WO2015002977A1 - Microorganismes modifiés et procédés d'utilisation de ces derniers pour la coproduction anaérobie d'isoprène et d'acide acétique - Google Patents

Microorganismes modifiés et procédés d'utilisation de ces derniers pour la coproduction anaérobie d'isoprène et d'acide acétique Download PDF

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WO2015002977A1
WO2015002977A1 PCT/US2014/045097 US2014045097W WO2015002977A1 WO 2015002977 A1 WO2015002977 A1 WO 2015002977A1 US 2014045097 W US2014045097 W US 2014045097W WO 2015002977 A1 WO2015002977 A1 WO 2015002977A1
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Prior art keywords
pathway
enzymes
conversion
coa
catalyze
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PCT/US2014/045097
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English (en)
Inventor
Daniel Johannes KOCH
Mateus Schreiner Garcez LOPES
Iuri Estrada GOUVEA
Ane Fernanda Beraldi Zeidler
Aline Silva Romao DUMARESQ
Marilene Elizabete Pavan RODRIGUES
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Braskem S/A Ap 09
Braskem America, Inc.
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Publication of WO2015002977A1 publication Critical patent/WO2015002977A1/fr

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    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54—Acetic acid
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes

Definitions

  • Isoprene is a critical starting material for a variety of synthetic polymers, most notably synthetic rubbers. Isoprene is naturally produced by a variety of microbial, plant and animal species. However, the yield of isoprene from naturally-occurring organism is commercially unattractive.
  • Isoprene is also copolymerized for use as a synthetic elastomer in other products such as footwear, mechanical products, medical products, sporting goods, and latex.
  • isoprene can be obtained by fractionating petroleum, the purification on this material is expensive and time-consuming. Petroleum cracking of the C5 stream of hydrocarbons produces only 15% isoprene. Isoprene could be also produced by isobutylene carbonylation with methanol and by isopentene dehydrogenation.
  • the present disclosure generally relates to microorganisms (e.g., modified or non- naturally occurring microorganisms) that comprise one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to isoprene and/or acetic acid and the use of such microorganisms for the production of isoprene and/or acetic acid.
  • the methods of the present disclosure are advantageous over prior methods in that they reduce (including eliminate) the need for toxic and expensive catalysts, and can be performed anaerobically thereby reducing (or eliminating) the risk of fire or explosion.
  • the present disclosure provides an anaerobic method of co-producing isoprene and acetic acid from a fermentable carbon source, the method comprising: providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to isoprene and acetic acid in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to isoprene and acetic acid, wherein the microorganism comprises one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and acetyl-CoA to lactoy
  • the bacteria is selected from the genera consisting of: Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus.
  • the eukaryote is a yeast, filamentous fungi, protozoa, or algae.
  • the yeast is Saccharomyces cerevisiae or Pichia pastoris.
  • the fermentable carbon source is sugarcane juice, sugarcane molasses, hydrolyzed starch, hydrolyzed lignocellulosic materials, glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, or glycerol in any form or mixture thereof.
  • the fermentable carbon source is a monosaccharide, oligosaccharide, or polysaccharide.
  • the produced isoprene and/or acetic acid is secreted by the microorganism into the fermentation media.
  • the methods further comprise recovering the produced isoprene and/or acetic acid from the fermentation media.
  • the microorganism has been genetically modified to express the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to isoprene and acetic acid.
  • the present disclosure also provides microorganisms comprising: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and acetyl-CoA to lactoyl-CoA and acetic acid; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and CoA to lactoyl-CoA; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA to propionyl-CoA; one or more polynucleotides coding for enzymes in
  • 2-methylacetoacetyl-CoA one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methylacetoacetyl-CoA to 3-hydroxy-2-methylbutanoyl-CoA; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy- 2-methylbutanoyl-CoA to 2-methyl-2-butenoyl-CoA; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-2-butenoyl-CoA to 2-methyl-2- butenal; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-2-butenal to 2-methyl-l-but-2-enol; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-2-butenal to 2-
  • the microorganism is an archea, bacteria, or eukaryote.
  • the bacteria is selected from the genera consisting of: Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus.
  • the eukaryote is a yeast, filamentous fungi, protozoa, or algae.
  • the yeast is Saccharomyces cerevisiae or Pichia pastoris.
  • the microorganism has been genetically modified to express the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to isoprene and acetic acid.
  • the present disclosure also provides methods of co-producing isoprene and acetic acid from a fermentable carbon source by providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and acetic acid, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to isoprene and acetic acid in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and acetic acid and one or more polynucleotides coding for enzymes in a pathway that cat
  • the microorganism is an archea, bacteria, or eukaryote.
  • the bacteria is selected from the genera consisting of: Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus.
  • the eukaryote is a yeast, filamentous fungi, protozoa, or algae.
  • the yeast is Saccharomyces cerevisiae or Pichia pastoris.
  • the fermentable carbon source is sugarcane juice, sugarcane molasses, hydrolyzed starch, hydrolyzed lignocellulosic materials, glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, or glycerol in any form or mixture thereof.
  • the fermentable carbon source is a monosaccharide, oligosaccharide, or polysaccharide.
  • the produced isoprene and/or acetic acid is secreted by the microorganism into the fermentation media.
  • the method further comprise recovering the produced isoprene and/or acetic acid from the fermentation media.
  • the microorganism has been genetically modified to express the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and acetic acid, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to isoprene and acetic acid.
  • the present disclosure also provides microorganisms comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of a fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and acetic acid, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to isoprene and acetic acid, wherein isoprene is produced via a 2-methylbutenoyl-CoA intermediate.
  • the microorganism is an archea, bacteria, or eukaryote.
  • the bacteria is selected from the genera consisting of: Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus.
  • the eukaryote is a yeast, filamentous fungi, protozoa, or algae.
  • the yeast is Saccharomyces cerevisiae or Pichia pastoris.
  • the microorganism has been genetically modified to express the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and acetic acid, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to isoprene and acetic acid.
  • Figure 1 depicts an exemplary pathway for the co-production of isoprene and acetic acid, where isoprene is produced via a 2-methylbutenoyl-CoA intermediate.
  • the present disclosure generally relates to microorganisms (e.g., non-naturally occurring microorganisms) that comprise a genetically modified pathway and uses of the microorganisms for the conversion of a fermentable carbon source to isoprene and/or acetic acid (see, Figure 1).
