WO2022261288A2 - Methods and compositions - Google Patents

Methods and compositions Download PDF

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Publication number
WO2022261288A2
WO2022261288A2 PCT/US2022/032784 US2022032784W WO2022261288A2 WO 2022261288 A2 WO2022261288 A2 WO 2022261288A2 US 2022032784 W US2022032784 W US 2022032784W WO 2022261288 A2 WO2022261288 A2 WO 2022261288A2
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Prior art keywords
optionally
tce
producing
seq
vcm
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PCT/US2022/032784
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French (fr)
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WO2022261288A3 (en
Inventor
Tahereh Karimi
Truong Huu NGUYEN
Miguel Eugenio CUEVA
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Cemvita Factory, Inc.
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Publication of WO2022261288A2 publication Critical patent/WO2022261288A2/en
Publication of WO2022261288A3 publication Critical patent/WO2022261288A3/en

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    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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    • C12Y114/12—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of two atoms of oxygen into one donor (1.14.12)
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    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
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    • C12Y121/00—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21)
    • C12Y121/99—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21) with other acceptors (1.21.99)
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    • C12Y204/00—Glycosyltransferases (2.4)
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase

Definitions

  • Embodiments of vinyl chloride monomer (YCM) and trichloroethylene (TCE) producing microorganisms having an improved VCM or TCE production ability are disclosed herein, as well as methods for the production of VCM and TCE producing microorganisms.
  • Biomanufacturing systems for producing VCM are disclosed herein, as well as methods of forming VCM from TCE, and methods of forming TCE.
  • a benefit to the various embodiments herein can be providing capabilities for the environmentally sustainable production of VCM and TCE on an industrial scale.
  • a benefit to the VCM and TCE producing microorganisms, and embodiments of biomanufacturing systems herein can include capabilities for the production of commercially useful quantities of TCE and VCM from microbial cultures.
  • Additional benefits of the microorganisms, systems and methods herein can include the use of carbon dioxide to produce bio-TCE and bio-VCM useful as a feedstock for the production of plastics, and for use in other applications; and reduction of excess carbon dioxide from the environment,
  • TCE trichloroethylene
  • VCM vinyl chloride monomer
  • Embodiments of a vinyl chloride monomer (VCM) producing recombinant microorganism having a VCM improved production ability are disclosed herein.
  • the VCM producing recombinant microorganism expresses at least one VCM producing enzyme by expressing i) at least one non-native VCM producing enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM producing enzyme nucleotide sequence, wherein an amount of the VCM producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native VCM producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native VCM producing enzyme, and wherein the VCM producing recombinant microorganism is capable of utilizing a chlorinated hydrocarbon source to produce VCM.
  • the at least one VCM producing enzyme includes TCE reductive dehalogenase (TceA), TceA anchor protein (TceB), tetrachloroethylene reductive dehalogenase (pceA), vinyl chloride reductase (vcrA), 1 ,2-dich!oropropane-to- propene reductive dehalogenase (dcpA), 1 ,2-trans-dishloroethene reductive dehalogenase catalytic A (TdrA), or a combination thereof.
  • TceA TCE reductive dehalogenase
  • TceB TceA anchor protein
  • pceA tetrachloroethylene reductive dehalogenase
  • vcrA vinyl chloride reductase
  • dcpA 1 ,2-dich!oropropane-to- propene reductive dehalogenase
  • TdrA 1,2-trans-dishlor
  • the VCM producing recombinant microorganism expresses TceA having an amino acid sequence at least 95% identical to SEQ ID NO: 1 by expressing a native or non-native TceA nucleotide sequence at least 95% identical to SEQ ID NO: 2; expresses TceB having an amino acid sequence at least 95% identical to SEQ ID NO: 3 by expressing a native or non-native TceB nucleotide sequence at least 95% Identical to SEQ ID NO: 4; expresses pceA having an amino acid sequence at least 95% identical to SEQ ID NO: 5 by expressing a native or non-native pceA nucleotide sequence at least 95% identical to SEQ ID NO: 6; expresses vcrA having an amino acid sequence at least 95% identical to SEQ ID NO: 7 by expressing a native or nonnative vcrA nucleotide sequence at least 95% identical to SEQ ID NO: 8; expresses dc
  • the chlorinated hydrocarbon source may be a chlorinated alkane or a chlorinated alkene.
  • the chlorinated hydrocarbon source may be a Cos chlorinated hydrocarbon, a Cw chlorinated hydrocarbon, a Ci4 chlorinated hydrocarbon or a C1-2 chlorinated hydrocarbon.
  • chlorinated hydrocarbons that may be employed in the present invention include trichloroethylene (TCE), dichlcroethylene (DCE) or chloroform.
  • the V ’ CM producing recombinant microorganism includes microorganisms of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehalococcoid.es strain PL2, Dehalococeoides mccarty e.g. strains KS, RC, IN .4, MB. 11 a or GY50. Dehalococcoides ethenogenes e.g.
  • strain 195 Dehalococcoides strain BA VI, Dehalococcoides strain VS, Dehalococcoides strain CBDB1, Dehalococcoides strain GT) Escherichia (optionally Escherichia coil ), Synechococcus (optionally Synechococcus elongatus), Suljurospirillum (optionally Sulfitrospirillum multivoram or Sulfurospirillum barnesii), Dehalobacter (optionally Dehalobacter restrictus), Desulfuromonas (optionally strain BB1 or Desulfuromonas ckloroeihenica), Desulfitobacterium (optionally Desulfitobacterium hafnien.se ), Geobacter (optionally Geobacter bemidjiensis, Geobacter lovieyi, Geobacter psychrophilus, Geobacter sp.
  • FRC-32 Geobacter sp. M2 !. Geobacter sulfurreducens, or Geobacter uraniireducens), Pelobacter (optionally Pelobacter propionicus) Dehalogenimonas (optionally sp. Strain WBC-2) Pseudomonas, Flavobacterium, Comamonas, Cytophaga, Acidovorax, Sphmgomonas, Bacillus, Acinetobacter or a combination thereof.
  • the TCE producing recombinant microorganism expresses at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and i or ii) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the TCE producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at feast one native TCE producing enzyme, and wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
  • the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, giyeera!dehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, ceilobiose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example Cj-g, CM, CM or Co? chlorinated alkanes (such as chloroform and / or carbon tetrachloride). In embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltito! and / or xylitol).
  • monosaccharides such as xylose, giyeera!dehyde, fructose, galactose, mannose and / or glucose
  • disaccharides
  • the chloride source may any source of chloride, including chloride salt ( NaCi, KC1, MgCb, CaCh or combinations thereof) and / or HO.
  • chloride salt NaCi, KC1, MgCb, CaCh or combinations thereof
  • HO HO
  • the at least one TCE producing enzyme includes phenol hydrase (PH), particulate methane monooxygenase (pMMO), benzene (and/or toluene) dioxygenase (BDO/ToD), toluene o-xylene monooxygenase oxygenase subunit (TouA), toluene-4-raonooxygenase system hydroxylase component subunit alpha, chlorobenzene dioxygenase, cis-chlorobenzene dihydrodioi dehydrogenase, toluene 2- monooxygenase, or a combination thereof, in certain embodiments, the TCE producing recombinant microorganism expresses PH having an amino acid sequence at least 95% identical to SEQ ID NO: 13 by expressing a native or non-native PH nucleotide sequence at least 95% identical to SEQ ID NO: 14; expresse
  • the TCE producing recombinant microorganism includes microorganisms of the genus Rhizobium (optionally Rhizobium meiiloii e.g. Rhizohium melUoti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emiliam hialeyi), Sinorhizobium (optionally Sinorhizobium meitioti), Calcidiscus (optionally Calcidiscus lep(oporus), Phaeodaciylum (optionally Phaeodactylutn tricornutum), Chaetoceros (optionally Chaetoceros neograciUs ).
  • Rhizobium Rhizobium meiiloii e.g. Rhizohium melUoti strain Dangeard
  • Porphyridium optionally Porphyridium purpureum
  • Emiliania optionally Emiliam hialeyi
  • Dumliella (optionally Duna!ieiia iertiolecta), Meristiella (optionally Meristiella gelidium), Ulva (optionally Ulva iactuca or Ulva rigida, e.g. Ulva rigida Agardh ), Enteromorpha (optionally Enieromorphia intestinalis, Cladophora (optionally Cladopkora rupestris), Fucus (optionally Fucus serratus), Laminaria (optionally Laminaria saccarina, e.g. Laminaria saccarina (L) Lamour or Laminaria digitata, e.g.
  • Porphyra umbilicalis (L) J. Ag.), Polysiphonia (optionally Polysiphonia nigrescens, e.g. Polys iphonia nigrescens (Huds.) Greville), Furceilaria (optionally Furcellaria lumbricalis, e.g. Furceliaria lumbricalis (Huds.) Lamour), Ceramium rubrum (optionally Ceramium rubrum, e.g.
  • Hypnea musciformis (Wulfen) Lamouroux), Asparagopsis (optionally Asparagopsis taxiformis, e.g. Asparagopsis taxifbrmis (Delik) Trev).
  • Gelidium (optionally Gelidium canadensis), Falkenbergia (optionally Falkenbergia hillebrandii, e.g. Falkenbergia hiliebrandii (Born.) Falkenb),
  • Coral lina (optionally Corallina officinalis), Gracilariopsis (optionally Gracilariopsis lemaneifbrmis), Gracilaria (optionally Gracilaria cornea, e.g. Gracilaria cornea J. Agardh), Methylosinus (optionally Methylosinus trichosporium, e g. Methylosinus trichosporium OB3h), Desulfitobacierium (optionally Desulfitobacterium frappieri, e.g. Desulfitobacterium frappieri TCE1, or Desulfitobacterium metal! ireducem), Methylomicrobium (optionally Methylomicrobium album, e.g Methylomicrobium a!
  • Meihylococcus (optionally Methylococcus capsulatus, e.g, Meihylococcus capsulaius (Bath)), Ralstonia (e.g. Ralstonia sp. KN 1-lOA), Pseudomonas (optionally Pseudomonas putida, e.g Pseudomonas putida FI, or Pseudomonas sp. M4), Rhodobacter (optionally Rhodobacter sphaeroldes or Rhodobacier capsulatus), Burkholderia (optionally Burkholderia cepacian , e.g. Burkholderia cepacian G4 ) or a combination thereof.
  • Meihylococcus optionally Methylococcus capsulatus, e.g, Meihylococcus capsulaius (Bath)
  • Ralstonia e.
  • the VCM producing recombinant microorganism expresses at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or is) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme nucleotide sequence, and wherein the VCM producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
  • the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, giyceraldehyde, fmcto.se, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, ceilobiose and / or trehalose).
  • the TCE feedstock may comprise chlorinated hydrocarbons, for example Cj-a, Cue, CM or Cue chlorinated alkanes (such as chloroform and / or carbon tetrachloride).
  • the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltitol and / or xylitof).
  • the chloride source may any source of chloride, including chloride salt (e.g. NaCl, KCI, MgCb, CaCh or combinations thereof) and / or HCi.
  • chloride salt e.g. NaCl, KCI, MgCb, CaCh or combinations thereof
  • the glucose producing microorganism expresses at least one glucose producing enzyme, wherein the glucose producing microorganism is capable of utilizing a carbon source and a hydrogen source to produce glucose.
  • the carbon source includes a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof
  • the hydrogen source comprises water.
  • the chloride source may any source of chloride, including chloride salt (e.g. NaCl, KCI, MgCb, CaCb or combinations thereof) and / or HCI.
  • the glucose producing enzyme includes sucrose permease (cscB), sucrose-phosphate synthase (sps), glucose- 1 -phosphate adeny ly Itransferase (glgC), sucrose phosphate phosphatase (spp), glycogen phophorylase (glgP), UDP-glucose pyrophosphorylase (galU), invertase, glucosy!gjycerol-phosphate synthase (ggpS), glycogen synthase (gigA); or combinations thereof.
  • the glucose producing microorganism expresses cscB having an amino acid sequence at least 95% identical to SEQ ID NO: 29 by expressing a non-native cscB nucleotide sequence at least 95% identical to SEQ ID NO: 30; expresses sps having an amino acid sequence at least 95% identical to SEQ ID NO: 31 by expressing a non-native sps nucleotide sequence at least 95% identical to SEQ ID NO: 32; expresses glgC having an amino acid sequence at least 95% identical to SEQ ID NO: 33 by expressing a non-native glgC nucleotide sequence at least 95% identical to SEQ ID NO: 34; expresses spp having an amino acid sequence at least 95% identical to SEQ ID NO: 35 by expressing a non-native spp nucleotide sequence at least 95% identical to SEQ ID NO: 36; expresses gigp having an amino add sequence at least 95% identical to SEQ ID NO:
  • the glucose producing recombinant organism includes a recombinant microorganism, a photosynthetic microorganism, a Cyanobacteria, a Syneehococeus, Symchococcus elongatas, Synechococcm leopoiiemis, Symchocystis, Anabaena .
  • a Pseudomonas Pseudomonas syringae, Pseudomonas savastanoi, Chlamydomonas, Chlamydomonas reinhardtii, algae, microalgae, electrosynthesis bacteria, a photosynthetic microorganism, yeast, filamentous fungi, or a plant cell.
  • Embodiments of methods of producing a vinyl chloride monomer (VCM) producing recombinant microorganism having an VCM improved producing ability or a. trichloroethylene (TCE) producing recombinant microorganism having an improved TCE producing ability are disclosed herein.
  • the method includes: producing the VCM producing recombinant microorganism by inserting at least one of a non- native VCM expressing nucleotide sequence or a non-native TCE expressing nucleotide sequence into a bacterial plasmid of a microorganism.
  • the method includes: producing the VCM producing recombinant microorganism by inserting at least one of a native VCM expressing nucleotide sequence, a nucleotide sequence which promotes the overexpression of a native VCM expressing nucleotide sequence, a native TCE expressing nucleotide sequence and / or a nucleotide sequence which promotes the overexpression of a native TCE expressing nucleotide sequence into a bacterial plasmid of a microorganism.
  • the native or non-native VCM expressing nucleotide sequence has a nucleotide sequence at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or a combination thereof in certain embodiments, the native or non-native TCE expressing nucleotide sequence has a nucleotide sequence at least 95% Identical to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or a combination thereof
  • the VCM producing recombinant microorganism includes microorganisms of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehalococcoides strain FL2, Dehalococcoides mccariy e.g.
  • strains KS, EC, INA, MB, 1 la or GY50 Dehalococcoides ethenogenes e.g strain 195, Dehalococcoides strain BAV1, Dehalococcoides strain VS, Dehalococcoides strain CBDBI, Dehalococcoides strain GT) Escherichia (optional ly Escherichia coli ), Synechococcus (optionally Synechococcus elongates), Stdfurospirillum (optionally Sulfurospirillum muhivoram or Sulfatrospir ilium bamesii), Dehalobacter (optionally Dehalobacter restrictus), Desulfuromonas (optionally strain BB1 or Desulfuromonas chloroethenicd), Desulfitobacterium (optionally Desuifitobacterium hafnieme), Geobacler (optionally Geobacter bemidjiensis, Geobacter lovleyi, Geobacter psychrophilus, Geobac
  • the TCE forming recombinant microorganism includes microorganisms of the genus Rhizobium (optionally Rhizobium meliioti e.g. Rhizobium meliioti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emil tana hvdeyi), Simrhizobium (optionally Sinorhizobium meliioti), Calcidiscus (optionally Calcidiscus lepioporus) Phaeodactyium (optionally Phaeodactylum tricornulum), Chaetoceros (optionally Chaetoceros neogracilis), DunaUella (optionally DumlieSla tertiolecta), Meristie.Ua (optionally Meristiella gelidium), Ulva (optionally Ulm iactuca or Ulva rigida, e.g.
  • Methylosinus trickosporium OB3b Desulfitobacterium (optionally Desulfitobacterium frappieri, e.g. Desulfitobacterium frappieri TCEl, or Desulfitobacterium metallireducens), Methylomicrobium (optionally Methylomicrobium album, e.g. Methylomicrobium album BG8), Methylococcus (optionally Methylococcus capsulatus, e.g. Methylococcus capsulatus (Bath)), Ralsionia (e.g. Ralstonia sp.
  • Desulfitobacterium optionally Desulfitobacterium frappieri, e.g. Desulfitobacterium frappieri TCEl, or Desulfitobacterium metallireducens
  • Methylomicrobium optionally Methylomicrobium album, e.g. Methylomicrobium album BG8
  • Methylococcus optionally Me
  • Pseudomonas optionally Pseudomonas putida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4
  • Rhodobacter optionally Rliodobacter sphaeroides or Rhodobacter capsulatus
  • Burkholderia optionally Burkholderia cepacian, e.g Burkholderia cepacian G4 or a combination thereof.
  • the VCM producing recombinant microorganism and the TCE producing recombinant microorganism are the same or different.
  • the method includes: producing TCE by reacting glucose with a chloride source in the presence of a TCE producing catalytic dement wherein the TCE producing catalytic element comprises a TCE bioreactor culture containing a TCE producing recombinant microorganism having an improved TCE producing ability, wherein the TCE producing recombinant microorganism expresses the at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or it) overexpressing at least one native TCE producing enzyme nucleotide sequence, and wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride
  • the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, glycera!dehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, cel!obiose and / or trehalose).
  • monosaccharides such as xylose, glycera!dehyde, fructose, galactose, mannose and / or glucose
  • disaccharides such as lactose, maltose, sucrose, cel!obiose and / or trehalose
  • the TCE feedstock may comprise chlorinated hydrocarbons, for example Cos, Cm,, CM or Cue chlorinated alkanes (such as chloroform and / or carbon tetrachloride), in embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltitol and / or xylitol).
  • chlorinated hydrocarbons for example Cos, Cm,, CM or Cue chlorinated alkanes (such as chloroform and / or carbon tetrachloride)
  • the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltitol and / or xylitol).
  • the chloride source may any source of chloride, including chloride salt (e.g. NaCl, KC!, MgCb, CaCk or combinations thereof) and / or HO,
  • chloride salt e.g. NaCl, KC!, MgCb, CaCk or combinations thereof
  • the method further includes: producing glucose by reacting a carbon source with a hydrogen source in the presence of a glucose producing catalytic element including at least one photosynthesis enzyme comprising Rubisco, wherein the glucose producing catalytic element Includes a glucose hioreactor culture containing a glucose producing microorganism, wherein the glucose producing microorganism expresses the at least one glucose producing enzyme, and wherein the glucose producing microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose.
  • a glucose producing catalytic element including at least one photosynthesis enzyme comprising Rubisco
  • the method further includes a VCM producing catalytic element, wherein the VCM producing catalytic dement includes a VCM hioreactor culture containing a VCM producing recombinant microorganism having an improved VCM producing ability, wherein the VCM producing recombinant microorganism expresses the at least one VCM producing enzyme by i) expressing at least one non-native VCM producing enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM producing enzyme, and wherein the VCM producing recombinant microorganism is capable of utilizing a chlorinated hydrocarbon to produce VCM, in embodiments of the invention, the chlorinated hydrocarbon may be a chlorinated alkane and / or a chlorinated alkene.
  • the chlorinated hydrocarbon may be a chlorinated alkane and / or a chlorinated alkene.
  • the chlorinated hydrocarbon may be a Cos chlorinated hydrocarbon, a Cos chlorinated hydrocarbon, a CM chlorinated hydrocarbon and / or a CM chlorinated hydrocarbon.
  • chlorinated hydrocarbon sources that may be employed in the present invention include trichloroethylene (TCE), diets loroethyieoe (DCE) and / or chloroform.
  • the carbon source includes a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof.
  • the hydrogen source includes water.
  • the chloride source may any source of chloride, including chloride salt (e.g , NaC!, KC!, MgCfe, CaCh or combinations thereof) and / or HCi.
  • VCM vinyl chloride monomer
  • VCM producing enzyme and collecting VCM front the reaction medium.
  • a method of producing trichloroethylene comprising: providing a reaction medium comprising a TCE producing enzyme and a TCE feedstock; maintaining the reaction medium under conditions which permit the production of TCE from the TCE feedstock by the TCE producing enzyme; and collecting TCE from the reaction medium,
  • Fig. 1 is a schematic illustration of a biomanufacturing system for producing vinyl chloride monomer (VCM) according to embodiments herein.
  • Fig. 2 is a series of graphs, including Fig.s 2(1), 2(2), 2(3), 2(4), and 2(5), which show retention times of chloride compounds and intermediates determined by gas chromatography, according to embodiments herein.
  • Fig. 3 A is a graph showing gas chromatography results for trichloroethylene (TCE) produced by the soil bacteria S. meli!oti, according to embodiments herein.
  • TCE trichloroethylene
  • Fig, 38 is a graph showing TCE production by S. me!ilofi compared to a media only control, according to embodiments herein.
  • Fig. 4A is a series of graphs, including Fig. 4(1 ), 4(2), and 4(3), which show gas chromatography results of VCM production from TCE in a Dehalococcoides culture, according to embodiments herein.
  • Fig. 4B is a graph showing gas chromatography results of ethylene, VCM, dichioroethyiene (DCE), and TCE production by a Dehalococcoides consortia culture, according to embodiments herein.
  • Fig. 4 € is a series of pie charts showing gas chromatography resul ts as percentages of the total production of ethylene, VCM, DCE, and TCE, according to embodiments herein.
  • Fig. 4D is a graph showing a standard curve of VCM yield from a Dehalococcoides consortia culture, according to embodiments herein.
  • Fig. 4E is a graph showing VCM yield from a Dehalococcoides consortia culture at different sampling times, according to embodiments herein.
  • Fig. 5 A is a series of graphs, including Fig.s 5(1), 5(2), and 5(3), which show gas chromatography results from engineered E. coll, cultures, according to embodiments herein,
  • Fig. 58 is a series of graphs, 5(4), 5(5), and 5(6), which show gas chromatography results from engineered £. coli cultures, according to embodiments herein.
  • Fig. 6A is a graph showing gas chromatography results (left) and corresponding peak retention tints data (right) from engineered Cyanobacteria cultures showing the production of VCM from TCE, according to embodiments herein,
  • Fig. 6B is a graph showing gas chromatography results for control levels of VCM production from a native Cyanobacteria culture, according to embodiments herein.
  • Fig. 6C is a graph showing gas chromatography results for VCM production from TCE by an engineered Cyanobacteria culture, according to embodiments herein.
  • Fig. 7 is a flow chart illustrating a method of producing TCE, according to embodiments herein.
  • “at least one of a non-native VCM expressing nucleotide sequence or a non-native TCE expressing nucleotide sequence” means a single non-native VCM expressing nucleotide sequence, or more than one nonnative VCM expressing nucleotide sequence, or a single a non-native TCE expressing nucleotide sequence, or more than one non-native TCE expressing nucleotide sequence, or any combination thereof
  • the term “about” refers to *10% of the nonpercentage number that is described, rounded to the nearest whole integer. For example, about 37 degrees Celsius, would include 33.3 to 40.7 degrees Celsius. Unless otherwise noted, the term “about” refers to ⁇ 5% of a percentage number. For example, about 80% would include 75% to 85%. When the term “about” is discussed in terms of a range, then the term refers to the appropriate amount less than the lower limit and more than the upper limit. For example, from about pH 4 to about pH 12 mm would include from pH 3,6 to pH 13.2.
  • the terras “provide”, “provided” or “providing” refer to the supply, production, purchase, manufacture, assembly, formation, selection, configuration, conversion, introduction, addition, or incorporation of any element, amount, component, reagent, quantity, measurement, or analysis of any method or system of any embodiment herein,
  • non-native refers to organisms or molecules that are occur in nature without human action.
  • non-native refers to organisms or molecules that are not naturally occurring and are produced by human actions.
  • An example of a non-native organism is a recombinant organism.
  • microorganism is intended to relate to both single microorganisms as well as populations of microorganisms which may belong to a single strain or multiple strains.
  • Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • sequence identity is determined by comparing the whole length of the sequences as identified herein.
  • Exemplary methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Exemplary computer program methods to determine identity and similarity between two sequences include e.g. the BestFit BLASTP (Protein Basic Local Alignment Search Tool), BLASTN (Nucleotide Basic Local Alignment Search Toot), and PASTA (AJischul, S. f. et a!., i. Mol. Biot. 215:403-410 (1990), publicly available from NCBI and other sources (BLAST.RTM. Manual, Aitschul, S., et ai., NCB! NLM NIH Bethesda, Md. 20894).
  • BestFit BLASTP Protein Basic Local Alignment Search Tool
  • BLASTN Nucleotide Basic Local Alignment Search Toot
  • PASTA AJischul, S. f. et a!., i. Mol. Biot.
  • EMBOSS European Molecular Biology Open Software Suite
  • Exemplary parameters for amino acid sequences comparison using EMBOSS are gap open 10.0, gap extend 0.5, B!osum matrix.
  • Exemplary parameters for nucleic acid sequences comparison using EMBOSS are gap open 10.0, gap extend 0.5, DNA full matrix (DNA identity matrix).
  • amino acid similarity the skilled person may also take into account so-called “conservative” amino acid substitutions, as will be clear to the skilled person.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino adds having sulphur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring arnino acids are as follows: Ala to ser; Arg to iys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or iie; Phe to met, leu or tyr; Ser to horr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Vai to He or leu.
  • Methods for determining the production of enzymes by recombinant VCM- and TCE-producing microorganisms as compared to reference microorganisms will be known to those skilled in art.
  • the recombinant microorganism and the reference microorganism can be reacted with the same starting materials under the same reaction conditions, and the resulting levels of enzymes produced may be assessed using gas chromatography techniques with which the skilled reader will be familiar and / or which are detailed in the protocols presented in the examples which follow.
  • Carbon dioxide emissions resulting from the use of fossil fuels continue to rise on a global scale. Reduction of atmospheric carbon dioxide levels is a key to mitigating or reversing climate change.
  • Carbon capture and storage (CCS) is a prominent technology for removal of industrial carbon dioxide from the atmosphere; it has been estimated that over 20 trillion tons of carbon dioxide captured from refining and other industrial processes can be transported and stored in various types of subterranean environments or storage tanks.
  • CCS is a cost effective and affordable way to reduce carbon dioxide emissions compared to other currently available methods, the problem remains that the carbon dioxide is merely being stored underground until it escapes. Therefore, CCS methods do not provide a sustainable solution to reduce excess carbon dioxide in the atmosphere.
  • Vinyl chloride monomer is one such chemical product that is almost exclusively used as a feedstock for the production of polyvinyl chloride (PVC), one of the most widely used plasties in the world. Over 40 million metric tons of PVC were produced worldwide in 2018, and the demand for PVC is expected to approach 60 million metric tons by 2025.
  • VCM is commercially produced by cracking natural gas or petroleum with chlorine, having a substantial impact on global energy consumption. With millions of metric tons of VCM being produced each year, more than enough carbon dioxide is produced by such processes to greatly contribute to the global carbon footprint. It has been shown that for each ton of VCM produced, 0.32 tons of CO? is emitted, A global production of 55 million tons of VCM per year corresponds to about 17.6 million tons of CO ?, released annually.
  • Trichloroethylene is widely used as a solvent for degreasing metal parts during manufacturing processes, and as a solvent in many consumer products, including adhesives, paint and stain removers, and wood finishes, and as a reagent for the production of other chemicals, including VCM.
  • VCM and TCE are widely used as a solvent for degreasing metal parts during manufacturing processes, and as a solvent in many consumer products, including adhesives, paint and stain removers, and wood finishes, and as a reagent for the production of other chemicals, including VCM.
  • Embodiments of the present disclosure can provide a benefit of removing carbon dioxide from the environment along with the benefit of producing valuable organic compounds capable of being sold commercially. Embodiments of the present disclosure can thus provide a renewable alternative to conventional carbon dioxide storage, by using recombinant microbial technology to convert the carbon dioxide into VCM and TCE as useful organic compounds.
  • One benefit of the embodiments of the present disclosure is that the methods can make it economically profitable for a chemical or an energy company to remove carbon dioxide from the environment.
  • An oil company could instead of pumping carbon dioxide into a subterranean environment or leaving the sequestered carbon dioxide underground, use the carbon dioxide to provide the chemical industry with a carbon source to use as a feedstock for a culture of recombinant microorganisms to convert the carbon dioxide to VCM and/or TCE in a eost-eftective way.
  • Embodiments of toe present disclosure can provide a benefit of engineering a TCE or a VCM producing microorganism, by adapting the relevant metabolic signaling pathways to produce VCM and/or TCE on an industrial scale. Such embodiments can make it profitable to remove carbon dioxide from the atmosphere and to passively generate valuable organic compounds while the microbes do the work - on a scale previously unimaginable.
  • system 100 includes glucose producing recombinant microorganism 102 that expresses at least one glucose producing enzy me 104 and is capable of utilizing carbon source 106 and hydrogen source 108 to produce glucose 110, biomass 112 and oxygen 114; TCE producing recombinant microorganism !
  • TCE trichloroethylene
  • VCM producing recombinant microorganism 117 that expresses at least one VCM producing recombinant enzyme 126 by expressing at least one non-native VCM producing enzyme nucleotide sequence 128, and is capable of utilizing the chlorinated hydrocarbon TCE 124 to produce VCM 130.
  • TCE producing recombinant microorganism 116 and VCM producing microorganism 117 can be different microorganisms, in certain embodiments, TCE producing recombinant microorganism i 16 and VCM producing recombinant microorganism 117 can be the same microorganism.
  • Embodiments of a biomanufacturing system herein can provide a benefit of a sustainable use of carbon sources, such as carbon dioxide, for the production of useful chemical compounds such as VCM and TCE. Such embodiments can also help to conserve natural resources. Biomanufacturing systems allowing chemical production by recombinant microorganisms can provide a benefit of reducing harm to the environment caused by conventional chemical manufacturing processes, thereby supporting long-term ecological balance.
  • Embodiments of a vinyl chloride monomer (VCM) producing recombinant microorganism having a VCM improved production ability are disclosed herein.
  • the VCM producing recombinant microorganism expresses at least one VCM producing enzyme by i) expressing at least one non-native VCM producing enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM producing enzyme, wherein an amount of the VCM producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native VCM producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at feast one native VCM producing enzyme nucleotide sequence, and wherein the VCM producing recombinant microorganism is capable of utilizing a chlorinated hydrocarbon source to produce VCM.
  • the VCM producing recombinant microorganism expresses at least one non-native VCM producing enzyme nucleotide sequence and produces VCM enzyme in a greater amount relative to a control microorganism lacking the at least one non-native VCM producing enzyme nucleotide sequence.
  • the VCM producing recombinant microorganism overexpresses at least one native VCM producing enzyme nucleotide sequence and produces VCM enzyme in a greater amount relative to a control microorganism which is not engineered to overexpress the at least one native VCM producing enzyme.
  • the VCM producing recombinant microorganism i) expresses at least one non-native VCM producing enzyme nucleotide sequence and ii) overexpresses at least one native VCM producing enzyme nucleotide sequence, and produces VCM enzyme in a greater amount relative to a control microorganism lacking the at least one non-native VCM producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native VCM producing enzyme.
  • Embodiments of VCM producing recombinant microorganisms can provide an important advantage over conventional VCM production, by avoiding much of the environmental pollution caused by chemical cracking processes, while providing for a sustainable way to produce VCM on a commercial scale.
  • the at least one VCM producing enzyme includes TCE reductive dehalogenase (TceA), TceA anchor protein (TeeB), tetrachloroethylene reductive dehalogenase (peeA), vinyl chloride reductase (vcrA), 1 ,2-dichSoropropane-io- propene reductive dehalogenase (dcpA), 1 ,2-trans ⁇ dishloroethene reductive dehalogenase catalytic A (TdrA), or a combination thereof.
  • the VCM producing recombinant microorganism expresses TceA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 by expressing a native or non-native TceA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.
  • the VCM producing recombinant microorganism expresses TeeB having an amino acid sequence at least 90%, 95%, 96%,
  • the VCM producing recombinant microorganism expresses pceA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5 by expressing a native or non-native pceA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6.
  • the VCM producing recombinant microorganism expresses vcrA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 by expressing a native or non-native vcrA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • the VCM producing recombinant microorganism expresses dcpA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 by expressing a native or non-native dcpA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.
  • the VCM producing recombinant microorganism expresses TdrA having an amino acid sequence 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11 by expressing a native or non-native TdrA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12.
  • the VCM producing recombinant microorganism includes microorganisms of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehaiococcoides strain FL2, Dehalococcoides mccarty e.g. strains KS, RC, IN A, MB, 1 la or GY50. Dehalococcoides ethenogenes e g. strain.
  • FRC-32 Geobacter sp. M21, Geobacter sulfurreducens, or Geobacter uraniireducens
  • Pelobacier optionally Pelobacier propionicus
  • Dehalogenimonas optionally sp. Strain WBC-2 Pseudomonas, Flavobacterium, Comamonas, Gytophaga, Acidavorax, Sphingomonas, Bacillus, Acineiobacter or a combination thereof.
  • the chlorinated hydrocarbon source may be a chlorinated alkane and / or a chlorinated alkene.
  • the chlorinated hydrocarbon source may be a Ci-g chlorinated hydrocarbon, a Cu chlorinated hydrocarbon, a CM chlorinated hydrocarbon and / or a C1-2 chlorinated hydrocarbon.
