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WO2014066892A1 - Voies métaboliques de novo pour la biosynthèse d'isoprène - Google Patents

Voies métaboliques de novo pour la biosynthèse d'isoprène Download PDF

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WO2014066892A1
WO2014066892A1 PCT/US2013/067079 US2013067079W WO2014066892A1 WO 2014066892 A1 WO2014066892 A1 WO 2014066892A1 US 2013067079 W US2013067079 W US 2013067079W WO 2014066892 A1 WO2014066892 A1 WO 2014066892A1
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microbial organism
naturally occurring
isoprene
occurring microbial
hydrolyase
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Pedro S. COELHO
Mary F. FARROW
Matthew A. SMITH
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01084—Dimethylmalate dehydrogenase (1.1.1.84)
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    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01076—Succinate-semialdehyde dehydrogenase (acetylating) (1.2.1.76)
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    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01033—Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase
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    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01009—Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase
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    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01005—Succinate-CoA ligase (ADP-forming) (6.2.1.5)

Definitions

  • the present invention relates generally to biosynthetic processes, and more specifically to the design of non-naturally occurring pathways for producing isoprene and the creation of organisms having such biosynthetic capability.
  • Isoprene is naturally produced by bacteria, animals, humans, and plants; these organisms collectively release an estimated 600 million tons of isoprene into the atmosphere each year.
  • Isoprene (2-methyl- 1,3 -butadiene) is an important commodity chemical used in a wide range of industrial products, such as synthetic rubber for tires and coatings, adhesives, and specialty elastomers.
  • Isoprene is also a versatile building block for the production of hydrocarbon fuels, including diesel, gasoline, and aviation fuels. Approximately 1 million tons of isoprene are made from petrochemical feedstocks every year. 1 Increasing global demand for isoprene and environmental concerns about greenhouse gas emissions have spurred interest in the
  • Isoprene consumers are also interested in isoprene bioproduction as a strategy to mitigate their vulnerability to uncertain supply and volatile prices.
  • Isoprene synthases catalyze the elimination of pyrophosphate from dimethylallyl pyrophosphate (DMAPP) to yield isoprene.
  • DMAPP dimethylallyl pyrophosphate
  • isoprene 's pervasiveness in the environment, efforts to identify prokaryotic isoprene synthases have met with limited success. 2
  • sequence data for these enzymes exist for two plant families: kudzu (the Asian vine, Pueraria montana) and poplar (Populus).
  • the catalytic efficiencies of the characterized isoprene synthases are sub-optimal for industrial applications (K M ⁇ 1-10 mM and k cat ⁇ 1 s "1 ).
  • IPP isopentenyl pyrophosphate
  • DMAPP DMAPP
  • MVA mevalonate
  • A-CoA ubiquitous intermediate acetyl-coenzyme A
  • prokaryotes use the l-deoxy-D-xylulose-5- phosphate/2-C-methyl-D-erythritol-4-phosphate (DXP/MEP) pathway to produce IPP and DMAPP from pyruvate and glyceraldehyde-3-phosphate derived from glycolysis.
  • Plants use both the MVA and the DXP/MEP pathways for achieving terpenoid biosynthesis. As shown in Figure 2, the MVA and DXP/MEP pathways have different yields and co-factor requirements.
  • the bottom operon was integrated into the chromosome under the control of a constitutive promoter.
  • the top operon, the isoprene synthase and an additional copy of an archaeal mevalonate kinase were expressed on two different plasmids driven from the inducible Vtrc promoter.
  • the reported parameters for this process consist of an isoprene yield of 0.1 1 g g "1 , volumetric productivity of 2.0 g L "1 h "1 and a titer of 60 g L -1.1
  • isoprene (b.p. 34 °C) allows gas phase recovery of the product, which simplifies purification and eliminates potential feedback inhibition by virtue of product accumulation. All of these factors drive equilibrium in favor of isoprene synthesis, and enable in situ product removal.