  • microorganisms may comprise one or more polynucleotides coding for enzymes that catalyze a conversion of a fermentable carbon source to isoprene and one or more polynucleotides coding for enzymes that catalyze a conversion of a fermentable carbon source to acetic acid.
  • isoprene is further converted to polyisoprene.
  • This disclosure provides, in part, the discovery of novel anaerobic enzymatic pathways including, for example, novel combinations of enzymatic pathways, for the production of isoprene and/or acetic acid from a carbon source (e.g., a fermentable carbon source).
  • a carbon source e.g., a fermentable carbon source
  • the methods provided herein provide end-results similar those of sterilization without the high capital expenditure and continuing higher management costs required to establish and maintain sterility throughout a production process.
  • most industrial- scale isoprene production processes are operated in the presence of measurable numbers of bacterial contaminants. It is believed that bacterial contamination of an isoprene production process causes a reduction in product yield and an inhibition of yeast growth (see, Chang et al., 1995, J. Microbiol. Biotechnol. 5:309-314; Ngang et al., 1990, Appl. Microbiol. Biotechnol. 33:490-493).
  • Such drawbacks of prior methods are avoided by the presently disclosed methods as the toxic nature of the produced isoprene reduces contaminants in the production process.
  • the enzymatic pathways disclosed herein are advantageous over prior known enzymatic pathways for the production of isoprene and/or acetic acid in that the enzymatic pathways disclosed herein are anaerobic. While it is possible to use aerobic processes to produce isoprene and/or acetic acid, anaerobic processes are preferred due risk incurred when olefins (which are by nature are explosive) are mixed with oxygen during the fermentation process. Moreover, the supplementation of oxygen and nitrogen in a fermenter requires an additional investment for aerobic process and another additional investment for the purification from the nitrogen from the isoprene and/or acetic acid.
  • the presence of oxygen can also catalyze the polymerization of isoprene and/or acetic acid and can promote the growth of aerobic contaminants in the fermentor broth.
  • aerobic fermentation processes for the production of isoprene and/or acetic acid present several drawbacks at industrial scale (where it is technically challenging to maintain aseptic conditions) such as the fact that: (i) greater biomass is obtained reducing overall yields on carbon; (ii) the presence and oxygen favors the growth of contaminants (Weusthuis et al., 2011, Trends in Biotechnology, 2011, Vol. 29, No.
  • the present disclosure provides an anaerobic method of co-producing isoprene and acetic acid from a fermentable carbon source, the method comprising: providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to isoprene and acetic acid in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to isoprene and acetic acid, wherein the microorganism comprises one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and acetyl-CoA to lactoy
  • the present disclosure also provides a microorganism comprising: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and acetyl-CoA to lactoyl-CoA and acetic acid; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and CoA to lactoyl-CoA; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA; one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA to propionyl-CoA; one or more polynucleotides coding for enzymes
  • microorganisms comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and acetic acid, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to isoprene and acetic acid in a fermentation media, wherein isoprene is produced via a 2-methylbutenoyl-CoA intermediate.
  • the present disclosure also provides methods of co-producing isoprene and acetic acid from a fermentable carbon source by providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and acetic acid, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to isoprene and acetic acid in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and acetic acid and one or more polynucleotides coding for enzymes in a pathway that cat
  • the present disclosure provides methods of co-producing isoprene and/or acetic acid from a fermentable carbon source, comprising: providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and/or acetic acid, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to isoprene and/or acetic acid in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a
  • expression of the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and/or acetic acid and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to isoprene and/or acetic acid in the microorganism to produce isoprene and/or acetic acid may be preformed prior to or after contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and/or acetic acid, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or
  • biological activity when referring to a protein, polypeptide or peptide, may mean that the protein, polypeptide or peptide exhibits a functionality or property that is useful as relating to some biological process, pathway or reaction.
  • Biological or functional activity can refer to, for example, an ability to interact or associate with (e.g. , bind to) another polypeptide or molecule, or it can refer to an ability to catalyze or regulate the interaction of other proteins or molecules (e.g., enzymatic reactions).
  • the term “culturing” may refer to growing a population of cells, e.g., microbial cells, under suitable conditions for growth, in a liquid or on solid medium.
  • derived from may encompass the terms originated from, obtained from, obtainable from, isolated from, and created from, and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to the another specified material.
  • exogenous polynucleotide refers to any deoxyribonucleic acid that originates outside of the microorganism.
  • an expression vector may refer to a DNA construct containing a polynucleotide or nucleic acid sequence encoding a polypeptide or protein, such as a DNA coding sequence (e.g. gene sequence) that is operably linked to one or more suitable control sequence(s) capable of affecting expression of the coding sequence in a host.
  • control sequences include a promoter to affect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
  • the vector may be a plasmid, cosmid, phage particle, bacterial artificial chromosome, or simply a potential genomic insert.
  • the vector may replicate and function independently of the host genome (e.g. , independent vector or plasmid), or may, in some instances, integrate into the genome itself (e.g. , integrated vector).
  • the plasmid is the most commonly used form of expression vector. However, the disclosure is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.
  • the term "expression” may refer to the process by which a polypeptide is produced based on a nucleic acid sequence encoding the polypeptides (e.g. , a gene). The process includes both transcription and translation.
  • the term "gene” may refer to a DNA segment that is involved in producing a polypeptide or protein (e.g., fusion protein) and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).
  • heterologous with reference to a nucleic acid, polynucleotide, protein or peptide, may refer to a nucleic acid, polynucleotide, protein or peptide that does not naturally occur in a specified cell, e.g., a host cell. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes.
  • homologous with reference to a nucleic acid, polynucleotide, protein or peptide, refers to a nucleic acid, polynucleotide, protein or peptide that occurs naturally in the cell.
  • a "host cell” may refer to a cell or cell line, including a cell such as a microorganism which a recombinant expression vector may be transfected for expression of a polypeptide or protein (e.g., fusion protein).
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • a host cell may include cells transfected or transformed in vivo with an expression vector.
  • the term "introduced,” in the context of inserting a nucleic acid sequence or a polynucleotide sequence into a cell, may include transfection, transformation, or transduction and refers to the incorporation of a nucleic acid sequence or polynucleotide sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence or polynucleotide sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.
  • the genome of the cell e.g., chromosome, plasmid, plastid, or mitochondrial DNA
  • non-naturally occurring when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species.
  • Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species.
  • Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon.
  • Non-naturally occurring microbial organisms of the disclosure can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration.
  • stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
  • Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway.
  • E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species.
  • Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.
  • operably linked may refer to a juxtaposition or arrangement of specified elements that allows them to perform in concert to bring about an effect.
  • a promoter may be operably linked to a coding sequence if it controls the transcription of the coding sequence.
  • a promoter may refer to a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene.
  • a promoter may be an inducible promoter or a constitutive promoter.
  • An inducible promoter is a promoter that is active under environmental or developmental regulatory conditions.
  • a polynucleotide or “nucleic acid sequence” may refer to a polymeric form of nucleotides of any length and any three-dimensional structure and single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs.
  • Such polynucleotides or nucleic acid sequences may encode amino acids (e.g., polypeptides or proteins such as fusion proteins).
  • polynucleotides which encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2'-0-Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin.
  • polynucleotide also includes peptide nucleic acids (PNA).
  • Polynucleotides may be naturally occurring or non-naturally occurring.
  • the terms polynucleotide, nucleic acid, and oligonucleotide are used herein interchangeably.
  • Polynucleotides may contain RNA, DNA, or both, and/or modified forms and/or analogs thereof.
  • a sequence of nucleotides may be interrupted by non-nucleotide components.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S (thioate), P(S)S (dithioate), (0)NPv 2 (amidate), P(0)R, P(0)OR', COCH 2 (formacetal), in which each R or R * is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (- 0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or comprise a combination of linear and circular portions.
  • a "protein” or “polypeptide” may refer to a composition comprised of amino acids and recognized as a protein by those of skill in the art.
  • the conventional one-letter or three-letter code for amino acid residues is used herein.
  • the terms protein and polypeptide are used interchangeably herein to refer to polymers of amino acids of any length, including those comprising linked (e.g., fused) peptides/polypeptides (e.g., fusion proteins).
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
  • related proteins, polypeptides or peptides may encompass variant proteins, polypeptides or peptides.
  • Variant proteins, polypeptides or peptides differ from a parent protein, polypeptide or peptide and/or from one another by a small number of amino acid residues. In some embodiments, the number of different amino acid residues is any of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, variants differ by about 1 to about 10 amino acids. Alternatively or additionally, variants may have a specified degree of sequence identity with a reference protein or nucleic acid, e.g.
  • variant proteins or nucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
  • the term “recovered,” “isolated,” “purified,” and “separated” may refer to a material (e.g., a protein, peptide, nucleic acid, polynucleotide or cell) that is removed from at least one component with which it is naturally associated.
  • these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.
  • the term “recombinant” may refer to nucleic acid sequences or polynucleotides, polypeptides or proteins, and cells based thereon, that have been manipulated by man such that they are not the same as nucleic acids, polypeptides, and cells as found in nature.
  • Recombinant may also refer to genetic material (e.g., nucleic acid sequences or polynucleotides, the polypeptides or proteins they encode, and vectors and cells comprising such nucleic acid sequences or polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another coding sequence or gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at decreased or elevated levels, expressing a gene conditionally or constitutively in manners different from its natural expression profile, and the like.
  • genetic material e.g., nucleic acid sequences or polynucleotides, the polypeptides or proteins they encode, and vectors and cells comprising such nucleic acid sequences or polynucleotides
  • selective marker may refer to a gene capable of expression in a host cell that allows for ease of selection of those hosts containing an introduced nucleic acid sequence, polynucleotide or vector.
  • selectable markers include but are not limited to antimicrobial substances (e.g. , hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage, on the host cell.
  • nucleic acid, polynucleotide, protein or polypeptide comprises a sequence that has at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference (e.g., wild-type) nucleic acid, polynucleotide, protein or polypeptide.
  • a reference e.g., wild-type
  • Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altshul et al. (1990) J. Mol. Biol. 215:403-410; Henikoff et al. (1989) Proc. Natl. Acad. Sci. 89: 10915; Karin et al. (1993) Proc. Natl. Acad. Sci. 90:5873; and Higgins et al. (1988) Gene 73:237). Software for performing
  • databases may be searched using FASTA (Person et al. (1988) Proc. Natl.
  • substantially identical polypeptides differ only by one or more conservative amino acid substitutions.
  • substantially identical polypeptides are immunologically cross-reactive.
  • substantially identical nucleic acid molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).
  • transfection may refer to the insertion of an exogenous nucleic acid or polynucleotide into a host cell.
  • the exogenous nucleic acid or polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.
  • transfecting or transfection is intended to encompass all conventional techniques for introducing nucleic acid or polynucleotide into host cells. Examples of transfection techniques include, but are not limited to, calcium phosphate precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, and microinjection.
  • transformed may refer to a cell that has a non-native (e.g., heterologous) nucleic acid sequence or polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.
  • non-native e.g., heterologous
  • vector may refer to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types.
  • Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, single and double stranded cassettes and the like.
  • wild-type As used herein, the term "wild-type,” “native,” or “naturally-occurring” proteins may refer to those proteins found in nature.
  • wild-type sequence refers to an amino acid or nucleic acid sequence that is found in nature or naturally occurring.
  • a wild-type sequence is the starting point of a protein engineering project, for example, production of variant proteins.
  • nucleic acids sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • a microorganism may be modified (e.g., genetically engineered) by any method known in the art to comprise and/or express one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to one or more intermediates in a pathway for the co-production of isoprene and acetic acid.
  • Such enzymes may include any of those enzymes as are forth in Figure 1.
  • the microorganism may be modified to comprise one or more polynucleotides coding for enzymes that catalyze a conversion of 2- methylbutenoyl-CoA to isoprene.