  • chlorinated hydrocarbon sources that may be employed in the present invention include trichloroethylene (ICE), dichloroethylene (DCE) and / or chloroform.
  • VCM vinyl chloride monomer
  • the reaction medium may he provided in a bioreactor. Any type of bioreactor known to one skilled in the art may be employed.
  • VCM may be collected from the reaction medium using conventional separation techniques e.g. distillation, evaporation or the like. In preferred embodiments, such techniques may be used to obtain a VCM stream comprising at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of VCM by weight.
  • the VCM producing microorganism which produces the VCM producing enzyme may also be provided in the reaction medium.
  • the VCM producing microorganism which produces the VCM producing enzyme is not present in the reaction mixture.
  • reaction medium The conditions under which the reaction medium is maintained to permit the production of VCM from the chlorinated hydrocarbon will depend upon nature of the VCM- produeing enzyme and / or VCM producing microorganism provided in the reaction medium.
  • the VCM producing enzyme may be produced by an organism which is native or recombinant.
  • the VCM producing organism may comprise a vinyl chloride monomer (VCM) producing recombinant microorganism as described herein.
  • VCM vinyl chloride monomer
  • the VCM producing microorganism includes microorganisms of the phylum Cyanobacteria or the genus Dekalococcoides (optionally Dehalococcoides strain FL2, Dehalococcoides mccarty e.g strains K.S, RC, JNA, MB, 11 a or GY50, Dehalococcoides ethsnogenes e.g.
  • strain 195 Dehalococcoides strain BAV1, Dehalococcoides strain VS, Dehalococcoides strain CBDB1, Dehalococcoides strain GT) Escherichia (optionally Escherichia coil), Synechococcus (optionally Synechococcus elongatus), Sulfurospirillum (optionally Sulfurospirillum multivoram or Sulfurospirillum harnesii), Dehalobacter (optionally Dehalobacter restrict*®), Desulfuromonas (optionally strain BB1 or De sulfur omonas cMoroethenica), Desulfitobacterium (optionally Desulfslobacierium hafhiense), Geobacter (optionally Geobacter bemidjiensis, Geobacter lovleyi, Geobacter psychrophilus, Geobacter sp.
  • FRC-32 Geobacter sp. M21, Geobacter sulfitrreducens, or Geobacter uraniireducens
  • Pelobacter optionally Pelobacter propionicus
  • Dehalogenimonas optionally sp. Strain WBC-2 Pseudomonas, Fiavobacterium, Comamonas. Cyiophaga . Acidovorax, Sphingomonas, Bacillus, Acinetobacter or a combination thereof.
  • the VCM producing microorganism may comprise a consortium of microorganisms.
  • the VCM producing microorganism may comprise a plurality of strains belonging to the genus Dehalococcoides. Such consortia have been demonstrated in the examples to be particularly efficient producers ofVCM
  • the YCM producing microorganism may express TceA having an amino acid sequence at least 95% identical to SEQ ID NO: I by expressing a TceA nucleotide sequence at least 95% identical to SEQ HI NO: 2; expresses TceB having an amino acid sequence at least 95% identical to SEQ ID NO: 3 by expressing a TceB nucleotide sequence at least 95% identical to SEQ ID NO: 4; expresses pce.4 having an amino acid sequence at least 95% identical to SEQ ID NO: 5 by expressing a peeA nucleotide sequence at least 95% identical to SEQ ID NO: 6; expresses vcrA having an amino add sequence at least 95% identical to SEQ ID NO: 7 by expressing a vcrA nucleotide sequence at least 95% identical to SEQ ID NO: 8; expresses depA having an amino add sequence at least 95% identical to SEQ ID NO: 9 by expressing a
  • Trichloroethylene (TCE) Producing Recombinant Microorganisms
  • Embodiments of a trichloroethylene (TCE) producing recombinant microorganism having an improved TCE producing ability are disclosed herein.
  • the TCE producing recombinant microorganism expresses at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or is) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the TCE producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme nucleotide sequence, and wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
  • the TCE producing recombinant microorganism expresses at least one non-native TCE producing enzyme nucleotide sequence and produces TCE enzyme in a greater amount relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence.
  • the TCE producing recombinant microorganism overexpresses at least one native TCE producing enzyme nucleotide sequence and produces TCE enzyme in a greater amount relative to a control microorganism which is not engineered to overexpress the at least one native TCE producing enzyme.
  • the TC producing recombinant microorganism i) expresses at least one non-native TCE producing enzyme nucleotide sequence and ii) overexpresses at least one native TCE producing enzyme nucleotide sequence, and produces TCE enzyme in a greater amount relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme.
  • Embodiments of a TCE producing recombinant microorganism can provide a benefit of sustainable TCE production on a commercial scale, while avoiding environmental harm caused by conventional TCE manufacturing processes.
  • the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, glyceraldehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, valobfose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example C?
  • the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltitol and / or xylitol).
  • the chloride source may any source of chloride, including chloride salt (e.g. NaC!, KC!, MgCh, CaCh or combinations thereof) and / or HCi.
  • chloride salt e.g. NaC!, KC!, MgCh, CaCh or combinations thereof
  • the at least one TCE producing enzyme includes phenol hydrase (PIT), particulate methane monooxygenase (pMMO), benzene (and/or toluene) dioxygenase ⁇ B DO/ToD), toluene o-xylene monooxygenase oxygenase subunit (TouA), toluene-4-monooxygenase system hydroxylase component subunit alpha, chlorobenzene dioxygenase, cis-chiorobenzene dihydrodiol dehydrogenase, toluene 2- monooxygenase, or a combination thereof, in certain embodiments, the TCE producing recombinant microorganism expresses PH having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13 by expressing a native or nonnative PH nucleotide
  • the TCE producing recombinant microorganism expresses pMMO having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15 by expressing a native or non-native pMMO nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16.
  • the TCE producing recombinant microorganism expresses ToD having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or !00% identical to SEQ ID NO: 17 by expressing a native or non-native ToD nucleotide sequence at least 90%, 95%. 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.
  • the TCE producing recombinant microorganism expresses TouA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SF.Q ID NO: 19 by expressing a native or non-native TouA nucleotide sequence at ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20.
  • the TCE producing recombinant microorganism expresses toiuene-4-monooxygenase system hydroxylase component subunit alpha having an amino acid sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21 by expressing & native or non- native toluene-4-monooxygenase system hydroxyla e component subunit alpha nucleotide sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.
  • the TCE producing recombinant microorganism expresses chlorobenzene dioxygenase having an amino acid sequence 90%, 95%, 96%, 97%, 98%,
  • the TCE producing recombinant microorganism expresses cis-chiorobenzene dihydrodioi dehydrogenase having an amino acid sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:
  • the TCE producing recombinant microorganism expresses toluene 2-monooxygenase having an amino add sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IP NO: 27 by expressing a native or non-native toluene 2-monooxygenase nucleotide sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28; or a combination thereof
  • the TCE producing recombinant microorganism includes microorganisms of the genus Rhizobium (optionally Rhizobium meliloti e.g Rhizobium meliloti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emiliana huxleyi), Simrhizobium (optionally Sinorhizobium meliloti), Calcidiscus (optionally Calcidiscus leptoporus), Phaeodactylum (optionally Phaeodactyhm tricormtum), Chaetoceros (optionally Chaetoceros neogracilis), Dunaliella (optionally Dunaliella tertiolectd), Meristiella (optionally Meristiella gelidium), Ulva (optionally Ulva lactuca or Ulva rigida, e.g.
  • Laminaria saccarina e.g. Laminaria saccarina (L) Lamonr or Laminaria digitaia, e.g. Laminaria digitata (Huds) Lamonr
  • Desmarestia optionally Desmarestia acuieata, e.g. Desmarestia aculeaia (L) Lamour
  • Chorda optionally Chorda filum.
  • Asparagopsis taxiformis (Deliie) Trev), Gelidium (optionally Gelidium camriemis), Faikenbergia (optionally Faikenbergia hiUebrandii, e.g. Faikenbergia hillebrandii (Born.) Falkenb), Corallina (optionally Corallina officinalis), Gracilariopsis (optionally Gracilariopsis lemaneifarmis), Graciiaria (optionally Gracilaria cornea, e g. Gracilaria cornea J. Agardh), Methylosinus (optionally Methylosinus trickasporium, e.g.
  • Methylosinus trichosporium OB3b Desuifitobacterium (optionally Desulfitobacterium frappieri. e.g. Desulfitobacterium fi-appieri TCE1, or Desulfitobacterium metallireducens), Meihylomicrobium (optionally Methylomicrobium album, e.g. Meihylomicrobium album BG8), Methylococcus (optionally Methylococcus capsulatus, e.g. Melhylococcus copsulaius (Bath)), Ralstonia (e.g. Ralstonia sp.
  • Desuifitobacterium optionally Desulfitobacterium frappieri. e.g. Desulfitobacterium fi-appieri TCE1, or Desulfitobacterium metallireducens
  • Meihylomicrobium optionally Methylomicrobium album, e.g. Meihylomicrobium album BG8
  • Pseudomonas optionally Pseudomonas putida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4
  • Rhodobacter optionally Rhodobacier sphaeroides or Rhodobacter capsulatus
  • Burkholderia optionally Burkholder ia cepacian, e.g. Burkhoideria cepacian G4 or a combination thereof.
  • the VCM producing recombinant microorganism and the TCE producing recombinant microorganism are the same or different organisms.
  • the VCM producing recombinant microorganism expresses at least one TCE producing enzyme by t) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or ii) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme nucleotide sequence, and wherein the VCM producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
  • Such embodiments can provide a benefit of a single recombinant microorganism having both a VCM and a TCE producing capability. Such embodiments can provide an advantage of a greater efficiency in VCM production from TCE.
  • the VCM producing recombinant microorganism and the TCE producing recombinant microorganism are different organisms. Such embodiments can provide an advantage of independently optimizing the manufacture of each organism and varying their ratio to one another during VCM or TCE production.
  • the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, giyeeraidehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, ee!iobiose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example CM, CM, CM or Co?, chlorinated alkanes (such as chloroform and / or carbon tetrachloride). In embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltUoi and / or xylitol).
  • monosaccharides such as xylose, giyeeraidehyde, fructose, galactose, mannose and / or glucose
  • disaccharides
  • the chloride source may any source of chloride, including chloride salt (e.g. NaCI, KCi, MgCb, CaCh or combinations thereof) and / or HC!.
  • a method of producing trichloroethylene comprising: providing a reaction medium comprising a TCE producing enzyme and a TCE feedstock; maintaining the reaction medium under conditions which permit the production of TCE from the TCE feedstock by the TCE producing enzyme; and collecting TCE from the reaction medium.
  • the reaction medium may be provided in a bioreactor. Any type of bioreactor known to one skilled in the art may be employed.
  • TCE may be collected from the reaction medium using conventional separation techniques e.g. distillation, evaporation or the like. In preferred embodiments, such techniques may be used to obtain a TCE stream comprising at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of TCE by weight.
  • the TCE producing microorganism which produces the TCE producing enzyme may also be provided in the reaction medium. In alternative, cell-free embodiments, the TCE producing microorganism which produces the TCE producing enzyme is not present in the reaction mixture. [0093] The conditions under which the reaction medium is maintained to permit the production of TCE from the chlorinated hydrocarbon by the TCE producing enzyme will depend upon the nature of the TCE producing enzyme and / or TCE producing microorganism provided in the reaction medium.
  • the TCE producing enzyme may be produced by a TCE producing microorganism may be native or recombinant.
  • the TCE producing organism may be a trichloroethylene (TCE) producing recombinant microorganism as described herein. j
  • the TCE feedstock may comprises a carbohydrate (e.g, a monosaccharide, optionally xylose, glyceraldehyde, fructose, galactose, mannose and / or glucose, or a disaccharide, optionally lactose, maltose, sucrose, cellobiose and / or trehalose), a chlorinated hydrocarbon (e.g. a Cw chlorinated alkane, optionally chloroform and / or carbon tetrachloride) and / or a polyol (e.g. glycerol, mannitol, sorbitol, ma!titol and / or xylitol).
  • the TCE feedstock comprises glucose.
  • the TCE producing microorganism may include microorganisms of the genus Rhizobium (optionally Rhizobium meliloii e.g Rhizobium meliloti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emilkma huxleyi), Sinorhisobium (optionally Sinorhizobhm meliloii), Calcidiscus (optionally Calcidiscm lepioporus), Phaeodactylum (optionally Phaeodactylum tricornutum), Chaetoceros (optionally Chaetoceros neogracilis), Dimaliella (optionally Dunaliella tertiolecta), Meristiella (optionally Meristiella gelidium), Ulva (optionally Ulva laciuca or Ulva rigida, e.g.
  • Chorda filum (L) Siackh), Chondrus (optionally Chondrm crispus, e.g. Chondrus crispus Stackh), Phyllophora (optionally Phyllophora psetidoceranoides, e.g PhyUophora pseudoceranoides (Gmelin)), Porphyra (optionally Porphyra umbilical is, e.g. Porphyra umbUkalis, (L) J. Ag), Polysiphonia (optionally Polys iphonia rtigrescens, e.g. Poiysiphonia nigrescens (Huds.) Greville), Furcellaria (optionally Furcellaria lumbricalis, e.g.
  • Hypnea musciformis (Wulfen) Lamouroux), Aspamgopsis (optionaUy Aspamgopsis taxiformis, e.g. Aspamgopsis iaxiformis (Delile) Trev), Gelidium (optionally Gelidium canariensis), Falkenbergia (optionaily Falkenbergia hillebrandii, e.g. Falkenbergia hillebrandii (Born.) Falkenti), Corallina (optionally Coraliim officinalis), Gracilariopsis (optionally Gracilariopsis lemaneiformis), Gracilaria (optionally Gracilaria cornea, e.g. Gracilaria cornea ,1.
  • Methylosinus (optionally Metltylosinus trichosporium, e.g. Meihylosinus trichosporium OB3b), Desulfitobacterium (optionally Desulfitobacterium frappieri, e.g. Desulfitobacterium frappieri TCE1, or Desulfitobacterium metallireducens), Meihylomicrobium (optionally Methylomicrobium album, e.g. Methylomicrobium album BGS), Methylococcus (optionally Methylococcus capsuiatus, e.g. Methylococcus capsuiatus (Bath)), Ralsioma (e.g. Ralstonia sp.
  • Methylosinus (optionally Metltylosinus trichosporium, e.g. Meihylosinus trichosporium OB3b), Desulfitobacterium (optionally Desulfitobacterium frappieri, e.g
  • Pseudomonas optionally Pseudomonas putida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4
  • Rhodobacter optionally Rliodobacter sphaeroides or Rhodobacter capsuiatus
  • Burkholderla optionally Burkholderia cepacian, e.g. Burkholderia cepacian G4
  • Burkholderla optionally Burkholderia cepacian, e.g. Burkholderia cepacian G4
  • the TCE producing microorganism may comprise a consortium of microorganisms.
  • the reaction medium comprising the TCE producing enzyme may additionally comprise a chloride source, for example a chloride salt (e.g. NaCI, KC!, MgCL, CaCb or combinations thereof) arsd / or HC!.
  • a chloride salt e.g. NaCI, KC!, MgCL, CaCb or combinations thereof
  • the TCE producing microorganism may express at least one TCE producing enzyme selected from phenol hydrase (PH), particulate methane monooxygenase (pMMO), benzene (and/brtofuene) dioxygenase (BDO/ToD), toluene o-xyfene monooxygenase oxygenase subunit (TouA), toluene-4-monooxygenase system hydroxylase component subunit alpha, chlorobenzene dioxygenase, ds-ch!orobenzene dihydrodiol dehydrogenase, toluene 2-monooxygenase, or a combination thereof
  • TCE producing enzyme selected from phenol hydrase (PH), particulate methane monooxygenase (pMMO), benzene (and/brtofuene) dioxygenase (BDO/ToD), toluene o
  • the TCE producing microorganism may express PH having an amino acid sequence at least 95% identical to SEQ ID NO: 13 by expressing a PH nucleotide sequence at least 95% identical to SEQ ID NO: 14; expresses pMMO having an amino acid sequence at least 95% identical to SEQ ID NO: 15 by expressing a pMMO nucleotide sequence at least 95% identical to SEQ ID NO: !
  • the glucose producing microorganism expresses at least one glucose producing enzyme, wherein the glucose producing microorganism is capable of utilizing a carbon source and a hydrogen source to produce glucose.
  • the carbon source includes a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof.
  • the hydrogen source comprises water.
  • the chloride source may any source of chloride, including chloride salt (e.g. NaCl, KCL MgCh, CaCb or combinations thereof) and / or HCI.
  • Embodiments of a glucose producing recombinant microorganism can provide a benefit of capturing a carbon source, such as carbon dioxide, into a chemical form, such as glucose, that can be utilized by the VCM producing or the TCE producing recombinant microorganism for the production of VCM or TCE.
  • a carbon source such as carbon dioxide
  • a chemical form such as glucose
  • the glucose producing enzyme includes sucrose permease (cscB), sucrose-phosphate synthase (sps), glucose- 1 -phosphate adenyly!iransferase (glgC), sucrose phosphate phosphatase (spp), glycogen phophorylase (gigP), UDP-gtucose pyrophosphoryia.se (ga!U), invertase, glucosylglycerol-phosphate synthase (ggpS), glycogen synthase (glgA); or combinations thereof.
  • sucrose permease cscB
  • sucrose-phosphate synthase sps
  • glgC glucose- 1 -phosphate adenyly!iransferase
  • spp sucrose phosphate phosphatase
  • gigP glycogen phophorylase
  • gigP UDP-gtucose pyrophosphoryia.se
  • the glucose producing microorganism expresses cscB having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29 by expressing a non-native cscB nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30.
  • the glucose producing microorganism expresses sps having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:
  • the glucose producing microorganism expresses glgC having an amino add sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to SEQ ID NO: 33 by expressing a non-native gigC nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34.
  • the glucose producing microorganism expresses spp having an amino add sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to SEQ ID NO:
  • the glucose producing 30epacian30nism expresses glgP having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37 by expressing a non-native g!gp nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38.
  • the glucose producing microorganism expresses ga!U having an amino acid sequence 90%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to SEQ ID NO: 39 by expressing a non-native ga!U nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40.
  • the glucose producing microorganism expresses invertase having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41 by expressing a non-native invertase nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42.
  • the glucose producing microorganism expresses ggpS having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43 by expressing a non-native ggpS nucleotide sequence at least 90%.
  • the glucose producing 30epacian30nism expresses gigA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to SEQ ID NO: 45 by expressing a non-native gigA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46,
  • the glucose producing recombinant organism includes a recombinant microorganism, a photosynthetic microorganism, a Cyanobacteria, a Symchococcus, Symchococcus elongates, Synechococcm leopoliensis Synechocystis, Anabaena, a Pseudomonas, Pseudomonas syringae, Pseudomonas savasianoi, Chlamydomonas, Chlamydomonas reinhardtii, algae, microalgae, electrosynthesis bacteria, a photosynthetic microorganism, yeast, filamentous fungi, or a plant cell.
  • a method of producing glucose comprising: providing a reaction medium comprising a glucose producing recombinant microorganism as described herein, a hydrogen source and a carbon source; maintaining the reaction medium under conditions which permit the production of glucose by the glucose producing microorganism; and collecting glucose from the reaction medium.
  • the hydrogen source may comprise water and / or the carbon source may comprise a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source.
  • Embodiments of methods of producing a vinyl chloride monomer (VCM) producing recombinant microorganism having an VCM improved producing ability or a trichloroethylene (TCE) producing recombinant microorganism having an improved TCE producing ability are disclosed herein.
  • the method includes: producing the VCM producing recombinant microorganism by inserting at least one of a nonnative VCM expressing nucleotide sequence or a non-native TCE expressing nucleotide sequence into a bacterial plasmid of a microorganism.
  • the method includes: producing the VCM producing recombinant microorganism by inserting at least one of a native VCM expressing nucleotide sequence, a nucleotide sequence which promotes the overexpression of a native VCM expressing nucleotide sequence, a native TCE expressing nucleotide sequence and / or a nucleotide sequence which promotes the overexpression of a native TCE expressing nucleotide sequence into a bacterial plasmid of a microorganism.
  • the native or non-native VCM expressing nucleotide sequence has a nucleotide sequence at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or a combination thereof
  • the native or non-native TCE expressing nucleotide sequence has a nucleotide sequence at least 95% identical to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or a combination thereof.
  • the embodied methods of producing VCM producing and TCE producing recombinant microorganisms can provide a benefit of enabling the production of microbial biomanufacturing systems that can produce VCM and TCE sustainably and on a commercial scale.
  • the VCM producing recombinant microorganism includes microorganisms of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehaiococcoides strain PL2, Dehaiococcoides mccarty e.g. strains KS. RC, JNA, MB, Ha or GY50, Dehaiococcoides ethenogenes e.g.
  • strain 195 Dehaiococcoides strain BAVI, Dehaiococcoides strain VS, Dehaiococcoides strain CBDB1, Dehaiococcoides strain GT) Escherichia (optionally Escherichia coli), Synechococcus (optionally Syneckococcus elongatus ), Sulfurospiriilum (optionally Sulfurospirillum multivorans or Sulfurospirillum bamesii), Dehalohacter (optionally Dehalobacter restrictus), Desulfuromonas (optionally strain BB1 or Destdfuromonas cMoroethenica), Desulfitobacterium (optionally Desulfiiobacterium hafriiense ), Geobacier (optionally Geobacter bemidjiemis, Geobacier lovleyi, Geobacter psychrophilus, Geobacier sp.
  • FRC-32 Geobacier sp. M21, Geobacter sulfiureducem, or Geobacier uraniireducens
  • Pelobacter optionally Pelobacter propionicus
  • Dehalogenimonas optionally sp. Strain WBC-2 Pseudomonas, Flavobacterium, Comamonas, Cytophaga, Acidovorax, Sp-hingomonas, Bacillus, Acinetobacter or a combination thereof
  • the TCE forming recombinant microorganism includes microorganisms of the genus Rhtobium (optionally Rhizohium meliloti e.g. Rhizobium meliloti strain Dangeard).
  • Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emiliam huxleyi), Simrhizobium (optionally Simrhizobium meliloti), Calcidiscus (optionally Calcidiscus leptoporus), Phoeodoctylum (optionally Phaeodactylum tricornutum), Chaetoceros (optionally Chaetoceros neogracilis), Dunalietla (optionally Dunaliella tertiolecta), Meristiella (optionally Merisiiella gelidium), Ulva (optionally Ulva laciuca or Ulva rigida, e.g.
  • Meihylosinus trichosporium OB 3b Destdfitobacterium (optionally Desulfltobacierium frappieri, e.g. Desulfitobacterium floppier i TCE1, or Desulfitobacterium metalUreducens), Methylomicrobium (optionally Methylomicrobium album, e.g. Methylomicrobium album BG8), Methylococcus (optionally Methylococcus capsulatus, e.g. Methylococcus capsulatus (Bath)), Ralstonia (e.g. Ralstonia sp.
  • Pseudomonas optionally Pseudomonas putida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4
  • Rlwdobacier optionally Rkodobacter sphaeroides or Rhodobacier capsulatus
  • Burkholderia optionally Burkholderia cepacian. e.g. Burkholder ia cepacian G4
  • Burkholderia optionally Burkholderia cepacian. e.g. Burkholder ia cepacian G4
  • the VCM producing recombinant microorganism and the TCE producing recombinant microorganism are the same or different.
  • Such embodimen ts can provide a benefit of versatility for the design of biomanufacturing systems and methods for the production of VCM and TCE.
  • method 700 includes: reacting a carbon source with a hydrogen source in the presence of a glucose producing catalytic element 702; producing glucose 704; reacting glucose with a chloride source in the presence of a TCE producing catalytic element 706; and producing TCE 708,
  • Embodiments of methods of producing trichloroethylene are disclosed herein.
  • the method includes: producing TCE by reacting glucose with a chloride source in the presence of a TCE producing catalytic element, wherein the TCE producing catalytic element comprises a TCE bioreaetor culture containing a TCE producing recombinant microorganism having an improved TCE producing ability, wherein the TCE producing recombinant microorganism expresses the at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or ii) overexpressing at least one native TCE producing enzyme nucleotide sequence, and wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
  • Embodiments of methods of producing TCE herein can provide a benefit of sustainable TCE production on a commercial scale, while avoiding environmental harm caused by
  • the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, giycera!dehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, cellobiose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example C os, Cos, CM or C1.2 chlorinated alkanes (such as chloroform and / or carbon tetrachloride). In embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltitoi and / or xylitol).
  • monosaccharides such as xylose, giycera!dehyde, fructose, galactose, mannose and / or glucose
  • disaccharides such
  • the chloride source may any source of chloride, including chloride salt (e.g. Nad, KCi, MgCk, CaCk or combinations thereof) and / or HC!.
  • chloride salt e.g. Nad, KCi, MgCk, CaCk or combinations thereof
  • the method further includes: producing glucose by reacting a carbon source with a hydrogen source in the presence of a glucose producing catalytic element including at least one photosynthesis enzyme comprising Rubisco, wherein the glucose producing catalytic element includes a glucose bioreactor culture containing a glucose producing microorganism, wherein the glucose producing microorganism expresses the at least one glucose producing enzyme, and wherein the glucose producing microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose.
  • a glucose producing catalytic element including at least one photosynthesis enzyme comprising Rubisco
  • the glucose producing catalytic element includes a glucose bioreactor culture containing a glucose producing microorganism, wherein the glucose producing microorganism expresses the at least one glucose producing enzyme, and wherein the glucose producing microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose.
  • the method farther includes a VCM producing catalytic element, wherein the VCM producing catalytic element includes a VCM bioreactor culture containing a VCM producing recombinant microorganism having an improved VCM producing ability, wherein the VCM producing recombinant microorganism expresses the at least one VCM producing enzyme by i) expressing at least one non-native VCM producing enzyme nucleotide sequence and i or ii) overexpressing the at least one native VCM producing enzyme, and wherein the VCM producing recombinant microorganism is capable of utilizing TCE to produce VCM.
  • the VCM producing catalytic element includes a VCM bioreactor culture containing a VCM producing recombinant microorganism having an improved VCM producing ability
  • the VCM producing recombinant microorganism expresses the at least one VCM producing enzyme by i) expressing at least one non-native VCM producing enzyme nucleotide sequence and i or ii) over
  • the carbon source includes a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof
  • the hydrogen source includes water.
  • the chloride source includes sodium chloride, HCI, or a combination thereof.
  • Embodiments of the methods herein can provide a benefit of a sustainable use of carbon sources for the production of useful chemical compounds such as VCM and TCE. Besides helping to conserve natural resources, the embodied methods can provide for chemical production by recombinant microorganisms that can provide a benefit of reducing harm to the environment caused by conventional chemical manufacturing processes, thereby supporting long-term ecological balance.
  • Embodiment 1 A method of forming vinyl chloride monomer (VCM) from trichloroethylene (TCE) comprising: reacting TCE in the presence of a VCM forming catalytic element, wherein the VCM forming catalytic element comprises at least one VCM forming enzyme.
  • VCM vinyl chloride monomer
  • TCE trichloroethylene
  • Embodiment 2 The method of embodiment 1, wherein the at least one VCM forming enzyme comprises TCE reductive dehalogenase (Tee A), TeeA anchor protein (TceB), tetrachloroethy lene reductive dehalogenase (peeA), or a combination thereof.
  • Tee A TCE reductive dehalogenase
  • TceB TeeA anchor protein
  • peeA tetrachloroethy lene reductive dehalogenase
  • Embodiment 3 The method of any of the above embodiments i-2, further comprising: forming TCE by reacting glucose with a chloride source in the presence of a TCE forming catalytic element, wherein the TCE forming catalytic element comprises at least one TCE forming enzyme.
  • Embodiment 4 The method of any of the above embodiments 1-3, wherein the at least one TCE forming enzyme comprises phenol hydrase (PH), particulate methane monooxygenase (pMMO), toluene dioxygenase (ToD), or a combination thereof.
  • PH phenol hydrase
  • pMMO particulate methane monooxygenase
  • ToD toluene dioxygenase
  • Embodiment 5 The method of any of the above embodiments 1-4, further comprising forming glucose by reacting a carbon source with a hydrogen source in the presence of a glucose forming catalytic element comprising at least one glucose forming enzyme.
  • Embodiment 6 The method of any of the above embodiments 1-5, wherein the carbon source comprises a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof; or the hydrogen source comprises water; or the chloride source comprises sodium chloride, HCI, or a combination thereof.
  • the carbon source comprises a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof
  • the hydrogen source comprises water
  • the chloride source comprises sodium chloride, HCI, or a combination thereof.
  • the VCM forming catalytic element comprises a VCM bioreactor culture containing a VCM forming recombinant microorganism having an improved VCM producing ability
  • the VCM forming recombinant microorganism expresses the at least one VCM forming enzyme by i) expressing at least one non-native VCM forming enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM forming enzyme nucleotide sequence, wherein the VCM forming recombinant microorganism is capable of utilizing TCE to produce VCM
  • Embodiment 8 The method of any of the above embodiments 1-7, wherein the VCM forming recombinant microorganism comprises Dehalococcoides sp., Dehalococcoides strain FL2, Dehalococcoides strain BAV1, Dehalococcoides consortia, E coli, a Cyanobacteria, Synechococcus elongatus, and combinations thereof; or further comprising maintaining a temperature in the VCM bioreactor culture of from about 15 degrees Celsius to about 65 degrees Celsius; or maintaining a pH in the VCM bioreactor culture of from about 4,5 to about 12,
  • Embodiment 9 The method of any of the above embodiments 1-8, wherein the TCE forming catalytic element comprises a TCE bioreactor culture containing a TCE forming recombinant microorganism having an improved TCE producing ability, wherein the TCE forming recombinant microorganism expresses the at least one TCE forming enzyme by i) expressing at least one non-native TCE forming enzyme nucleotide sequence and i or ii) overexpressing at least one native TCE forming enzyme nucleotide sequence, wherein the TCE forming recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
  • Embodiment 10 The method of any of the above embodiments 1-9, wherein the TCE forming recombinant microorganism comprises Rhizohhm mdiloti , Rhkobium meliloti strain Dangeard, Porphyridium purpureum, Emiliania huxleyi, Sinorhizobium mdiloti , and combinations thereof,
  • Embodiment 11 The method of any of the above embodiments 1-10, wherein the VCM bioreactor culture and foe TCE bioreactor culture are the same, or wherein the VCM forming recombinant microorganism and the TCE forming recombinant microorganism are the same.
  • Embodiment 12 The method of any of the above embodiments 1-1 1, wherein the glucose forming catalytic element comprises a glucose bioreactor culture containing a glucose forming microorganism, wherein foe glucose forming microorganism expresses the at least one glucose forming enzyme, wherein the glucose forming microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose.
  • the glucose forming microorganism comprises a recombinant microorganism, a photosynthetic microorganism, a Cyanobacteria, a Syneckococcus, Synechococcm elongates, Synechocoecus leopoliensis, Synechocystis, Anahaena, a Pseudomonas, Pseudomonas syringae, Pseudomonas savastanoi, Chlamydomonas, Chlamydomoms remhardtii, Escherichia, Escherichia coli, Geobacteria, algae, microalgae, electrosynthesis bacteria, a photosynthetic microorganism, yeast, filamentous fungi, and a plant cell.
  • Embodiment 14 The method of any of the above embodiments 1-13, further comprising generating biomass and oxygen in the glucose bioreactor culture and removing an amount of the biomass from the glucose forming bioreactor culture, the TCE forming bioreactor culture, the VCM forming bioreactor culture, or a combination thereof; or maintaining an amount of oxygen in the glucose forming bioreactor culture of from about 20 % by volume to about 250 % by volume or less, based on a total internal volume of the glucose forming bioreactor culture; or converting the carbon source to glucose at an efficiency of from about 30% to about 80%; or maintaining a temperature in the glucose forming bioreactor of from about 15 degrees Celsius to about 45 degrees Celsius; or maintaining a pH in the glucose forming bioreactor of from about 4 to about 12.
  • Embodiment 15 The method of any of the above embodiments 1-14, further comprising converting glucose to glyoxylaie, and reacting glyoxylate with the chloride source to form TCE; or increasing a rate of TCE formation by maintaining a concentration of glucose in the TCE hioreactor culture of from about 5 g/L to about 16 g/L; or maintaining a temperature in the TCE bioreactor culture of from about 30 degrees Celsius to about 37 degrees Celsius; or maintaining a pH in the TCE hioreactor culture of from about 6.5 to about 8.5.
  • a biomanufacturing system for producing vinyl chloride monomer comprising: at least one VCM forming recombinant microorganism having an improved VCM producing ability, wherein the at least one recombinant organism expresses at least one VCM forming enzyme by i) expressing at least one non-native VCM forming enzyme nucleotide sequence and / or is) overexpressing at least one native VCM forming enzyme, wherein the at least one recombinant organism is capable of utilizing a chlorinated hydrocarbon source to produce VCM.
  • VCM vinyl chloride monomer
  • Embodiment 16 further comprising: at least one TCE forming recombinant microorganism having an improved TCE producing ability, wherein the at least one recombinant organism expresses at least one TCE forming enzyme by i) expressing at least one non-native VCM forming enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM forming enzyme nucleotide sequence, wherein the at least one recombinant organism is capable of utilizing a chlorinated hydrocarbon source to produce VCM.