  • an anaerobic pathway for isoprene whilst unprecedented, would enable lower fermentation costs compared to the MVA and DXP/MEP pathways that have thus far been pursued in industry. Since an anaerobic pathway for isoprene at 0.324 g g " yield has not been reported in nature, we sought to formulate such a pathway based on existing reaction classes rather than limiting our search to enzymes working on their native substrates.
  • DHIV common metabolite 2,3-dihydroxyisovalerate
  • Fig 5-1 1 we propose 4 major isoprene biosynthetic pathways (Fig 5-1 1), comprising a series of reduction and dehydration steps to produce isoprene from DHIV.
  • the proposed pathways are evaluated with respect to yield, redox balance, ATP balance, number of steps from glucose and number of unknown enzymes. This evaluation is summarized in Table 1.
  • Pathway 1 requires the fewest new enzymes to be engineered, and is the most
  • FIG. 1 Mevalonate (MVA) and l-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4- phosphate (DXP/MEP) pathways for isoprenoid biosynthesis.
  • Figure 2 Theoretical yield calculations for isoprene biosynthesis via the MVA and the
  • Figure 3 Calculations of the maximum theoretical yield for isoprene fermentation from glucose. (1) Maximal yield if electrons are supplied exogenously (i.e. electrochemically or
  • Figure 6 Transformations of linaool dehydratase-isomerase.
  • Figure 7 Biosynthesis of isoprene from DHIV via (3-methyl-2-oxobutanoyl)-CoA (pathway 2).
  • Figure 8 Alternative syntheses of isoprene from KIV (pathway 2).
  • Figure 9 Biosynthesis of isoprene from DHIV via (2,3-dihydroxy-3-methylbutanoyl)-CoA.
  • Figure 10 Biosynthesis of isoprene from DHIV via 2-hydroxy-3-methyl-3-butenoic acid.
  • Figure 11 Alternative syntheses of isoprene from DHIV. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is directed to the design and production of cells and organisms that have the ability to produce isoprene.
  • the invention in particular, relates to the design of a microbial organism capable of producing isoprene by introducing one or more exogenous nucleic acids encoding an isoprene pathway enzyme.
  • microbial As used herein, the terms "microbial,” “microbial organism” or “microorganism” are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.
  • 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.
  • exemplary metabolic polypeptides include enzymes or proteins within an isoprene biosynthetic pathway.
  • a metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic
  • CoA or "coenzyme A” is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system.
  • substantially anaerobic when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media.
  • the term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.
  • Exogenous as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism.
  • the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism.
  • the term refers to an activity that is introduced into the host reference organism.
  • endogenous refers to a referenced molecule or activity that is present in the host.
  • the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism.
  • heterologous refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
  • the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above.
  • the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.
  • Pathway 1 converts DHIV to isoprene via the sequence of steps described in Figure 5.
  • DHIV is first dehydrated to 2-ketoisovalerate (KIV).
  • KIV undergoes an NAD(P)H- dependent carboxylation to (i?)-3,3-dimethylmalate, which then undergoes ATP-dependent decarboxylation and dehydration to 3-methylbut-2-enoic acid.
  • 3-methylbut-2-enoic acid is subsequently transformed to (3-methylbut-2-enoyl)-CoA in an ATP-dependent reaction.
  • the theoretical yield of isoprene from glucose in this embodiment is 0.324 g g "1 .
  • the first reaction in this sequence (step 1.1 in Fig 5) is catalyzed by the enzyme dihydroxyacid dehydratase (EC 4.2.1.9), which acts on DHIV as its natural substrate, and is a key enzyme in the biosynthesis of branched amino acids (L-valine, L-isoleucine, L-leucine).
  • This enzyme is endogenous to industrially relevant hosts such as E. coli (gene ilvD), S. cerevisiae and
  • Step 1.2 is the reverse of the reaction catalyzed by dimethylmalate dehydrogenase (DMMD; EC 1.1.1.84) on its native substrate, (i?)-3,3-dimethylmalate.
