where the pathway is specific to the production of the compound of interest, and that can diffuse or be transported through the first mieobe's cell membrane or cell wall to the fermentation broth. The impurity or byproduct compound can be highly oxidized such as an organic acid or reduced such as an alcohol, e.g. ethanoL For example, 4ITB is such a compound of interest while CoA -conjugates such as 4HB-CoA are not. For example, for butadiene production, crotooate, crotyl-alcohol and/or crotonyl-CoA would be product specific pathway synthesis intermediates, but crotonate and crotyl-alcohol would be by-products subject to the present invention whereas crotonyl-CoA would not. The COT synthesis organic impurity or a COI synthesis toxic or growth-inhibiting byproduct can include and comprise, as non-limiting examples, an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4-Hydroxybutyric acid (4HB), acetate, succinate, alanine, alp a-ketoglutatrate, fumarate, crotonate, crotyl alcohol, and gamma-ammobutyric acid (GAB A). In alternative embodiments, compounds of interest made by a co-culture of the invention, comprise: an alcohol; a diol; a triol; a monohydric, a dihydric or a trihydric alcohol; a lower monohydric, a dihydric or a trihydric alcohol; a butanediol; a germinal or a vicinal diol; or a 1,4-Butanediol (BDO), and the like.
In alternative embodiments, compounds of interest made by a co-culture of the invention, comprise: 1,3 -Propanediol (1,3-PDO) and other glycols including ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol, dipropylene glycol, tripropylene glycol, neopentyl glycol and bisphenol A.
In alternative embodiments, compounds of interest made by a co-culture of the invention, comprise: alkanes, butadiene and alkenes, alkynes, an adipate, a 6-aminocaproic acid, a caprolactam, a hexane - 1,6 diamine (HMD A), caprolactam, an organic acid, a succinic acid, and the like, e.g., as described in U.S. Pat. App. Pub. No. 20120309062.
In alternative embodiments, compounds of interest made by a co-culture of the invention, comprise: a hexametheylenediamine or levulinic acid, e.g., as described in U.S. Pat. App. Pub. No. 20120282661.
In alternative embodiments, compounds of interest made by a co-culture of the invention, comprise: a 2-hydroxyisobutyric acid, a 3-hydroxyisobutyric acid or a methacrylic acid, e.g., as described in U.S. Pat. App. Pub. No. 20120276605.
In alternative embodiments, compounds of interest made by a co-culture of the invention, comprise: butadiene, crotyl alcohol, 1,3-butanediol, and the additional compounds and their metabolic pathways as described in U.S. Patent US8268607, WIPO App. Pubs. WO2013036764A1, WO2013028519A1, WO2013012975A1, and WO2012177710A1.
Production Co-Cultures
In alternative embodiments, the first microbial organism produces a 1 ,4-Butanediol (BDO), and the at least second microbial organism is capable of metabolizing or converting a BDO synthesis organic impurity or a BDO synthesis toxic or growth-inhibiting byproduct to a BDO. For example, in alternative embodiments, the organic impurity generated by synthesis or production of the BDO, or the toxic, growth- inhibiting or synthesis-inhibiting byproduct generated by synthesis of the BDO, comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4-
Hydroxybutyric acid (4HB),more preferably acetate, 4HB and/or glutamate, most preferably glutamate and/or 4HB. The organic acid cars include one or more acetate, pyruvate, succinate, alanine, glutamate, alpha-ketoglutatrate, fumarate, and gamma-aminoburyric acid (GABA) In alternative embodiments, the method incorporates use of compositions and apparatus that can efficiently detect one or more COI synthesis organic impurity or COI synthesis toxic or growth-inhibiting byproduct produced as a product of a biosynthetic process, e.g., as a product of the first microbial organism biosynthetic process. In alternative embodiments, this allows data on when to add nutrients and/or the at least second microorganism, e.g., when adding the at least second microorganism after the first microorganism, or when the at least second microorganism is added only after the synthesis or production of the compound is substantially complete, and the like.
In alternative embodiments, the first microbial organism comprises a methanol metabolic pathway, e.g., where the microorganism has at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol. In certain embodiments, the methanol metabolic pathway comprises one or more enzymes selected from the group consisting of a methanol methyltransferase; a methylenetetrahydrofolate reductase; a methylenetetrahydrofolate dehydrogenase; a methenyltetrahydrofolate cyclohydrolase; a formyltetrahydrofolate deformylase; a formyltetrahydrofolate synthetase; a formate hydrogen lyase; a hydrogenase; a formate dehydrogenase; a methanol dehydrogenase; a formaldehyde activating enzyme; a formaldehyde dehydrogenase; a S-(hydroxymethyl)glutathione synthase; a glutathione-dependent formaldehyde dehydrogenase; and an S-formylglutathione hydrolase. Such organisms advantageously allow for the production of reducing equivalents, which can then be used by the organism for the production of BDO using any one of the BDO pathways.