  • Embodiment 18 The system of any of the above embodiments 16-17, wherein the at least one VCM forming recombinant microorganism and the at least one TCE forming recombinant microorganism are the same; or further comprising a glucose forming microorganism,
  • Embodiment 19 The system of any of the above embodiments 16-18, wherein the at least one VCM forming recombinant microorganism comprises Dehalococcoid.es sp y ., Dehalococcoides strain FL2, Dehahcoccoides strain BAV1 , Dehalococcoides consortia, E.
  • the at least one TCE forming recombinant microorganism comprises Rhizobium meliloti, Rhizobium meiiloti strain Dangeard, Porphyridium purpureum, Emiliania huxleyi, Sinorhizobium meliloti, and combinations thereof or wherein the glucose forming microorganism comprises a recombinant microorganism, a photosynthetic microorganism, a Cyanobacteria, & Synechococcus, Synechococcus elongaius, Synechococcus ieopoliensis, Synechocystis, Anabaena, a Pseudomonas, Pseudomonas syringae, Pseudomonas savastanoi, Chiamydomonas, Chlamydomonas
  • Embodiment 20 A method of forming vinyl chloride monomer (VCM) from trichloroethylene (TCE) comprising: forming glucose by reacting a carbon source with a hydrogen source in the presence of a glucose forming catalytic element comprising at least one glucose forming enzyme, wherein the glucose forming catalytic element comprises a glucose bioreactor culture containing a glucose forming microorganism, wherein the glucose forming microorganism expresses the at least one glucose forming enzyme, wherein the glucose forming microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose; forming TCE by reacting glucose with a chloride source in the presence of a TCE forming catalytic element, wherein the TCE forming catalytic element comprises a TCE bioreactor culture containing a TCE forming recombinant microorganism having an improved ICE producing ability, wherein the TCE forming recombinant microorganism expresses the at least one TCE forming
  • Embodiment 21 The method of Embodiment 20, wherein the carbon source comprises a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof; or the hydrogen source comprises water; or the chloride source comprises sodium chloride, HC1, or a combination thereof.
  • a genetic engineering approach was designed for the production of VCM from C02 (see Fig. 1).
  • a glucose producing recombinant microorganism of the invention (“microbe A”) performs carbon capture and utilization through utilizing a carbon dioxide source to produce glucose and biomass.
  • the glucose produced is utilized by one or more genetically engineered VCM or TCE producing microbes (“microbe B”) to produce TCE and VCM.
  • the bioVCM phase 1 focused on the genetic engineering of microbe B.
  • two microbes are engineered to be capable of performing each of the two reactions separately, ch!oride/giyoxyiate/formate to TCE (“Reaction 1”), and TCE to VCM (“Reaction 2”).
  • Reaction 1 ch!oride/giyoxyiate/formate to TCE
  • Reaction 2 TCE to VCM
  • the capabilities of the microbes engineered to perform Reaction 1 and Reaction 2 separately will also be combined into a single microbe 8 capable of performing both reactions,
  • E. col cells and Cyanobacteria Syneckococcus elongalus UTEX 2434 are maintained and grown on LB and BG11 media respectively, supplemented with the appropriate antibiotics.
  • DNA constructs were synthesized by Genewiz. Primers for PCR (polymerase chain reaction) were designed using Bench! ing and PrimerS software, and synthesized by MiiliporeSigma.
  • TceA (SEQ ID NO: 2), TceAB (SEQ ID NO: 47), pH (SEQ ID NO: 14), pMMQ (SEQ ID NO: 16), ToD (SEQ ID NO: 18), and peeA (SEQ ID NO: 6). All gBlocks were flanked with BamHI (SEQ IDNO: 48) and Mindlll (SEQ ID NO: 49) recognition sites for adequate cloning. Construction of plasmids pET30a_TeeA, pET30a_pH, pET30a_pMMO, pET30a_ToD and pET30a peeA was achieved by digesting the pET30a plasmid and gBlocks with BamHI and Hindi!
  • NEB DNA Ligase
  • the resulting ligation was transformed into DHSaipha competent cells (NEB5alpha) and plated onto LB medium plates supplemented with spectinomycin.
  • the confirmation of the correct assembly and insertion of the aforementioned genes into either the pET30a or pSyn6 plasmids was ascertained by Sanger sequencing. The sequencing results matched the desired sequence.
  • the cells were resuspended with 100 uL ofBGl 1.
  • a plasmid concentration between 200 to 500 ng was added, and the culture was gently flicked.
  • the mixture was incubated in a 34°C water bath with a dark lid for at least 4 hours.
  • a volume of 0,5 mL 13 ( 311 was added to the transformation and transferred to a conical tube and incubated overnight at room temperature, ensuring even and continuous illumination by covering the conical tube with kim wipes.
  • I rnL of BG i 1 media supplemented with spectinomycin was added to a final concentration of 10 ug/mL,
  • the culture was then induced by adding Isopropyl b-D-l- thiogalaetopyranoside (IPTG) to a final concentration of 0.5 to 2 mM.
  • IPTG Isopropyl b-D-l- thiogalaetopyranoside
  • the culture was grown for an additional 2-4 hours.
  • Ceil biomass was obtained by centrifugation and stored at 4°C until purification.
  • For engineered Cyanobacteria ceils were grown in fresh media to an OD of 0.6-1. Cell biomass was then obtained by centrifugation and stored at 4°C until purification.
  • Induction assays For the induction assay, after the engineered E. coli cells were induced by IPTG and grown for an additional 2-4 hours, samples were collected from the main cel! culture for gas chromatography (GC) analysis. Cell culture samples (SOOgX.) were added to 2mL gas chromatography (GC) vials. Substrates (TCE, NaCi or HC1) were added to the vial at different concentrations. The vials were crimped and incubated for different time periods. Once done, vials were stored at -20°C until GC analysis,
  • VCM Native bacteria and consortia. Since VCM was shown to be involved in the natural process, we first determined whether there are native microbes that can produce or metabolize trichloroethylene (TCE) or vinyl chloride monomer (VCM). The goal was to understand the natural chlorination pathway for production of VCM. from which enzyme candidates could be identified for genetic engineering.
  • TCE trichloroethylene
  • VCM vinyl chloride monomer
  • Reaction 1 chloride to TCE, microbe 81
  • TCE Rkizobium meiiloti Dangeard
  • a ICC Porphyridium purpureum
  • UTEX Emiliania huxleyi
  • PH phenol hydrolase
  • pMMO particulate methane monooxygenase
  • To D toluene d ioxygena.se
  • Sequencing results confirmed the presence of the phenol hydrolase gene in the p£T30 vector to form the rET30-RH plasmid, and the presence of the particulate methane monooxygenase gene in the pET3Q vector to form the pE ' DO-pMMO plasmid. Sequencing results also confirmed the presence of the Toluene dioxygenase gene in the pET30 vector to form the pET30-ToD plasmid.
  • microbe B2 a gene encoding TCE reductive dehalogenase (TeeA, SEQ ID NO: 2) and its anchor protein (TceB, SEQ ID NO: 4), originated from Dehalococcoides , were obtained from GenBank.
  • TeeA TCE reductive dehalogenase
  • TceB anchor protein
  • peeA tetrachioroethyiene reductive dehalogenase
  • peeA was also obtained from GenBank.
  • the genes were cloned into plasmids to generated plasmids pSyn6-TceAB, pSyn6-TceA, pET30-TceA, and pET30-PceA.
  • pSyn6- TceAB contains both the TCE reductive dehalogenase gene (TceA) and its anchor protein (SEQ I ' D NO: 47), whereas pSyn6-TceA contains only the gene encoding the enzyme (SEQ ID NO: 2).
  • TceA TCE reductive dehalogenase gene
  • SEQ I ' D NO: 47 anchor protein
  • pSyn6-TceA contains only the gene encoding the enzyme (SEQ ID NO: 2).
  • the two plasmids were created and their sequences were confirmed by gene sequencing.
  • the two plasmids were transformed into Cyanobacteria Symckococcus elongatus UTEX 2434 (henceforth, S2434) to create two new engineered Cyanobacteria, S2434-TceAB and S2434-TceA.
  • Several engineered cell cultures were created. The engineered cells were then confirmed using polymerase chain reaction (PCR). Gel electrophoresis confirmed the engineered Cyanobacteria 82434-Tce.AB and S2434-TceA.
  • graph 201 TCE, tetrachloroethylene
  • graph 202 TCE, trichloroethylene
  • graph 203 DCE, dichloroethyiene
  • graph 204 VCM, vinyl chloride monomer
  • graph 205 Ethylene. All compounds show distinct retention times.
  • Y axis peak height (pA)
  • TCE production was tested from the soil bacteria S. meliloti. Data showed that the bacteria has the potential to produce TCE from the sample.
  • a protocol was adapted from a protocol in which S. meliloti was grown in undefined media (soil extracts). In the protocol, cells were grown and maintained in soil extract medium or Vincents minimal media with 20 mmol/L succinate as carbon source at 28°C under rotation. For the experiment, 1 OmL of cell culture were grown to an ODeoonm of i .0. If the OD measured was higher than 1, the culture was diluted with fresh media. For detection of TCE, 0.5mL of culture was added into each GC vial.
  • the vials were incubated at 30°C for different time periods (up to 48 hours). The vials were removed at appropriate times and stored at -20°C until testing.
  • Fig, SB shows that VCM production started before the induction due to leaky expression of Tee A enzyme.
  • Methanol was used as a solvent.
  • Cell cultures initially were fed with 250 pg/snL of TCE. It has been shown that enzyme expression under the IPTG-inducibie T7 promoter can be leaky, meaning that the enzyme can be produced (at low level) even without the inducer.
  • VCM yield from two cultures of engineered A. colt BL2l-TceA cultures is shown In Table 5, The data show that in E. coii BL21-TceA culture #2, 202.9 gg/mL of VCM was produced, The ratio indicated that more than 80% of the TCE is converted to VCM. With higher substrate amounts, the production capacity of the engineered microbe can be significantly increased.
  • Fig, 6A shows GC data from engineered Cyanobacteria (S2434-TceAB) and shows the production of VCM from TCE. Left, chromatogram of the GC. Right, gas composition from the sample. Peak #1 is VCM, #2 methanol, #3 DCE, and #4 TCE.
  • TCE was fed to the native, non- engmeered S2434 and several cultures of engineered Cyanobacteria (82434-TceAB arid S2434-TceA).
  • Fig. 6B shows GC data from native Cyanobacteria S2434, and did not show any VCM production.
  • Fig, 6C shows GC data from engineered Cyanobacteria S2434- TceAB, and shows a VCM peak, in the graphs, TCE, tetrachloroethylene; VCM, vinyl chloride monomer.
  • Table 6 shows that the yield of engineered Cyanobacteria is 10 times higher than that of the Dehalococcoides consortia. An amount of 144 pg/mL VCM was shown to be produced from 250 pg/mL TCE fed. That value is equal to 58% of TCE conversion.
  • SEQ ID. NO. 6- Dehalococcoides - peeA
  • SEQ ID NO. 10 Uncultured bacterium ⁇ cicpA
  • CTGCGAGC AGCCG CGTGCGTTCTGGCTGGCC A ACCGTCCCGGCG ATG ACCTGGTG
  • SEQ ID NO. 17 -- Pseudomonas - benzene (toluene) dioxygenase iDSANRADVFLRKPAPVAPELQHEVEQFYYWEAKLLNDRRFEEWFALLAEDiHYFM
  • SEQ ID NO, 18 - Pseudomonas - benzene (toluene) dioxygenase
  • CATCTCGT GAGCAACGTGATGATCGTCGGCGCAGAGGCAGAAGGGGAGTACGAA
  • SEQ ID NO. 20 - Pseudomonas - TouA
  • CTGCAATCGCCCAGTATT CGGTTACAGCTATCGTTTCTAAAGGAGATAATAATAA
  • GAAAATGCTAATAAGACCCTGAAT ’ ATTTAGTTTTAGAAATAGCAATGTGGAAAG
  • GACTG TTCGGGAAACCGTACCCACGATCTGTAACATGTGCAATCTCCCGATTGCC
  • SEQ ID NO. 21 - Pseudomonas - to!uene»4-monooxygenase system hydroxylase component subunit alpha
  • SEQ ID NO, 22 - Pseudomonas - toluene-4-monooxygenase system hydroxylase component subunit alpha
  • SEQ ID NO. 27 ⁇ Pseudomonas MSYQVTIEPTGEQIEVEEGQTILEAALRQGVWLPFACGHGTCATCKCQVLEGEVDLG
  • GAAGGCTCCCGTGCG IYTTCCCTGGCCAACCCGCCCCAGCAGGCCGACGAGGTG
  • SEQ ID MO. 30 ⁇ Escherichia cols - escB

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Abstract

Embodiments of vinyl chloride monomer (VCM) and trichloroethylene (TCE) producing microorganisms having an improved VCM or TCE production ability are disclosed herein, as well as methods for the production of VCM and TCE producing microorganisms. Biomanufacturing systems for producing VCM are disclosed herein, as well as methods of forming VCM from TCE, and methods of forming TCE. A benefit to the various embodiments herein can be providing capabilities for the environmentally sustainable production of VCM and TCE on an industrial scale. A benefit to the VCM and TCE producing microorganisms, and embodiments of biomanufacturing systems herein, can include capabilities for the production of commercially useful quantities of TCE and VCM from microbial cultures. Additional benefits of the microorganisms, systems and methods herein can include the use of carbon dioxide to produce bio-TCE and bio-VCM useful as a feedstock for the production of plastics.

Description

METHODS AND COMPOSITIONS
[0001] This application claims priority to Provisional Application No. 63/208,640, filed June 9, 2021 , The entirety of the aforementioned application is incorporated herein by reference.
FIELD
[0002] Embodiments of vinyl chloride monomer (YCM) and trichloroethylene (TCE) producing microorganisms having an improved VCM or TCE production ability are disclosed herein, as well as methods for the production of VCM and TCE producing microorganisms. Biomanufacturing systems for producing VCM are disclosed herein, as well as methods of forming VCM from TCE, and methods of forming TCE. A benefit to the various embodiments herein can be providing capabilities for the environmentally sustainable production of VCM and TCE on an industrial scale. A benefit to the VCM and TCE producing microorganisms, and embodiments of biomanufacturing systems herein, can include capabilities for the production of commercially useful quantities of TCE and VCM from microbial cultures. Additional benefits of the microorganisms, systems and methods herein can include the use of carbon dioxide to produce bio-TCE and bio-VCM useful as a feedstock for the production of plastics, and for use in other applications; and reduction of excess carbon dioxide from the environment,
BACKGROUND
[6003] The increased demands on the chemical industry worldwide have led to an excess of carbon dioxide from burning fossil fuels such as oil and gas, indirect emissions generated by using electricity and heat and the production of some chemicals, contributing substantially to what many are calling a global warming crisis. Industry is so desperate to prevent carbon dioxide from entering the atmosphere that they have resorted to sequestering carbon dioxide from exhaust streams and the atmosphere. They then store the carbon dioxide in subterranean environments. However, these sequestration methods just remove carbon dioxide from the atmosphere by storing it under ground. They do not actually convert the carbon dioxide back into any other useful material.
[6604] The harmful effects of industrial chemical production on the environment have prompted developments in renewable sources of chemical reagents. Two commercially important chemical reagents include trichloroethylene (TCE) and vinyl chloride monomer (VCM). TCE is widely used as a degreaser and chemical solvent in many consumer products, and as a reagent for the production of other chemicals, including VCM. VCM is almost exclusively used as a feedstock for the production of polyvinyl chloride (PVC), which has a wide range of industrial uses. Currently, VCM is commercially produced by cracking natural gas or petroleum with chlorine. With millions of metric tons of VCM being produced each year, more than enough carbon dioxide is produced by such processes to greatly contribute to the global carbon footprint. Producing TCE and VCM through sustainable, renewable methods would accordingly help to meet the huge demand from chemical industries, while also helping to protect the environment.
SUMMARY
[0005] Embodiments of a vinyl chloride monomer (VCM) producing recombinant microorganism having a VCM improved production ability are disclosed herein. In various embodiments, the VCM producing recombinant microorganism expresses at least one VCM producing enzyme by expressing i) at least one non-native VCM producing enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM producing enzyme nucleotide sequence, wherein an amount of the VCM producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native VCM producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native VCM producing enzyme, and wherein the VCM producing recombinant microorganism is capable of utilizing a chlorinated hydrocarbon source to produce VCM.
[0006] in certain embodiments, the at least one VCM producing enzyme includes TCE reductive dehalogenase (TceA), TceA anchor protein (TceB), tetrachloroethylene reductive dehalogenase (pceA), vinyl chloride reductase (vcrA), 1 ,2-dich!oropropane-to- propene reductive dehalogenase (dcpA), 1 ,2-trans-dishloroethene reductive dehalogenase catalytic A (TdrA), or a combination thereof. In certain embodiments, the VCM producing recombinant microorganism expresses TceA having an amino acid sequence at least 95% identical to SEQ ID NO: 1 by expressing a native or non-native TceA nucleotide sequence at least 95% identical to SEQ ID NO: 2; expresses TceB having an amino acid sequence at least 95% identical to SEQ ID NO: 3 by expressing a native or non-native TceB nucleotide sequence at least 95% Identical to SEQ ID NO: 4; expresses pceA having an amino acid sequence at least 95% identical to SEQ ID NO: 5 by expressing a native or non-native pceA nucleotide sequence at least 95% identical to SEQ ID NO: 6; expresses vcrA having an amino acid sequence at least 95% identical to SEQ ID NO: 7 by expressing a native or nonnative vcrA nucleotide sequence at least 95% identical to SEQ ID NO: 8; expresses dcpA having an amino acid sequence at least 95% identical to SEQ ID NO: 9 by expressing a native or non-native dcpA nucleotide sequence at least 95% identical to SEQ ID NO: 10; expresses TdrA having an amino acid sequence 95% identical to SEQ ID NO: 11 by expressing a native or non-native TdrA nucleotide sequence at least 95% identical to SEQ ID NO: 12; ora combination thereof.
[0007J In embod iments of the invention, the chlorinated hydrocarbon source may be a chlorinated alkane or a chlorinated alkene. In embodiments, the chlorinated hydrocarbon source may be a Cos chlorinated hydrocarbon, a Cw chlorinated hydrocarbon, a Ci4 chlorinated hydrocarbon or a C1-2 chlorinated hydrocarbon. Examples of chlorinated hydrocarbons that may be employed in the present invention include trichloroethylene (TCE), dichlcroethylene (DCE) or chloroform.
[0898] In embodiments of the invention, the V’CM producing recombinant microorganism includes microorganisms of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehalococcoid.es strain PL2, Dehalococeoides mccarty e.g. strains KS, RC, IN .4, MB. 11 a or GY50. Dehalococcoides ethenogenes e.g. strain 195, Dehalococcoides strain BA VI, Dehalococcoides strain VS, Dehalococcoides strain CBDB1, Dehalococcoides strain GT) Escherichia (optionally Escherichia coil ), Synechococcus (optionally Synechococcus elongatus), Suljurospirillum (optionally Sulfitrospirillum multivoram or Sulfurospirillum barnesii), Dehalobacter (optionally Dehalobacter restrictus), Desulfuromonas (optionally strain BB1 or Desulfuromonas ckloroeihenica), Desulfitobacterium (optionally Desulfitobacterium hafnien.se ), Geobacter (optionally Geobacter bemidjiensis, Geobacter lovieyi, Geobacter psychrophilus, Geobacter sp. FRC-32, Geobacter sp. M2 !. Geobacter sulfurreducens, or Geobacter uraniireducens), Pelobacter (optionally Pelobacter propionicus) Dehalogenimonas (optionally sp. Strain WBC-2) Pseudomonas, Flavobacterium, Comamonas, Cytophaga, Acidovorax, Sphmgomonas, Bacillus, Acinetobacter or a combination thereof.
[00091 Embodiments of a trichloroethylene (TCE) producing recombinant microorganism having an improved TCE producing ability are disclosed herein. In various embodiments, the TCE producing recombinant microorganism expresses at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and i or ii) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the TCE producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at feast one native TCE producing enzyme, and wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
10010! hi embodiments of the invention, the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, giyeera!dehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, ceilobiose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example Cj-g, CM, CM or Co? chlorinated alkanes (such as chloroform and / or carbon tetrachloride). In embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltito! and / or xylitol).
[0011] in embodiments, the chloride source may any source of chloride, including chloride salt (
Figure imgf000005_0001
NaCi, KC1, MgCb, CaCh or combinations thereof) and / or HO.
[0012] in certain embodiments, the at least one TCE producing enzyme includes phenol hydrase (PH), particulate methane monooxygenase (pMMO), benzene (and/or toluene) dioxygenase (BDO/ToD), toluene o-xylene monooxygenase oxygenase subunit (TouA), toluene-4-raonooxygenase system hydroxylase component subunit alpha, chlorobenzene dioxygenase, cis-chlorobenzene dihydrodioi dehydrogenase, toluene 2- monooxygenase, or a combination thereof, in certain embodiments, the TCE producing recombinant microorganism expresses PH having an amino acid sequence at least 95% identical to SEQ ID NO: 13 by expressing a native or non-native PH nucleotide sequence at least 95% identical to SEQ ID NO: 14; expresses pMMO having an amino acid sequence at least 95% identical to SEQ ID NO: 15 by expressing a native or non-native pMMO nucleotide sequence at least 95% identical to SEQ ID NO: 16; expresses ToD having an amino acid sequence at least 95% identical to SEQ ID NO; 17 by expressing a native or nonnative ToD nucleotide sequence at least 95% identical to SEQ ID NO: 18; expresses TouA having an amino acid sequence at least 95% identical to SEQ ID NO: 19 by expressing a native or non-native TouA nucleotide sequence at least 95% identical to SEQ ID NO: 20; expresses toluene-4-monooxygenase system hydroxylase component subunit alpha having an amino acid sequence at least 95% identical to SEQ ID NO: 21 by expressing a native or nonnative toiuene~4-monooxygenase system hydroxylase component subunit alpha nucleotide sequence at least 95% identical to SEQ ID NO: 22; expresses chlorobenzene dioxygenase having an amino acid sequence 95% identical to SEQ ID NO: 23 by expressing a native or non-native chlorobenzene dioxygenase nucleotide sequence at least 95% identical to SEQ ID NO: 24; expresses cis-chlorobenzene dihydrodioi dehydrogenase having an amino acid sequence at least 95% identical to SEQ ID NO: 25 by expressing a native or non-native cis- chlorobenzene dihydrodiol dehydrogenase nucleotide sequence at least 95% identical to SEQ ID NO: 26; expresses toluene 2-monooxygenase having an amino acid sequence at least 95% identical to SEQ ID NO: 27 by expressing a native or non-native toluene 2-monooxygenase nucleotide sequence at least 95% identical to SEQ ID NO: 28; or a combination thereof.
[0613] 'In certain embodiments, the TCE producing recombinant microorganism includes microorganisms of the genus Rhizobium (optionally Rhizobium meiiloii e.g. Rhizohium melUoti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emiliam hialeyi), Sinorhizobium (optionally Sinorhizobium meitioti), Calcidiscus (optionally Calcidiscus lep(oporus), Phaeodaciylum (optionally Phaeodactylutn tricornutum), Chaetoceros (optionally Chaetoceros neograciUs ). Dumliella (optionally Duna!ieiia iertiolecta), Meristiella (optionally Meristiella gelidium), Ulva (optionally Ulva iactuca or Ulva rigida, e.g. Ulva rigida Agardh ), Enteromorpha (optionally Enieromorphia intestinalis, Cladophora (optionally Cladopkora rupestris), Fucus (optionally Fucus serratus), Laminaria (optionally Laminaria saccarina, e.g. Laminaria saccarina (L) Lamour or Laminaria digitata, e.g. Laminaria digitata (Ends) Lamour), Desmaresiia (optionally Desmaresiia aculeata, e.g. Desmaresiia aculeata (L) Lamour), Chorda (optionally Chorda fllum, e.g. Chorda filum (L) Stackh), Chondrus (optionally Chondrus crispus, e.g. Chondrus crispus Stackh), Phyllophora (optionally Phyllophora pseudoceranoides, e.g. PhyUophora pseudoceranotdes (Gmelinj), Porphyra (optionally Porpkyra umbilicalis, e.g. Porphyra umbilicalis, (L) J. Ag.), Polysiphonia (optionally Polysiphonia nigrescens, e.g. Polys iphonia nigrescens (Huds.) Greville), Furceilaria (optionally Furcellaria lumbricalis, e.g. Furceliaria lumbricalis (Huds.) Lamour), Ceramium rubrum (optionally Ceramium rubrum, e.g. Ceramium rubrum (Huds.) Agardh), Ahnfeltia (optionally Ahnfeltia plicata, e.g Ahnfeliia plicata (Hudson) Fries), Laurencia (optionally Laurencia pinnaiifida, e.g. Laurencia pinnaiifida (Huds.) Lamour, or Laurencia obtuse, e.g. Laurencia obtusa (Huds) Lamour), Caulerpa, Hypnea (optionally i/gpueo musciformis, e.g. Hypnea musciformis (Wulfen) Lamouroux), Asparagopsis (optionally Asparagopsis taxiformis, e.g. Asparagopsis taxifbrmis (Delik) Trev). Gelidium (optionally Gelidium canadensis), Falkenbergia (optionally Falkenbergia hillebrandii, e.g. Falkenbergia hiliebrandii (Born.) Falkenb),
Coral lina (optionally Corallina officinalis), Gracilariopsis (optionally Gracilariopsis lemaneifbrmis), Gracilaria (optionally Gracilaria cornea, e.g. Gracilaria cornea J. Agardh), Methylosinus (optionally Methylosinus trichosporium, e g. Methylosinus trichosporium OB3h), Desulfitobacierium (optionally Desulfitobacterium frappieri, e.g. Desulfitobacterium frappieri TCE1, or Desulfitobacterium metal! ireducem), Methylomicrobium (optionally Methylomicrobium album, e.g Methylomicrobium a! bum BG8), Meihylococcus (optionally Methylococcus capsulatus, e.g, Meihylococcus capsulaius (Bath)), Ralstonia (e.g. Ralstonia sp. KN 1-lOA), Pseudomonas (optionally Pseudomonas putida, e.g Pseudomonas putida FI, or Pseudomonas sp. M4), Rhodobacter (optionally Rhodobacter sphaeroldes or Rhodobacier capsulatus), Burkholderia (optionally Burkholderia cepacian , e.g. Burkholderia cepacian G4 ) or a combination thereof.
[0014J In some aspects, the VCM producing recombinant microorganism expresses at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or is) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme nucleotide sequence, and wherein the VCM producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
[0015] in embodiments of the invention, the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, giyceraldehyde, fmcto.se, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, ceilobiose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example Cj-a, Cue, CM or Cue chlorinated alkanes (such as chloroform and / or carbon tetrachloride). In embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltitol and / or xylitof).
[0016] In embodiments, the chloride source may any source of chloride, including chloride salt (e.g. NaCl, KCI, MgCb, CaCh or combinations thereof) and / or HCi.
[0017] Embodiments of a glucose producing recombinant organism are disclosed herein. In various embodiments, the glucose producing microorganism expresses at least one glucose producing enzyme, wherein the glucose producing microorganism is capable of utilizing a carbon source and a hydrogen source to produce glucose. In certain embodiments, the carbon source includes a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof, in certain embodiments, the hydrogen source comprises water. In embodiments, the chloride source may any source of chloride, including chloride salt (e.g. NaCl, KCI, MgCb, CaCb or combinations thereof) and / or HCI. [OOlSjj in certain embodiments, the glucose producing enzyme includes sucrose permease (cscB), sucrose-phosphate synthase (sps), glucose- 1 -phosphate adeny ly Itransferase (glgC), sucrose phosphate phosphatase (spp), glycogen phophorylase (glgP), UDP-glucose pyrophosphorylase (galU), invertase, glucosy!gjycerol-phosphate synthase (ggpS), glycogen synthase (gigA); or combinations thereof. In certain embodiments, the glucose producing microorganism expresses cscB having an amino acid sequence at least 95% identical to SEQ ID NO: 29 by expressing a non-native cscB nucleotide sequence at least 95% identical to SEQ ID NO: 30; expresses sps having an amino acid sequence at least 95% identical to SEQ ID NO: 31 by expressing a non-native sps nucleotide sequence at least 95% identical to SEQ ID NO: 32; expresses glgC having an amino acid sequence at least 95% identical to SEQ ID NO: 33 by expressing a non-native glgC nucleotide sequence at least 95% identical to SEQ ID NO: 34; expresses spp having an amino acid sequence at least 95% identical to SEQ ID NO: 35 by expressing a non-native spp nucleotide sequence at least 95% identical to SEQ ID NO: 36; expresses gigp having an amino add sequence at least 95% identical to SEQ ID NO: 37 by expressing a non-native glgP nucleotide sequence at least 95% identical to SEQ ID NO: 38; expresses galU having an amino acid sequence 95% identical to SEQ ID NO: 39 by expressing a non-native gaili nucleotide sequence at least 95% identical to SEQ ID NO: 40; expresses invertase having an amino acid sequence at least 95% identical to SEQ ID NO: 41 by expressing a non-native invertase nucleotide sequence at least 95% identical to SEQ ID NO: 42; expresses ggpS having an amino acid sequence at least 95% identical to SEQ ID NO: 43 by expressing a non-native ggpS nucleotide sequence at least 95% identical to SEQ ID NO: 44; expresses gigA hav ing an amino acid sequence at least 95% identical to SEQ ID NO: 45 by expressing a non-native gigA nucleotide sequence at least 95% identical to SEQ ID NO: 46; or a combination thereof.
[6019] In certain embodiments, the glucose producing recombinant organism includes a recombinant microorganism, a photosynthetic microorganism, a Cyanobacteria, a Syneehococeus, Symchococcus elongatas, Synechococcm leopoiiemis, Symchocystis, Anabaena . a Pseudomonas, Pseudomonas syringae, Pseudomonas savastanoi, Chlamydomonas, Chlamydomonas reinhardtii, algae, microalgae, electrosynthesis bacteria, a photosynthetic microorganism, yeast, filamentous fungi, or a plant cell.
[0020] Embodiments of methods of producing a vinyl chloride monomer (VCM) producing recombinant microorganism having an VCM improved producing ability or a. trichloroethylene (TCE) producing recombinant microorganism having an improved TCE producing ability are disclosed herein. In various embodiments, the method includes: producing the VCM producing recombinant microorganism by inserting at least one of a non- native VCM expressing nucleotide sequence or a non-native TCE expressing nucleotide sequence into a bacterial plasmid of a microorganism. Additionally or alternatively, the method includes: producing the VCM producing recombinant microorganism by inserting at least one of a native VCM expressing nucleotide sequence, a nucleotide sequence which promotes the overexpression of a native VCM expressing nucleotide sequence, a native TCE expressing nucleotide sequence and / or a nucleotide sequence which promotes the overexpression of a native TCE expressing nucleotide sequence into a bacterial plasmid of a microorganism. In certain embodiments, the native or non-native VCM expressing nucleotide sequence has a nucleotide sequence at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or a combination thereof in certain embodiments, the native or non-native TCE expressing nucleotide sequence has a nucleotide sequence at least 95% Identical to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or a combination thereof
[1)621] In embodiments of the invention, the VCM producing recombinant microorganism includes microorganisms of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehalococcoides strain FL2, Dehalococcoides mccariy e.g. strains KS, EC, INA, MB, 1 la or GY50, Dehalococcoides ethenogenes e.g strain 195, Dehalococcoides strain BAV1, Dehalococcoides strain VS, Dehalococcoides strain CBDBI, Dehalococcoides strain GT) Escherichia (optional ly Escherichia coli ), Synechococcus (optionally Synechococcus elongates), Stdfurospirillum (optionally Sulfurospirillum muhivoram or Sulfatrospir ilium bamesii), Dehalobacter (optionally Dehalobacter restrictus), Desulfuromonas (optionally strain BB1 or Desulfuromonas chloroethenicd), Desulfitobacterium (optionally Desuifitobacterium hafnieme), Geobacler (optionally Geobacter bemidjiensis, Geobacter lovleyi, Geobacter psychrophilus, Geobacler sp. FRC-32, Geobacter sp M21, Geobacter sulfurreducens, or Geobacter uraniireducens), Pelobacter (optionally Pelobacter propionlcus) Dehalogenimonas (optionally sp. Strain WBC-2) Pseudomonas, Flavobacterium, Comamonas, Cytophaga, Acidovorax, Sphingomonas, Bacillus, Acinetobacter or a combination thereof
[0022] in certain embodiments, the TCE forming recombinant microorganism includes microorganisms of the genus Rhizobium (optionally Rhizobium meliioti e.g. Rhizobium meliioti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emil tana hvdeyi), Simrhizobium (optionally Sinorhizobium meliioti), Calcidiscus (optionally Calcidiscus lepioporus) Phaeodactyium (optionally Phaeodactylum tricornulum), Chaetoceros (optionally Chaetoceros neogracilis), DunaUella (optionally DumlieSla tertiolecta), Meristie.Ua (optionally Meristiella gelidium), Ulva (optionally Ulm iactuca or Ulva rigida, e.g. Ulva rigida Agardh), Enteromorpha (optionally Enteromorphia intestinalis, Cladophora (optionally Cladophora rupestris), fucus (optionally Fucus serratus), Laminaria (optionally Laminaria saccarina, e.g. Laminaria saccarina (L) Lamour or Laminaria digitata, e.g. Laminaria digitata (Huds) Lamour), Desmarestia (optionally Desmarestia aculeaia, e.g. Desmarestia acideaia (L) Lamout), Chorda (optionally Chorda filum, e.g. Chorda filum (L) Stackh), Chondrus (optionally Chondrus crispus, e.g. Chondrus crispus Stackh), Phyllophora (optionally Phyllophora pseudoceranoides, e.g. Phyllophora pseudoceranoides (Gmelin)), Porphyra (optionally Porphyra umbdicalis , e.g. Porphyra umbtiicalis, (L) J. Ag ), Polysiphonia (optionally Polysipkonia nigrescens, e.g. Polysiphonia nigrescens (Huds.) Grevite), Furcellaria (optionally Furcellaria lumbricalis, e.g.