  • DMMD dimethylmalate dehydrogenase
  • DMMD is a 4 subunit, NADH-dependent enzyme, whose activity on (i?)-3,3-dimethylmalate, n- propyl malate, and malate has been identified in purified protein from Pseudomonas fluorescens and other Pseudomonas species. 10 Since the reverse reaction shown in step 1.2 is
  • Candidate enzymes will be screened for activity on (i?)-3,3-dimethylmalate and KIV production, and engineered by directed evolution for higher affinity for KIV.
  • Step 1.3 describes the decarboxylation and dehydration of a 3-hydroxy acid.
  • Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) catalyzes the ATP-dependent decarboxylation of mevalonate diphosphate to IPP, as part of the MVA pathway.
  • This enzyme (gene MDD, MVD1) is endogenous to many organisms, including the industrially relevant host S. cerevisiae. MDD from S. cerevisiae has been reported to accept at least one nonnative substrate, 3-hydroxy- 3-methylbutyrate. 11 In order to find a decarboxylase that is active on (i?)-3,3-dimethylmalate, i) S.
  • steps 1.2 and 1.3 convert KlV to 3-methyl-2- butenoic acid, with the overall effect of a reduction and a dehydration.
  • Step 1.4 utilizes a CoA synthetase to make (3-methylbut-2-enoyl)-CoA, which can then be reduced to the aldehyde by a CoA-dependent semialdehyde dehydrogenase in step 1.5.
  • ADP- forming succinate-CoA ligase (EC 6.2.1.5) is a candidate enzyme for catalyzing step 1.4, or for serving as a starting point for directed evolution experiments to create a (3-methylbut-2-enoyl)- CoA ligase. This enzyme is endogenous to many organisms, including E.
  • Pseudomonas fluorescens Psedomonas stutzeri, Rattus norvegicus, Rhodobacter sphaeroides, Salmonella enterica subsp. Arizonae, Serratia marcescens, Spinacia oleracea, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sus scrofa, Thermococcus kodakarensis, Theroplasma acidophilum, Thermus aquaticus, thermos thermophilus, Toxoplasma gondii, and Trichomonas vaginalis.
  • Carboxylate reductase is a 128 kDa monomeric protein comprising three domains: adenylating, phosphopantetheine attachment and reductase domains. Bound carboxylic acids are first converted to acyl adenylate intermediates in the adenylating domain and are subsequently reduced to aldehydes by NADPH in the reductase domain.
  • Carboxylate reductase exhibits broad substrate acceptance, including various aromatic and aliphatic carboxylic acids and 2-oxoacids (e.g. benzoate, vanilic acid, ferulic acid, a-ketoglutarate, aconitate, citrate, maleate). 13 At least two strategies can be implemented in order to find a carboxylate reductase that is active on 3- methyl-2-butenoic acid: i) engineer the Nocardia enzyme towards 3-methyl-2-butenoic acid or ii) screen several homologous enzymes, identified through a protein database search such as BLAST for activity on 3-methyl-2-butenoic acid.
  • a protein database search such as BLAST for activity on 3-methyl-2-butenoic acid.
  • Step 1.6 aldehyde reduction to an alcohol, can be achieved by alcohol dehydrogenases (EC 1.1.1.1 and 1.1.1.2). These enzymes typically exhibit broad substrate specificity. 14 Furthermore, E. coli has a large number of genes annotated as alcohol dehydrogenases: E. coli K-12 has about 100 dehydrogenase genes (10% of all enzymes). 15 Accordingly, others have reported that native E. coli enzymes are often capable of achieving the desired reduction. For instance, Lamm et al. used carboxylate reductase to reduce vanillic acid to vanillin and found that alcohol
  • cerevisiae has demonstrated activity on a number of straight chain and branched aldehydes
  • step 1.6 is not likely to be a significant bottleneck in the overall effort to engineer the proposed artificial isoprene pathway.