In alternative embodiments, the first microbial organism comprises a BDO synthetic pathway (a product specific pathway); e.g., where the first microorganism comprises at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO. In certain embodiments, the BDO pathway enzyme is: a succinyl- CoA transferase or a succinyl-CoA synthetase (or succinyl-CoA ligase); a succinyl-CoA reductase (aldehyde forming); a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde forming); a 1 ,4-butanediol dehydrogenase; a succinate reductase; a succinyl-CoA reductase (alcohol forming); a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyrate reductase; a 4-hydroxybutyryl-phosphate reductase; and/or a 4-hydroxybutyryl-CoA reductase (alcohol forming). In alternative embodiments, the first microbial organism comprises a methanol metabolic pathway, either alone or in combination with a BDO pathway, as provided herein, and can further comprise a formaldehyde assimilation pathway that utilizes formaldehyde, e.g., obtained from the oxidation of methanol, in the formation of intermediates of certain central metabolic pathways that can be used, for example, in the formation of biomass. In some of the embodiments, the formaldehyde assimilation pathway comprises a hexulose-6- phosphate synthase, 6-phospho-3-hexuloisomerase, dihydroxyacetone synthase or dihydroxyacetone kinase. In certain embodiments, the first microbial organism comprises a methanol metabolic pathway, e.g., comprising at least one exogenous nucleic acid encoding a methanol dehydrogenase expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol and/or expressed in a sufficient amount to convert methanol to formaldehyde. In some embodiments, the microbial organism further comprises a formaldehyde assimilation pathway. In certain embodiments, the first microbial organism further comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of glycolysis. In certain embodiments, the formaldehyde assimilation pathway enzyme is a hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, dihydroxyacetone synthase and/or dihydroxyacetone kinase.
In some embodiments, the first microbial organism comprises one or more gene disruptions, occurring in one or more endogenous genes encoding protein(s) or enzyme(s) involved in native production of ethanol, glycerol, acetate, lactate, formate, CO2, and/or amino acids by said microbial organism, wherein said one or more gene disruptions confer increased production of BDO in said microbial organism. In some embodiments, one or more endogenous enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, CO2 and/or amino acids by the microbial organism, has attenuated enzyme activity or expression levels. In alternative embodiments, the organism any number of gene disruptions, e.g., from one to about twenty-five gene disruptions, or more.
The figures are intended to clarify the invention, and to demonstrate and further illustrate certain preferred embodiments and aspects without restricting the subject of the invention to the examples and figures. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other
embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A co-culture method for improving the yield, economy or efficiency of a biosynthesis of a compound by a microbial organism., comprising:
(b) providing a co-culture nutrient source, a biomass, or a feedstock,
wherein optionally the feedstock comprises a carbohydrate-comprising feedstock, and optionally the carbohydrate-comprising feedstock comprises an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D-xylose, a trehalose, a biomass- derived mixed sugar and/or a biomass-derived mixed sugar stream,
and optionally the nutrient source comprises a glycerol, a crude glycerol, or a glycerol and a methanol,
and optionally the nutrient source comprises a carbon source, and optionally the carbon source is a gas or a syngas, a carbon dioxide gas or a carbon monoxide gas, a methanol, an ethanol, a formate or a glycerol, and optionally the gas or syngas comprises an H2 gas, which can be provided alone or as part of a syngas, or a synthesis gas from a burning waste: such as a CO + H2, a C02 + H2, or a CO + C02 + H2); or a CO conversion to a C02, and optionally the nutrient source or feedstock comprises a biomass, wherein optionally the biomass comprises: a plant or a microbial biomass; a biomass derived from or comprising bacteria, cyanobacteria, yeast, fungi, or algae;
and optionally the nutrient source or feedstock comprises a polyhydroxyalkanoate (PHA)-comprising biomass, and optionally the PHA comprises a polyglycolide, a poly-3- hydroxypropionate, a poly-3-hydroxybutyrate, a poly-4-hydroxybutyrate, a poly-5- hydroxyvalerate, or a co-polymer thereof;