Furcellaria lumbricalis (Huds.) Lamour), Ceramium rubrum (optionally Ceramium rubrum, e.g. Ceramium rubrum (Huds.) Agardh), Ahnfeltia (optionally Ahnfeliia plicata, e.g.
Ahnfeltia plicata (Hudson) Fries), Laurencia (optionally Laurencia pinnatifida, e.g. Laurencia pinnatifida (Huds.) Lamour, or Laurencia obtuse, e.g. Laurencia obtusa (Huds.) Lamour), Caulerpa, Hypnea (optionally Hypnea musciformis, e.g. Hypnea musciformis (Wulfen) Lamouroux), Asparagopsis (optionally Asparagopsis taxiformis, e.g. Asparagopsis taxiformis (Delile) Trev), Gelidium (optionally Gelidium canariemis), Faihenbergia (optionally Faihenbergia hillebrandii, e.g. Faihenbergia hillebrandii (Born.) Falkenb), Corallina (optionally Corallina officinalis )s Gracilariopsis (optionally Gracilariopsis lemaneiformis), Gracilaria (optionally Gracilaria cornea, e.g. Gracdaria cornea J. Agardh), Methylosinus (optionally Methylosinus trichosporium, e.g. Methylosinus trickosporium OB3b), Desulfitobacterium (optionally Desulfitobacterium frappieri, e.g. Desulfitobacterium frappieri TCEl, or Desulfitobacterium metallireducens), Methylomicrobium (optionally Methylomicrobium album, e.g. Methylomicrobium album BG8), Methylococcus (optionally Methylococcus capsulatus, e.g. Methylococcus capsulatus (Bath)), Ralsionia (e.g. Ralstonia sp. KN 1-iOA), Pseudomonas (optionally Pseudomonas putida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4), Rhodobacter (optionally Rliodobacter sphaeroides or Rhodobacter capsulatus), Burkholderia (optionally Burkholderia cepacian, e.g Burkholderia cepacian G4) or a combination thereof.
(0023] In some aspects of methods herein, the VCM producing recombinant microorganism and the TCE producing recombinant microorganism are the same or different. [90241 Embodiments of methods of producing trichloroethylene (TCE) are disclosed herein, in various embodiments, the method includes: producing TCE by reacting glucose with a chloride source in the presence of a TCE producing catalytic dement wherein the TCE producing catalytic element comprises a TCE bioreactor culture containing a TCE producing recombinant microorganism having an improved TCE producing ability, wherein the TCE producing recombinant microorganism expresses the at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or it) overexpressing at least one native TCE producing enzyme nucleotide sequence, and wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE,
[1)9251 in embodiments of the invention, the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, glycera!dehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, cel!obiose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example Cos, Cm,, CM or Cue chlorinated alkanes (such as chloroform and / or carbon tetrachloride), in embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltitol and / or xylitol).
[90261 In embodiments, the chloride source may any source of chloride, including chloride salt (e.g. NaCl, KC!, MgCb, CaCk or combinations thereof) and / or HO,
[90271 in certain embodiments, the method further includes: producing glucose by reacting a carbon source with a hydrogen source in the presence of a glucose producing catalytic element including at least one photosynthesis enzyme comprising Rubisco, wherein the glucose producing catalytic element Includes a glucose hioreactor culture containing a glucose producing microorganism, wherein the glucose producing microorganism expresses the at least one glucose producing enzyme, and wherein the glucose producing microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose.
[0828] In certain embodiments, the method further includes a VCM producing catalytic element, wherein the VCM producing catalytic dement includes a VCM hioreactor culture containing a VCM producing recombinant microorganism having an improved VCM producing ability, wherein the VCM producing recombinant microorganism expresses the at least one VCM producing enzyme by i) expressing at least one non-native VCM producing enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM producing enzyme, and wherein the VCM producing recombinant microorganism is capable of utilizing a chlorinated hydrocarbon to produce VCM, in embodiments of the invention,, the chlorinated hydrocarbon may be a chlorinated alkane and / or a chlorinated alkene. In embodiments, the chlorinated hydrocarbon may be a Cos chlorinated hydrocarbon, a Cos chlorinated hydrocarbon, a CM chlorinated hydrocarbon and / or a CM chlorinated hydrocarbon. Examples of chlorinated hydrocarbon sources that may be employed in the present invention include trichloroethylene (TCE), diets loroethyieoe (DCE) and / or chloroform. In certain embodiments, the carbon source includes a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof. In certain embodiments, the hydrogen source includes water. In embodiments, the chloride source may any source of chloride, including chloride salt ( e.g , NaC!, KC!, MgCfe, CaCh or combinations thereof) and / or HCi.
[0029] in one aspect of the invention, there is provided a method of producing vinyl chloride monomer (VCM) comprising: providing a reaction medium comprising a VCM producing enzyme and a chlorinated hydrocarbon; maintaining the reaction medium under conditions which permit the production of VCM from the chlorinated hydrocarbon by the
VCM producing enzyme; and collecting VCM front the reaction medium.
[0(136] In a further aspect of the invention, there is provided a method of producing trichloroethylene (TCE) comprising: providing a reaction medium comprising a TCE producing enzyme and a TCE feedstock; maintaining the reaction medium under conditions which permit the production of TCE from the TCE feedstock by the TCE producing enzyme; and collecting TCE from the reaction medium,
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The foregoing summary, as well as the following detailed description of the embodiments, will be better understood when read in conjunction with the attached drawings. For the purpose of illustration, there are shown in the drawings some embodiments, which may be preferable, it should be understood that the embodiments depicted are not limited to the precise details shown. Unless otherwise noted, the drawings are not to scale.
[0632] Fig. 1 is a schematic illustration of a biomanufacturing system for producing vinyl chloride monomer (VCM) according to embodiments herein.
[0633] Fig. 2 is a series of graphs, including Fig.s 2(1), 2(2), 2(3), 2(4), and 2(5), which show retention times of chloride compounds and intermediates determined by gas chromatography, according to embodiments herein.
[0034] Fig. 3 A is a graph showing gas chromatography results for trichloroethylene (TCE) produced by the soil bacteria S. meli!oti, according to embodiments herein.
I I [0035] Fig, 38 is a graph showing TCE production by S. me!ilofi compared to a media only control, according to embodiments herein.
[§0361 Fig. 4A is a series of graphs, including Fig. 4(1 ), 4(2), and 4(3), which show gas chromatography results of VCM production from TCE in a Dehalococcoides culture, according to embodiments herein.
[0037] Fig. 4B is a graph showing gas chromatography results of ethylene, VCM, dichioroethyiene (DCE), and TCE production by a Dehalococcoides consortia culture, according to embodiments herein.
[0638] Fig. 4€ is a series of pie charts showing gas chromatography resul ts as percentages of the total production of ethylene, VCM, DCE, and TCE, according to embodiments herein.
[6839) Fig. 4D is a graph showing a standard curve of VCM yield from a Dehalococcoides consortia culture, according to embodiments herein.
[0046] Fig. 4E is a graph showing VCM yield from a Dehalococcoides consortia culture at different sampling times, according to embodiments herein.
[0041] Fig. 5 A is a series of graphs, including Fig.s 5(1), 5(2), and 5(3), which show gas chromatography results from engineered E. coll, cultures, according to embodiments herein,
[0042] Fig. 58 is a series of graphs, 5(4), 5(5), and 5(6), which show gas chromatography results from engineered £. coli cultures, according to embodiments herein.
[0043] Fig. 6A is a graph showing gas chromatography results (left) and corresponding peak retention tints data (right) from engineered Cyanobacteria cultures showing the production of VCM from TCE, according to embodiments herein,
[0944] Fig. 6B is a graph showing gas chromatography results for control levels of VCM production from a native Cyanobacteria culture, according to embodiments herein.
[0045] Fig. 6C is a graph showing gas chromatography results for VCM production from TCE by an engineered Cyanobacteria culture, according to embodiments herein.
[0046] Fig. 7 is a flow chart illustrating a method of producing TCE, according to embodiments herein.
DETAILED DESCRIPTION
[0047] Unless otherwise noted, all measurements are in standard metric units.
[0048] Unless otherwise noted, all instances of the words “a,” “an,” or “the” can refer to one or more titan one of the word that they modify. [0049] Unless otherwise noted, the phrase “at least one of’ means one or more than one of multiple objects, or a combination thereof. For example, “at least one of a non-native VCM expressing nucleotide sequence or a non-native TCE expressing nucleotide sequence” means a single non-native VCM expressing nucleotide sequence, or more than one nonnative VCM expressing nucleotide sequence, or a single a non-native TCE expressing nucleotide sequence, or more than one non-native TCE expressing nucleotide sequence, or any combination thereof
[0050] Unless otherwise noted, the term “about” refers to *10% of the nonpercentage number that is described, rounded to the nearest whole integer. For example, about 37 degrees Celsius, would include 33.3 to 40.7 degrees Celsius. Unless otherwise noted, the term “about” refers to ±5% of a percentage number. For example, about 80% would include 75% to 85%. When the term “about” is discussed in terms of a range, then the term refers to the appropriate amount less than the lower limit and more than the upper limit. For example, from about pH 4 to about pH 12 mm would include from pH 3,6 to pH 13.2.
[0051] Unless otherwise noted, properties (height, width, length, ratio etc.) as described herein are understood to he averaged measurements.
[0052] Unless otherwise noted, the terras “provide”, “provided” or “providing” refer to the supply, production, purchase, manufacture, assembly, formation, selection, configuration, conversion, introduction, addition, or incorporation of any element, amount, component, reagent, quantity, measurement, or analysis of any method or system of any embodiment herein,
[6053] Unless otherwise noted, the terms “natural” or “native” refer to organisms or molecules that are occur in nature without human action. Unless otherwise noted, the terms “non-native” or “synthetic” refer to organisms or molecules that are not naturally occurring and are produced by human actions. An example of a non-native organism is a recombinant organism.
[0054] As used herein “microorganism” is intended to relate to both single microorganisms as well as populations of microorganisms which may belong to a single strain or multiple strains.
[0055] Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide, "identity" and "similarity” can be readily calculated by various methods, known to those skilled in the art. In an embodiment sequence identity is determined by comparing the whole length of the sequences as identified herein.
[0056] Exemplary methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Exemplary computer program methods to determine identity and similarity between two sequences include e.g. the BestFit BLASTP (Protein Basic Local Alignment Search Tool), BLASTN (Nucleotide Basic Local Alignment Search Toot), and PASTA (AJischul, S. f. et a!., i. Mol. Biot. 215:403-410 (1990), publicly available from NCBI and other sources (BLAST.RTM. Manual, Aitschul, S., et ai., NCB! NLM NIH Bethesda, Md. 20894). A most exemplary algorithm used is EMBOSS (European Molecular Biology Open Software Suite). Exemplary parameters for amino acid sequences comparison using EMBOSS are gap open 10.0, gap extend 0.5, B!osum matrix. Exemplary parameters for nucleic acid sequences comparison using EMBOSS are gap open 10.0, gap extend 0.5, DNA full matrix (DNA identity matrix). In embodiments, it is possible to compare the DNA/ protein sequences among different species to determine the homology of sequences using online data such as Gene bank, KEG, BLAST and Ensemble.
[0057] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino adds having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring arnino acids are as follows: Ala to ser; Arg to iys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or iie; Phe to met, leu or tyr; Ser to ihr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Vai to He or leu.
10058] Methods for determining the production of enzymes by recombinant VCM- and TCE-producing microorganisms as compared to reference microorganisms will be known to those skilled in art. For example the recombinant microorganism and the reference microorganism can be reacted with the same starting materials under the same reaction conditions, and the resulting levels of enzymes produced may be assessed using gas chromatography techniques with which the skilled reader will be familiar and / or which are detailed in the protocols presented in the examples which follow.
[0659] Carbon dioxide emissions resulting from the use of fossil fuels continue to rise on a global scale. Reduction of atmospheric carbon dioxide levels is a key to mitigating or reversing climate change. Carbon capture and storage (CCS) is a prominent technology for removal of industrial carbon dioxide from the atmosphere; it has been estimated that over 20 trillion tons of carbon dioxide captured from refining and other industrial processes can be transported and stored in various types of subterranean environments or storage tanks. Although CCS is a cost effective and affordable way to reduce carbon dioxide emissions compared to other currently available methods, the problem remains that the carbon dioxide is merely being stored underground until it escapes. Therefore, CCS methods do not provide a sustainable solution to reduce excess carbon dioxide in the atmosphere. Also, there is little financial incentive for industries to pump carbon dioxide into subterranean environments, unless they are forced to by environmental regulations, or they are paid to do it as part of their business model. Arguably, global warming is a crisis because it is more lucrative to produce carbon dioxide than to dispose of carbon dioxide.
[0060] There remains a need to remove excess carbon dioxide from the atmosphere in more efficient and sustainable ways. There remains a need for technologies that can harness the over-abundance of carbon dioxide to make useful products, and for other applications that are beneficial to industry and the environment.
[0061] The challenges of the limited supply of fuel resources, and the harmful effects of industrial chemical manufacturing on the environment, have prompted a growing emphasis on maximizing output from existing resources, and in developing renewable sources of chemicals that can minimize environmental impacts. There has been a great deal of interest in developing technologies to produce chemical compounds from renewable sources, such as carbon dioxide and biomass.
[0062] Vinyl chloride monomer (VCM) is one such chemical product that is almost exclusively used as a feedstock for the production of polyvinyl chloride (PVC), one of the most widely used plasties in the world. Over 40 million metric tons of PVC were produced worldwide in 2018, and the demand for PVC is expected to approach 60 million metric tons by 2025. Currently, VCM is commercially produced by cracking natural gas or petroleum with chlorine, having a substantial impact on global energy consumption. With millions of metric tons of VCM being produced each year, more than enough carbon dioxide is produced by such processes to greatly contribute to the global carbon footprint. It has been shown that for each ton of VCM produced, 0.32 tons of CO? is emitted, A global production of 55 million tons of VCM per year corresponds to about 17.6 million tons of CO?, released annually.
[0663] Trichloroethylene (TCE) is widely used as a solvent for degreasing metal parts during manufacturing processes, and as a solvent in many consumer products, including adhesives, paint and stain removers, and wood finishes, and as a reagent for the production of other chemicals, including VCM. Producing VCM and TCE through renewable and sustainable methods would accordingly help to meet the huge worldwide demand for products such as PVC that are made using these chemicals, while also helping to protect the environment.
[0064] Based on modern history, it is fair to say that excess carbon dioxide in the atmosphere will not be reduced until it becomes profitable to reduce it. There remains a need for systems and processes that can produce VCM and TCE from environmentally sustainable sources at a commercial scale. There remains a need to remove excess carbon dioxide from the atmosphere. There remains a need for improved methods to produce VCM and TCE from renewable feedstocks for use in industrial and commercial applications.
[0065] Embodiments of the present disclosure can provide a benefit of removing carbon dioxide from the environment along with the benefit of producing valuable organic compounds capable of being sold commercially. Embodiments of the present disclosure can thus provide a renewable alternative to conventional carbon dioxide storage, by using recombinant microbial technology to convert the carbon dioxide into VCM and TCE as useful organic compounds. One benefit of the embodiments of the present disclosure is that the methods can make it economically profitable for a chemical or an energy company to remove carbon dioxide from the environment. An oil company, or a contractor thereof, could instead of pumping carbon dioxide into a subterranean environment or leaving the sequestered carbon dioxide underground, use the carbon dioxide to provide the chemical industry with a carbon source to use as a feedstock for a culture of recombinant microorganisms to convert the carbon dioxide to VCM and/or TCE in a eost-eftective way.
[0666] The most effective methods for protecting the environment are those methods that people actually use. The more profitable those methods are; the more likely people are to use them. One of the benefits of the methods disclosed herein is the cost-effectiveness of using a bioreactor system. Embodiments of toe present disclosure can provide a benefit of engineering a TCE or a VCM producing microorganism, by adapting the relevant metabolic signaling pathways to produce VCM and/or TCE on an industrial scale. Such embodiments can make it profitable to remove carbon dioxide from the atmosphere and to passively generate valuable organic compounds while the microbes do the work - on a scale previously unimaginable.
[0067] What would happen to the global warming crisis if it became more profitable, or just as profitable, to convert carbon dioxide into valuable organic compounds as it did to generate the carbon dioxide in the first place? The presently disclosed methods might transform chemical and energy producers from global warming companies to global cooling companies.
Embodiments of" Bfcmauufeetursng Systems for Producing Vinyl Chloride Monomer (VCM)
[0068] Embodiments of a biomanufacturing system for producing vinyl chloride monomer (VCM) are disclosed herein, in an embodiment of a biomanufacturing system, referring to Fig. i, system 100 includes glucose producing recombinant microorganism 102 that expresses at least one glucose producing enzy me 104 and is capable of utilizing carbon source 106 and hydrogen source 108 to produce glucose 110, biomass 112 and oxygen 114; TCE producing recombinant microorganism ! 16 that expresses at least one trichloroethylene (TCE) producing recombinant enzyme 118 by expressing at least one non-native TCE producing enzyme nucleotide sequence 120, and is capable of utilizing chloride source 122 to produce TCE 124; VCM producing recombinant microorganism 117 that expresses at least one VCM producing recombinant enzyme 126 by expressing at least one non-native VCM producing enzyme nucleotide sequence 128, and is capable of utilizing the chlorinated hydrocarbon TCE 124 to produce VCM 130. In certain embodiments, TCE producing recombinant microorganism 116 and VCM producing microorganism 117 can be different microorganisms, in certain embodiments, TCE producing recombinant microorganism i 16 and VCM producing recombinant microorganism 117 can be the same microorganism.
(0069] Embodiments of a biomanufacturing system herein can provide a benefit of a sustainable use of carbon sources, such as carbon dioxide, for the production of useful chemical compounds such as VCM and TCE. Such embodiments can also help to conserve natural resources. Biomanufacturing systems allowing chemical production by recombinant microorganisms can provide a benefit of reducing harm to the environment caused by conventional chemical manufacturing processes, thereby supporting long-term ecological balance.
Embodiments of Vinyl Chloride Monomer (VCM) Producing Recombinant Microorganisms
[0070] Embodiments of a vinyl chloride monomer (VCM) producing recombinant microorganism having a VCM improved production ability are disclosed herein. In various embodiments, the VCM producing recombinant microorganism expresses at least one VCM producing enzyme by i) expressing at least one non-native VCM producing enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM producing enzyme, wherein an amount of the VCM producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native VCM producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at feast one native VCM producing enzyme nucleotide sequence, and wherein the VCM producing recombinant microorganism is capable of utilizing a chlorinated hydrocarbon source to produce VCM. in embodiments of the invention, the VCM producing recombinant microorganism expresses at least one non-native VCM producing enzyme nucleotide sequence and produces VCM enzyme in a greater amount relative to a control microorganism lacking the at least one non-native VCM producing enzyme nucleotide sequence. In certain embodiments, the VCM producing recombinant microorganism overexpresses at least one native VCM producing enzyme nucleotide sequence and produces VCM enzyme in a greater amount relative to a control microorganism which is not engineered to overexpress the at least one native VCM producing enzyme. In embodiments of the invention, the VCM producing recombinant microorganism i) expresses at least one non-native VCM producing enzyme nucleotide sequence and ii) overexpresses at least one native VCM producing enzyme nucleotide sequence, and produces VCM enzyme in a greater amount relative to a control microorganism lacking the at least one non-native VCM producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native VCM producing enzyme. Embodiments of VCM producing recombinant microorganisms can provide an important advantage over conventional VCM production, by avoiding much of the environmental pollution caused by chemical cracking processes, while providing for a sustainable way to produce VCM on a commercial scale.
[0071 J in certain embodiments, the at least one VCM producing enzyme includes TCE reductive dehalogenase (TceA), TceA anchor protein (TeeB), tetrachloroethylene reductive dehalogenase (peeA), vinyl chloride reductase (vcrA), 1 ,2-dichSoropropane-io- propene reductive dehalogenase (dcpA), 1 ,2-trans~dishloroethene reductive dehalogenase catalytic A (TdrA), or a combination thereof. In certain embodiments, the VCM producing recombinant microorganism expresses TceA having an amino acid sequence at feast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 by expressing a native or non-native TceA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In certain embodiments, the VCM producing recombinant microorganism expresses TeeB having an amino acid sequence at least 90%, 95%, 96%,
97%, 98%, 99%, or i0G% identical to SEQ ID NO: 3 by expressing a native or non-native TeeB nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. in certain embodiments, the VCM producing recombinant microorganism expresses pceA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5 by expressing a native or non-native pceA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. to certain embodiments, the VCM producing recombinant microorganism expresses vcrA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 by expressing a native or non-native vcrA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. In certain embodiments, the VCM producing recombinant microorganism expresses dcpA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 by expressing a native or non-native dcpA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. In certain embodiments, the VCM producing recombinant microorganism expresses TdrA having an amino acid sequence 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11 by expressing a native or non-native TdrA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12. [0072] !iii embodiments of the invention, the VCM producing recombinant microorganism includes microorganisms of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehaiococcoides strain FL2, Dehalococcoides mccarty e.g. strains KS, RC, IN A, MB, 1 la or GY50. Dehalococcoides ethenogenes e g. strain. 195, Dehalococcoides strain BA VI, Dehalococcoides strain VS, Dehalococcoides strain CBDB1, Dehalococcoides strain GT) Escherichia (optionally Escherichia coli ), Symchococcus (optionally Symchococcus elongatus), Suljuraspirillum (optionally Sulfurospir ilium multivoram or Sulfurospirillum barnesii), Dehalobacter (optionally Dehalobacter restrictus), Desulfuromonas (optionally strain BB1 or Desulfuromonas chloroeikenica), Desulfitobacterium (optionally Desuifiiohacterium hafniense), Geobacter (optionally Geobacter bemidjiensis, Geobacter lovleyi, Geobacter psychrophilus, Geobacter sp. FRC-32, Geobacter sp. M21, Geobacter sulfurreducens, or Geobacter uraniireducens), Pelobacier (optionally Pelobacier propionicus) Dehalogenimonas (optionally sp. Strain WBC-2) Pseudomonas, Flavobacterium, Comamonas, Gytophaga, Acidavorax, Sphingomonas, Bacillus, Acineiobacter or a combination thereof.
[0073J In embodiments of the invention, the chlorinated hydrocarbon source may be a chlorinated alkane and / or a chlorinated alkene. In embodiments, the chlorinated hydrocarbon source may be a Ci-g chlorinated hydrocarbon, a Cu chlorinated hydrocarbon, a CM chlorinated hydrocarbon and / or a C1-2 chlorinated hydrocarbon. Examples of chlorinated hydrocarbon sources that may be employed in the present invention include trichloroethylene (ICE), dichloroethylene (DCE) and / or chloroform.
Embodiments of VCM Production Methods
[0074] To the inventors' knowledge, the ose of microorganisms to produce VCM, whether native or recombinant microorganisms, has not been atempted let alone achieved previously. As demonstrated in the examples provided herein, the production of VCM using microorganisms has now been successfully performed. Thus, according to a further aspect of the present invention, there is provided a method of producing vinyl chloride monomer (VCM) comprising: providing a reaction medium comprising a VCM producing enzyme and a chlorinated hydrocarbon; maintaining the reaction medium under conditions which permit the production of VCM front the chlorinated hydrocarbon by the VCM producing enzyme; and collecting VCM from the reaction medium.
[0075] in embodiments of the invention, the reaction medium may he provided in a bioreactor. Any type of bioreactor known to one skilled in the art may be employed. VCM may be collected from the reaction medium using conventional separation techniques e.g. distillation, evaporation or the like. In preferred embodiments, such techniques may be used to obtain a VCM stream comprising at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of VCM by weight.
[00761 hi embodiments of the invention, the VCM producing microorganism which produces the VCM producing enzyme may also be provided in the reaction medium. In alternative, cell-free embodiments, the VCM producing microorganism which produces the VCM producing enzyme is not present in the reaction mixture.
[6077] The conditions under which the reaction medium is maintained to permit the production of VCM from the chlorinated hydrocarbon will depend upon nature of the VCM- produeing enzyme and / or VCM producing microorganism provided in the reaction medium.
[0078] In embodiments of this aspect of the invention, the VCM producing enzyme may be produced by an organism which is native or recombinant. In embodiments in which the VCM producing organism is recombinant, it may comprise a vinyl chloride monomer (VCM) producing recombinant microorganism as described herein.
[0979] Whether the VCM producing microorganism is recombinant or native, in embodiments, it includes microorganisms of the phylum Cyanobacteria or the genus Dekalococcoides (optionally Dehalococcoides strain FL2, Dehalococcoides mccarty e.g strains K.S, RC, JNA, MB, 11 a or GY50, Dehalococcoides ethsnogenes e.g. strain 195, Dehalococcoides strain BAV1, Dehalococcoides strain VS, Dehalococcoides strain CBDB1, Dehalococcoides strain GT) Escherichia (optionally Escherichia coil), Synechococcus (optionally Synechococcus elongatus), Sulfurospirillum (optionally Sulfurospirillum multivoram or Sulfurospirillum harnesii), Dehalobacter (optionally Dehalobacter restrict*®), Desulfuromonas (optionally strain BB1 or De sulfur omonas cMoroethenica), Desulfitobacterium (optionally Desulfslobacierium hafhiense), Geobacter (optionally Geobacter bemidjiensis, Geobacter lovleyi, Geobacter psychrophilus, Geobacter sp. FRC-32, Geobacter sp. M21, Geobacter sulfitrreducens, or Geobacter uraniireducens), Pelobacter (optionally Pelobacter propionicus) Dehalogenimonas (optionally sp. Strain WBC-2) Pseudomonas, Fiavobacterium, Comamonas. Cyiophaga . Acidovorax, Sphingomonas, Bacillus, Acinetobacter or a combination thereof.
[0080J in ail embodiments of the invention, the VCM producing microorganism may comprise a consortium of microorganisms. In certain embodiments, the VCM producing microorganism may comprise a plurality of strains belonging to the genus Dehalococcoides. Such consortia have been demonstrated in the examples to be particularly efficient producers ofVCM
[Q081] In embodiments of the invention, the YCM producing microorganism may express TceA having an amino acid sequence at least 95% identical to SEQ ID NO: I by expressing a TceA nucleotide sequence at least 95% identical to SEQ HI NO: 2; expresses TceB having an amino acid sequence at least 95% identical to SEQ ID NO: 3 by expressing a TceB nucleotide sequence at least 95% identical to SEQ ID NO: 4; expresses pce.4 having an amino acid sequence at least 95% identical to SEQ ID NO: 5 by expressing a peeA nucleotide sequence at least 95% identical to SEQ ID NO: 6; expresses vcrA having an amino add sequence at least 95% identical to SEQ ID NO: 7 by expressing a vcrA nucleotide sequence at least 95% identical to SEQ ID NO: 8; expresses depA having an amino add sequence at least 95% identical to SEQ ID NO: 9 by expressing a dcpA nucleotide sequence at least 95% identical to SEQ ID NO: 10; and / or expresses TdrA having an amino acid sequence 95% identical to SEQ ID NO: 11 by expressing a TdrA nucleotide sequence at least 95% identical to SEQ ID NO; 12.
Embodiments of Trichloroethylene (TCE) Producing Recombinant Microorganisms
{0082) Embodiments of a trichloroethylene (TCE) producing recombinant microorganism having an improved TCE producing ability are disclosed herein. In various embodiments, the TCE producing recombinant microorganism expresses at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or is) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the TCE producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme nucleotide sequence, and wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE. In embodiments of the invention, the TCE producing recombinant microorganism expresses at least one non-native TCE producing enzyme nucleotide sequence and produces TCE enzyme in a greater amount relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence. In certain embodiments, the TCE producing recombinant microorganism overexpresses at least one native TCE producing enzyme nucleotide sequence and produces TCE enzyme in a greater amount relative to a control microorganism which is not engineered to overexpress the at least one native TCE producing enzyme. In embodiments of the invention, the TC producing recombinant microorganism i) expresses at least one non-native TCE producing enzyme nucleotide sequence and ii) overexpresses at least one native TCE producing enzyme nucleotide sequence, and produces TCE enzyme in a greater amount relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme. Embodiments of a TCE producing recombinant microorganism can provide a benefit of sustainable TCE production on a commercial scale, while avoiding environmental harm caused by conventional TCE manufacturing processes.
[0083] l.n embodiments of the invention, the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, glyceraldehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, ceilobfose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example C? .g, Ci~e , C·.* or Cs-2 chlorinated alkanes (such as chloroform and / or carbon tetrachloride), !n embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltitol and / or xylitol).
[0084] In embodiments, the chloride source may any source of chloride, including chloride salt (e.g. NaC!, KC!, MgCh, CaCh or combinations thereof) and / or HCi.
[0085] In certain embodiments, the at least one TCE producing enzyme includes phenol hydrase (PIT), particulate methane monooxygenase (pMMO), benzene (and/or toluene) dioxygenase <B DO/ToD), toluene o-xylene monooxygenase oxygenase subunit (TouA), toluene-4-monooxygenase system hydroxylase component subunit alpha, chlorobenzene dioxygenase, cis-chiorobenzene dihydrodiol dehydrogenase, toluene 2- monooxygenase, or a combination thereof, in certain embodiments, the TCE producing recombinant microorganism expresses PH having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13 by expressing a native or nonnative PH nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14. In certain embodiments, the TCE producing recombinant microorganism expresses pMMO having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15 by expressing a native or non-native pMMO nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16. in certain embodiments, the TCE producing recombinant microorganism expresses ToD having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or !00% identical to SEQ ID NO: 17 by expressing a native or non-native ToD nucleotide sequence at least 90%, 95%. 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18. in certain embodiments, the TCE producing recombinant microorganism expresses TouA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SF.Q ID NO: 19 by expressing a native or non-native TouA nucleotide sequence at ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20. in certain embodiments, the TCE producing recombinant microorganism expresses toiuene-4-monooxygenase system hydroxylase component subunit alpha having an amino acid sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21 by expressing & native or non- native toluene-4-monooxygenase system hydroxyla e component subunit alpha nucleotide sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22. in certain embodiments, the TCE producing recombinant microorganism expresses chlorobenzene dioxygenase having an amino acid sequence 90%, 95%, 96%, 97%, 98%,
99%, or 100% identical to SEQ ID NO: 23 by expressing a native or non-native chlorobenzene dioxygenase nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24. In certain embodiments, the TCE producing recombinant microorganism expresses cis-chiorobenzene dihydrodioi dehydrogenase having an amino acid sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:
25 by expressing a native or non-native cis-chiorobenzene dihydrodioi dehydrogenase nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26, In certain embodiments, the TCE producing recombinant microorganism expresses toluene 2-monooxygenase having an amino add sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IP NO: 27 by expressing a native or non-native toluene 2-monooxygenase nucleotide sequence at Ieast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28; or a combination thereof
[0086] In certain embodiments, the TCE producing recombinant microorganism includes microorganisms of the genus Rhizobium (optionally Rhizobium meliloti e.g Rhizobium meliloti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emiliana huxleyi), Simrhizobium (optionally Sinorhizobium meliloti), Calcidiscus (optionally Calcidiscus leptoporus), Phaeodactylum (optionally Phaeodactyhm tricormtum), Chaetoceros (optionally Chaetoceros neogracilis), Dunaliella (optionally Dunaliella tertiolectd), Meristiella (optionally Meristiella gelidium), Ulva (optionally Ulva lactuca or Ulva rigida, e.g. Ulva rigida Agardh), Enieromorpha (optionally Enieromorphia intestinalis, Cladophora (optionally Cladophom rupestris), Fucus (optionally Fttcus serratus). Laminaria (optionally Laminaria saccarina, e.g. Laminaria saccarina (L) Lamonr or Laminaria digitaia, e.g. Laminaria digitata (Huds) Lamonr), Desmarestia (optionally Desmarestia acuieata, e.g. Desmarestia aculeaia (L) Lamour), Chorda (optionally Chorda filum. e.g Chorda filum (L) Stackh), Chondrus (optionally Chondrus crispus, e.g. Chondrus crispus Stackh), Phyllophora (optionally Phyllophora pseudoceranoides, e.g. PhyUophora pseudoceranoides (Gmelin)), Porphyra (optionally Porphyra umbticdis, e.g. Porphyra umbilicalis, (L) J. Ag.}, Polysiphonia (optionally Polysiphonia nigrescens, e.g. Polysiphonia nigrescens (Huds.) Greville), Furcellaria (optionally Furcellaria iumbricalis, e g.