  • step 1.6 3-methylbut-2-en-l-ol, the product of step 1.6, is at the same redox level as isoprene, such that a single dehydration (step 1.7) can convert this intermediate into the final product.
  • meningoseptica sequence that had been traced by Bevers et al.
  • dehydration activity on several isoprene precursors 22
  • Information about the types of enzyme or amounts of isoprene were not disclosed.
  • a drawback of this approach is that the oleate hydratase required a
  • cofactor e.g. octanoic acid
  • octanoic acid for steric and/or electronic complementation in its catalytic site since the alcohol used as the substrate was significantly shorter than the fatty acid which functions as the natural substrate (e.g. oleate).
  • oleate hydratase towards 3-methylbut-2-en-l-ol via a substrate walk approach, where the enzyme is progressively evolved to accept shorter and shorter alcohols, will be able to eliminate the need for the "cofactor” whilst maintain (maintaining? retain or retaining?) wild-type-like catalytic parameters.
  • Prenyl isoflavanoid hydratases can be used instead of oleate hydratase to catalyze step 1.7. These fungal enzymes are involved in secondary metabolism (defense mechanism) and are thus thought to be more accommodating in terms of substrate acceptance. 19 Marliere et al. have described using kievitone hydratase (EC 4.2.1.95) and phaseollidin hydratase (EC 4.2.1.97) for making isoprene, but again this approach has the added complication of requiring added "cofactors" to fill the active site. 23
  • Linalool dehydratase-isomerase (EC 4.2.1.127) to catalyze step 1.7.
  • Linalool dehydratase-isomerase from Castellaniella defragrans is a 40 kDa enzyme with no cofactors that catalyzes two reactions: i) the isomerization of geraniol to linalool and ii) the dehydration of linalool to myrcene ( Figure 6).
  • the enzyme has been overexpressed in E. coli and is well- characterized biochemically.
  • the target product isoprene has the same geometry as myrcene but is 5 carbons shorter (i.e. it is a hemiterpene instead of a monoterpene).
  • Pathway 2 converts DHIV to isoprene via the sequence of steps described in Figures 7 and 8.
  • DHIV is dehydrated to KIV, which is then converted to (3-methyl-2- oxobutanoyl)-CoA in an ATP-dependent reaction.
  • (3-methyl-2-oxobutanoyl)-CoA undergoes three sequential NAD(P)H-dependent reductions to 3-methyl-2-oxobutanal, 1 -hydroxy- 3 -methyl- butan-2-one and 3-methylbutane-l ,2-diol.
  • Two subsequent dehydrations convert 3- methylbutane-l ,2-diol to 3-methylbut-2-en-l-ol and finally to isoprene. From DHIV, the net equation to isoprene in this embodiment is:
  • the theoretical yield of isoprene from glucose in this embodiment is 0.324 g g 1 .
  • the overall transformation from glucose to isoprene in Pathway 2 yields 1 equivalent of ATP per isoprene formed, which will allow pathway optimization by metabolic evolution.
  • the first reaction in this sequence (step 2.1 in Fig 7) is catalyzed by the enzyme dihydroxyacid dehydratase (EC 4.2.1.9), which acts on DHIV as its natural substrate.
  • Dihydroxyacid dehydratase is endogenous to industrially relevant hosts and is suitable to operate under the anaerobic conditions described in the envisioned process.
  • Steps 2.2 and 2.3 represent the NAD(P)H and ATP-dependent net reduction of a 2-oxoacid to a 2-oxoaldehyde.
  • ADP-forming succinate-CoA ligase (EC 6.2.1.5) and acylating succinate semialdehyde dehydrogenase (EC 1.2.1.76) are candidate enzymes for catalyzing these steps, or for serving as a starting point for directed evolution experiments.
  • the CoA-dependent succinate semialdehyde dehydrogenase from Clostridium kluyveri or Metallosphaera sedula can potentially catalyze the reduction of (3-methylbut-2-oxobutanoyl)-CoA to 3-methyl-2- oxobutanal.