  • a modified microorganism as provided herein may comprise:
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate e.g., a lactate dehydrogenase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and acetyl-CoA to lactoyl-CoA and acetic acid e.g., a propionate CoA- transferase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and CoA to lactoyl-CoA e.g., an acetyl coenzyme A synthetase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA e.g., a lactoyl-CoA dehydratase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA to propionyl-CoA e.g., an acryloyl-CoA reductase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA e.g., a pyruvate formate lyase and pyruvate formate lyase activating enzyme
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetyl-CoA and propionyl-CoA to 2-methylacetoacetyl-CoA (e.g., a thiolase);
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methylacetoacetyl-CoA to 3-hydroxy-2-methylbutanoyl-CoA (e.g., a 3-hydroxy- 2-methylbutyryl-CoA dehydrogenase);
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-2-methylbutanoyl-CoA to 2-methyl-2-butenoyl-CoA e.g., an enoyl- CoA hydratase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-2-butenoyl-CoA to 2-methyl-2-butenal e.g., a succinate semialdehyde dehydrogenase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-2-butenal to 2-methyl-l-but-2-enol e.g., an alcohol dehydrogenase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-l-but-2-enol to isoprene (e.g., linalool dehydratase/isomerase);
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-2-butenoyl-CoA to 2-methyl-l-but-2-enol e.g., a HMG-CoA reductase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-l-but-2-enol to 2-methyl-l-butenyl monophosphate (e.g., a mevalonate kinase);
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-l-butenyl monophosphate to 2-methyl-l-butenyl diphosphate e.g., a phosphomevalonate kinase
  • polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methyl-l-butenyl diphosphate to isoprene e.g., an isoprene synthase.
  • Exemplary enzymes which convert a fermentable carbon source to isoprene and/or acetic acid are presented in Table 1 below, as well as, the substrates that they act upon and product that they produce.
  • the enzyme number represented in Table 1 correlates with the enzyme numbering used in Figure 1 which schematically represents the enzymatic conversion of a fermentable carbon source to isoprene through a 2-methylbutenoyl-CoA intermediate and acetic acid.
  • Table 1 indicates a gene identifier (GI) number(s) that corresponds to an exemplary amino acid sequence(s) for the listed enzyme.
  • GI gene identifier
  • the disclosure contemplates the modification (e.g., engineering) of one or more of the enzymes provided herein. Such modification may be performed to redesign the substrate specificity of the enzyme and/or to modify (e.g., reduce) its activity against others substrates in order to increase its selectivity for a given substrate.
  • one or more enzymes as provided herein may be engineered to alter (e.g., enhance including, for example, increase its catalytic activity or its substrate specificity) one or more of its properties.
  • the one or more enzymes are expressed in a microorganism selected from an archea, bacteria, or eukaryote.
  • the bacteria is a Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus including, for example, Pelobacter propionicus, Clostridium propionicum, Clostridium acetobutylicum, Lactobacillus, Propionibacterium acidipropionici or Propionibacterium freudenreichii.
  • the eukaryote is a yeast, filamentous fungi, protozoa, or algae.
  • the yeast is Saccharomyces cerevisiae or Pichia pastoris.
  • sequence alignment and comparative modeling of proteins may be used to alter one or more of the enzymes disclosed herein.
  • Homology modeling or comparative modeling refers to building an atomic-resolution model of the desired protein from its primary amino acid sequence and an experimental three-dimensional structure of a similar protein. This model may allow for the enzyme substrate binding site to be defined, and the identification of specific amino acid positions that may be replaced to other natural amino acid in order to redesign its substrate specificity.
  • variants or modified sequences having substantial identity or homology with the polynucleotides encoding enzymes as disclosed herein may be utilized in the practice of the disclosure. Such sequences can be referred to as variants or modified sequences. That is, a polynucleotide sequence may be modified yet still retain the ability to encode a polypeptide exhibiting the desired activity. Such variants or modified sequences are thus equivalents. Generally, the variant or modified sequence may comprise at least about 40%-60%, preferably about 60%-80%, more preferably about 80%-90%, and even more preferably about 90%-95% sequence identity with the native sequence.
  • the microorganism may be modified by genetic engineering techniques (i.e., recombinant technology), classical microbiological techniques, or a combination of such techniques and can also include naturally occurring genetic variants to produce a genetically modified microorganism.
  • genetic engineering techniques i.e., recombinant technology
  • classical microbiological techniques or a combination of such techniques and can also include naturally occurring genetic variants to produce a genetically modified microorganism.
  • a genetically modified microorganism may include a microorganism in which a polynucleotide has been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect of expression (e.g., over-expression) of one or more enzymes as provided herein within the microorganism.
  • Genetic modifications which result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene.
  • Addition of cloned genes to increase gene expression can include maintaining the cloned gene(s) on replicating plasmids or integrating the cloned gene(s) into the genome of the production organism. Furthermore, increasing the expression of desired cloned genes can include operatively linking the cloned gene(s) to native or heterologous transcriptional control elements.
  • the expression of one or more of the enzymes provided herein is under the control of a regulatory sequence that controls directly or indirectly the enzyme expression in a time-dependent fashion during the fermentation.
  • a microorganism is transformed or transfected with a genetic vehicle, such as an expression vector comprising an exogenous polynucleotide sequence coding for the enzymes provided herein.
  • Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may typically, but not always, comprise a replication system (i.e. vector) recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and may preferably, but not necessarily, also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment.
  • a replication system i.e. vector
  • Expression systems may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome -binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, mRNA stabilizing sequences, nucleotide sequences homologous to host chromosomal DNA, and/or a multiple cloning site.
  • Signal peptides may also be included where appropriate, preferably from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.
  • the vectors can be constructed using standard methods (see, e.g.,Sambrooket al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor, N.Y. 1989; and Ausubel, et al., Current Protocols in Molecular Biology, Greene Publishing, Co. N.Y, 1995).
  • polynucleotides that encode the enzymes disclosed herein are typically carried out in recombinant vectors.
  • Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes, episomal vectors and gene expression vectors, which can all be employed.
  • a vector may be selected to accommodate a polynucleotide encoding a protein of a desired size.
  • a suitable host cell is transfected or transformed with the vector.
  • Host cells may be prokaryotic, such as any of a number of bacterial strains, or may be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells including, for example, rodent, simian or human cells.
  • Each vector contains various functional components, which generally include a cloning site, an origin of replication and at least one selectable marker gene.
  • a vector may additionally possess one or more of the following elements: an enhancer, promoter, and transcription termination and/or other signal sequences.
  • sequence elements may be optimized for the selected host species (e.g. humanized) Such sequence elements may be positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a preselected enzyme.
  • Vectors may contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells.
  • the sequence may be one that enables the vector to replicate independently of the host chromosomal DNA and may include origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria
  • the 2 micron plasmid origin is suitable for yeast
  • various viral origins e.g.SV 40, adenovirus
  • the origin of replication is not needed for mammalian expression vectors unless these are used in mammalian cells able to replicate high levels of DNA, such as COS cells.