Purcellaria Iumbricalis (Huds.) Lamour), Ceramium rubrum (optionally Ceramium rubrum, e.g. Ceramium rubrum (Huds.) Agardh), Ahnfeltia (optionally Ahnfeltia piicala, e.g
Ahnfeitia plicate (Hudson) Fries), Laurencia (optionally Laurencia pinnalifida, e.g. Laurencia pinnatifida (Huds.) Lamour, or Laurencia obtuse, e.g. Laurencia obtusa (Huds.) Lamour), Caulerpa, Hypnea (optionally Hypnea musciformis, e.g. Hypnea musciformis (Wulfen) Lamouroux), Asparagopsis (optionally Asparagopsis taxiformis, e.g. Asparagopsis taxiformis (Deliie) Trev), Gelidium (optionally Gelidium camriemis), Faikenbergia (optionally Faikenbergia hiUebrandii, e.g. Faikenbergia hillebrandii (Born.) Falkenb), Corallina (optionally Corallina officinalis), Gracilariopsis (optionally Gracilariopsis lemaneifarmis), Graciiaria (optionally Gracilaria cornea, e g. Gracilaria cornea J. Agardh), Methylosinus (optionally Methylosinus trickasporium, e.g. Methylosinus trichosporium OB3b), Desuifitobacterium (optionally Desulfitobacterium frappieri. e.g. Desulfitobacterium fi-appieri TCE1, or Desulfitobacterium metallireducens), Meihylomicrobium (optionally Methylomicrobium album, e.g. Meihylomicrobium album BG8), Methylococcus (optionally Methylococcus capsulatus, e.g. Melhylococcus copsulaius (Bath)), Ralstonia (e.g. Ralstonia sp. KN l-!OA), Pseudomonas (optionally Pseudomonas putida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4), Rhodobacter (optionally Rhodobacier sphaeroides or Rhodobacter capsulatus), Burkholderia (optionally Burkholder ia cepacian, e.g. Burkhoideria cepacian G4) or a combination thereof.
[0087} in some embodiments, the VCM producing recombinant microorganism and the TCE producing recombinant microorganism are the same or different organisms. In some aspects, the VCM producing recombinant microorganism expresses at least one TCE producing enzyme by t) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or ii) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme nucleotide sequence, and wherein the VCM producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE. Such embodiments can provide a benefit of a single recombinant microorganism having both a VCM and a TCE producing capability. Such embodiments can provide an advantage of a greater efficiency in VCM production from TCE. In some embodiments, the VCM producing recombinant microorganism and the TCE producing recombinant microorganism are different organisms. Such embodiments can provide an advantage of independently optimizing the manufacture of each organism and varying their ratio to one another during VCM or TCE production.
[0088] In embodiments of the Invention, the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, giyeeraidehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, ee!iobiose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example CM, CM, CM or Co?, chlorinated alkanes (such as chloroform and / or carbon tetrachloride). In embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltUoi and / or xylitol).
[0089] In embodiments, the chloride source may any source of chloride, including chloride salt (e.g. NaCI, KCi, MgCb, CaCh or combinations thereof) and / or HC!. Embodiments of TCE Production Methods
[0090] According to a further aspect of the present invention, there is provided a method of producing trichloroethylene (TCE) comprising: providing a reaction medium comprising a TCE producing enzyme and a TCE feedstock; maintaining the reaction medium under conditions which permit the production of TCE from the TCE feedstock by the TCE producing enzyme; and collecting TCE from the reaction medium.
[0091] In embodiments of the invention, the reaction medium may be provided in a bioreactor. Any type of bioreactor known to one skilled in the art may be employed. TCE may be collected from the reaction medium using conventional separation techniques e.g. distillation, evaporation or the like. In preferred embodiments, such techniques may be used to obtain a TCE stream comprising at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of TCE by weight.
[0092] In embodiments of the invention, the TCE producing microorganism which produces the TCE producing enzyme may also be provided in the reaction medium. In alternative, cell-free embodiments, the TCE producing microorganism which produces the TCE producing enzyme is not present in the reaction mixture. [0093] The conditions under which the reaction medium is maintained to permit the production of TCE from the chlorinated hydrocarbon by the TCE producing enzyme will depend upon the nature of the TCE producing enzyme and / or TCE producing microorganism provided in the reaction medium.
[0094] In embodiments of this aspect of the invention, the TCE producing enzyme may be produced by a TCE producing microorganism may be native or recombinant. In arrangements in which the TCE producing organism is recombinant, it may be a trichloroethylene (TCE) producing recombinant microorganism as described herein. j|Ci89§! In embodiments of the invention, the TCE feedstock may comprises a carbohydrate (e.g, a monosaccharide, optionally xylose, glyceraldehyde, fructose, galactose, mannose and / or glucose, or a disaccharide, optionally lactose, maltose, sucrose, cellobiose and / or trehalose), a chlorinated hydrocarbon (e.g. a Cw chlorinated alkane, optionally chloroform and / or carbon tetrachloride) and / or a polyol (e.g. glycerol, mannitol, sorbitol, ma!titol and / or xylitol). in preferred embodiments, the TCE feedstock comprises glucose.
[0096] Whether the TCE producing microorganism is recombinant or native, it may include microorganisms of the genus Rhizobium (optionally Rhizobium meliloii e.g Rhizobium meliloti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emilkma huxleyi), Sinorhisobium (optionally Sinorhizobhm meliloii), Calcidiscus (optionally Calcidiscm lepioporus), Phaeodactylum (optionally Phaeodactylum tricornutum), Chaetoceros (optionally Chaetoceros neogracilis), Dimaliella (optionally Dunaliella tertiolecta), Meristiella (optionally Meristiella gelidium), Ulva (optionally Ulva laciuca or Ulva rigida, e.g. Ulva rigida Agardh), Enieromorpha (optionally ErUeromorphia intestinalis, Cladophora (optionally Cladophora rupestris), Fucus (optionally Fucus serratus), Laminaria (optionally Laminaria saccarina, e.g. Laminaria saccarina (L) Lamour or Laminaria digitata, e.g. Laminaria digitaia (Muds) Lamour), Desmarestia (optionally Desmarestia aculeaia, e.g. Desmarestia aculeaia (L) Lamour), Chorda (optionally Chorda filum, e.g. Chorda filum (L) Siackh), Chondrus (optionally Chondrm crispus, e.g. Chondrus crispus Stackh), Phyllophora (optionally Phyllophora psetidoceranoides, e.g PhyUophora pseudoceranoides (Gmelin)), Porphyra (optionally Porphyra umbilical is, e.g. Porphyra umbUkalis, (L) J. Ag), Polysiphonia (optionally Polys iphonia rtigrescens, e.g. Poiysiphonia nigrescens (Huds.) Greville), Furcellaria (optionally Furcellaria lumbricalis, e.g. Furcellaria lumbricalis (Huds.) Lamour), Ceramium rubrum (optionally Ceramium rubrum, e.g. Ceramium rubrum (Huds.) Agardh), Ahnfeltia (optionally Ahnfeltia plicata, e.g. Ahnfeltia plicata (Hudson) Fries), Laurencia (optionally Laurencia pinnatifida, e.g. Laurencia pinnatifida (Huds.) Lamour, or Laurencia obtuse, e,g. Laurencia obtusa (Huds.) Lamour), Caulerpa, Hypma (optionally Hypnea musciformis, e g. Hypnea musciformis (Wulfen) Lamouroux), Aspamgopsis (optionaUy Aspamgopsis taxiformis, e.g. Aspamgopsis iaxiformis (Delile) Trev), Gelidium (optionally Gelidium canariensis), Falkenbergia (optionaily Falkenbergia hillebrandii, e.g. Falkenbergia hillebrandii (Born.) Falkenti), Corallina (optionally Coraliim officinalis), Gracilariopsis (optionally Gracilariopsis lemaneiformis), Gracilaria (optionally Gracilaria cornea, e.g. Gracilaria cornea ,1. Agardh), Methylosinus (optionally Metltylosinus trichosporium, e.g. Meihylosinus trichosporium OB3b), Desulfitobacterium (optionally Desulfitobacterium frappieri, e.g. Desulfitobacterium frappieri TCE1, or Desulfitobacterium metallireducens), Meihylomicrobium (optionally Methylomicrobium album, e.g. Methylomicrobium album BGS), Methylococcus (optionally Methylococcus capsuiatus, e.g. Methylococcus capsuiatus (Bath)), Ralsioma (e.g. Ralstonia sp. KN 1-iOA), Pseudomonas (optionally Pseudomonas putida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4), Rhodobacter (optionally Rliodobacter sphaeroides or Rhodobacter capsuiatus), Burkholderla (optionally Burkholderia cepacian, e.g. Burkholderia cepacian G4 ) or a combination thereof
[009?| In all embodiments of the invention, the TCE producing microorganism may comprise a consortium of microorganisms.
[0098] 1st embodiments of the Invention, the reaction medium comprising the TCE producing enzyme may additionally comprise a chloride source, for example a chloride salt (e.g. NaCI, KC!, MgCL, CaCb or combinations thereof) arsd / or HC!.
[0099] in embodiments of the invention, the TCE producing microorganism may express at least one TCE producing enzyme selected from phenol hydrase (PH), particulate methane monooxygenase (pMMO), benzene (and/brtofuene) dioxygenase (BDO/ToD), toluene o-xyfene monooxygenase oxygenase subunit (TouA), toluene-4-monooxygenase system hydroxylase component subunit alpha, chlorobenzene dioxygenase, ds-ch!orobenzene dihydrodiol dehydrogenase, toluene 2-monooxygenase, or a combination thereof
[0100] In certain embodiments, the TCE producing microorganism may express PH having an amino acid sequence at least 95% identical to SEQ ID NO: 13 by expressing a PH nucleotide sequence at least 95% identical to SEQ ID NO: 14; expresses pMMO having an amino acid sequence at least 95% identical to SEQ ID NO: 15 by expressing a pMMO nucleotide sequence at least 95% identical to SEQ ID NO: ! 6; expresses ToD having an amino acid sequence at least 95% identical to SEQ ID NO: 17 by expressing a ToD nucleotide sequence at least 95% identical to SEQ ID NO: 18; expresses TouA having an amino acid sequence at least 95% identical to SEQ ID NO: 19 by expressing a TouA nucleotide sequence at least 95% identical to SEQ ID NO: 20; expresses toluene-4- monooxygenase system hydroxylase component subunit alpha having an amino acid sequence at least 95% identical to SEQ ID NO: 21 by expressing a toluene-4-monooxygenase system hydroxylase component subunit alpha nucleotide sequence at least 95% identical to SEQ ID NO: 22; expresses chlorobenzene dioxygenase having an amino acid sequence 95% identical to SEQ ID NO: 23 by expressing a chlorobenzene dioxygenase nucleotide sequence at least 95% identical to SEQ ID NO: 24; expresses cis-chlorobenzene dihydrodiol dehydrogenase having an amino acid sequence at least 95% identical to SEQ ID NO: 25 by- expressing a cis-chlorobenzene dihydrodiol dehydrogenase nucleotide sequence at least 95% identical to SEQ ID NO: 26; or expresses toluene 2-monooxygenase having an amino acid sequence at least 95% identical to SEQ ID NO: 27 by expressing a toluene 2-monooxygenase nucleotide sequence at least 95% identical to SEQ ID NO: 28; or a combination thereof. Embodiments of Glucose Producing Recombinant Microorganisms
[01011 Embodiments of a glucose producing recombinant microorganism are disclosed herein. In various embodiments, the glucose producing microorganism expresses at least one glucose producing enzyme, wherein the glucose producing microorganism is capable of utilizing a carbon source and a hydrogen source to produce glucose. In certain embodiments, the carbon source includes a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof. In certain embodiments, the hydrogen source comprises water. In embodiments, the chloride source may any source of chloride, including chloride salt (e.g. NaCl, KCL MgCh, CaCb or combinations thereof) and / or HCI. Embodiments of a glucose producing recombinant microorganism can provide a benefit of capturing a carbon source, such as carbon dioxide, into a chemical form, such as glucose, that can be utilized by the VCM producing or the TCE producing recombinant microorganism for the production of VCM or TCE.
[0102| In certain embodiments, the glucose producing enzyme includes sucrose permease (cscB), sucrose-phosphate synthase (sps), glucose- 1 -phosphate adenyly!iransferase (glgC), sucrose phosphate phosphatase (spp), glycogen phophorylase (gigP), UDP-gtucose pyrophosphoryia.se (ga!U), invertase, glucosylglycerol-phosphate synthase (ggpS), glycogen synthase (glgA); or combinations thereof. In certain embodiments, the glucose producing microorganism expresses cscB having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29 by expressing a non-native cscB nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. in certain embodiments, the glucose producing microorganism expresses sps having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:
31 by expressing a non-native sps nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32, In certain embodiments, the glucose producing microorganism expresses glgC having an amino add sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to SEQ ID NO: 33 by expressing a non-native gigC nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. In certain embodiments, the glucose producing microorganism expresses spp having an amino add sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to SEQ ID NO:
35 by expressing a non-native spp nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36. In certain embodiments, the glucose producing 30epacian30nism expresses glgP having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37 by expressing a non-native g!gp nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38. in certain embodiments, the glucose producing microorganism expresses ga!U having an amino acid sequence 90%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to SEQ ID NO: 39 by expressing a non-native ga!U nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40. in certain embodiments, the glucose producing microorganism expresses invertase having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41 by expressing a non-native invertase nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42. In certain embodiments, the glucose producing microorganism expresses ggpS having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43 by expressing a non-native ggpS nucleotide sequence at least 90%. 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44, In certain embodiments, the glucose producing 30epacian30nism expresses gigA having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to SEQ ID NO: 45 by expressing a non-native gigA nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46,
[0103] In certain embodiments, the glucose producing recombinant organism includes a recombinant microorganism, a photosynthetic microorganism, a Cyanobacteria, a Symchococcus, Symchococcus elongates, Synechococcm leopoliensis Synechocystis, Anabaena, a Pseudomonas, Pseudomonas syringae, Pseudomonas savasianoi, Chlamydomonas, Chlamydomonas reinhardtii, algae, microalgae, electrosynthesis bacteria, a photosynthetic microorganism, yeast, filamentous fungi, or a plant cell.
[0104] The use of the recombinant glucose producing microorganisms of the present invention advantageously enables the efficient production of glucose using carbon capture technology. Thus, according to a further aspect of the present invention, there Is provided: a method of producing glucose comprising: providing a reaction medium comprising a glucose producing recombinant microorganism as described herein, a hydrogen source and a carbon source; maintaining the reaction medium under conditions which permit the production of glucose by the glucose producing microorganism; and collecting glucose from the reaction medium.
(01051 to embodiments, the hydrogen source may comprise water and / or the carbon source may comprise a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source.
Embodiments of Methods of Producing a Vinyl Chloride Monomer (VCM) Producing
Recombinant Microorganism
[0106] Embodiments of methods of producing a vinyl chloride monomer (VCM) producing recombinant microorganism having an VCM improved producing ability or a trichloroethylene (TCE) producing recombinant microorganism having an improved TCE producing ability are disclosed herein. In various embodiments, the method includes: producing the VCM producing recombinant microorganism by inserting at least one of a nonnative VCM expressing nucleotide sequence or a non-native TCE expressing nucleotide sequence into a bacterial plasmid of a microorganism. Additionally or alternatively, the method includes: producing the VCM producing recombinant microorganism by inserting at least one of a native VCM expressing nucleotide sequence, a nucleotide sequence which promotes the overexpression of a native VCM expressing nucleotide sequence, a native TCE expressing nucleotide sequence and / or a nucleotide sequence which promotes the overexpression of a native TCE expressing nucleotide sequence into a bacterial plasmid of a microorganism. In certain embodiments, the native or non-native VCM expressing nucleotide sequence has a nucleotide sequence at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or a combination thereof, in certain embodiments, the native or non-native TCE expressing nucleotide sequence has a nucleotide sequence at least 95% identical to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or a combination thereof. The embodied methods of producing VCM producing and TCE producing recombinant microorganisms can provide a benefit of enabling the production of microbial biomanufacturing systems that can produce VCM and TCE sustainably and on a commercial scale.
[0107] in embodiments of the invention, the VCM producing recombinant microorganism includes microorganisms of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehaiococcoides strain PL2, Dehaiococcoides mccarty e.g. strains KS. RC, JNA, MB, Ha or GY50, Dehaiococcoides ethenogenes e.g. strain 195, Dehaiococcoides strain BAVI, Dehaiococcoides strain VS, Dehaiococcoides strain CBDB1, Dehaiococcoides strain GT) Escherichia (optionally Escherichia coli), Synechococcus (optionally Syneckococcus elongatus ), Sulfurospiriilum (optionally Sulfurospirillum multivorans or Sulfurospirillum bamesii), Dehalohacter (optionally Dehalobacter restrictus), Desulfuromonas (optionally strain BB1 or Destdfuromonas cMoroethenica), Desulfitobacterium (optionally Desulfiiobacterium hafriiense ), Geobacier (optionally Geobacter bemidjiemis, Geobacier lovleyi, Geobacter psychrophilus, Geobacier sp. FRC-32, Geobacier sp. M21, Geobacter sulfiureducem, or Geobacier uraniireducens), Pelobacter (optionally Pelobacter propionicus) Dehalogenimonas (optionally sp. Strain WBC-2) Pseudomonas, Flavobacterium, Comamonas, Cytophaga, Acidovorax, Sp-hingomonas, Bacillus, Acinetobacter or a combination thereof
[0108] in certain method embodiments, the TCE forming recombinant microorganism includes microorganisms of the genus Rhtobium (optionally Rhizohium meliloti e.g. Rhizobium meliloti strain Dangeard). Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emiliam huxleyi), Simrhizobium (optionally Simrhizobium meliloti), Calcidiscus (optionally Calcidiscus leptoporus), Phoeodoctylum (optionally Phaeodactylum tricornutum), Chaetoceros (optionally Chaetoceros neogracilis), Dunalietla (optionally Dunaliella tertiolecta), Meristiella (optionally Merisiiella gelidium), Ulva (optionally Ulva laciuca or Ulva rigida, e.g. Ulva rigida Agardh), Enteromorpha (optionally Enteromorphia mtesiinalis, Cladophora (optionally Cladophora rupestris), Fucus (optionally Fucus serratus), Laminaria (optionally Laminaria saccarina. e.g Laminaria saccarina (L) Lamour or Laminaria digiiata, e.g. Laminaria digitate (Muds) Lamour). Desmarestia (optionally Desmarestia aculeata, e.g. Desmarestia aculeata (L) Lamour ), Chorda (optionally Chorda filum, e.g. Chorda filum (L) Staekh), Chondrus (optionally Ckondrus crispus, e.g Chondrus crispus Staekh), Phyliophora (optionally Phyllophora pseudoceranoides, e.g. Phyllophora pseudoceranoi'des. (Gmelin) ), Porphyra (optionally Porphyra umbilicalis, e g. Porphyra umbilicalis, (L) J. Ag.), Polysiphonia (optionally Polysiphonia nigrescens, e..g. Polysiphonia nigrescem (Ends.) Greviile), Furcellaria (optionally Furcellaria lumbricalis, e.g.
Furcellaria lumbricalis (Muds.) Lamour), Ceramium rubrum (optionally Ceramium rubriim, e.g. Ceramium rubrum (Huds.) Agardh), Ahnfeltia (optionally Ahnfeltia plicata, e g.
Ahnfeltia plicata (Hudson) Fries), Laurencia (optionally Laurencia pinnatiflda, e.g. Laurencia pinnatiflda (Huds.) Lamour, or Laurencia obtuse, e g. Laurencia obiusa (Huds) Lamour), Caulerpa, Hypnea (optionally Hypnea musciformis, e g. Hypnea musciformis (Wutfen) Lamouroux), Asparagopsis (optionally Asparagopsis taxiformis, eg. Asparagopsis (axiformis (Delile) Trev), Gelidium (optionally Gelidium camriensis), Falkenbergia (optionally Falkenbergia hillebrandii. e.g. Falkenbergia hillebrandii (Bom) Falkenb), Coralline (optionally Corallina officinalis), Gracilariopsis (optionally Gracilariopsis lemaneijbrmis), Graci!aria (optionally Gracilaria cornea, e g. Gracilaria cornea J. Agardh), Methylosinus (optionally Methylosinus trichosporium, e.g. Meihylosinus trichosporium OB 3b), Destdfitobacterium (optionally Desulfltobacierium frappieri, e.g. Desulfitobacterium floppier i TCE1, or Desulfitobacterium metalUreducens), Methylomicrobium (optionally Methylomicrobium album, e.g. Methylomicrobium album BG8), Methylococcus (optionally Methylococcus capsulatus, e.g. Methylococcus capsulatus (Bath)), Ralstonia (e.g. Ralstonia sp. KN I-IOA), Pseudomonas (optionally Pseudomonas putida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4), Rlwdobacier (optionally Rkodobacter sphaeroides or Rhodobacier capsulatus), Burkholderia (optionally Burkholderia cepacian. e.g. Burkholder ia cepacian G4 ) or a combination thereof
|0I09| In some aspects of methods herein, the VCM producing recombinant microorganism and the TCE producing recombinant microorganism are the same or different. Such embodimen ts can provide a benefit of versatility for the design of biomanufacturing systems and methods for the production of VCM and TCE.
Embodi ments of Methods of Producing Trichloroethylene (TCE) as Shown in Figure 7
[0110] Embodiments of methods of producing trichloroethylene (TCE) are disclosed herein. In an embodiment, referring to Fig. 7, method 700 includes: reacting a carbon source with a hydrogen source in the presence of a glucose producing catalytic element 702; producing glucose 704; reacting glucose with a chloride source in the presence of a TCE producing catalytic element 706; and producing TCE 708,
[0111] Embodiments of methods of producing trichloroethylene (TCE) are disclosed herein. In various embodiments, the method includes: producing TCE by reacting glucose with a chloride source in the presence of a TCE producing catalytic element, wherein the TCE producing catalytic element comprises a TCE bioreaetor culture containing a TCE producing recombinant microorganism having an improved TCE producing ability, wherein the TCE producing recombinant microorganism expresses the at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or ii) overexpressing at least one native TCE producing enzyme nucleotide sequence, and wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE. Embodiments of methods of producing TCE herein can provide a benefit of sustainable TCE production on a commercial scale, while avoiding environmental harm caused by conventional TCE manufacturing processes.
[0H2] In embodiments of the invention, the TCE feedstock may comprise carbohydrates including monosaccharides (such as xylose, giycera!dehyde, fructose, galactose, mannose and / or glucose) and / or disaccharides (such as lactose, maltose, sucrose, cellobiose and / or trehalose). Additionally or alternatively, the TCE feedstock may comprise chlorinated hydrocarbons, for example C os, Cos, CM or C1.2 chlorinated alkanes (such as chloroform and / or carbon tetrachloride). In embodiments, the TCE feedstock may comprise polyol compounds (such as glycerol, mannitol, sorbitol, maltitoi and / or xylitol).
[0113] in embodiments, the chloride source may any source of chloride, including chloride salt (e.g. Nad, KCi, MgCk, CaCk or combinations thereof) and / or HC!.
[0114] in certain embodiments, the method further includes: producing glucose by reacting a carbon source with a hydrogen source in the presence of a glucose producing catalytic element including at least one photosynthesis enzyme comprising Rubisco, wherein the glucose producing catalytic element includes a glucose bioreactor culture containing a glucose producing microorganism, wherein the glucose producing microorganism expresses the at least one glucose producing enzyme, and wherein the glucose producing microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose. Such embodiments can provide a benefit of allowing the capture of a carbon source into glucose that can be provided to a TCE bioreactor culture to be utilized by a TCE producing recombinant microorganism for TCE production.
[OHS] In certain embodiments, the method farther includes a VCM producing catalytic element, wherein the VCM producing catalytic element includes a VCM bioreactor culture containing a VCM producing recombinant microorganism having an improved VCM producing ability, wherein the VCM producing recombinant microorganism expresses the at least one VCM producing enzyme by i) expressing at least one non-native VCM producing enzyme nucleotide sequence and i or ii) overexpressing the at least one native VCM producing enzyme, and wherein the VCM producing recombinant microorganism is capable of utilizing TCE to produce VCM. In certain embodiments, the carbon source includes a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof, in certain embodiments, the hydrogen source includes water. In certain embodiments, the chloride source includes sodium chloride, HCI, or a combination thereof.
[0.1.16] Embodiments of the methods herein can provide a benefit of a sustainable use of carbon sources for the production of useful chemical compounds such as VCM and TCE. Besides helping to conserve natural resources, the embodied methods can provide for chemical production by recombinant microorganisms that can provide a benefit of reducing harm to the environment caused by conventional chemical manufacturing processes, thereby supporting long-term ecological balance.
Additional Embodiments;
[0117] Embodiment 1. A method of forming vinyl chloride monomer (VCM) from trichloroethylene (TCE) comprising: reacting TCE in the presence of a VCM forming catalytic element, wherein the VCM forming catalytic element comprises at least one VCM forming enzyme.
[0118] Embodiment 2. The method of embodiment 1, wherein the at least one VCM forming enzyme comprises TCE reductive dehalogenase (Tee A), TeeA anchor protein (TceB), tetrachloroethy lene reductive dehalogenase (peeA), or a combination thereof.
[0119] Embodiment 3. The method of any of the above embodiments i-2, further comprising: forming TCE by reacting glucose with a chloride source in the presence of a TCE forming catalytic element, wherein the TCE forming catalytic element comprises at least one TCE forming enzyme.
[0120] Embodiment 4. The method of any of the above embodiments 1-3, wherein the at least one TCE forming enzyme comprises phenol hydrase (PH), particulate methane monooxygenase (pMMO), toluene dioxygenase (ToD), or a combination thereof.
[0121] Embodiment 5. The method of any of the above embodiments 1-4, further comprising forming glucose by reacting a carbon source with a hydrogen source in the presence of a glucose forming catalytic element comprising at least one glucose forming enzyme.
[0122] Embodiment 6. The method of any of the above embodiments 1-5, wherein the carbon source comprises a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof; or the hydrogen source comprises water; or the chloride source comprises sodium chloride, HCI, or a combination thereof. [9123] Embodiment 7. The method of any of the above embodiments 1-6, wherein the VCM forming catalytic element comprises a VCM bioreactor culture containing a VCM forming recombinant microorganism having an improved VCM producing ability, wherein the VCM forming recombinant microorganism expresses the at least one VCM forming enzyme by i) expressing at least one non-native VCM forming enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM forming enzyme nucleotide sequence, wherein the VCM forming recombinant microorganism is capable of utilizing TCE to produce VCM,
[0124] Embodiment 8. The method of any of the above embodiments 1-7, wherein the VCM forming recombinant microorganism comprises Dehalococcoides sp., Dehalococcoides strain FL2, Dehalococcoides strain BAV1, Dehalococcoides consortia, E coli, a Cyanobacteria, Synechococcus elongatus, and combinations thereof; or further comprising maintaining a temperature in the VCM bioreactor culture of from about 15 degrees Celsius to about 65 degrees Celsius; or maintaining a pH in the VCM bioreactor culture of from about 4,5 to about 12,
[0125] Embodiment 9. The method of any of the above embodiments 1-8, wherein the TCE forming catalytic element comprises a TCE bioreactor culture containing a TCE forming recombinant microorganism having an improved TCE producing ability, wherein the TCE forming recombinant microorganism expresses the at least one TCE forming enzyme by i) expressing at least one non-native TCE forming enzyme nucleotide sequence and i or ii) overexpressing at least one native TCE forming enzyme nucleotide sequence, wherein the TCE forming recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
[0126] Embodiment 10. The method of any of the above embodiments 1-9, wherein the TCE forming recombinant microorganism comprises Rhizohhm mdiloti , Rhkobium meliloti strain Dangeard, Porphyridium purpureum, Emiliania huxleyi, Sinorhizobium mdiloti , and combinations thereof,
[0127] Embodiment 11. The method of any of the above embodiments 1-10, wherein the VCM bioreactor culture and foe TCE bioreactor culture are the same, or wherein the VCM forming recombinant microorganism and the TCE forming recombinant microorganism are the same.
[0128] Embodiment 12. The method of any of the above embodiments 1-1 1, wherein the glucose forming catalytic element comprises a glucose bioreactor culture containing a glucose forming microorganism, wherein foe glucose forming microorganism expresses the at least one glucose forming enzyme, wherein the glucose forming microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose.
{0I29J Embodiment 13. The method of any of the above embodiments 1-12. wherein the glucose forming microorganism comprises a recombinant microorganism, a photosynthetic microorganism, a Cyanobacteria, a Syneckococcus, Synechococcm elongates, Synechocoecus leopoliensis, Synechocystis, Anahaena, a Pseudomonas, Pseudomonas syringae, Pseudomonas savastanoi, Chlamydomonas, Chlamydomoms remhardtii, Escherichia, Escherichia coli, Geobacteria, algae, microalgae, electrosynthesis bacteria, a photosynthetic microorganism, yeast, filamentous fungi, and a plant cell.
|0130] Embodiment 14. The method of any of the above embodiments 1-13, further comprising generating biomass and oxygen in the glucose bioreactor culture and removing an amount of the biomass from the glucose forming bioreactor culture, the TCE forming bioreactor culture, the VCM forming bioreactor culture, or a combination thereof; or maintaining an amount of oxygen in the glucose forming bioreactor culture of from about 20 % by volume to about 250 % by volume or less, based on a total internal volume of the glucose forming bioreactor culture; or converting the carbon source to glucose at an efficiency of from about 30% to about 80%; or maintaining a temperature in the glucose forming bioreactor of from about 15 degrees Celsius to about 45 degrees Celsius; or maintaining a pH in the glucose forming bioreactor of from about 4 to about 12.
[0131| Embodiment 15. The method of any of the above embodiments 1-14, further comprising converting glucose to glyoxylaie, and reacting glyoxylate with the chloride source to form TCE; or increasing a rate of TCE formation by maintaining a concentration of glucose in the TCE hioreactor culture of from about 5 g/L to about 16 g/L; or maintaining a temperature in the TCE bioreactor culture of from about 30 degrees Celsius to about 37 degrees Celsius; or maintaining a pH in the TCE hioreactor culture of from about 6.5 to about 8.5.
1)3132) Embodiment 16, A biomanufacturing system for producing vinyl chloride monomer (VCM) comprising: at least one VCM forming recombinant microorganism having an improved VCM producing ability, wherein the at least one recombinant organism expresses at least one VCM forming enzyme by i) expressing at least one non-native VCM forming enzyme nucleotide sequence and / or is) overexpressing at least one native VCM forming enzyme, wherein the at least one recombinant organism is capable of utilizing a chlorinated hydrocarbon source to produce VCM. [9133] Embodiment 17. The system of Embodiment 16, further comprising: at least one TCE forming recombinant microorganism having an improved TCE producing ability, wherein the at least one recombinant organism expresses at least one TCE forming enzyme by i) expressing at least one non-native VCM forming enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM forming enzyme nucleotide sequence, wherein the at least one recombinant organism is capable of utilizing a chlorinated hydrocarbon source to produce VCM.
[0134] Embodiment 18 The system of any of the above embodiments 16-17, wherein the at least one VCM forming recombinant microorganism and the at least one TCE forming recombinant microorganism are the same; or further comprising a glucose forming microorganism,
[0135] Embodiment 19. The system of any of the above embodiments 16-18, wherein the at least one VCM forming recombinant microorganism comprises Dehalococcoid.es spy., Dehalococcoides strain FL2, Dehahcoccoides strain BAV1 , Dehalococcoides consortia, E. coli , a Cyanobacteria, Synechococcus ehmgatus, and combinations thereof, or the at least one TCE forming recombinant microorganism comprises Rhizobium meliloti, Rhizobium meiiloti strain Dangeard, Porphyridium purpureum, Emiliania huxleyi, Sinorhizobium meliloti, and combinations thereof or wherein the glucose forming microorganism comprises a recombinant microorganism, a photosynthetic microorganism, a Cyanobacteria, & Synechococcus, Synechococcus elongaius, Synechococcus ieopoliensis, Synechocystis, Anabaena, a Pseudomonas, Pseudomonas syringae, Pseudomonas savastanoi, Chiamydomonas, Chlamydomonas reinhardiii, Escherichia, Escherichia coli, Geobacteria, algae, microalgae, electrosynthesis bacteria, a photosynthetic microorganism, yeast filamentous fungi, and a plant cell.
[0136] Embodiment 20. A method of forming vinyl chloride monomer (VCM) from trichloroethylene (TCE) comprising: forming glucose by reacting a carbon source with a hydrogen source in the presence of a glucose forming catalytic element comprising at least one glucose forming enzyme, wherein the glucose forming catalytic element comprises a glucose bioreactor culture containing a glucose forming microorganism, wherein the glucose forming microorganism expresses the at least one glucose forming enzyme, wherein the glucose forming microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose; forming TCE by reacting glucose with a chloride source in the presence of a TCE forming catalytic element, wherein the TCE forming catalytic element comprises a TCE bioreactor culture containing a TCE forming recombinant microorganism having an improved ICE producing ability, wherein the TCE forming recombinant microorganism expresses the at least one TCE forming enzyme by i) expressing at least one non-native TCE forming enzyme nucleotide sequence and / or ii) overexpressing at least one native TCE forming enzyme nucleotide sequence, wherein the TCE forming recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE; and reacting TCE in the presence of a VCM forming catalytic element, the VCM forming catalytic element comprises a VCM bioreactor culture containing a VCM forming recombinant microorganism having an improved VCM producing ability, wherein the VCM forming recombinant microorganism expresses the at least one VCM forming enzyme by i) expressing at least one non-native VCM forming enzyme nucleotide sequence and / or ii) overexpressing at least one native VCM forming enzyme nucleotide sequence, wherein the VCM forming recombinant microorganism is capable of utilizing TCE to produce VCM.