  • Clostridium kluyveri acylating succinate semialdehyde dehydrogenase may be sensitive to oxygen, this is compatible with the anaerobic process described herein.
  • the AMP-forming enzyme carboxylate reductase from Nocardia sp. (EC 1.2.99.6) or its homologs may be engineered for activity on KIV to produce 3-methyl-2-oxobutanal in one step. This route is not ideal for the overall biotransformation of glucose to isoprene, however, because the ATP hydrolysis required for AMP phosphorylation lowers the net ATP yield to 0.
  • Steps 2.4 and 2.5, aldehyde and ketone reductions to alcohols can be achieved by alcohol dehydrogenases (EC 1.1.1.1 and 1.1.1.2), or 3-methylbutanal reductase (EC 1.1.1.265).
  • step 2.5 3-methylbutane-l ,2-diol
  • Hydro lyases including oleate hydratase (EC 4.2.1.53), kievitone hydratase (EC 4.2.1.95), phaseollidin hydratase (EC 4.2.1.97), and their homologs may be engineered towards activity on 3-methylbutane-l,2-diol and 3-methylbut-2-en-l-ol.
  • Linalool dehydratase-isomerase (EC 4.2.1.127) may be ideal to catalyze step 2.7, given its biochemical characterization in E. coli and lack of cofactor requirement. We thus propose to engineer linalool dehydratase-isomerase to accept 3-methylbut-2-en-l-ol as a substrate.
  • Figure 8 shows several alternative versions for pathway 2 that convert KIV to isoprene via different permutations of reductions and dehydrations. Proposed enzymes for each step are summarized in Table 2. All versions of pathway 2 yield 1 equivalent of ATP per isoprene formed. Table 2. Enzymes for alternative syntheses of isoprene from KIV.
  • Pathway 3 converts DHIV to isoprene via the sequence of steps described in Figure 9.
  • DHIV is first converted to (2,3-dihydroxy-3-methylbutanoyl)-CoA in an ATP- dependent reaction.
  • (2,3-dihydroxy-3-methylbutanoyl)-CoA undergoes an NAD(P)H-dependent reduction to 2,3-dihydroxy-3-methylbutanal, which is dehydrated to 3-methyl-2-oxobutanal.
  • 3- methyl-2-oxobutanal undergoes two sequential NAD(P)H-dependent reductions to 2-hydroxy-3- methylbutanal and 3-methylbutane-l ,2-diol.
  • Two subsequent dehydrations convert 3- methylbutane-l ,2-diol to 3-methylbut-2-en-l-ol and finally to isoprene. From DHIV, the net equation to isoprene in this embodiment is:
  • Steps 3.1 and 3.2 are catalyzed by the enzymes ADP-forming succinate-CoA ligase (EC 6.2.1.5), acylating succinate semialdehyde dehydrogenase (EC 1.2.1.76), or their homologs, engineered for activity on DHIV and (2,3-dihydroxy-3-methylbutanoyl)-CoA, respectively.
  • 2,3-dihydroxy- 3-methylbutanal may be formed directly from DHIV by the AMP-forming enzyme carboxylate reductase from Nocardia sp. (EC 1.2.99.6) or its homologs. This route is not ideal for the overall biotransformation of glucose to isoprene, however, because the ATP hydrolysis required for AMP phosphorylation lowers the net ATP yield to 0.
  • Step 3.3 is the dehydration of an a-hydroxy aldehyde to a dialdehyde, which may be catalyzed by the enzymes dihydroxyacid dehydratase (EC 4.2.1.9) or dioldehydratase (EC 4.2.1.28).
  • dihydroxyacid dehydratase EC 4.2.1.9
  • dioldehydratase EC 4.2.1.28
  • 2,3- dihydroxy-3-methylbutanal, the substrate for step 3.3 is very similar to DHIV, the natural dihydroxyacid dehydratase substrate. Therefore, it is likely that this enzyme can be engineered for activity on 2,3-dihydroxy-3-methylbutanal. This is particularly advantageous, as
  • dihydroxyacid dehydratase is present in industrially relevant hosts such as E. coli and S.