  • a cloning or expression vector may contain a selection gene (also referred to as a selectable marker). This gene encodes a protein necessary for the survival or growth of transformed host cells in a selective culture medium. Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium.
  • Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate, hygromycin, thiostrepton, apramycin or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.
  • the replication of vectors may be performed in E. coli (e.g., strain TBI or TGI, DH5a, ⁇ , JM110).
  • E. coli-selectable marker for example, the ⁇ -lactamase gene that confers resistance to the antibiotic ampicillin, may be of use.
  • selectable markers can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19, or pUC119.
  • Expression vectors may contain a promoter that is recognized by the host organism.
  • the promoter may be operably linked to a coding sequence of interest. Such a promoter may be inducible or constitutive.
  • Polynucleotides are operably linked when the polynucleotides are in a relationship permitting them to function in their intended manner.
  • Promoters suitable for use with prokaryotic hosts may include, for example, the a-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, the erythromycin promoter, apramycin promoter, hygromycin promoter, methylenomycin promoter and hybrid promoters such as the tac promoter. Moreover, host constitutive or inducible promoters may be used. Promoters for use in bacterial systems will also generally contain a Shine-Dalgarno sequence operably linked to the coding sequence.
  • Viral promoters obtained from the genomes of viruses include promoters from polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2 or 5), herpes simplex virus (thymidine kinase promoter), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus (e.g.,MoMLV, or RSV LTR), Hepatitis-B virus, Myeloproliferative sarcoma virus promoter (MPSV), VISNA, and Simian Virus 40 (SV40).
  • Heterologous mammalian promoters include, e.g., the actin promoter, immunoglobulin promoter, heat-shock protein promoters.
  • the early and late promoters of the SV40 virus are conveniently obtained as a restriction fragment that also contains the SV40 viral origin of replication (see, e.g.,Fierset al., Nature, 273: 113 (1978); Mulligan and Berg, Science, 209: 1422-1427 (1980); and Pavlakiset al., Proc. Natl. Acad. Sci. USA, 78:7398-7402 (1981)).
  • the immediate early promoter of the human cytomegalovirus (CMV) is conveniently obtained as a Hind III E restriction fragment (see, e.g., Greenaway et al., Gene, 18:355-360 (1982)).
  • a broad host range promoter such as the SV40 early promoter or the Rous sarcoma virus LTR, is suitable for use in the present expression vectors.
  • a strong promoter may be employed to provide for high level transcription and expression of the desired product.
  • the eukaryotic promoters that have been identified as strong promoters for high-level expression are the SV40 early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus long terminal repeat, and human cytomegalovirus immediate early promoter (CMV or CMV IE).
  • the promoter is a SV40 or a CMV early promoter.
  • the promoters employed may be constitutive or regulatable, e.g., inducible.
  • exemplary inducible promoters include jun, fos and metallothionein and heat shock promoters.
  • One or both promoters of the transcription units can be an inducible promoter.
  • the GFP is expressed from a constitutive promoter while an inducible promoter drives transcription of the gene coding for one or more enzymes as disclosed herein and/or the amplifiable selectable marker.
  • the transcriptional regulatory region in higher eukaryotes may comprise an enhancer sequence.
  • enhancer sequences from mammalian genes are known e.g., from globin, elastase, albumin, a-fetoprotein and insulin genes.
  • a suitable enhancer is an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the enhancer of the cytomegalovirus immediate early promoter
  • the enhancer sequences may be introduced into the vector at a position 5' or 3' to the gene of interest, but is preferably located at a site 5' to the promoter.
  • Yeast and mammalian expression vectors may contain prokaryotic sequences that facilitate the propagation of the vector in bacteria. Therefore, the vector may have other components such as an origin of replication ⁇ e.g., a nucleic acid sequence that enables the vector to replicate in one or more selected host cells), antibiotic resistance genes for selection in bacteria, and/or an amber stop codon which can permit translation to read through the codon. Additional eukaryotic selectable gene(s) may be incorporated.
  • the origin of replication is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known, e.g., the ColEl origin of replication in bacteria.
  • SV40 SV40
  • polyoma adenovirus
  • VSV or BPV adenovirus
  • BPV BPV
  • SV40 origin may typically be used only because it contains the early promoter
  • the constructs may be designed with at least one cloning site for insertion of any gene coding for any enzyme disclosed herein.
  • the cloning site may be a multiple cloning site, e.g., containing multiple restriction sites.
  • the plasmids may be propagated in bacterial host cells to prepare DNA stocks for subcloning steps or for introduction into eukaryotic host cells.
  • Transfection of eukaryotic host cells can be any performed by any method well known in the art. Transfection methods include lipofection, electroporation, calcium phosphate co-precipitation, rubidium chloride or polycation mediated transfection, protoplast fusion and microinjection.
  • the transfection is a stable transfection.
  • the transfection method that provides optimal transfection frequency and expression of the construct in the particular host cell line and type is favored. Suitable methods can be determined by routine procedures. For stable transfectants, the constructs are integrated so as to be stably maintained within the host chromosome.
  • Vectors may be introduced to selected host cells by any of a number of suitable methods known to those skilled in the art.
  • vector constructs may be introduced to appropriate cells by any of a number of transformation methods for plasmid vectors.
  • standard calcium-chloride-mediated bacterial transformation is still commonly used to introduce naked DNA to bacteria (see, e.g.,Sambrooket al, 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), but electroporation and conjugation may also be used (see, e.g.,Ausubelet al, 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).
  • yeast or other fungal cells For the introduction of vector constructs to yeast or other fungal cells, chemical transformation methods may be used ⁇ e.g., Rose et al, 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Transformed cells may be isolated on selective media appropriate to the selectable marker used. Alternatively, or in addition, plates or filters lifted from plates may be scanned for GFP fluorescence to identify transformed clones.
  • Plasmid vectors may be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection ("lipofection"), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation (see, e.g.,Ausubelet al, 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).
  • Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture.
  • LipofectAMINETM Life Technologies
  • LipoTaxiTM LipoTaxiTM kits
  • Other companies offering reagents and methods for lipofection include Bio-Rad Laboratories, CLONTECH, Glen Research, InVitrogen, JBL Scientific, MBIFermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.