[0137} Embodiment 21. The method of Embodiment 20, wherein the carbon source comprises a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof; or the hydrogen source comprises water; or the chloride source comprises sodium chloride, HC1, or a combination thereof.
EXAMPLES
Example i. Genetic Engineering Approach
[0138] A genetic engineering approach was designed for the production of VCM from C02 (see Fig. 1). A glucose producing recombinant microorganism of the invention (“microbe A”) performs carbon capture and utilization through utilizing a carbon dioxide source to produce glucose and biomass. The glucose produced is utilized by one or more genetically engineered VCM or TCE producing microbes (“microbe B”) to produce TCE and VCM.
[0139] The bioVCM phase 1 focused on the genetic engineering of microbe B. To further enhance the yield of the process, two microbes are engineered to be capable of performing each of the two reactions separately, ch!oride/giyoxyiate/formate to TCE (“Reaction 1”), and TCE to VCM (“Reaction 2”). The capabilities of the microbes engineered to perform Reaction 1 and Reaction 2 separately will also be combined into a single microbe 8 capable of performing both reactions,
[0140] The following outlines the overall reactions to be taking place in bioreactor cultures:
C02 capture/utilization; Reaction:
6CO?. + 6 H2O ® C6H12O6 ■:· 6O2
10 g/L Glucose
60% Reaction Efficiency
(Including selectivity)
Nutrients:
NaNCb 17.6 mM
K2HPO4 0.23 mM
Ferric Ammonium Citrate 0.021 mM
N2CO3 0.19 mM
Citric Acid H2O 0.031 mM
NaCi 150 mM
Reaction 1:
Glucose to G!yoxy!ate:
Figure imgf000041_0001
Glyoxyiate to TCE:
C2H2O3 ·+ 3 HCL C2HCI3 + O2 H2O Glucose to TCE: 30% Reaction Efficiency (including selectivity)
CftHnOs + 6 HC! . 2 CjHCb + 2 CO2 ÷ 2 FfeO + 61 h
Reaction 2:
TCE to DCE:
C2HCI3 + H2 ■■® C2H2CI2 + HO DCE to VCM:
C2H2CI2 t H2 -® C2H3O + HO
TCE to VCM: 80% Reaction Efficiency (including selectivity)
C2HCI3 + H2 ··® C2H3C! 4· 2HC1 Combined Reaction: 30% Efficiency (including selectivity)
CeHaOe + 2 HC1 -® 2C2iI3Ci + 2 CO2 + 2 H2O ÷ 2 ih
Example 2. Materials and Methods
[0141] Bacteria growth and maintenance. For Reaction 1 (chloride to TCE), three native bacteria were obtained: Rhizobium melHoti Dangeard (ATCC), Porphyridium purpureum , and Emiliania huxleyi (ll'TEX). For Reaction 2 (TCE to VCM), two native bacteria were obtained: Dehaiocwcoides sp. Strain FL2 (ATCC 2098) and strain BA VI (ATCC2100). A Dehalococcoides consortia was also obtained from RNAS Remediation Produets. Aii the cells are grown and maintained following standard protocols and media.
[0142] For genetic engineering, E. col: cells and Cyanobacteria Syneckococcus elongalus UTEX 2434 are maintained and grown on LB and BG11 media respectively, supplemented with the appropriate antibiotics.
[0143] Bioinfortnatics and genetic design. Research on new metabolic pathways was done using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Genes and protein sequences were obtained from various databases, including KEGG and UniProt.
DNA constructs were synthesized by Genewiz. Primers for PCR (polymerase chain reaction) were designed using Bench! ing and PrimerS software, and synthesized by MiiliporeSigma.
[0144] Cloning and genetic confirmation. The list of plasmids used in this study are listed in Table 1 below. The following genes were synthesized as gBlocks by Genewiz:
TceA (SEQ ID NO: 2), TceAB (SEQ ID NO: 47), pH (SEQ ID NO: 14), pMMQ (SEQ ID NO: 16), ToD (SEQ ID NO: 18), and peeA (SEQ ID NO: 6). All gBlocks were flanked with BamHI (SEQ IDNO: 48) and Mindlll (SEQ ID NO: 49) recognition sites for adequate cloning. Construction of plasmids pET30a_TeeA, pET30a_pH, pET30a_pMMO, pET30a_ToD and pET30a peeA was achieved by digesting the pET30a plasmid and gBlocks with BamHI and Hindi!! enzymes (NEB), and then ligated (T4 DNA Ligase, NEB), The resulting ligation was transformed into either DH5alpha or BI..21 competent cells (NEB5aIpha, NEB BL21) and plated onto LB medium plates supplemented with kanamycin.
[0145] Construction of the plasmids pSyn6 pH, pSyn6_pMMO, pSyn6_TceA and pSyn6_TceAB was achieved by amplifying the pH, pMMO, TceA and TceAB genes (Phusion® High Fidelity PCR Kit, NEB) from gBlocks using primers flanked by HindlH and Kpnl (SEQ ID NO: 50) recognition sites. The amplified fragments and pSyn6 plasmid were digested with HindlH and Kpnl restriction enzymes (NEB), and then ligated (T4 DNA Ligase, NEB). The resulting ligation was transformed into DHSaipha competent cells (NEB5alpha) and plated onto LB medium plates supplemented with spectinomycin. The confirmation of the correct assembly and insertion of the aforementioned genes into either the pET30a or pSyn6 plasmids was ascertained by Sanger sequencing. The sequencing results matched the desired sequence.
Table 1
Figure imgf000042_0001
Figure imgf000043_0001
{0146] The transformation of Cyanobacteria with the newly engineered plasmids was achieved by inoculating a 10 ml, culture of BG1 1 media with at least 3 mL of dense S. elongaius UTEX 2434 culture and grown at 30°C with continuous illumination. Once the subculture reached an optical density (750 nm) between 1 and 2, 1 ,5 ml, of the cells (per transformation) were harvested by centrifugation at 14,000 rpm for 3 minutes at room temperature. The supernatant was removed by pipetting and resuspending the cells with 1 mL 8G11 media. The cells were centrifuged at 14,000 rpm for 1 minute at room temperature, the supernatant was removed by pipetting. The cells were resuspended with 100 uL ofBGl 1. A plasmid concentration between 200 to 500 ng was added, and the culture was gently flicked. The mixture was incubated in a 34°C water bath with a dark lid for at least 4 hours. A volume of 0,5 mL 13(311 was added to the transformation and transferred to a conical tube and incubated overnight at room temperature, ensuring even and continuous illumination by covering the conical tube with kim wipes. I rnL of BG i 1 media supplemented with spectinomycin was added to a final concentration of 10 ug/mL,
[0147] Cultures were maintained by incubating at 30 °C with continuous illumination and adding BG11 supplemented with spectinomycin (10 ug/mL) every second day until it reached the desired final volume. [0148] Protein analysis. Expressed enzymes from engineered E. coli and Cyanobacteria were purified and confirmed by SDS-PAGE. In brief, fori?, coli cells containing plasmid pET30, a single colony was picked and grown overnight in LB media with antibiotics. In the next day, cells were transferred io fresh LB media and grown to an optical density' ((3D) of 0.6. The culture was then induced by adding Isopropyl b-D-l- thiogalaetopyranoside (IPTG) to a final concentration of 0.5 to 2 mM. The culture was grown for an additional 2-4 hours. Ceil biomass was obtained by centrifugation and stored at 4°C until purification. For engineered Cyanobacteria, ceils were grown in fresh media to an OD of 0.6-1. Cell biomass was then obtained by centrifugation and stored at 4°C until purification.
|6149] Total protein from the cell was extracted using a CelLytic B Plus kit (Mi!!iporeSigma) following the supplier protocol. The target enzyme was then purified using a His-Spin Protein Minipreps kit (ZymoResearch).
[0150] The presence of the enzyme was then confirmed using SDS-PAGE. In brief, 40 uL of protein samples (unreduced or reduced with Novex Bolt reducing agent) was loaded into NuPAGE™ 4-12% Bis-Trts Protein Gels (ThermoFisher Scientific) along with SeeBIue Plus2 Prestained Protein Standard (ThermoFisher Scientific). Samples were run at 200V for 22 min. Gels were removed, washed with water and stained with Imperial™ Protein Stain (ThermoFisher Scientific), images of the protein were recorded by mobile camera.
[0151] Induction assays. For the induction assay, after the engineered E. coli cells were induced by IPTG and grown for an additional 2-4 hours, samples were collected from the main cel! culture for gas chromatography (GC) analysis. Cell culture samples (SOOgX.) were added to 2mL gas chromatography (GC) vials. Substrates (TCE, NaCi or HC1) were added to the vial at different concentrations. The vials were crimped and incubated for different time periods. Once done, vials were stored at -20°C until GC analysis,
[0152] For engineered Cyanobacteria, cells were grown in fresh media to an OD of 0.6-1. Samples were collected from the bulk cells cultures and were added to GC vials and subsequent steps were done similar to the protocol for E. coli.
[0153] Gas chromatography. Analysis of headspace gas was done using an Agilent 7890A gas chromatography system, in brief, 150 mΐ, of gas sample was injected into the system. The detailed setup for the GC is listed in Table 2 below.
Table 2 Oven
Equilibration Time 0,2. min Max Temperature 250 degrees C Oven Program On 50 °C for 8 min then 20 °C/msn to 200 °C for 1 min
Run Time 16.5 min
Front SS Inlet H2
Mode Splitless
Heater Off Pressure Off
Total Flow Off Septum Purge Flow Off Gas Saver Off
Purge Flow to Split Vent 40 mL/min at 0.2 min Back SS Inlet H2
***Exciuded from Affecting GC’s Readiness State
Figure imgf000045_0001
Mode Split
Heater On 180 °C
Pressure On 17.897 psi
Total Flow On 13 J mL/min
Septum Purge Flow On 3 mL/min Gas Saver On 20 mL/min After 0 non
Split Ratio 2:1 Split Flow 7.2567 mL/min Column #1
J&W 122-1334; 5000.55454
DB-624
240 eC: 30 m x 250 mhi x 1.4 pro In; Back SS Inlet H2 Out: Back Detector FID (initial) 50 °C
Pressure 17.999 psi
Flow 3.6283 mL/min
Average Velocity 80.812 cm/sec Holdup Time 0.61872 min Flow Program Off
3.6283 roL/min for 0 min
Run Time 16.5 min
Front Detector FID
Heater Off
H2 Flow Off
Air Flow Off
Makeup Flow Off
Const Col + Makeup Off
Flame Off
Back Detector FID
* 1,1 * Excluded from Affecting GC's Readiness State***
Heater On 240 CC
H2 Flow On 32 mL/m in
Air Flow On 400 mL/min
Makeup Flow On 25.183 mL/min
Const Col + Makeup Off Flarne On
Example 3. Recombinant Microbial Production of TCE and VCM
[0154] Native bacteria and consortia. Since VCM was shown to be involved in the natural process, we first determined whether there are native microbes that can produce or metabolize trichloroethylene (TCE) or vinyl chloride monomer (VCM). The goal was to understand the natural chlorination pathway for production of VCM. from which enzyme candidates could be identified for genetic engineering.
[0155] For Reaction 1 (chloride to TCE, microbe 81), we identified three native bacteria: Rkizobium meiiloti Dangeard (A ICC), Porphyridium purpureum, and Emiliania huxleyi (UTEX), Each of these bacteria was shown to be able to produce TCE from natural resources. These cells were prepared and successfully grown and maintained. For Reaction 2 (TCE to VCM, microbe 82), we identified bacteria in the Dehalococcoides family that can metabolize TCE to VCM and ethylene. We obtained two strains, Dehalococcoides FI.2 and BAV1, as they were shown to produce VCM as the main product from TCE. We also obtained a Dehalococcoides consortia as they were shown to be more resilient and to have a high capacity for TCE metabolism. While the two strains were successfully maintained, we decided to focus on the Dehalococcoides consortia since they have a higher growth rate for the experimental setup.
Figure imgf000047_0001
chloride compounds to trichloroethylene (TCE). Three gene candidates were identified after a hioinformatic study and literature review for conversion of chloride compounds to TCE.
They are phenol hydrolase (PH), particulate methane monooxygenase (pMMO) and toluene d ioxygena.se (To D) .
[0157] These three genes (PH, SEQ ID NO: 14; pMMO, SEQ ID NO: 16; ToD, SEQ ID NO: 18) were synthesized and cloned into plasmid pET30a to generate three new plasmids pET30-PH, pET3G-pMMO, and pET30-ToD. The plasmids were transformed into E. coli BL21 cells for the expression of these enzymes under IPTG induction. The presence of the genes in p.ET30 plasmids were confirmed through gene sequencing. Gel electrophoresis results confirmed cloning PH and pMMO genes into pET30a The expected band size for vector pET30a is 5422 base pairs (bp), pMMO 1266 bp, and PH 1089 bp. Engineered E. coli BL2I cells containing plasmids pET30-PH and pET3Q-pMMO were successfully grown in plate cultures,
[0158] Sequencing results confirmed the presence of the phenol hydrolase gene in the p£T30 vector to form the rET30-RH plasmid, and the presence of the particulate methane monooxygenase gene in the pET3Q vector to form the pE'DO-pMMO plasmid. Sequencing results also confirmed the presence of the Toluene dioxygenase gene in the pET30 vector to form the pET30-ToD plasmid.
[0159] In conclusion, we have successfully engineered three E. coli strains containing three enzyme candidates for the conversion of chloride compounds to trichloroethylene. The production of PH and pMMO enzymes were confirmed by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE).
Figure imgf000047_0002
microbe B2, a gene encoding TCE reductive dehalogenase (TeeA, SEQ ID NO: 2) and its anchor protein (TceB, SEQ ID NO: 4), originated from Dehalococcoides , were obtained from GenBank. Another gene candidate, tetrachioroethyiene reductive dehalogenase (peeA, SEQ ID NO: 6), was also obtained from GenBank. The genes were cloned into plasmids to generated plasmids pSyn6-TceAB, pSyn6-TceA, pET30-TceA, and pET30-PceA.
[0161] Cloning of the Tee A genes into pET30a was confirmed by gel electrophoresis. Plasmid pET30-TceA was transformed into E. coli BL21 cells. The presence of this plasmid was confirmed by gene sequencing. Engineered E. coii B!.,21 with plasmid pET30-TceA was successfully grown in plate cultures. Sequencing results confirmed the presence of the TCE reductive dehydrogenase (Tee A) gene in the pET30 vector.
[0162] Two plasmids were generated for the engineering of Cyanobacteria, pSyn6- TceAB and pSyn6-TceA. pSyn6-TceAB contains both the TCE reductive dehalogenase gene (TceA) and its anchor protein (SEQ I'D NO: 47), whereas pSyn6-TceA contains only the gene encoding the enzyme (SEQ ID NO: 2). The two plasmids were created and their sequences were confirmed by gene sequencing.
[0163] The two plasmids were transformed into Cyanobacteria Symckococcus elongatus UTEX 2434 (henceforth, S2434) to create two new engineered Cyanobacteria, S2434-TceAB and S2434-TceA. Several engineered cell cultures were created. The engineered cells were then confirmed using polymerase chain reaction (PCR). Gel electrophoresis confirmed the engineered Cyanobacteria 82434-Tce.AB and S2434-TceA.
The results confirmed the success of the Cyanobacteria genetic engineering.
Figure imgf000048_0001
new process, there was no established method for detecting and measuring VCM and other chloride compounds from biological samples. Thus, we have developed a new protocol for measuring these compounds from the headspace of biological cultures. Each compound was separately injected into a gas chromatograph. Chromatograms from the analysis showed a clear distinction between VCM and other compounds. Retention times of chloride compounds and intermediates determined by gas chromatography are shown in the graphs Fig. 2. In Fig. 2, graph 201 : TCE, tetrachloroethylene; graph 202, TCE, trichloroethylene; graph 203: DCE, dichloroethyiene; graph 204: VCM, vinyl chloride monomer; graph 205: Ethylene. All compounds show distinct retention times. X axis: minutes, Y axis: peak height (pA),
[0165] For Reaction i, TCE production was tested from the soil bacteria S. meliloti. Data showed that the bacteria has the potential to produce TCE from the sample. A protocol was adapted from a protocol in which S. meliloti was grown in undefined media (soil extracts). In the protocol, cells were grown and maintained in soil extract medium or Vincents minimal media with 20 mmol/L succinate as carbon source at 28°C under rotation. For the experiment, 1 OmL of cell culture were grown to an ODeoonm of i .0. If the OD measured was higher than 1, the culture was diluted with fresh media. For detection of TCE, 0.5mL of culture was added into each GC vial. Different amounts of hydrogen peroxide were then added to a final concentration of 10 - 20 mmoi/L. The vial was closed with septa and crimped tightly. GC vials were incubated in the dark at room temperature up to 8 hrs. The viais were removed at appropriate times and stored at -20°C until testing., TCE production of S. meliloii will be tested using HCi orNaCl as the substrates.
[0166] Gas chromatography results from the soil bacteria S. meliloii are shown in Fig. 3A and Fig. 3B, in Fig. 3.4. the chromatogram (302) shows the TCE peak from a 1 hr sample ofS. meliloti. In the Fig. 3B graph, there is seen a significant increase in the TCE peak area in the S. meliloti sample at t =lhour compared to the media only sample at t = 24 hours.
[6167] Another protocol was also developed for testing TCE production using HCI or NaCI as substrates. This protocol was initially tested with engineered E. coli expressing the enzyme phenol hydrolase (PH) or particulate methane monooxygenase (pMMO) under an IPTG-inducible promoter. Ceils were grown in LB media with appropriate antibiotics overnight under rotation at 37°C. In the next day, a stock culture was diluted in fresh media to an ODsoonm of 0.1 -0.2. The fresh cell culture was grown at 37°C until the OD reached 0.5- 0.6. IPTG was then added to the culture, and the culture was grown at 30°C for an additional 2-4 hours. Culture samples were added into gas chromatography vials for analysis (0.5 ml... for each vial). Amounts of 10% HCI or 10% NaCI, or a combination of both, were added into the GC vials for a final concentration of 1% HCI, or 1 % NaCI, or 1 % of the combination.
The vials were incubated at 30°C for different time periods (up to 48 hours). The vials were removed at appropriate times and stored at -20°C until testing.
[6168] For Reaction 2, after confirming the successful genetic engineering, the cells were tested for their capability in producing VCM and TCE for the process. For VCM production, the native Deh.alocoecoi.des and engineered Cyanobacteria and E. coli were fed with different concentrations of T CE. Gas samples were collected and the presence of TCE, VCM and other intermediates were confirmed using gas chromatography.
[0169] Results from GC are shown in the graphs in Fig. 4A. These results showed that the Dehalococcoides culture significantly consumed TCE and produced VCM in a 24- hour period. In Fig. 4 A, X axis: minutes, Y axis: peak height (pA); graph 402: TCE, tetrachloroethylene; VCM, vinyl chloride monomer; DCE, dichioroethylene; peaks in graphs 404 and 406 correspond to the labeled peaks in graph 402. In addition to VCM, two other products from the process were identified: ethylene and dichioroethylene (DCE). DCE was shown to be an intermediate in the chain reactions from TCE to VCM, whereas ethylene is an end product of the process. Since the experiment was done using the Dehalococcoides consortia, the high ethylene peak likely indicated the accumulation of ethylene in the container, rather than the actual reaction process. The percentage of the area of each compound versus the total area of every peak in the chromatograms shown in Fig. 4 A was calculated. The results showed that the percentage of DCE and VCM significantly increased from 0 to 24 hours, whereas the TCE percentage was decreased (see the graphs in Fig. 4B, Fig. 4C). As seen in Fig. 4B, the area of the GC peaks showed an increase in VCM and DCE concentration and a decrease in TCE concentration. Fig, 4C shows percentages in relation to the total area of the four compounds. These results farther indicated that TCE is metabolized into VCM by the Dehalococcoides consortia.
[0170] To calculate the yield of VCM production, we established a standard curve of VCM (Fig. 4D, 'Fable 3). Table 3 shows peak areas of VCM standards obtained horn gas chromatography. Fig, 4D shoves the standard curve showing a linear relationship between the standards and peak areas. The equation in Fig. 4D was used to calculate VCM yield.
Table 3
Figure imgf000050_0001
Using the standard curve, the yield of VCM production from Dehalococcoides was calculated to be 8E-04 Ib/gai in the 48 hr sampling sample (Fig. 4E, Table 4), For Fig. 4E, VCM yield from Dehalococcoides consortia was measured at different sampling times. Samples were collected in duplicates. Yields were calculated in pg/mL, ft is noted that the yield is dependent on the amount of the TCE substrate, and that higher yields can be achieved If more TCE is fed to the culture.
Table 4
Figure imgf000050_0002
[0172] For the engineered E, coil (BL21-TceA) cultures, GC data showed that the production of VCM from TCE happens even before the IPTG induction (see the graphs in Fig, 5A and Fig, 5B). in Fig, 5A arid Fig. 5B, graphs 502 and 508 are Before Induction; graphs 504 and 50 are T = 4 hours; graphs 506 and 512 are T = 24 hours; VCM, vinyl chloride monomer; DCE, dichioroethy!ene; TCE, trichloroethylene. Fig. 5A shows that VCM production started before the induction and was maintained after 4 hr of induction.
Fig, SB shows that VCM production started before the induction due to leaky expression of Tee A enzyme. Methanol was used as a solvent. Cell cultures initially were fed with 250 pg/snL of TCE. It has been shown that enzyme expression under the IPTG-inducibie T7 promoter can be leaky, meaning that the enzyme can be produced (at low level) even without the inducer. In the experiment, we fed 250 pg/mi, of TCE into the engineered A, end culture. VCM yield from two cultures of engineered A. colt BL2l-TceA cultures is shown In Table 5, The data show that in E. coii BL21-TceA culture #2, 202.9 gg/mL of VCM was produced, The ratio indicated that more than 80% of the TCE is converted to VCM. With higher substrate amounts, the production capacity of the engineered microbe can be significantly increased.
Table 5 j
Figure imgf000051_0001
[0173] For Cyanobacteria, TCE was fed to the cultures, and gas samples were collected at different time points. The GC data showed different intermediates were produced from the culture (see the graph 602 in Fig, 6A), Fig, 6A shows GC data from engineered Cyanobacteria (S2434-TceAB) and shows the production of VCM from TCE. Left, chromatogram of the GC. Right, gas composition from the sample. Peak #1 is VCM, #2 methanol, #3 DCE, and #4 TCE. In another experiment, TCE was fed to the native, non- engmeered S2434 and several cultures of engineered Cyanobacteria (82434-TceAB arid S2434-TceA). Gas samples were collected after 24 hours and analyzed by gas chromatography. The data showed that up to 144 pg/mL VCM is produced from 250 pg/mL TCE fed. That value is equal to 58% of TCE conversion (see graph 604 in Fig, 6B, graph 606 in Fig, 6C). Fig. 6B shows GC data from native Cyanobacteria S2434, and did not show any VCM production. Fig, 6C shows GC data from engineered Cyanobacteria S2434- TceAB, and shows a VCM peak, in the graphs, TCE, tetrachloroethylene; VCM, vinyl chloride monomer. Table 6 shows that the yield of engineered Cyanobacteria is 10 times higher than that of the Dehalococcoides consortia. An amount of 144 pg/mL VCM was shown to be produced from 250 pg/mL TCE fed. That value is equal to 58% of TCE conversion.