  • Dioldehydratases are coenzyme B12-dependent, and have been identified in many organisms, including Acetobacterium sp., Citrobacter freundii, Clostridium glycolicum,
  • Flavobacterium sp. Klebsiella pneumonia, Lactobacillus brevis, Lactobacillus buchneri,
  • Dioldehydratases have been expressed in E. coli, and have been reported to accept a range of substrates, including glycerol, 1 ,2 propanediol, 1,2-butanediol, and 2,3-butanediol. 24 Dioldehydratase has been reported to be sensitive to oxygen. Since the proposed pathway is designed to operate under anaerobic conditions, this enzyme should be compatible with the envisioned process.
  • Steps 3.4 and 3.5, aldehyde and ketone reductions to alcohols can be achieved by alcohol dehydrogenases (EC 1.1.1.1 and 1.1.1.2) or 3-methylbutanal reductase (EC 1.1.1.265).
  • alcohol dehydrogenases EC 1.1.1.1 and 1.1.1.2
  • 3-methylbutanal reductase EC 1.1.1.265
  • Hydrolyases including oleate hydratase (EC 4.2.1.53), kievitone hydratase (EC 4.2.1.95), phaseollidin hydratase (EC 4.2.1.97), and linalool dehydratase-isomerase (EC 4.2.1.127) may be used to catalyze the sequential dehydration reactions of steps 3.6 and 3.7. Pathway 4 (via 2-hydroxy-3-methyl-3-butenoic acid)
  • Pathway 4 converts DHIV to isoprene via the sequence of steps described in Figure 10.
  • DHIV is first dehydrated to 2-hydroxy-3-methyl-3-butenoic acid (step 4.1), which is then converted to (2-hydroxy-3-methylbut-3-enoyl)-CoA in an ATP and CoA-dependent reaction (step 4.2).
  • (2-hydroxy-3-methylbut-3-enoyl)-CoA undergoes two sequential NAD(P)H- dependent reductions to 2-hydroxy-3-methyl-3-butenal (step 4.3) and 3-methyl-3-buten-l ,2-diol (step 4.4).
  • 3-methyl-3-buten-l ,2-diol is dehydrated to 3-methyl-3-butenal (step 4.5), which is then reduced in an NAD(P)H-dependent reaction to 3-methylbut-3-en-l-ol (step 4.6).
  • 3- methylbut-3-en-l-ol is dehydrated to produce isoprene (step 4.7).
  • the enzymes used for transformation of identified substrates to products include: 4.1) hydrolyases, including oleate hydratase (EC 4.2.1.53), kievitone hydratase (EC 4.2.1.95), phaseollidin hydratase (EC 4.2.1.97), or linalool dehydratase-isomerase (EC 4.2.1.127); 4.2) ADP-forming succinate-CoA ligase (EC 6.2.1.5); 4.3) acylating succinate semialdehyde dehydrogenase (EC 1.2.1.76); 4.4) alcohol dehydrogenase (EC 1.1.1.1 and 1.1.1.2) or 3-methylbutanal reductase (1.1.1.265); 4.5) dihydroxyacid dehydratase (4.2.1.9) or dioldehydratase (EC 4.2.1.28); 4.6) alcohol
  • dehydrogenase EC 1.1.1.1 and 1.1.1.2
  • 3-methylbutanal reductase 1.1.1.265
  • hydrolyases including oleate hydratase (EC 4.21.53), kievitone hydratase (EC 4.2.1.95), phaseollidin hydratase (EC 4.2.1.97), or linalool dehydratase-isomerase (EC 4.2.1.127).
  • DHIV the net equation to isoprene in this embodiment is: (6)
  • the theoretical yield of isoprene from glucose in this embodiment is 0.324 g g "1 .