  • the host cell may be capable of expressing the construct encoding the desired protein, processing the protein and transporting a secreted protein to the cell surface for secretion. Processing includes co- and post-translational modification such as leader peptide cleavage, GPI attachment, glycosylation, ubiquitination, and disulfide bond formation.
  • Immortalized host cell cultures amenable to transfection and in vitro cell culture and of the kind typically employed in genetic engineering are preferred. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line
  • mice Sertoli cells TM4, Mather, Biol. Reprod., 23:243-251 (1980)
  • monkey kidney cells CV1 ATCC CCL 70
  • African green monkey kidney cells VRO-76, ATCC CRL-
  • HELA human cervical carcinoma cells
  • MDCK canine kidney cells
  • FS4 cells human hepatoma cell line (Hep G2), human HT1080 cells, KB cells, JW-2 cells,
  • Embryonic cells used for generating transgenic animals are also suitable (e.g., zygotes and embryonic stem cells).
  • Suitable host cells for cloning or expressing polynucleotides (e.g., DNA) in vectors may include, for example, prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g.,E. coli, Enter obacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratiamarcescans, and Shigella, as well as Bacilli such as B.
  • subtilis and B. licheniformis e.g.,B. licheniformis 41 P disclosed in DD 266,710 published Apr. 12, 1989
  • Pseudomonas such as P. aeruginosa
  • Streptomyces a preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli XI 776 (ATCC 31,537), E. coli JM110 (ATCC 47,013) and E. coli W3110 (ATCC 27,325) are suitable.
  • eukaryotic microbes such as filamentous fungi or yeast may be suitable cloning or expression hosts for vectors comprising polynucleotides coding for one or more enzymes.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • Kluyveromyces hosts such as, e.g.,K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus(ATCC 16,045), K.
  • wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastors (EP 183,070); Candida; Trichodermareesia (EP 244,234); Neurosporacrassa; Schwanniomycessuch as Schwanniomycesoccidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • suitable host cells for expression may be derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculo viral strains and variants and corresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedesaegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori (silk moth) have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of AutographacalifornicaNPV and the Bm-5 strain of BombyxmoriNPV, and such viruses may be used as the virus herein according to the present disclosure, particularly for transfection of Spodopterafrugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, tobacco, lemna, and other plant cells can also be utilized as host cells.
  • Examples of useful mammalian host cells are Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells/- DHFR (CHO, Urlaubet al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al, J. Gen Virol. 36: 59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, (Biol. Reprod.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed or transfected with the above-described expression or cloning vectors for production of one or more enzymes as disclosed herein or with polynucleotides coding for one or more enzymes as disclosed herein and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Host cells containing desired nucleic acid sequences coding for the disclosed enzymes may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • Such polynucleotides may be modified ⁇ e.g., genetically engineered) to modulate ⁇ e.g., increase or decrease) the substrate specificity of an encoded enzyme, or the polynucleotides may be modified to change the substrate specificity of the encoded enzyme ⁇ e.g., a polynucleotide that codes for an enzyme with specificity for a substrate may be modified such that the enzyme has specificity for an alternative substrate).
  • Preferred microorganisms may comprise polynucleotides coding for one or more of the enzymes as set forth in Tables 1- 2 and Figure 1.
  • the microorganism may comprise a pyruvate formate lyase and pyruvate formate lyase activating enzyme as set forth in EC 2.3.1.54 or 1.97.1.4 including, for example, any one of SEQ ID NOS: 1-2 (see, Table 2).
  • the microorganism may comprise a lactate dehydrogenase as set forth in EC 1.1.1.28 including, for example, SEQ ID NO: 3 (see, Table 2).
  • the microorganism may comprise an propionate CoA-transferase as set forth in EC 2.8.3.1 including, for example, SEQ ID NO: 4 (see, Table 2).
  • the microorganism may comprise an acetyl coenzyme A synthetase as set forth in EC 6.2.1.1 including, for example, SEQ ID NO: 5 (see, Table 2).
  • the microorganism may comprise a lactoyl-CoA dehydratase as set forth in EC 4.2.1.54 including, for example, any one of SEQ ID NOS: 6-8 (see, Table 2).
  • the microorganism may comprise an acryloyl-CoA reductase as set forth in EC 1.3.1.95 including, for example, any one of SEQ ID NOS: 9-11 (see, Table 2).
  • the microorganism may comprise a thiolase as set forth in EC 2.3.1.9 including, for example, SEQ ID NO: 12 (see, Table 2).
  • the microorganism may comprise a 3-hydroxy-2-methylbutyryl-CoA dehydrogenase as set forth in EC 1.1.1.178 including, for example, SEQ ID NO: 13 (see, Table 2).
  • the microorganism may comprise an enoyl-CoA hydratase as set forth in EC 4.2.1.17 including, for example, SEQ ID NO: 14 (see, Table 2).
  • the microorganism may comprise a succinate semialdehyde dehydrogenase as set forth in EC 1.2.1.76 including, for example, SEQ ID NO: 15 (see, Table 2).
  • the microorganism may comprise an alcohol dehydrogenase as set forth in EC 1.1.1.1 or 1.1.1.72 including, for example, SEQ ID NO: 16 (see, Table 2).
  • the microorganism may comprise a HMG-CoA reductase as set forth in EC 1.1.1.34 including, for example, SEQ ID NO: 17 (see, Table 2).
  • the microorganism may comprise a linalool dehydratase/isomerase as set forth in EC 4.2.1.127 including, for example, SEQ ID NO: 18 (see, Table 2).
  • the microorganism may comprise a mevalonate kinase as set forth in EC 2.7.1.36 including, for example, SEQ ID NO: 19 (see, Table 2).
  • the microorganism may comprise a phosphomevalonate kinase as set forth in EC 2.7.4.2 including, for example, SEQ ID NO: 20 (see, Table 2).
  • the microorganism may comprise an isoprene synthase as set forth in EC 4.2.3.27 including, for example, SEQ ID NO: 21 (see, Table 2).
  • O 21 ispS GL 13539551 Q50L36 Populus alba
  • Isoprene and/or acetic acid may be produced by contacting any of the genetically modified microorganisms provided herein with a fermentable carbon source.