Table 6
Figure imgf000052_0001
APPENDIX
SEQ ID NO: 1 - Dehalococcoides - Tee A
MSEKYHSTVTRRDFMKRLGLAGAGAGALGAAVLAENNLPHEFKDVDDLLSAGKAL
EGDHANKVNNHPWWVTTRDMEDPTCNiDWSUKRYSGWNNQGAYFLPEDYLSFT'Y
TGRRHTiVDSKLElFiLQGKKYRDSAFiESGIDWMKENiDPDYDPGELGYGDRREDALI
YAATNGSHNCWENPLYGRYEGSRPYLSMRTMNGiNGLHBFGHADTKTTNYPKWEG
TPEENLLIMRTAARYFGASSVGAIKiTDNVKKlFYAKAQFFCLGPWYTITNMAEYIEY
PVPVDNYAIPIVFEDIPADQGHYSYK-RFGGDDKiAVPNALDNIFTYTIMLPEKRFKYA
HSIPMDPCSCIAYPLPTEVEARIQQFIAGLGYNSMGGGVEAWGPGSAFGNLSGLGEQS
RVSST1EPRYGSNTKGS1..RMLTDLPLAPTKPIDAGIREPCKTCG1CAEHCPTQAISHEGP
RYDSPHWDCVSGYEGWHL,DYHKCTNCTICEAYCPFFTMSNNSWVKNLVKSTVATT
PVFNGFFK.NMEEAFGYGPRYSPSRDEWWASENPXR.GASVDIF
SEQ ID NO: 2 --- Deha!oeoceoides -TceA
ATGAGTGAAAAATACCACTCCACCGTGACCCGTCGCGATTTTATGAAACGCCTGG
GCCTCGCCGGTGCCGGTGCCGGTGCCCTGGGTGCCGCCGTGCTCGCCGAAAACA
ATCTGCCGGACGAGTTTAAAGATGTGGATGATGTCCTGAGGGCCGGCAAAGCGGT
GGAGGGTGATCACGCCAATAAGGTGAACAATCACCCCTGGTGGGTGACCACCCG
CGATCACGAAGATCCCACCTGCAACATTGATTGGAGCCTGATTAAGCGC'TACAGT
GGCTGGAACAATCAAGGTGCCTATTTTCTCCCCGAAGATTATCTCAGCCCCACCT
ATACCGGCCGCCGCCACACCATTGTGGATAGCAAACTGGAAATTGAACTGCAAG
GCAAAAAGTATCGCGATAGCGCCTTTATTGAAAGTGGTATTGATTGGATGAAGG
AGAATATTGATCCCGATTACGATCCCGGCGAACTCGGCTATGGTGATCGTCGCGA
GGA7GCCCTGATTTACGCCGCCACCAACGGTTCCCACAATTGCTGGGAAAACCCC
CTCTACGGCCGCTATGAGGGTT'CCCGCCCCTATCTGAGCATGCGCACCATGAACG
GCATTAATGGTCTCCACGAGTTTGGCCACGCCGATATIAAAACCACCAATTACCC
CAAGTGGGAAGGCACCCCCGAAGAGAACCTGCTCATTATGCGCACCGCCQCCCG
CTATITTGGTGCCTCCAGCGTGGGTGCCATCAAGATCACCGATAACGTGAAGAAA
ATTITCTACGCCAAGGCCCAACCCTnTGCCTGGGCCCCTGGTATACCATTACCA
ATATGGCCGAATACATTGAGTATCCCGTGCCCGTGGATAACTACGCCATTCCCAT
TGTGTYTGAGGATATTCCCGCCGATCAAGGCCACTACAGCTATAAACGCTTTGGC
GGTGATGATAAGATTGCCGTGCCCAATGCCCTCGATAACATTTTTACCTACACCA
TT'ATGCTGCCCGAAAAACGCTTTAAGTATGCCCACTCCATTCCTATGGACCCCTG
CAGGTGC ATTGCCT AGCGCCTGT1T ACCG A AGTGG AGGCCGGC ATTCA AC A ATΪΎ
ATTGCCGGCCTCGGTTATAATAGCATGGGCGGCQGCGTGGAAGCCTGGGGTCCC
GGTTCCGCCTTTGGTAACCTGAGCGGCCTCGGTGAACAAAGTCGCGTGAGTTCCA
CCATTGAGCCCCGCTACGGCAGTAACACCAAAGGTTCCCTGCGCATGCTCACCGA
TCTGCCCCTCGCCCCCACCAAACCCATfGATGCCGGCATTCGCGAATTTTGCAAG
ACCTGCGGTAITTGCGC'CGAGCACTGCCCCACCCAAGCCATTAGTCACGAAGGCC
CCCGCTATGATAGTCCCCACTGGGATTGCGTGTCCGGCTACGAGGGTFGGCACCT
GGATTATCACAAGTGCACCAATTGCACCATTTGCGAAGCCGTGTGCCCCTTTTTC
ACCATGTCCAACAATAGCTGGGTGCACAACCTCGTGAAATCCACCGTGGCCACC ACCGCCGTGTTTAATGGCTTTTTCAAGAAGATGGAAGAGGCGITTGGCTAGGGTC
CCCGCTATAGCCCCAGTCGCGATGAATGGTGGGCCAGCGAGAACCCCATTCGCG
GTGCCAGTGTGGATAITITTTGA
SEQ ID NO, 3 ■ Deha!ococcoides - TceB
MGGAi,YYFI,VGMI,lGGAAIWl'ITYTQFKNISFKWWEWSI,MALSLi.,I,V'SSIFQHMYSS
MSYEMEYQSAFMYLGVFOTLAV!LNLIVWRTYSGRKE
SEQ ID NO. 4-· Dehalococcoides - TceB
ATGGGCGGTGCCCTCTACTATTTTCTGGTGGGCATGCTCATTGGCGGTGCCGCCA
TTTGGTFCATCACCTACACCCAATTCAAGAACATTAGTTTTAAGTGGTGGGAATG
GAGCCTCATGGCCCTGAGTCTGCTCCTGGTGTCCAGCATTTTT'CAACACATGTAT
AGTTCCATGTCCGTGGAAATGGAGTACCAAAGCGCCTiTATGTATCTGGGCGTGT
TTGGCACCCTGGCCGTGATTCTCAATCTGAITGTGTGGCGCACCTACAGCGGTCG
CAAAGAGTAG
SEQ ID NO. 5 - Dehalococcoides - pceA
SNFHSTLTRKDFLKGIGMAGAGLGAASAVTPMFHDLDELVASTPSTRNLPWFVKER
ElIGD?>TTPIDWDMlQRR?YTWARMDFTLFVYDNL,KAiGAPYARWLDWEDKKAEDE
ILYAKAREEFPGFEPGiDGFGDiRTTALTHASEMFAFGQFPQRMNLGGNMVDLVAAV
RAAGGYLGSTDSYAGP.KMVHTPEEMGGTK.YQGTPEDNLRTLKAGIRYFGGEDVGA
LELDDNLKKLVFTVDQYGKTLEFGDVEECIETPRKVTIPNKCKYIFLWTMRQPYEWT
RRQSGRFEGAATETSYERAYNTKAHFQDFARGLGYQM1SAGNNSLSPAGAWAVLG
GLGELSRASYVNHPLYGITVRVTWGFLTDMPLPPSRPiDFGARRFCESCGICAEACPF
GAiNPGEP'rWRDDNTFGNAGFLGWRCDYTKCPHCPiCQGTCPFNSHPGSFIHDVVKG
TV STEP VFNTFFK.NMEKSFKY GRKNPATW WDEVDDYPY GVDTSY
SEQ ID. NO. 6- Dehalococcoides - peeA
ATGTCGAACTTTCACAGTACACTTACCCGTAAGGATTTCCTGAAAGGGATAGGTA
TGGCCGGAGCCGGATTGGGTGCCGCCTCAGCCGTAACCCCTATGTTCCATGACCT
TGATGAGCTCGTAGCTTCAACACCCAGCACCCGCAATTTACCCTGGTTTGTCAAA
GAGCGGGAGCACGGAGACCCTACCACCCCCATAGATTGGGATATGATTCAAAGA
CGCCCTTATACCTGGGCGCGCATGGACCCTACCCTGCCGGTTTATGACAATTTGA
AGGCTATCGGTGCGCCGGTGGCCAGATGGCIGGATTGGGAAGATAAAAAAGCCG
AAGACGAAATTCTGTATGCCAAAGCCCG'IGAAGAATTTCCCGGTTTTGAGCCGG
GTATAGACGGGTTCGGGGATATCCGGACTACCGCTCTTACTCATGCTTCGGAAAT
GrrCGCTTFCGGTCAGTTTCCCCAGAGGATGAATTTGGGCGGCAATATGGTAGAC
CTGGTAGCGGCAGTTCGGGCGGCCGGCGGTTACCTGGGTAGTACCGATAGCTAT
GCGGGACCTAAAATGGTGCATAGCCCTGAAGAGATGGGCGGCAGGAAGTATGAG
GGTACTCCCGAAGAT'AACCTCCGAACCCTGAAAGCCGGTATCCGTTATTTTGGCG GTGAAGACGTGGGTGCTiTGGAACTTGATGATAACCTTAAAAAACTTGTCTTTAC
TGTTGACCAGTACGGCAAAACACITGAATTCGGGGATGTTGAGGAATGTATTGA
AACTCCCCGAAAAGTTACTATTCCCAACAAATGCAAATATATTTTCCTCTGGACT
ATGCGTCAGCCATACGAATGGACCCQCCGCCAGTCCGGCAGGTTTGAAGGAGCG
GCTACAGAGACCAGTTACGAACGCGCCTACAATACCAAAGCCCACTTCCAGGAT
TTTGCCCGCGGCTTGGGCTAGCAGATGATAAGTGCGGGGAAGAACAGCCTTTCTC
CGGCTGGTGCTTGGGCGGTTCTGGGCGGTCTGGGTGAACTTTCCCGTGCTTCATA
TGTAA ACCACCCGCTTTACGGCATTACCGTCAGAGTTACCTGGGGTTTCCTGACT
GATATGCCGCTGCCACCCAGCCGCCCCATTGATTTYGGTGCCCGCAGATTCTGTG
AAAGCTGCGGTATCTGTGCCGAGGCTTGTCCTTTCGGGGCTATCAATCCCGGTGA
ACCTACATGGAGAGATGATAATAGCTITGGCAAGGCAGGTTTCCTGGGCTGGCGC
TGTGACTATACCAAGTGCCCCCACTGTCCCATTTGCCAGGGTACCTGTCCCTI'CA
ATTCCCACCCCGGCTCGTTTATACATGACGTAGTCAAGGGCACTGTTTCCACCAC
ACCTGTGTI'CAATACTTTCTTCAAGAATATGGAAAAGTGCrn'AAGTAGGGGCGG
AAGAATCCTGCTACCTGGTGGGACGAAGTGGATGACTATCCGTATGGGGTCGAT
ACGAGTTACTAA
SEQ ID NO. ?■■ Dehalococcoides - vcrA
MSKFHKTISRRDFMK.GLGLAGAGIGAVAASAPVFHI)?'DEFVSSEANST'KDQPWYVK
HREHFDPTITVDWDSFDRYDGYQHKGVYEGPPDAPFTSWGNRLQMRMSGEEQKKRi
LAAICKERFPGWDGGLHGRGDQRADALFYAVTQPFPGSGEEGHGLFQPYPDQPGKF
YARWGLYGPPHDSAPPDGSVPKWEGTFEDNFLML.RAAAKYFGAGGVGALNF.ADPK
CKKLJYKKAQPMTF,GKG1YS.EIGGFGML0AKFYPKVPDI-IAVPINFKEADYSYYNDA
EWVIPTKCESiFTFTLPQPQELNKRTGGIAGAGSYTWKDFARVGTL-VQMFlKNLGY
HALYWPIGWGPGGCFTTFDGQGEQGRTGAAIHWKFGSSQRGSERVITDLPIAPTPPFD
AGMFEFCKTCYICRDVCVSGGVHQEDEPTWDSGNWWNVQGYLGYRTDWSGCHNQ
CGMCQSSCPFTYLGLENASLVHKIVKGVVANYrVFNSFFTNMEKALGYGDLTMENS
NWWKEEGPIYGFDPGT
SEQ ID NO, 8: ·· Dehalococcoides - vcrA
ATGAGTAAATTTCATAAAAGGATTAGGCGCCGAGATITCATGAAAGGACTAGGA
TTAGCCGGGGCAGGCATAGGCGGTGTTGCGGCGTCAGCTCCGGTTTTTCATGACA
TT’GATGAATTTGTTTCAAGCGAAGCAAATTCTACTAAAGATCAACCTTGGTACGT
TAAGCATCGAGAGCATTTTGACCCTACGATTACAG7TGACTGGGATATTTTTGAT
AGATATGACGGGTATCAGCATAAGGGTGTCTATGAAGGCCCTCCAGATGCTCCCT
TTACATCATGGGGCAATAGGGTTCAGATGAGAATGTCAGGTGAAGAGCAAAAGA
AGCGAATTTTGGCCGCTAAAAAAGAGAGGTTCCCTGGTTGGGACGGTGGGTTAC
ACGGGAGAGGGGATCAGCGGGCGGATGCACTATTTTACGCAGTAACTCAACCAT
'TTCGTGGTAGTGGTGAGGAAGGGCACGGAGTAITCCAACCTTATCGTGATCAACC
CGGTAAGTTYTACGCGAGATGGGGTTTGTATGGTCCGCCACATGATTCAGCGCCA
CCTGATGGGAGCGTACCAAAATGGGAGGGTACTCCAGAAGACAATTTTCTAATG
CTGAGGGCAGCTGCAAAATATTTTGGTGCTGGTGGCGTTGGTGCTCTTAACCTGG
CAGATCCCAAATGCAAAAAACTAATATATAAGAAAGCTCAGCCGATGACTCTAG GAAAAGGAACATACAGTGAAATAGGTGGACCAGGAATGATCGATGCAAAATTTT
ATCCCAAGGTTGCTGAGCATGCCGTACCTATTAACTTTAAGGAAGCGGA'ITATAG
CTACTACAATGAI'GCAGAGTGGGTIATTCCAACAAAGTGTGAATCCATTITCACT
TT'CACGGTACGTCAACCACAAGAAGTCAATAAGAGGACGGGTGGTATAGCAGGT
GCTGGATCATATACTGTATACAAAGATTTCGCTAGGGTAGGCACT1TAGTCCAAA
TGTTFATTAAGAATCTAGGTTATCACGCTTTATATTGGCCAATTGGATGGGGACC
GGGTGGTTGCTTTACCACTTTTGACGGGCAAGGTGAACAGGGTAGAACAGGTGC
TGCTATCCA'lTGGAAGTITGGTTCirTCAGAACGTGGTrCTGAAAGAGTAATAACT
GATTTACCGATAGCTCCTACCCCGCCAATFGATGCAGGTATGTTYGAGTTTfGCA
AAACCTGTTATATATGCCGTGACGTTTGCGTCTGTGGGGGTGTGCACCAAGAAGA
CGAACCAACTTGGGATTCAGGTAATTGGTGGAATGTACAAGGATATCTCGGCT'A
CCGAACGGATTGGAGTGGTYGCCATAACCAGTGCGGTATGTGTCAATCCTCCTGC
GCTTTTACTTATiTAGGTTTGGAAAATGGTTCATTAGTGCACAAAATAGTAAAAG
GTGTTGTTGCTAACACGACTGTTTTTAATAGTTTTFFTACCAATATGGAGAAAGCA
'ITAGGATATGGTGATTTAACCATGGAAAATTCTAACTGGTGGAAAGAAGAAGGA
CCGATATACGGCTTTGATCCCGGTACTTAG
SEQ ID NO. 9: - Uncultured bacterium - depA
LRMQlGAWASLDRGQTGYLKYPPEGFRTiKVTHETLGVPKWEGSETENAFMiRTFLR
QFGAGAiGYARVDDNSVGPRKPLFNTHVRLENNSDYKYDTNGTFVMPEKCKYAlVI
YDRSPRDPNNYRRTVNSPQAFVSNMEKCEYGHKLQNFLWC3LGYQSYWFEDGTTSK
FTGTPTNVWGILSGVGEYNRlHNAVSQPEGESGNFASILFTDLPLPTiKPiDFGALEFC
KTCGfCADVCPAGAlPTVEEYREPTWNRATGPWSASNDHRGYPNKSfECVKWYFSN
AITAFAFASRPVGVCRRCASHCVFSKDI-IEAWIHEVVKGVVSITPVMNSFFTKMDRL
SGYSDVISDEGRAEYWHQYLPAI
SEQ ID NO. 10: - Uncultured bacterium ·· cicpA
TTGCGTATGCAAAITGGAGCCTGGGCTTCTTTGGATAGAGGACAAACAGGATATC
TAAAATATCCACCTGAAGGTTTTCGAACAATAAAAGITACTCATGAAACCTTAGG
TGTACCGAAGTGGGAAGGCTCCGAAACGGAAAACGCA'ITCAI'GATTCGAACTTT
CCTGCGGCAATTTGGTGCAGGGGCGATTGGCFATGCAAGAGTGGATGATAACAG
CGTAGGACCACGTAAACCCCTTTTTAATACTCATGTGAGATTGGAAAACAATI'CA
G ATT AΪA AGTATG ATACT A ATGG A AC ATTTGTC ATGCCCG AG A A GT GC A AATATG
CCATCGTGATTTACGATAGAAGTCCCCGAGATCCCAATAACTATCGTCGTACTGT
CAATAGTCCTCAAGCCTTTGTATCAAACATGGAGAAATGCGAGTATGGTCATAA
GCTCCAAAACTTCCTTTGGGG'ITTAGGCTACCAGTCCTATTGGTTTGAAGATGGC
ACAACTAGCAAATTCACAGGGACTCCGACGAATGTTTGGGGTATTCTCTCAGGTG
TAGGCGAATACAATCGGATTCAGAATGCGGTGTGAGAACGCGAAGGCGAAAGTG
GTAAlTiTGCCAGTATTCTCTlTACGGATTlGCClTTGCCTACCACTAAACCGATA
GACTTTGGGGCACTAGAATrCTGGAAGAGCTGCGGTATTTGTGCTGATGTTTGGG
CAGCCGGAGGGATTCCAAGAGTGGAGGAGTATAGAGAGGCAAGTTGGAAGGGAG
CCACAGGACCTIGGAGCGCTTCGAATGACCACAGAGGTTACCCTAATAAATCTAT
TGAATGTGTGAAGTGGTATTTTTCTAATGCAATTACAGCCTTCGCTCCTGCCTCTC GCCCAGTTGGAGTGTGTCGCCGATGTGCCAGCCATTGTGTCTTTAGTAAAGATCA
TGAAGCTTGGATTCATGAAGTAGTTAAGGGTGTAGTTTCCACTACCCCTGTGATG
AACAGCTTCTTTACTAAAATGGATAGACTATCCGGTTACAGTGACGTCATCTCAG
ATGAAGGCAGGGCTGAATATTGGCACCAGTACCTGCCCGCTATTTAA
SEQ ID NO. 11 : ·· Dehalogenimonas -■ TdrA
MQlAjiRERPEKSAQIPBERRRNKSMSNFHSSlSRRDFMKGLGLAGAGGLGAAVLANN
NLFVEFKDLDDVLSAGKALEGDHANKVNMDPW'WVA'rRDHENPTCNlDWSQITRYS
GWNNQGAYVMPDNYLSSDYSGRRYTiiDSViAAANGTKPiDTLRMQQGINWM!ANI
DPNYYAGYQGYGDRKEDALLYAATNGSHNCWVNPLYGSDRYNGSRPYLSLRSMT
GTLGMHEFGYNDVKLQDYPNWQGTFEENULMRAVSRYFGASSVGAIKrrDNVKKl
FFAKAQPFLPAPWHHLLKTAEYVEYPVPVDNYPIPIVFDDVPADQGHYSYKRfGGDD
KIVVPNALDNiFTYTVMLPETRFKYVCSiGMDPCSSiAYPLFTEIEQRLQTFIAGLGYN
SMGGGVSAWGPGGAFGNLSGLGEQGRVSSTIEPRYGSGTKGALRMLSDLPLAPTKP1
DAG!REFCKTCGICATKCPGQAiSYEGPRYDSPFWDCVSGYEGWHLDYSKOGCTNC
ESYCPFFTMSNNSWVHGLVKATIASTPTFNGFFKRTEEAFGYGPVYSPNHDQWWAK
ENPIRGASVDVF
SEQ ID NO. 12: - Dehalogenimonas - TdrA
GTGCAGCTTGGCATCCGGGAGAGACCGGAAAAATCAGCACAGATACCAGA AGA
AAGAAGGAGAAATAAATCCATGTCCAATTTCCATTCAAGCATAAGCCGAAGAGA
TTTTATGAAGGGCTTGGGTCTGGCAGGTGCAGGTGGGCTGGGTGCCGCTGTACTT
GCGAAGAATAATGTGCCGGTTGAGTTTAAGGATCTTGATGACGTGCTATCTGCAG
GCAAAGCCCTAGAGGGTGACCACGCTAATAAAGTGAACAATGACCCGTGGTGGG
TTGCAAGCAGAGACCATGAAAAGCGCACCTGTAAGATTGACTGGAGGGAGATAA
CCAGATATAGCGGATGGAATAACCAAGGGGCCTATG1TATGCCTGACAATTACC
TGTCATCAGATTATAGCGGAAGGCGATATACAATTATTGATTCGGTAATAGCAGC
TGCCAATGGAACGAAACCTATTGATACTCTCCGCATGCAACAGGGGATAAACTG
GATGA'ITGCGAATATTGATCCGAACTACTACGCTGGTiATCAGGGCTATGGTGAC
CGCAAGGAAGATGCCTT'ACTTTATGCCGCCACGAATGGCTCGCATAATTGCTGGG
TCAATCCGCTTTACGGGAGTGATCGTTATAATGGCTCTCGTCCCTACCTCTCACTG
CGCTCCATGACCGGAACACTTGGCATGCATGAATTCGGCTATAATGACGTCAAGC
TTCAGGA'n'ACCCGAAGTGGCAGGGTACGCCTGAGGAAAACTTGATTTTAATGC
GCGCCGTAAGCCGATACTTTGGAGCGTCTrCCGTCGGTGCCATTAAGATAACAGA
TAACGTGAAGAAAATATTCTiCGGCAAAGCTCAACCCTTTCTCTTTGCGCCCTGG
CATCACCTTTTGAAAACGGCTGAGTATGTTGAATACCCGGTCCCGGTGGATAATT
ATCCCATAGCTATTGTGTTTGACGATGTACCCGGAGACCAAGGCCATTACAGCTA
T'AAGCGTTTCGGCGGTGATGATAAGATAGTAGTTCCCAACGCGCTGGATAACATC
TYCACCTATACCGTTATGCTTCCCGAGACGCGCTTTAAATATGTATGTTCGATAG
GTATGGATCCTTGTTCCAGCATTGCCTATCCGCTGTYTACCGAAATAGAACAACG
CCTGCAGACTTFGAITGCAGGCTTGGGATATAATTCCATGGGCGGCGGTGTCTGC
GCTTGGGGACCAGGTGGCGCCTTCGGTAATCTCAGCGGCCTTGGCGAACAAGGC
CGCGTATCTAGTACTATTGAGCCACGGTACGGCTCCGGTACCAAGGGTGCTTTAA
GAATGCTCTCAGATCTGCCTCTCGCCCCCACTAAACCGATTGATGCCGGTATCCG
AGAGTTCTGCAAGACCTGTGGAATCTGCGCGACGAAATGTGGCGGACAGGCCAT CTCCTACXiAGGGCCCACGCTACGACTCACCTTTTTGGGATTGCGTGAGTGGTTAT
GAAGGATGGCACCTTGATTATTGCAAGTGTATAGGTTGTAGCAATTGTGAGTGAT
ACTGCCCCTTTTTCACTATGTCTAACAACTCTTGGGTGCATGGTTTGGTAAAGGC
GACAATTGCCTCTACACCCACTTTCAATGGCTTCTTTAAGAGAACGGAAGAAGCC
TTCGGATACGGCCCGGTYFATTCCCCAAACCATGATCAATGGTGGGCTAAAGAAA
ACCCAA'rrCGCGGCGCAAGTGTAGATGTTTTTTAA
SEQ ID NO. 13: - Ra!stonia - PH
MYSLTIEPIGQTIPfAPGQTVLDACLRNGVWLPHACCHGLCATCKVQVVEOEFEHGE
ASSFALMDFERDSGQCLACCATAQSDMVIEADIEEDADSLGLPLADYRAEVVEARA
LTPTIRGIWLRVKGGAAAAFQAGQYLNLRVPGCDQPRAFSLANRPGDDLVELHVRR
VEGGQATGYLHDQLSVGDELGFSAPYGRFFYRKSAQK.PMLFLAGGSGLSSPRAMIL
DMLAAGETLPiTLVGGARNRTELYYDEAFRALAGAHPNFRYYPALSDEPADSGWDG
ARGYVHBVLHGLYANGATADFRGHKAYLCGPPPMlEACIRTLMQGRLFEEDiHTEK
FIS AG DAQNS A RS FLFK I
SEQ ID NO, 14: - Cupriavidus - PH
ATGTATFCCCTGACCATTGAACCGATCGGGCAGACCATCCCCATCGCGCCGGGCC
AGACCGTGCTGGATGCCTGCCTGGGCAACGGGGTGTGGCTGCCGCACGCCTGCTG
CCACGGGCTGTGCGCCACCTGCAAGGTGCAGGTGGTGGAGGGCGAATTCGAGCA
TGGAGAGGCCTCCAGCTTCGCGCTGATGGACTTCGAGCGCGACAGCGGGCAGTG
CCTGGCTTGCTGCGCCACCGCGCAGTCCGACA'T'GGTGATCGAGGCCGATATCGA
GGAAGACGCCGACTCGCTCGGCCTGCCGCTGGCTGACTATCGTGCCGAGGT'GGT
GGAGGCCCGCGCGCTGACCCCCACCATCCGCGGCAl'CTGGCTGCGCGTGAAGGG
GGGGGCCGCGGCTGCGTTCCAGGGGGGCCAGTACCTCAACCTGCGGGTGCCGGG
CTGCGAGC AGCCG CGTGCGTTCTGGCTGGCC A ACCGTCCCGGCG ATG ACCTGGTG
GAGCTGCATGTGCGGGGGGTGGAAGGCGGGCAGGCTACGGGGTACCTGGACGAT
CAGCTGTCGGTGGGTGACGAACTCGGGTTTTCCGCGCCTTACGGCCGCTTCTTCG
TGCGCAAGTCAGCGCAAAAGCCGATGCTGTTCCTGGCGGGCGGCTCGGGCTTGT
CCAGCGCGCGCGCCATGATCCTGGACATGCTGGCTGCCGGCGAGACCCTGCCGA
TGAGGCTGGTGCAAGGGGCGGGCAAGCGCAGGGAGGTGTACTAGGAGGAGGGGT
TCCGTGCACTGGCCGGCGCGCACCCCAACTTCCGCTAT'GTGCCCGCGCTCTCCGA
CGAACCGGCGGACAGCGGCTGGGACGGCGCGCGCGGCTATGTGCATGACGTCCT
GCACGGCCTTTACGCCAATGGCGCGACCGCCGACTTCCGTGGCCACAAGGCCTAT
CTGTGCGGCCCGCCGCCGATGATCGAAGCCTGCATCCGCACGTTGATGCAGGGC
CGGCTGTTCGAGGAGGACATCCACACCGAGAAATiCATCTCGGCCGGCGACGCA
CAGAACAGCGCGCGCAGCCCGCTGTTCAAGATCTGA
SEQ ID NO, 15: - Methylococcus - pMMO
MKTIKDRIAKWSAIGLLSAVAATAFYAPSASAHGEKSQAAFMRMRTIHWYDLSWSK
EKVKiNETVEIKGKFHVFEGWPETVDEPDVAFLNVGMPGPVFIRKESYiGGQLVPRSV
RLE!GKTYDFRVVLKAREPGDWHVHTMMNVQGGGPIIGPGKWITVEGSMSEFRNPV
5? TTLTGQTVDLENYNEGNTYFWI-iAFWFAIGVAWlGYWSRRPiFIPRLLMVDAGRADE LVSATDRKVAMGFLAATfLliVVMAMSSANSKYPrnPLQAGTMRGMKPLELPAPWS VKVEDATYRVPGRAMRMKLTiTNHGNSPiRLGEFYTASVRFLDSDVYKDTTGYFED LLAEDGLS V SDN SPLAPGETRTV DVTASD A AWEVYRLSDH YDPDSRF AGLLFFFD AT GNRQWQlDAPUPSFNt
SEQ ID NO. 16: ·■ Methyiococcus - pMMO
ATGAA A ACAATAAAGG ACCGGATTGCA A A ATGGTCTGCAATCGGACTGCTGTCC
GCCGTGGCAGCGACCGCCTTCTATGCGCCGAGCGCCAGCGCCCACGGTGAGAAA
TCGCAGGCCGCGTTCATGCGTATGCGTACCATCCACTGGTACGACCTGAGCTGGT
CGAAAGAGAAAGTCA AGATCAACGAGACCGTGGAAATCAAAGGCAAGTTCCAC
GTGTTCGAAGGCTGGCCGGAAACGGTCGACGAACCGGATGTGGCGTTCCTGAAC
GTGGGGATGCCGGGTCCGGTGTTGATCGGCAAGGAATCGTACATCGGCGGTGAG
CTGGTGCCGC-GTTCCGTACGTCTGGAAATCGGCAAGACCTATGACTTCCGGGTTG
TCCTCAAAGCCCGTCGTCCGGGTGACTGGCACGTTCACACCATGATGAACGTCCA
GGGCGGTGGACCGATCATCGGTCCCGGCAAAl'GGATCACCGTGGAAGGCTCCAT
GAGTGAATTCCGCAACCCCGTCACCACCCTGACCGGTCAGACGGTGGACCTGGA
GAACTACAACGAAGGCAACACCTATTTCTGGCACGCC'TTCTGGTTCGCCAT'CGGA
GTTGCCTGGATCGGGTAGTGGTCGCGTCGACCGATCTTCATCCCCCGTCTGCTGA
TGGTGG ATGCCGGT'CGTGCGG ACG A ACTGGTGTCCGCG ACCG ACCGC A AGGTGG
CGATGGGCTTCCTGGCCGCCACCATCCTGATCGT'GGTCATGGCCATGTCCAGCGC
CAACAGC A AGTACCCG ATC ACC ATCCGGCTGC AGGCCGGC ACC ATGCGTGGC AT
GAAGCCGCTGGAACTGCCGGCGCCGACGGTATCGGTGAAAGTGGAAGACGCCAC
CTACCGGGTACCGGGCCGCGCCATGCGGATGAAGCTGACCATCACCAACCACGG
CAACAGCCCGATCCGGCTGGG’I'GAGTTCTACACCGCCTCGGTGCGTTTCCTGGAT
TCCGACGTGTACAAGGACACCACGGGCTATCCGGAAGACCTGCTGGGCGAAGAC
GGCCTGAGCGTCAGCGACAACAGCCCGCTGGCTCCGGGTGAGACGCGCACGGT'C
GACGTGACGGCGIGCGACGCGGCGTGGGAAGTGTACCGTCTGTCCGACATCATC
TACGATCCGGACAGCCGITFCGCCGGTCTGCTGITCTTCTTCGACGCCACTGGCA
ACCGCCAGGTCGT'CCAGATCGACGCACCGCTGATGCCGTCGTTCATGTAA
SEQ ID NO. 17: -- Pseudomonas - benzene (toluene) dioxygenase iDSANRADVFLRKPAPVAPELQHEVEQFYYWEAKLLNDRRFEEWFALLAEDiHYFM
PIRTTRlMRDSRLEYSGSREYAHFDDDATMMKGRLRKfTSDVSWSENPASRTRHLVS
NVMIVGAEAEGEYEISSAF1VYRNRLERQLDIPAGERRDTLRRNTSEAGFE1VNRTIL1
DQST1LANNLSFFFATNPSLLK.QAGDVEENPGPNQTDTSPIR.LRRSWNTSEIEALFDEH
AGRIDPRIYTDEDLYQLELERVFARSWLLLGHETQIRKPGDYrFITMGEDPVVVVRQ
KDASIAVFLNQCRHRGMR1CRADAGNAKAFTCSYHGWAYDTAGNLVNVPYEAESF
ACLNKXEWSPLKARYETYKGLIFANWDENAVDLDTYLGEAKFYMDBMLDRTEAGT
EAiPGVQKWVIPCNWKFAAEQFCSDMYHAGTTSHLSGH-AGLPEDLEMADLAPPTV
GKQYRASWGGHGSGFYVGDPNLMLAIMGPKVTSYWTEGPASEKAAERLGSVERGS
KLMVEHMTVFPTCSFLPGiNTVRTWHPRGPNEVEVWAFTVVDADAPDDIKEEFRRQ TIJiTFSAGGVFEQDDGBNWVBIQHItRGBKARSRPPNAEMSMDQTVDNDPYYPGEI
SNNVYSEEAARGLYAHWLRMMTSPDWDALKATR
SEQ ID NO, 18: - Pseudomonas - benzene (toluene) dioxygenase
ATTGATTCAGCCAACAGAGCCGACGTCTTTCTCCGCAAGCCGGCACCCGTAGCGC
CCGAAGTGGAGCAGGAAGTCGAGCAGTTCTAGTATTGGGAGGGCAAGCTCCTCA
ACGATCGCCGCTTCGAGGAGTGGTTCGCGCTGC/rCGCGGAAGACATTCACTACrr
CATGCCCATTCGCACCACGCGGAT'CATGCGGGACTCGCGCCTTGAATACTCAGGC
TCCCGAGAGTACGCGCACTTCGATOACGACGCCACGATGATGAAGGGACGCTTG
CGCAAGATCACGTCCGACGTGAGCTGGTCCGAGAACCCCGCATCGCGGACCCGG
CATCTCGT’GAGCAACGTGATGATCGTCGGCGCAGAGGCAGAAGGGGAGTACGAA
ATCTCAAGCGCCTTGATTGTGTACGGCAATCGTCTGGAGCGGCAGCTCGACATCT
TTGCCGGTGAGCGTCGCGATACGTTGCGCCGTAACACGAGCGAGGCCGGGTTCG
AGATCGTCAATCGGACCATCCTGATCGACCAGAGCACCATCCT'GGCCAATAACCT
CAGTTTCTTCTTCGCCACCAACTTTAGCCTGCTCAAACAAGCCGGCGATGTGGAA
GAGAACCCCGGTCCCAATCAGACCGACACATCACCTATCAGGCTGCGCAGGAGC
TGGAACACCAGCGAGATAGAAGCGCTCTTTGACGAGCATGCCGGACGTATCGAT
CCGCGCATTTATACCGATGAGGATCTGTACCAACTCGAACTGGAGCGTGTCTTCG
CCCGGTCCTGGCTGCTG'rrGGGGCATGAAACCCAGATTCGCAAGCCGGGCGATT
ACATCACGAGCTACATGGGTGAAGACCGTGTCGTGGTCGTCCGGCAGAAAGACG
CCAGCATTGCCGTGITCCTGAACCAGTGCCGCCACCGTGGCATGCGCATCTGCCG
CGCGGATGCCGGAAACGCGAAGGCGTTCACITGCAGCTACCACGGGTGGGCTTA
CGACACCGCCGGCAATCTTGTCAAT'GTGCCTTACGAGGCCGAATCCTTCGCGTGC
CTGAACAAGAAGGAATGGAGCCCGCTGAAGGCCCGGGTAGAAACCTACAAGGG
CCTGATTfTCGCCAACTGGGATGAGAACGCTGTAGACCTCGACACGTATCTGGGC
GAGGCGAAGTTCTACATGGACCACATGCTCGACCGCACCGAGGCCGGCACCGAA
GCGATCCCGGGCGTGCAGAAGTGGGTCATTCCCTGTAACTGGAAATTCGCCGCA
GAGCAGTTTTGCAGCGACATGTACCATGCCGGGACGACCTCGCATC'FGTCTGGCA
TCCTGGCAGGCCTGCCAGAAGACCTTGAAATGGCCGACCTTGCTCCGCCGACAGT
TGGCAAGCAGTACCGTGCGTCATGGGGCGGACATGGAAGTGGCTTCTATGTCGG
CGACCCCAAI'CTGATGCTTGCCATCATGGGGCCAAAGGTCACCAGCTACTGGACC
GAAGGCCCCGCGTCGGAAAAGGGGGCCGAACGTGTGGGTAGCGTGGAGCGCGG
CTCGAAACTCATGGTCGAGCACATGACCGTCTTCCCCACGTGTTGCTTCCTCCCA
GGTATCAATACGGTCCGGACATGGCATCCGCGCGGGCCGAACGAGGTCGAGGTA
TGGGCGTTTACGGTGGTCGATGCTGATGCTCCTGACGATATCAAGGAAGAGTTCC
GGCGCCAGACGC!GCGCACCTTCTCTGCCGGTGGCGTGTTCGAGCAGGACGACG
GGGAGAACTGGGTCGAGATCCAGCACATCCTGCGAGGCCACAAGGCGCGGAGCC
GCCCTTTCAATGCCGAGATGAGCAI'GGACCAGACCGTCGACAACGACCCGGTTT
ACCCCGGGCGGATCAGCAACAACGTCTACAGCGAGGAAGCTGCC'CGCGGGCTCT
ATGCCCATTGGCTGCGGATGATGACATCCCCCGACTGGGACGCGCTGAAGGCGA
CACGC
SEQ ID NO, ! 9: - Pseudomonas -■ TouA MSMLKREDWYDi;rRrrNWTPKYVTENELPPEEMSGARGiSMEAWEKYDEi>YK.rrY
PEYVSIQREKDSGAYSIKAALERDGFVDRADPGWVSTMQLHFGAIALEEYAASTAEA
RMARFAKAPGNRNMAITGMMDENRHGQIQLYFPYANVKRSRKWDWAHKAIHTNE
WAAIAARSFFDDMMMTRDSVAVSIMLTFAFETGfTNMQFLGLAADAAEAGDHTFA
SUSSiQTDESRiiAQQGGPSLKlLYENGKKDEAQQMYDVAIWRSWKLFSVLTGP!MD