  • the overall transformation from glucose to isoprene in Pathway 4 yields 1 equivalent of ATP per isoprene formed, which will allow pathway optimization by metabolic evolution.
  • 9 Figure 11 shows several alternative versions for pathway 4 that convert DHIV to isoprene via different permutations of reductions and dehydrations. Proposed enzymes for each step are summarized in Table 3. All versions of pathway 4 yield 1 equivalent of ATP per isoprene formed, which will allow pathway optimization by metabolic evolution. 9 Table 3. Enzymes for alternative syntheses of isoprene from DHIV.

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Abstract

L'invention concerne des voies métaboliques non naturelles pour la fermentation anaérobie d'isoprène à partir de glucose à un rendement théorique de 0,324 g g-1. L'invention concerne également des procédés de clonage de micro-organismes avec lesdites voies pour produire de l'isoprène et des dérivés de celui-ci.
PCT/US2013/067079 2012-10-26 2013-10-28 Voies métaboliques de novo pour la biosynthèse d'isoprène Ceased WO2014066892A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10662415B2 (en) 2017-12-07 2020-05-26 Zymergen Inc. Engineered biosynthetic pathways for production of (6E)-8-hydroxygeraniol by fermentation
US10696991B2 (en) 2017-12-21 2020-06-30 Zymergen Inc. Nepetalactol oxidoreductases, nepetalactol synthases, and microbes capable of producing nepetalactone

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* Cited by examiner, † Cited by third party
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WO2000078935A1 (fr) * 1999-06-22 2000-12-28 Smithkline Beecham Corporation Genes de voie du mevalonate
US20100003716A1 (en) * 2008-04-23 2010-01-07 Cervin Marguerite A Isoprene synthase variants for improved microbial production of isoprene
US20100086978A1 (en) * 2008-09-15 2010-04-08 Beck Zachary Q Increased isoprene production using the archaeal lower mevalonate pathway
US20100261942A1 (en) * 2009-03-03 2010-10-14 Amyris Biotechnologies, Inc. Microbial derived isoprene and methods for making the same
US20110014672A1 (en) * 2009-06-17 2011-01-20 Chotani Gopal K Isoprene production using the dxp and mva pathway
US20120021478A1 (en) * 2010-07-26 2012-01-26 Osterhout Robin E Microorganisms and methods for the biosynthesis of aromatics, 2,4-pentadienoate and 1,3-butadiene

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078935A1 (fr) * 1999-06-22 2000-12-28 Smithkline Beecham Corporation Genes de voie du mevalonate
US20100003716A1 (en) * 2008-04-23 2010-01-07 Cervin Marguerite A Isoprene synthase variants for improved microbial production of isoprene
US20100086978A1 (en) * 2008-09-15 2010-04-08 Beck Zachary Q Increased isoprene production using the archaeal lower mevalonate pathway
US20100261942A1 (en) * 2009-03-03 2010-10-14 Amyris Biotechnologies, Inc. Microbial derived isoprene and methods for making the same
US20110014672A1 (en) * 2009-06-17 2011-01-20 Chotani Gopal K Isoprene production using the dxp and mva pathway
US20120021478A1 (en) * 2010-07-26 2012-01-26 Osterhout Robin E Microorganisms and methods for the biosynthesis of aromatics, 2,4-pentadienoate and 1,3-butadiene

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10662415B2 (en) 2017-12-07 2020-05-26 Zymergen Inc. Engineered biosynthetic pathways for production of (6E)-8-hydroxygeraniol by fermentation
US10696991B2 (en) 2017-12-21 2020-06-30 Zymergen Inc. Nepetalactol oxidoreductases, nepetalactol synthases, and microbes capable of producing nepetalactone
US11193150B2 (en) 2017-12-21 2021-12-07 Zymergen Inc. Nepetalactol oxidoreductases, nepetalactol synthases, and microbes capable of producing nepetalactone

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