(b) providing a first microbial organism capable of synthesizing, producing or making the compound by metabolic or enzymatic conversion or biosynthesis of the nutrient source, carbon source or carbohydrate to the compound,
wherein optionally the compound comprises or is: an alcohol; a diol; a triol; a monohydric, a dihydric or a trihydric alcohol; a lower monohydric, a dihydric or a trihydric alcohol; a butanediol; a germinal or a vicinal diol; or a 1 ,4-Butanediol (BDO);
(c) providing at least a second microbial organism capable of metabolizing or converting: an organic impurity generated by synthesis or production of the compound; or, a toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound, to:
a non-toxic composition,
a non-growth-inhibiting composition,
a non-synthesis-inhibiting composition,
a carbon dioxide,
a biomass or cell mass of the microorganism, wherein optionally the biomass comprises a cell wall, a protein or a nucleic acid, and/or
the compound or a precursor thereof,
and optionally the organic impurity generated by synthesis or production of the compound, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the compound, comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4-Hydroxybutyric acid (4HB),
and optionally, when the compound is BDO, the organic impurity generated by synthesis or production of the BDO, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the BDO, comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4-
Hydroxybutyric acid (4HB),
and optionally the at least second microbial organism:
is an obligate aerobe,
is a Generally Recognized As Safe (GRAS) organism,
has an affinity for oxygen equal to or greater than the compound-synthesizing first microorganism,
cannot grow on, metabolize or oxidize, or substantially grow on, metabolize or oxidize the compound,
cannot excrete or produce, or substantially cannot excrete or produce, a substance or composition that negatively affects growth or compound product formation by the co-culture,
wherein optionally: cannot excrete or produce, or substantially cannot excrete or produce: an organic acid, an acetate, a glutamate, a pyrroloquinoline-quinone ( PQQ) and/or a 4-Hydroxybutyric acid (-IS I B ): and
(d) mixing, fermenting, culturing or incubating the carbohydrate-comprising feedstock, the first microbial organism of (b) and the at least second microbial organism of (c) under conditions such that the compound is synthesized or produced by the first microbial organism of (b),
wherein optionally the at least second microbial organism of claim 1 step (c) is added to the mixing, fermenting, culturing:
before, during, and/or after the synthesis or production of the compound by the first microbial organism of claim 1 step (b), or
after substantially most of the synthesis or production of the compound by the first microbial organism of claim 1 step (b), or
when production of the compound is substantially complete,
and optionally the culture conditions comprise: aerobic or micro- aerobic conditions, or substantially anaerobic conditions, or use of a liquid culture or a fermentation broth,
and optionally, when the at least second microbial organism of claim 1 step (c) is added to the mixing, fermenting, culturing after the first microbial organism of (b) begins synthesizing or producing the compound, or is added only after the synthesis or production of the compound is substantially complete,
and optionally the first microbial organism of (b) is mixed, fermented or cultured under micro-aerobic conditions or substantially anaerobic conditions, and after the second microbial organism of claim 1 step (c) is added, the conditions are changed to increase the amount of oxygen in the mixing, fermenting, culturing conditions.
2. The co-culture method of claim 1 , wherein the first microbial organism produces a 1,4-Butanediol (BDO), and the at least second microbial organism is capable of metabolizing or converting a BDO synthesis organic impurity or a BDO synthesis toxic or growth- inhibiting byproduct to a BDO.
3. The co-culture method of claim 1, wherein the first microbial organism of (b) and the at least second microbial organism of (c), are in syntrophy, or artificial syntrophy,
and optionally the first microbial organism of (b) is auxotrophic for a composition made only by the at least second microbial organism of (c), and/or
the at least second microbial organism of (c) is auxotrophic for a composition made only by the first microbial organism of (b),
wherein optionally the composition is an amino acid.