  • Such methods may preferably comprise contacting a fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to any of the intermediates provided in Figure 1 (tables 1) and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates provided in Figure (table 1) to isoprene and/or acetic acid in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to the one or more intermediates provided in Figure 1 (tables 1) and one or more polynucleotides coding for enzymes in a pathway that catalyze
  • oxidation-reduction (redox) reactions For example, during fermentation, glucose is oxidized in a series of enzymatic reactions into smaller molecules with the concomitant release of energy. The electrons released are transferred from one reaction to another through universal electron carriers, such Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NAD(P)), which act as cofactors for oxidoreductase enzymes.
  • NAD Nicotinamide Adenine Dinucleotide
  • NAD(P) Nicotinamide Adenine Dinucleotide Phosphate
  • glucose is oxidized by enzymes using the oxidized form of the cofactors (NAD(P)+ and/or NAD+) as cofactor thus generating reducing equivalents in the form of the reduced cofactor (NAD(P)H and NADH).
  • NAD(P)+ and/or NAD+ the cofactors
  • NAD(P)H and NADH the reduced cofactor
  • redox-balanced metabolism is required, i.e., the cofactors must be regenerated by the reduction of microbial cell metabolic compounds.
  • Microorganism-catalyzed fermentation for the production of natural products is a widely known application of biocatalysis.
  • Industrial microorganisms can affect multistep conversions of renewable feedstocks to high value chemical products in a single reactor.
  • Products of microorganism-catalyzed fermentation processes range from chemicals such as ethanol, lactic acid, amino acids and vitamins, to high value small molecule pharmaceuticals, protein pharmaceuticals, and industrial enzymes.
  • the biocatalysts are whole-cell microorganisms, including microorganisms that have been genetically modified to express heterologous genes.
  • Some key parameters for efficient microorganism-catalyzed fermentation processes include the ability to grow microorganisms to a greater cell density, increased yield of desired products, increased amount of volumetric productivity, removal of unwanted co- metabolites, improved utilization of inexpensive carbon and nitrogen sources, adaptation to varying fermenter conditions, increased production of a primary metabolite, increased production of a secondary metabolite, increased tolerance to acidic conditions, increased tolerance to basic conditions, increased tolerance to organic solvents, increased tolerance to high salt conditions and increased tolerance to high or low temperatures. Inefficiencies in any of these parameters can result in high manufacturing costs, inability to capture or maintain market share, and/or failure to bring fermented end-products to market.
  • compositions of the present disclosure can be adapted to conventional fermentation bioreactors ⁇ e.g., batch, fed-batch, cell recycle, and continuous fermentation).
  • a microorganism ⁇ e.g., a genetically modified microorganism as provided herein is cultivated in liquid fermentation media (i.e., a submerged culture) which leads to excretion of the fermented product(s) into the fermentation media.
  • the fermented end product(s) can be isolated from the fermentation media using any suitable method known in the art.
  • formation of the fermented product occurs during an initial, fast growth period of the microorganism. In one embodiment, formation of the fermented product occurs during a second period in which the culture is maintained in a slow-growing or non-growing state. In one embodiment, formation of the fermented product occurs during more than one growth period of the microorganism. In such embodiments, the amount of fermented product formed per unit of time is generally a function of the metabolic activity of the microorganism, the physiological culture conditions (e.g., pH, temperature, medium composition), and the amount of microorganisms present in the fermentation process.
  • the physiological culture conditions e.g., pH, temperature, medium composition
  • the fermentation product is recovered from the periplasm or culture medium as a secreted metabolite.
  • the fermentation product is extracted from the microorganism, for example when the microorganism lacks a secretory signal corresponding to the fermentation product.
  • the microorganisms are ruptured and the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions may then be separated if necessary.
  • the fermentation product of interest may then be purified from the remaining supernatant solution or suspension by, for example, distillation, fractionation, chromatography, precipitation, filtration, and the like.
  • the methods of the present disclosure are preferably preformed under anaerobic conditions. Both the degree of reduction of a product as well as the ATP requirement of its synthesis determines whether a production process is able to proceed aerobically or anaerobically. To produce isoprene and/or acetic acid via anaerobic microbial conversion, or at least by using a process with reduced oxygen consumption, redox imbalances should be avoided.
  • redox reactions including some of the conversions as set forth in Figure 1. Such redox reactions involve electron transfer mediated by the participation of redox cofactors such as NADH, NADPH and ferredoxin.
  • redox cofactors Since the amounts of redox cofactors in the cell are limited to permit the continuation of metabolic processes, the cofactors have to be regenerated. In order to avoid such redox imbalances, alternative ways of cofactor regeneration may be engineered, and in some cases additional sources of ATP generation may be provided. Alternatively, oxidation and reduction processes may be separated spatially in bioelectrochemical systems (Rabaey and. Rozendal, 2010, Nature reviews, Microbiology, vol 8: 706-716).
  • redox imbalances may be avoided by using substrates (e.g., fermentable carbon sources) that are more oxidized or more reduced, for example, if the utilization of a substrate results in a deficit or surplus of electrons, a requirement for oxygen can be circumvented by using substrates that are more reduced or oxidized, respectively.
  • substrates e.g., fermentable carbon sources
  • glycerol which is a major byproduct of biodiesel production is more reduced than sugars, and is therefore more suitable for the synthesis of compounds whose production from sugar results in cofactor oxidation, such as succinic acid.
  • co-substrates can be added that function as electron donors (Babel 2009, Eng.
  • Isoprene produced via any of the disclosed processes or methods may be converted to polyisoprene, lattices of polyisoprene, Styrene-Isoprene-Styrene (SIS) Block Copolymer, and styrene/isoprene/ butadiene rubber (SIBR).
  • SIS Styrene-Isoprene-Styrene
  • SIBR styrene/isoprene/ butadiene rubber
  • Example 1 Modification of microorganism for co-production of isoprene and/or acetic acid.
  • a microorganism such as a bacterium is genetically modified to co-produce isoprene and/or acetic acid from a fermentable carbon source including, for example, glucose.
  • a microorganism may be genetically engineered by any methods known in the art to comprise: i.) one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the fermentable carbon source to 2-methylbutenoyl-CoA and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-methylbutenoyl-CoA to isoprene.
  • the microorganism is modified to comprise one or more polynucleotides coding for enzymes that catalyze a conversion of the fermentable carbon source to acetic acid.
  • microorganism may be modified to exhibit increased activity in the enzymatic reactions (A) and (B) of Figure 1, i.e., through overexpression of E. coli pyruvate formate lyase (pflAB) and lactate dehydrogenase (IdhA).