4. The method of any of claim 1 to claim 3, wherein: (a) the compound is made by metabolizing or converting an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D-xylose, a biomass-derived mixed sugar and/or a biomass-derived mixed sugar stream to the compound;
(b) the microorganism is a bacteria, a Saccharomycetales , or a yeast, or a member of the genus Saccharomyces, Escherichia, Pseudomonas or Acinetobacter,
or the bacteria or a yeast is a: Saccharomyces cerevisiae, Pseudomonas putida, Escherichia coli ox Acinetobacter calcoaceticus ,
and optionally the first microbial organism of (b) is a bacteria and the second microbial organism is a yeast;
(c) the at least second microbial organism of claim 1 step (c) itself cannot or substantially cannot grow on or metabolize: the nutrient, feedstock or biomass, or a sugar or a carbohydrate or any biomass-derived mixed sugar, or at least one sugar, carbohydrate or biomass-derived mixed sugar,
(d) the at least second microbial organism of claim 1 step (c) cannot or substantially cannot grow on or metabolize an L-arabinose, an arabinose, a sucrose, a glucose, a fructose, a xylose, a D-xylose, and/or a biomass-derived mixed sugar,
(e) the at least second microbial organism of claim 1 step (c) that cannot or substantially cannot grow on or metabolize a sugar or a carbohydrate or any biomass-derived mixed sugar:
lacks an enzyme, enzymes or operon involved in synthesizing a pyrroloquinoline- quinone ( PQQ) or an alcohol dehydrogenase, or
comprises a disabled or a non-functioning enzyme, enzymes or operon involved in synthesizing a pyrroloquinoline-quinone ( PQQ) or an alcohol dehydrogenase, or
lacks or comprises a disabled or non-functioning PQQ operon;
(f) if the at least second microbial organism of claim 1 step (c) is a Saccharomyces or a Saccharomyces cerevisiae, then a hexokinase or hexokinases and a glucokinase, and a galactokinase if galactose is in the feedstock, is deleted;
(g) if the at least second microbial organism of claim 1 step (c) is a Pseudomonas or a Pseudomonas putida , or a P. putida SI 2, which comprise a glucokinase and at least two PQQ-glucose dehydrogenases, then the glucokinase and at least two PQQ-glucose dehydrogenases must be deleted, inactivated or disabled or substantially inactivated or disabled, or the operon for PQQ biosynthesis is deleted, inactivated or disabled or substantially inactivated or disabled,
and optionally if a fructose-containing feedstock is used a fructose PTS or a fructose- 1-P kinase must be deleted, inactivated or disabled or substantially inactivated or disabled; or
(h) the first microbial organism of (b) and/or the at least second microbial organism of (c) are genetically engineered or modified to comprise or contain and express a heterologous protein or enzyme.
5. The method of any of claims 1 to 4, further comprising measuring or monitoring conductivity of the liquid culture or fermentation broth during the culture or fermentation to determine the amount or level or rate of consumption of all organic acids or an organic acid.
6. The method of any of claims 1 to 5, wherein when the at least second microbial organism of claim 1 step (c) is added to the mixing, fermenting, culturing during and/or after the production of the compound by the first microbial organism of claim 1 step (b) has begun or is complete or substantially complete, the ratio of the amount of time the first microbial organism of claim 1 step (b) is mixed, fermented or cultured to synthesize or produce the compound (without the second microbial organism of claim 1 step (c)) (the so-called "ferment period") to the amount of time the first microbial organism of claim 1 step (b) is co- cultured with the at least second microbial organism of claim 1 step (c) (the so-called "burn- off period") is 2: 1, 3 : 1, 4: 1, 5: 1, or 6: 1, or between about 2: 1 and 6: 1 (the so-called "burn-off period ratio"),
wherein optionally the ferment period:burn-off period is about 30 hours:about 6 hours, or is about 25 to 30 hours (ferment period):about 2 to 10 hours (burn-off period).
7. The method of claim 1, when the compound is BDO, the organic impurity generated by synthesis or production of the BDO, or the toxic, growth-inhibiting or synthesis- inhibiting byproduct generated by synthesis of the BDO, comprises: an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ) and/or a 4- Hydroxybutyric acid (4HB).
8. The method of claim 1, when the compound is an adipate, a 6-aminocaproic acid or a caprolactam, the organic impurity generated by synthesis or production of the adipate, 6- aminocaproic acid or caprolactam, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the adipate, a 6-aminocaproic acid or a caprolactam, comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a
pyrroloquinoline-quinone (PQQ), an adipate semialdehyde, an alpha-ketoadipate, an alpha- ketohydroxyadipate, and/or a hexa-2-enedionate.
9. The method of claim 1, when the compound is a hexametheylenediamine, the organic impurity generated by synthesis or production of the hexametheylenediamine or levulinic acid, or the toxic, growth-inhibiting or synthesis-inhibiting byproduct generated by synthesis of the hexametheylenediamine or levulinic acid,, comprises an organic acid, an acetate, a malonate, a pyruvate, a glutamate, a pyrroloquinoline-quinone (PQQ), an adipate, an adipate semialdehyde, a 3-oxoadipate, a 5-carboxy-2-pettenoate, a 5-carboxy-2- pentenoate, an 6-aminocaproate semialdehyde, and/or a caprolactam.
PCT/US2014/028638 2013-03-15 2014-03-14 Development and use of microbes for impurity reduction in biomass hydrolysates and fermentation broths WO2014144293A2 (en)

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