  • the microorganism may be modified to comprise one or more acrylate pathway genes, which occur naturally in organisms like Clostridium propionicum, Megasphaera elsdenii and Prevotella ruminicola. These genes include pet (FIG.
  • a CoA synthetase with broad substrate specificity like the Saccharomyces cerevisiae AMP forming acetyl coenzyme A ligase (ACS1), might function appropriately or can be modified for a high specificity for lactate as preferred main substrate.
  • ACS1 Saccharomyces cerevisiae AMP forming acetyl coenzyme A ligase
  • acetyl-CoA Through overproduction of acetyl-CoA and use of a lactoyl-CoA synthetase or use of transferase with preference for acetyl-CoA over propionyl-CoA, the engineered organism will not accumulate propionate as end-product, as is seen in the natural acrylate pathway, but a combination of acetyl- CoA, propionyl-CoA and acetate. While acetate is one of the desired end products, acetyl-CoA and propionyl-CoA are further utilized through reactions from the naturally occurring 2- methylbutyrate pathway (FIG. 1FGH). This pathway occurs naturally in Ascaris lumbricoides and Ascaris suum.
  • FGH 2- methylbutyrate pathway
  • a thiolase found in these organisms (3-ketoacyl-CoA thiolase) can catalyze the conversion of propionyl-CoA and acetyl-CoA to 2-methylacetoacetyl-CoA.
  • a dehydrogenase capable of reducing 2-methylacetoacetyl-CoA to 3-hydroxy-2- methylbutanoyl-CoA (FIG. 1G), such as encoded by Pseudomonas putida fadB2x or a similar dehydratase gene, followed by dehydration (FIG. 1H) through a dehydratase, as encoded by ech from Pseudomonas putida, will yield the intermediate 2-methylbutenoyl-CoA.
  • the intermediate 2-methylbutenoyl-CoA is the entry point into non-natural, isoprene specific biosynthetic pathways.
  • the activated acid group of the intermediate is reduced by successive reduction (FIG. 1IJ) with an acid-CoA reductase, for instance a succinyl-CoA reductase (sucD, Clostridium kluyveri) evolved towards specificity for 2-methyl-2-butenoyl- CoA, and an appropriate primary alcohol dehydrogenase, for instance one with broad substrate acceptance (i.e., E. coliyqhD).
  • an acid-CoA reductase for instance a succinyl-CoA reductase (sucD, Clostridium kluyveri) evolved towards specificity for 2-methyl-2-butenoyl- CoA, and an appropriate primary alcohol dehydrogenase, for instance one with broad substrate acceptance (i.e., E. coliyqhD).
  • a bifunctional reductase like the HMG1 encoded 3- hydroxy-3-methylglutaryl-CoA reductase from S. cerevisiae may be found or evolved to perform the reaction depicted in figure IK.
  • the resulting alcohol 2-methyl-l-but-2-enol can, in one embodiment of the invention, be dehydrated through linalool dehydratase (Idi, Castellaniella defragrans) or a natural or evolved enzyme with equivalent functionality on the desired substrate (FIG. 1L).
  • 2-methyl-l-but-2-enol is successively phosphorylated to the mono- and diphosphate compound (FIG. IMN) by a natural or evolved alcohol kinase and alcohol monophosphate kinase.
  • a natural or evolved alcohol kinase and alcohol monophosphate kinase are mevalonate kinase and phosphomevalonate kinase from S. cerevisiae (ergl2 and erg8, respectively).
  • Final isoprene synthesis from the diphosphate compound is achieved through dehydration by an isoprene synthase (i.e., ispS, Populus alba).
  • a microorganism that lacks one or more enzymes e.g., one or more functional enzymes that are catalytically active
  • a microorganism that lacks one or more enzymes may be genetically modified to comprise one or more polynucleotides coding for enzymes (e.g., functional enzymes including, for example any enzyme disclosed herein) in a pathway that the microorganism lacks to catalyze a conversion of the fermentable carbon source to isoprene and/or acetic acid.
  • Example 2 Fermentation of glucose by genetically modified microorganism to produce isoprene and/or acetic acid.
  • a genetically modified microorganism, as produced in Example 1 above, is used to ferment a carbon source, to produce isoprene and/or acetic acid.
  • a previously-sterilized culture medium comprising a fermentable carbon source (e.g., 9 g/L glucose, 1 g/L KH 2 P0 4 , 2 g/L ( ⁇ 4 ) 2 ⁇ 0 4 , 5 mg/L FeS0 4 '7H 2 0, 10 mg/L MgS0 4 » 7H 2 0, 2.5 mg/L MnS0 4 » H 2 0, 10 mg/L CaCl 2 » 6H 2 0, 10 mg/L CoCl 2 » 6H 2 0, and 10 g/L yeast extract) is charged in a bioreactor.
  • a fermentable carbon source e.g., 9 g/L glucose, 1 g/L KH 2 P0 4 , 2 g/L ( ⁇ 4 ) 2 ⁇ 0 4 , 5 mg/L FeS0 4 '7H 2 0, 10 mg/L MgS0 4 » 7H 2 0, 2.5 mg/L MnS0 4 » H 2 0, 10 mg/L CaCl 2 » 6H 2 0, 10 mg/L
  • anaerobic conditions are maintained by, for example, sparging nitrogen through the culture medium.
  • a suitable temperature for fermentation e.g., about 30 °C
  • a near physiological pH e.g., about 6.5
  • Fermentation is allowed to run to completion.
  • isoprene and/or acetic acid may be removed (e.g., separated from the fermentable carbon source) from the bioreactor.

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Abstract

L'invention se rapporte généralement à des microorganismes (p.ex., des microorganismes non naturellement présents) qui comprennent un ou plusieurs polynucléotides codant pour des enzymes dans une voie, qui catalysent la conversion d'une source de carbone en isoprène et/ou en acide acétique. L'invention concerne également l'utilisation desdits microorganismes pour la production d'isoprène et/ou d'acide acétique.
PCT/US2014/045097 2013-07-01 2014-07-01 Microorganismes modifiés et procédés d'utilisation de ces derniers pour la coproduction anaérobie d'isoprène et d'acide acétique WO2015002977A1 (fr)

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US10774347B2 (en) 2016-03-09 2020-09-15 Braskem S.A. Microorganisms and methods for the co-production of ethylene glycol and three carbon compounds
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