YYTPLESRNQSFKEFMLEWIVAQFERQLl.DI.-GI.DKPWYWDQFMQDLDETHHGMHL
GYWYWRPTVWWDPAAGYSPEEREWLEEKYPGWNDTWGQCWDVITDNLVNGKPE
LTVFETLPT1CNMCNLPIAHTPGNKWNVKDYQLEYHGR1..YHFGSEADRWCFQIDPER
YKNHINLVDRFLKGEIQPADLAGALMYMSLEPGVMGDDAHDYEWVKAYQKKTNA
A
SEQ ID NO. 20: - Pseudomonas - TouA
TTTAATCATCTGGTGAAACTTAAAATCAAGTTAACCAATTAATAACATTCAGATC
GTTACCAACCTTGGGGTTGAAAGCCTATAAATAGGTAACTFGATGATTAAAAGGT
ATTTTTGGGTATTGGCATATACATTGCTTCAGATACAGATATCATAACAACAAAC
AGAGACAAACCATGAAATATGGAAACGGGGTGGACGGTTTGCTGGTGGTGGTAG
AAGATGTAATGGTGGACITTTCATTCGATGACACCGCCCTGACCGGAYFGATGGC
CGGCCGTGACTATCC’GCGCACGTACCGCGAGTTCGTTGAGATGTATCCCGACGAT
GCGGCCTGCGCTGCGGGGTTGGGCCAATTGCGCTGGCCCAGTGGCTTTGTCTGTC
CCGCGTGTAGGACTGCTGCGTCGCCGTGGCAGGATTCGCGGGGGCACCTCGTGT
GCTCGTCCTGCCGCCATCAAGGAACAGTGACCGCTGGTACCCTCCTGGACAAGA
CGCGGACAeCGCTGACCAeiTGGTTTGAGGCCGCCTGGCATGTGACTGCCGCAA
AGAATGGTCTGCCTGCGAAGACACTTGAGCGAACACTTGGCGTCAGATATCGAG
TGGCGTGGACGATGTACAACCCAGCAACGCTTGGTrfAATAGAAGAGGGGCCTG
GGCTTTATATCGAGTTGGATCTTATCGGAACCGATATCGATACTGTCAACCAGGC
GACAGGAGAGAAGGCCAAATCCAACAAAAATAGTGAAAGCAACAACCGAGGCC
CAATTTATTACGCTCCGCAACTGGCGTACACCAACAATGTTGGCAACCTGACACT
CGGAGTAGAGGCGCATTCGCCCAAGGTGGCCTGGGTACGGAATTCGGTAAGACC
AGCTITCTGTCCAGGACCTCCAGAAAGAATGTGGATACAGGCCTTGATAAGTGAT
CTCGTYGATTGAACCTGCGTATTCCCTTTGCAGCCGCCTACAAGGTGAATGATTG
CCTGATCGTCGGAGGGT'CTGTCGATGCCGTTTGGACTCAACTGAATCTGGAACTC
CTGCAATCGCCCAGTATT'CGGTTACAGCTATCGTTTCTAAAGGAGATAATAATAA
ATGAGAGCTCTATTTGACTACGGACCTGAAGATATTCGGGCAACAAATATCCAG
GATCCCAGGTTGAACOATGATCACGCAATGCTGGTTGAGGTCTCAGCCACATCTA
TCTGTGGATCTGATTTACACGTGTATCGGGGAGITCTCGATTCAATGATGGAAAA
AGGGGGCTCGCAGACTGGCGATGAGCTAATGGGGCGAGTCAAAGAAATTGGTAA
GGACGTAGGACGCTTCAAGCCGGGCGACCGAGTCTCCATGGCTTATTCGTGCTCC
TGTGGTGATTGTTACATGTGYAATGTCGGCCAAACCGCACACTGCGAGACGACG A AGA AGGC AGT AT ACGGCTTTGGTGTCCCTnTGGTG ATCTA A ACGGT ACGC ATG CCGAGGCCTTGATrCTTCCCATGCGGAGGGGCACACCATCAAGGTGCCGGACGCA
ATTTCAGATTCGAGCCGGCTTAATGCTGTCCTGCAATCTACCGTCAGCGATCATT
GCCAACAAACTGGCCGATATCGCGCCCGGAGAGAATGTTGCGCTGATTGGTTGT
GGACCGACCGGA1TAATGTGCCTCGATATTGCGTTACAAAAGTCGGCGGGCACG
GTTGTGGCAATGGATAAGGTGGCCCACCGCrrGAGTGTGGCTGCAAAAAAGGGA
GCTGTACCGATCAACCC'ITCCGATAGCGACTGGATGGAAAAGCACTCGCGGAAA CAGGCGCGCGGGGTTTTGACAAAGTGATFQAAGTGGTTGGGTATCCCGAAAGGC
TACAGATGGCA'TTAGATATCGTTCGGCCCGGTGGCACGATTGCTGCTATTGGAGT
TTTCTGCGACCAAGAGTTTAAITTGGTGTTAGCGGATGTATTCCTGCGCGATATTA
CACTGCATATGAATGGCTTCGCAACGTACAGCCGTTTATGTGGGAAGGGCTACGT
CTGATGGAGCGCGGGGTGCTFTCACCCGAAGAGTATTT'CTCTCATGACTTTAAAC
TiGATGAGATCCACAAAGCCTATTCGACTTTTCACACCAAGGATGATAATGCAAT
GAAAATGCTAATAAGACCCTGAAT’ATTTAGTTTTAGAAATAGCAATGTGGAAAG
CAAAATCCCGTATCGCAGGATGTTGGATCAATGGATCCTTAAAATAACAGGTGCT
GTAGCAGGCAITAGGGAAGAGGGTAACAGGAGGTAATTATGTCAATGCTAAAAC
GTGAAGATTGGTACGATC’ITACGCGTACTACCAACTGGACGCCTAAATATGTCAC
TGAAAAGGAACTGTTTGCGGAGGAAATGAGCGGAGCACGTGGGATTTCTATGGA
AGCTTGGGAAAAATACGATGAACGGTACAAGATAACTTATCCGGAATACGTCAG
TATTCAGCGAGAAAAGGACTCCGGCGCATATTCAATCAAAGCGGCACTGGAGCG
TGATGGTTTCGTTGATCGAGCCGATCCAGGCTGGGTTAGCACTATGCAACTTCAC
TTCGGAGCGATCGCACTTGAAGAATACGCCGCAAGCACT'GCTGAAGCCCGTATG
GGGGGATTCQCGAAGGCACGGGGAAACGGGAATATGGCGACTTTCGGAATGATG
GATGAAAACCGCCATGGGCAAATCCAACTTTACTTrCCGTATGCCAATGTCAAGC
GGAGCAGGAAATGGGATFGGGCGCACAAAGCCATTCATACTAACGAATGGGCCG
CAATCGCTGCACGGTCTTTCTTTGACGACATGATGATGACCCGCGATYCCGTGGC
GGTCTCTATCATGCTGAGGTTCGGATTCGAAACAGGCTFCACCAATATGCAGTTT
CTCGGTTTGGCCGCTG AGGGTGCTG AGGCCGGTG ACC AT ACCTTTGCC AGCCTG A
TFTCAAGCATACAGACGGACGAATGACG’FCACGCACAGCAAGGTGGACCGTCGC
TCAAGATCCTGGTGGAGAAIGGTAAAAAAGACGAAGCACAACAAATGGTCGATG
1'CGCAATCTGGCGATCTTGGAAGCTTTTCTCGGTACTCACCGGCCCCATCATGGA
TTATTACACGCCACTGGAATCGCGTAATCAGTCCTTCAAGGAGTTTATGCTTGAG
TGGATTGTGGCTGAATTTGAACGTCAGTTGCTTGATCTAGGGCTCGACAAACGCT
GGTATTGGGATCAGTTCATGCAAGACCTCGATGAAACACACCATGGCATGCACC
TGGGCGTGTGGTA'ITGGCGCCCCACAGTCTGGTGGGATCCGGCAGCTGGTGTGTC
TCCTGAAGAGCGGGAATGGCTGGAAGAGAAATATCCCGGTTGGAATGATACCTG
GGGCCAGTGrrGGGATGTGATTACCGATAACCTAGTGAATGGTAAACCAGAGTT
GACTG'TTCGGGAAACCGTACCCACGATCTGTAACATGTGCAATCTCCCGATTGCC
CATACTCCAGGTAATAAATGGAATGTAAAGGACTACCAGCTCGAATATGAGGGA
CGCCTTTACCAGTTCGGCTCTGAGGCCGACCGCTGGTGTTTCCAGATCGACCCGG
AACGTTACAAAAACCATACGAACCTTGTCGACCGATTCCTGAAAGGTGAAATTC
AGCCTGCGGATTTAGCGGGTGCCCTAATGTACATGAGTCTTGAGCCGGGCG!TAT
GGGTGAGGACGCTCATGATTATGAGTGGGTCAAGGCGTAGCAGAAAAAAACCAA
CGCGGCTTGATTTGATTTAACAAACAATITAACAGGAGGTGGGACATGGCGACG
TTGCCGATTATGTCTAATIYTGAGCGTGATTTTGTTATCCAGTTGGTGCCCGTTGA
GACGGAGGATAC'GATGGATCAGGTAGCAGAAAAATGTGCCT'ACCACTCCAITAA
TCGCAGGGTACATCCGCAACCGGAAAAGATTFTACG’FGT'FCGCCGGCACGAGGA
TGGGACGCTGTTTCCTCGAGGCAT'GATCG'TATCGGATGCGGGGCTTCGGCCTACC
GAAACACTCGATATTATTITTATGGATAACTGAGGGTCTCTCGCICTATGGCATFC
GAAAAAATTTGCACATTGGATGA'TGTFTGGGAGTGTGAAATGGAGACCTTCACTA
CATCGACCGGCG'FTGACATATTACTCGTGGGCGTTGAAGGCGGCGATATGAAGG
CTFTTCAGGGCATGTGCCCACATCAGGAAATGGAATTGGTFGAAGGTGAATTCGA
TGGCAAGGTGCTGACTTGCAAGGCACACCITTGGCAGTTTGATTGCCATAATGGT GAAGGGATTAACCCTTCCGACTGCCGGATCGCTGAATACCCGGTCAAGATCGAG
GGTGAAGACGT'GTTCGTGGATGTGGAAGGGGTTGAACCGTTCAAGT'CCCACAGC
TGACATGGACGTCATAAGGCCAGGCTCTCCGAAAAAGGGGGGAACCCCTGAATG
GAGAGCCTAAGCTITGGTTCTTATGATGAGTCGTGGGAAGGAGAAACATTTACAT
AACGGTAATAAGTGACCATATGTTn'GGGTACAAGCAACGCAACAAAGGTTATT
C A ATC A A AC A ATAT AG AG A A ACC A A A ATGAC A ACC A AC AC AGTTC A A AC ATT AT
CCGCATCGGATAATGCACTTAACAATAACATGGTAGGGCCAGTCCTCAGGGCGG
GGGATGTGGCGATTGGGGTAGGGGAGGGCGCAGAAATGGACAATCCTGGCAAAG
AAATCAAGGTCG ATGAC AAACTTGCCTATGTTCGTATTGGCGCCGAAGATGAATT
AATTCTCAGAAAAGAAACCATTGAAGAATGCCTGGGCCGACCATTTCGCATGCA
GGAGTTAGAGATCAATCTAAGCTCTFTCGCTGGAATTATAGACATGGACTTCGAC
CGGGTCGGTiTCTACTTCAATAAGCACCTTTGAAGCGGCCACACTATAAAGGAGA
ACTGAACAATGTCAGAACAACAACCTGAAGCATTAAAACCACTGAAAACCTGGT
CGCACCTGGCTGGCAATCGGCGACGTCCGAGTGAATATGAGGTTGTATCGACCA
ATCTTCACTACTTC ACGG ATA ATCGGGA A AG AGCATGGGAGTTAG ACTC A AAGGT
CCCAATGCAAACATGGTATAAAAAATATTGCTTTGATAGCCCACTCAAACACGAT
GACTGGAAT'GCTnTCGCGATCCTGACCAGCTCGTCT'ACAGAACTTATAATCTAC
TTCAAGACGGTCAAGAGTCGTACGTTCAGGGATTATTTGATCAACTCAATGACC-G
CGGCCACGATCAGATGCTGACTCGTGAATGGGTAGAAACATTAGCCCGTTTTTAT
ACACCAGCAAGGTATCTiTTTCACGCCTTACAAATGGGCTCAGTATATATCCATC
AAATTGC ACCGGCC AGT ACC ATC AC A A ACTGTGC A ACTTACG A G ACGGC AG ACC
ATTTGCGCTGGCTAACTCACACCGCATACCGTACCAGGGAGCTCGCTAATTGTTA
TCCTGATGTGGGTITCGGGAAAAGAGAACGAGATGTTTGGGAAAATGATCCTGC
GTGGCAGGGCTTCCGAGAATTGATAGAGAAAGCCCTGATCGCCTGGGATTGGGG
TGAAGCTTTTACTGCCATTAACCTCGTTACCAAACCGGCTGTAGAAGAAGCTCTA
TTGCAACAACTAGGTAGTCTTGCCCAATCCGAAGGCGATACTCTGCTCGGTCTCT
TGGCTCAGGCACAAAAACGTGACGCTGAGCGGCATCGGAGATGGTCTTCTGCAT
TGGTCAAAATGGCTCTGGAGAAAGAAGGCAACCGTGAAGTGTTGCAGAAATGGG
TTGCCAAATGGGAACCATTAGCCGACAAAGCCATAGAGGCATATTGCAGCGCAT
TACCTGACGGTGAAAACGCCATCGTCGAAGCGAAAAGCGCGTCGCGGTATGTT'C
GCCAAATGATGGGGCTGTAATCTTTGTAAGCACTAAATGAACCGGTGATGGTrGT
CTCGCTATGACCGGTTTCTTGATGAGTAGACACAGGCATAAACAATATGAGCAAT
A AG AT A A A A ATTGCCG ATAC AG ATGTCG A AGP Ά CC ATCTCCG AC AG GG ATACG
A1TCTGAGGGCGGCTTTACGTGATGGAATACCAATCTCATACGAGTGCAACTCCG
GCGGGTGTGGGTCTTGTAAAATAGACGTTGTTGAAGGACAGGTTGAAACCCTAT
GGGGGGAAGCGCCCGGTCTAAGCCCAAGGGACAAAAGAAAAAGCAGAAAACTT
GCCTGCCAGTGTCTCGCCAGTGGACCTGTCACCATCAAGGCTCAATTGACCGATA
ATAAACTACCCGAAATACGCCCCTTTCGTCGTCGGGTTCGGTATGTGGGCCGACG
TGATCTAACGTCCGACATGGCTGAATTTTCITT'rCAGGCCGAGGAGCCAGCGCTT
TTTCTTGCGGGACAATATGGCATGC'T'GACTGTCCCGGGTATTGAAGGAGATAGGG
CTTATrCCATGTCCAATG'IT'l'CTAACGATTCCGGACGCTGGCAGTTTATTATAAAG
CGAATGCCGGGAGGTAAGGCATCCAACTGGCTTTTCGACGAACTGAAACCGGGG
GGCATGATCGAGATTGACGGACCTTTCGGTCT'CGCCTACCTTCATCCGGAAATTC
AACGTGATGTGGTTTGCATIGCCGGCGGGTCAGGGCTCTCTCCGGTAATGTCCAT
TGTGCGAGCGATTACAGGGGATCCACGCCTCTCCGAACGCAAGGl'CCACCTTTTT
TATGGGGGCCGCACTCCACAAGACCTTTGCACGTCGGAATTGCTG'T’CAGAGATCG AACCTTTAAACACGAAAGCGAAAGTAATATCTAAGACGGCGGTTTCAGATCATC
AGTCTGCAGAGAAGGAAAATTGGGAGGGCCCTTGTTGCTACATACATGAACTGG
CCGAGCAGACACTGGGCGATCATATGATGGAATTTGAGTACTATTTCTGCGGCCC
GCCGCGACTGACGGAAGCGG'TTCAACGCATGCTGATGATCGATCACAAAGTGCG
GTTTGATCGGATTCATTrCGATCGTITTTTCTGAAGAAGAGGTACGTTGGCCGCG
ATACGTTACATGGTCAGCCTTGCGAAAAAAATCTGAACATCAACAGAAAACTTT
AACCATCGGGCATTAACAGGGGCGGTTGCAGGAGCTGAGTGCAACACGGTCATT
GAATTGGAGTTGGAGCATCATGCCGCATAGCCATGGCCTTCTGCATTACCTTGCC
CATAAGTTCGATTGGGCATTTGAAGTCGAAGGGGTTAGGGGGTCGCATGTTGAGT
TGCAGCGCGATGGCATT'CAACTCCTCCTGGCTGTGTACCGACAGGTCTGTACCCT
TGGGCAAGTACTGGCGGATCAGACCATTGATGTTTTCGTTGCTGCCGCGCTGCCA
AGGGCT'GTGCGGGTCGCAGAAGAAGATTGCCACGCCGGTTCTCI'GGGTGATCTC
GGCATGCCGCGCCATCTCCCGCCCCTGGTCGTAGGTCATGCTCTTGCGCATCGCC
AGCGGCATGCCATTGAGCGCGGCACTGAAGCCTTCCATCGCCGAGGTCGCCGTC
GCATCGTTCATCTTCACCAGCATCAGGTAGCCACTGGTGCGCTCCACCAACGTGC
CTACGGACGAGGCGTTGGCC'FTACCCTTGATCAGGTCGCCTTCCCAATGCCCCGG
CATCAGCCGGTCTTCGATCTCCGGCGGGCGCACATGAATGCTGACCATCTCGGGG
A'l'CTGCCCGCGCCGATCCACGCCGCCCGAGCGCGGCCTGCGCGTCGTCTTGCTTT
GGCGCAGGCAGATGATCAGCTCCTrACGCAGCTCGCCGACCGGCAAGGCATAGA
TCGCGTTATAGATCGTCTCGCGACAGACGTAGGCCTCTCTGAGGCTGGGTATGTT
CATGCTGCGTAGCTTGCCGGCAATCTGCTCGGGAGACAAACGCTCACGCAGCAT
ATGGGCCACCAACTCGAAGCGCTCGCTACCCGGCAGCAGCTTACGCATTGGCCG
ACAGACTCGGCGGCTGGCCCTCATCTGTTGTTGAGCCACATGGGCCGAGTAGCCG
CCGCGGACATCTCGGTTGCGACGCACCTCTCGACTAATGGTCGAAGGCGATCTAT
GGATCAGACGAGCAATCCTGCGCAAGCTGAAGCCTTGGGCCTGGCCAATTTGGA
TGGTGGCGCGCTCITCAACGCTGAGTTCGGTATAAGACATAGGGGCAGCACCGT
ACCGGAAAGGTCTGGTGTTGCACTCAGITTCCGCGGCCGCCCTI'ATCGGGGAGTT
GGCT'GAGCGAGTACGTGAACTGGTAAGGCAGACATGTGAGGCGTTTGAGATTCG
CATGGTGAAAGGTGTGGTGAGGAAAAATCACGTCCACGTTCTGGTGAGCAGCGC
GCCGGGTTTGGCACCGAGCCGAGTCCGAACTATAATTTTGAGATGGAGGCGGAC
TGATGGGGGGGGTCGTTCAGTCGACGCGTATCCGGACTTTCAGTCCGTAGATCAC
TAACCCACCGACrrCAGTCGGTGGlTGTTGAGTGATTTTTGTTTCCAGTCAGGTGT
TATAGAGCGCGTAACATTTTTCGCTGGCACCCTGAGA1TTAGTTCCGGGAACCCC
CCACCCCACACAACAACAAACGGGGTTCTTGACATGGCAACCAGCTATAAGCCC
CAGCTGAAATATTC'T'GATTFTCAGGATGITACGGAGCAGATCCAGTTCCAAAGCA
CGGAAGGCAAAATCTGGTTAGGCGAGAAACGCATGCTACTGATGCAGCTCTCCG
GGCTGGCGGCATTTCGCCGGGAAATGGTCAAGACGATCGGTATCGAGCGGGCAA
AAGGTTTCTTTCTTGGCCTCGGTTACCAGTCCGGCCTTAGGGATGCTGAACTCGC
CCGCAAGCTGCGCCCGCACTGCAGTGAGCTCGATATATTTCTGGCCGGACCACAA
CTCCACTCGCTGACGGGTATGGTGAAAGTGGTTCGTATCGAGATCGATATCGATG
AGGAAACAGGGGGTTHTACGGTGAGTTGGACTGGATi'GAlTCGTTCGAGGTGG
AGATCTGCCAAACCGAGCTCGGCCAGATGAACGAACCCGTCTGCTGGTCCCTGCT
AGGGTATGCTrGCGGGTATAGCTCCTGATFGAGGGGGCGTCAGATCGTCTTTAGA
GAAGTCACCTGT'CGCGGCTGCGGGGATGAAAAGTGCCATATCGTCGGCAAACCA
GCAGAAGAGTGGGAGGACGCCGAAGAATTGAGGCAGTACrT'GAAAGGAGACCCG
ATGATCGAAGAGCTCTACGACCTGCAGTCACAGGTGAGTTCGCTGCGCAGCAGT CTTGAGAAACAGCAGGGCCAGTACTACGGGATTGGCCAGTCCGCGTCGTATAAC AAGGTCTGCAAAATGATCGACAAGGCCGCTCTGGGCAAGGTCTCGGTGCTTCTTC TGGGTGAGACTGGI'GIGGGCAAAGAGGTTATTGCCAGAAGCG’ITCA'TTTACGCA GTGAGCGGGCGGAACAACCTTTTATTGCCGTGAA1TGCGCGGCCATACCGCCCG ATCTCATTGAAGCGGAATTGTTTGGCGTCGAGAAGGGAGCCTACACTGGCGCAA ATCAGTCCCGGCCGGGGCGTTTTGAGCGAGCCCA'IGGCGGCACCATATTCCTGGA CGAGGTAGTAGAGCTTACGCCCCGAGCCC \GGCAACCTTGCTGAGGGTCCTGCA GGAAGGCGAACTGGAAGGGGTGGGTGACY .ATCGCACGCGCTCAGTCAACGTCCG TGTAATCGCCGCCACCAATGAGAGCCTTGOCGAGGCGGTTGAGAGCGGCAAGTT CCGGGC AG ATCOTACT ACCGTCTC AATG rTITCCCGGTAAAGATTCCACCCC'IG CGGGAGCGGGTGGAAGACCTGCCA TATTGGCGGAACATTTCCTCAAGAAGTTCC ACACGGAATACAACAAACGGACCC'.. GGGTCTTTCAGACAAAGCCCTCGCCCTGT GCCTGAATTACCGCTGGCCAGGGAA GAIYCGGGAGCTTGAAAACGTAATCGAGC GGGGCGTAATCCTTACCGACAACAA* GAATCCATCAGCCAGGACTCCCTATTCGC A( IT Cm CCGGGC A A TTGC'l C i'G A A A . AGGCTATGAGTCAGTGGATGTTGAGGGA CATCTGGTACAGGAGGGCGCCTCCTCt 'AAAGTTGGAGGGTGGGCGGAGGAAATT CTTGAAAGTGCGATCAGCCTCGATGAA GTGGAAGAGACGCTCATGCACAGAGCC ATGGAGCAGGCCGATCACAACGTATCCAGAGCAGCCCCJCATTCTTGGACTGACA GGCCCGGCTCTGGCCT AC AGAGTG A AG A XAAAAACGGGCATTTTTTCTGAAGCCT GAAAACCAAAACGGCATTACGGCATTAC'lACATl'ACGGCAACAGGGATGTTTCG CCTTCCCCCTYCTGCCTCACGAAATAAGCAAACAGAGTCAAAGTTTGATCACTT
SEQ ID NO. 21 : - Pseudomonas - to!uene»4-monooxygenase system hydroxylase component subunit alpha
MAMHPRKDWYELTRATNWTPSYVTEEQLFPERMSGHMGIPLEKWESYDEPYKTSY
PEYVSiQREKDAGAYSVKAALERAKIYENSDPGWISTLKSHYGA!AVGEYAAVTGEG
RMARFSKAPGNRNMATFGMMDELRHGQLQLFFPHEYCKKDRQFDWAWRAYHSNE
WAAIAAKHFFDDIlTGRDAiSVAlMI/rFSFETGFTNMQFLGLAADAAEAGDYTFANLl
SSiQTDESRHAQQGGPALQLLIENGKREEAQKKVDMAiWRAWRLFAVLTGPVMDY
YTPLEDRSQSFKEFMYEWIIGQFERSUDLGLDKPWYWDLFLKDIDELHHSYHMGV
WYWRTTAWWNPAAGVTPEERDWLEEKYPGWNKRWGRCWDVITENVLNDRMDL
VSPETLPSVCNMSQIPLVGVPGDDWNIEVFSLEHNGRLYHFGSEVDRWVFQQDPVQ
YQNHMNIVDRFLAGQiQPMTLEGALKYMGFQSIEEMGKDAH'DFAWADKCKPAlviK
KSA
SEQ ID NO, 22: - Pseudomonas - toluene-4-monooxygenase system hydroxylase component subunit alpha
ATGGCGATGCACCCACGTAAAGACTGGTATGAACTGACCAGGGCGACAAATTGG
ACACCTAGCTATGTTACCGAAGAGCAGCTTTTCCCAGAGCGGATGTCCGGTCATA
TGGGTATCCCGCTGGAAAAATGGGAAAGCTATGATGAGCCCTATAAGACATCCT
ATCCGGAGTACGTAAGTATCCAACGT'GAAAAGGATGCAGGTGCTTATTCGGTGA
AGGCGGCACTTGAGCG'T'GCAAA AATITATGAGAACTCTGACCCAGGITGGATCA
GCACTTTGAAATCCCATTACGGCGCCATCGCAGTTGGTGAATATGCAGCCGTAAC CGGTGAAGGTCGTATGGCCCGTTTTTCAAAAGCACCGGGAAATCGCAACATGGC
TACGTTTGGCATGATGGATGAACTGCGCCATGGCCAGTTACAGCTGTTTTTCCCG
CAIGAATACTGTAAGAAGGATCGCCAGT ITGATTGGGCATGGCGGGCCTATCAC
AGTAACGAATGGGCAGCCATTGCTGCAAAGCATTTCTTTGATGACATCATTACCG
GACGTGATGCGATCAGCGTTGCGATCATGITGACGTTTTCATTCGAAACCGGCTT
CACCAACATGCAGTTTCTTGGGTTGGCGGCAGATGCCGCAGAAGCAGGTGACTA
CACGTTTGCAAACCTGATCTCCAGCATTCAAACCGATGAGTCGCGTCATGCACAA
CAGGGCGGCCCCGCATTACAGTTGCTGATCGAAAACGGAAAAAGAGAAGAAGC
CCAAAAGAAAGTCGACATGGCAATTTGGCGT 3CCTGGCGTCTATTTGCGGTACTA
ACCGGGCCGGTTATGGATTACTACACGCCGTI G "!AGGACCGCAGCCAGTCATTC
AAGGAGTTTATGTACGAGTGGATCATCGGACAG ITCGAACGCTCGTTGATAGATC
TGGGGTTGGACAAGCCCTGGTACTGGGATCTAT FCCTCAAGGATATTGATGAGCT
TCACCATAGTTATCACATGGGTGTTTTGGAC TGGCGTACAACCGCTTGGTGGAAC
CCTGGTGCCGGGGTCACTCGTGAGGAGGGTGAGTGGGTGGAAGAAAAGTATCCA
GGATGGAATAAACGTTGGGGTCGTTGCTGGGATGTGATCACCGAAAACGTTCTC
AATGACCGTATGGATCrrGTCTCTCCAGAAACCriGCCCAGCGTGTGCAACATGA
GCCAGATACCGCTGGTAGGTGTTCCTGGTGATGAOTGGAATATGGAAGTTTTCAG
TCTTGAGCACAATGGGCGTCTTTATCATTTTGGCTCTGAAGTGGATCGCTGGGTA
TTCCAGCAAGATCCGGTTCAGTATCAAAATCATATGAATATCGTCGACCGCTTCC
TCGCAGGTCAGATACAGCCGATGACTTTGCAAGGTGCCCTCAA ATATATGGGCTT
CCAATCTATTGAAGAGATGGGCAAAGACGCCCACGACTTTGCATGGGCTGACAA
GTGCAAGCCTGCTATGAAGAAATCGGCCTGA
SEQ ID NO. 23: - Pseudomonas - chlorobenzene dioxygenase
MATHVAlfGNGVAOFTTAQALRAEGFEGRISLIGNEPHLPYDRPSLSKAVLGGSLEHP
PVLAEADWYGEARIDMLSGRSV'rNLNVDARTISLDDGSTFAADAlVlATGSRARTLA
LPOSQLTGWTLRTYDDVRPLCRGWTPATRLVfAGGGUGCEVArrARKLGLAVTIL
ESADELLVRVLGRR1GAWLRGLLTELGVRFELGTGVAGFSGDDRLEEVLASDGRRFA
ADNALVCIGAEPEDQLARQAGLSCDRGViVDDHGATHAEGVPAVGDAASWPLRDG
GRRSLETYMNAQRQAAAVAAAJLGKHGSAPQVPVSWTEIAGHRMQMAGDIEGPGE
FVLRGTLGDGAALLFR.I,RIX3H.IQAVVAVDAPRDFAMAPRLVEARAAiEPARLADPS
NSMRDLVRAQQGDSA
SEQ ID NO. 24: - Unknown - chlorobenzene dioxygenase
ATGGCGACCCATGTGGCGATTATTGGCAACGGCGTGGCGGGCTTTACCACCGCG
CAGGCGCTGCGCGCGGAAGGCTTTGAAGGCCGCATTAGCCTGATTGGCAACGAA
CCGC ATCTGCCGT Al’G ATGGGCGG AGCCTG AGC A A AGCGGTGCTGGGCGGC AGC
CTGGAACATCCGCCGGTGCTGGCGGAAGCGGAITGGTATGGCGAAGCGCGCAIT
GATATGCTGAGCGGCCGCAGCGTGACCAACCTGAACGTGGATGCGCGCACCATT
AGCCTGGATGATGGCAGCACCTFTGCGGCGGATGCGATTGTGATTGCGACCGGC
AGCCGCGCGCGCACCCTGGCGCTGCCGGGCAGCCAGCTGACCGGCGTGGTGACC
GTGCGCACGTATGATGATGTGCGCCCGCTGTGCGGCGGGTGGACCCCGGGGACGG
GCCTGGTGATTGCGGGCGGCGGCCTGATTGGCTGCGAAGTGGCGACCACCGCGC GCAAACTGGGCCTGGCGGTGACCATTCTGGAAAGCGCGGATGAACTGCTGGTGC
GCGTGCTGGGCCGCCGCATTGGCGCGTGGCTGCGCGGCCTGCTGACCGAACTGG
GCGTGCGCTTTGAACTGGGCACCGGCGTGGCGGGCTTTAGCGGCGATGATGGGCT
GGAAGAAGTGCTGGCGAGCGATGGCCGCCGCTTTGCGGCGGATAACGCGCTGGT
GTGCATTGGGGCGGAACCGGAAGATCAGCTGGCGCGCCAGGCGGGCCTGAGCTG
CGATCGCGGCGTGATTGTGGATGATCATGGCGCGACCCAT'GCGGAAGGCGTGTT
TGCGGTGGGCGATGCGGCGAGCTGGCCGCTGCGCGATGGCGGCCGCCGCAGCCT'
GGAAACCTATATGAACGCGCAGCGCCAGGCGGCGGCGGTGGCGGCGGCGATTCT
GGGCAAACATGGCAGCGCGCCGCAGGTGCCGGTGAGCTGGACCGAAATTGCGGG
CCATCGCATGCAGATGGCGGGCGATATTGAAGGCCCGGGCGAAT'ITGTGCTGCG
CGGCACCCTGGGCGATGGCGCGGCGCTGCTGTTTCGCCTGCGCGATGGCCGCATT
CAGGCGGTGGTGGCGGTGGATGCGCCGCGCGATTTTGCGATGGCGCCGCGCCTG
GTGGAAGCGCGCGCGGCGATTGAACCGGCGCGCCTGGCGGATTTTAGCAACAGC
ATGCGCG AT CTGGTGCGCGCGC AGC AGGGCGATAGCGC
SEQ ID NO, 25: ■■ Cupriavidus -cis-chlorobenzene dihydrodioi dehydrogenase
MKLKGEVALVl’GGGAGLGRAIVDRYVAEGARVAVLDKSAAGLEEIRKRHGDAVVG lEGDVRSLDSHREAVARCVETFGKLDCUGNAGVWDYQTQLADJPDNGlSEAFDEMF
AIIVKGYILAAKAALPALYKSKGSAIFTVSNAGFYPGGGGVLYTAGKHAViGLVKQL
AHEWGPRIRVNGlAPGGILGSDiRGLKTLGLQDQTIATMPLADMLGPVLPTGRVATA
EEYAGAYVFFATRADTVPLTGSVLNiDGGMGVRGLFEASLGAQLDKHFA
SEQ ID NO. 26: - Unknown - cis-chiorobenzene dihydrodioi dehydrogenase
ATGAAACTGAAAGGCGAAGTGGCGCTGGTGACCGGCGGCGGCGCGGGCCTGGG
CCGCGCGATTGTGGATCGCTAIGTGGCGGAAGGCGCGCGCG'T'GGCGGTGCTGGA
TAAAAGGGCGGGGGGGGTGGAAGAAATTCGCAAACGCCATGGGGATGCGGTGGT
GGGCATT'GAAGGCGATGTGCGCAGCCTGGATAGCCATCGCGAAGCGGTGGCGCG
CTGCGTGGAAACCTTTGGCAAACTGGA'TTGCCTGATTGGCAACGCGGGGGTGTG
GGATFATCAGACCCAGCTGGCGGATATI'CCGGATAACGGCATTAGCGAAGCGTT
TGATGAAATGTTTGCGATTAITGTGAAAGGCTATATTCTGGCGGCGAAAGCGGCG
CTGGCGGGGCTGTATAAAAGCAAAGGCAGCGCGATTnTACCGTGAGCAACGCG
GGCTTTTATCCGGGCGGCGGCGGCGTGCTGTATACCGCGGGCAAACATGCGGTG
ATT'GGCCTGGTGAAACAGCTGGCGCATGA ATGGGGCCCGCGCATTCGCGTGAAC
GGCATTGCGCCGGGCGGCAITCTGGGCAGCGATATTCGCGGCCTGAAAACCCTG
GGCCTGCAGGATCAGACCATTGCGACCATGCCGCTGGCGGATATGCTGGGCCCG
GTGCTGCCGACGGGCCGCGTGGCGACCGCGGAAGAATATGCGGGCGCGTATGTG
TTTTTTGCGACCCGCGCGGATACCGTGCCGCTGACCGGCAGCGTGCTGAACATTG
ATGGCGGCATGGGCGTGGGCGGGCTGTTiGAAGCGAGCCTGGGCGCGGAGCTGG
ATAAACATTTTGGG
SEQ ID NO. 27: ■■ Pseudomonas MSYQVTIEPTGEQIEVEEGQTILEAALRQGVWLPFACGHGTCATCKCQVLEGEVDLG
AASSFALMOMERDEGKVLACCAIPQSDLVIEADIDVDPOFAGLPVQDYRAVVTQLV
ELSPTiRGVHLRlDRPMAFQAGQYVNLQLPGIEGSRAFSLANPPQQADEVELUVRLV
EGGVATGYIHQQLKVGDALALSGPYGQFFVRGSQFGDLIFIAGGSGLSSPQSMILDLL
ARGDTRRiTLFQGARTRAELYNRELPEALAERHAMFSYVPALSQAAEiDEQWQGVRG
YVHDAARQHFDGRFNGHKAYLGGPPPMIDAAITCLMQGRLFERDIFMERFYSAADG
TAEGQRSALFKR1
SEQ LO NG. 28: · Pseudomonas
ATGAGCTATCAAGTCACCATCGAACCTACCGGTGAGCAGATCGAAGTGGAGGAG
GGCCAGACCATCCTGGAAGCCGCCCTGCGCCAGGGTGTCTGGCTGCCGTTTGCCT
GTGGCCACGGCACTIGTGCCACCTGCAAATGCCAGGTGCTGGAAGGCGAAGTCG
ACCTCGGCGCTGGATCGTCCTTCGCCCTGATGGACATGGAACGCGACGAGGGGA
AGGTGCTGGCCTGCTGCGCCATCCCGCAGAGTGACCTGGTGATCGAGGCCGACA
TCGATGTCGAGCCGGACTTCGCCGGCCTGCCGGTGCAGGACTACCGTGCGGTGGT
CAGCCAGCTGGTGGAACTGTCGCCGACGATTCGCGGCGTGCACCTCAAGGTCGAC
CGGCCGATGGCCT'TCCAGGCCGGGCAGTACGTGAACTTGCAGCTGCCAGGTATC
GAAGGCTCCCGTGCG'IYTTCCCTGGCCAACCCGCCCCAGCAGGCCGACGAGGTG
GAACTGCACGTGCGTCTGGTAGAAGGCGGCGTGGCCACCGGGI'ACATCCACCAG
CAGCTCAAGGTGGGCGATGCGCTGGCGCTGTCGGGCCCrrACGGGCAGTTCTTCG
TGCGCGGCTCACAGCCGGGTGACCTGATTTTCATCGCTGGCGGCTCGGGGCTYTC
CAGCCCGCAGTCGATGATTCTTGACTTGCTGGCGCGTGGCGATACCCGGCGCATC
ACACTGTTCCAGGGCGCCCGTACTCGAGCTGAACTGTACAACCGCGAGCTGTTCG
AGGCGCTGGCCGAGCGCCATGCCAACTTCAGCTACGTGCCTGCTCTYAGCCAGGC
TGCTGAGGACGAGCAGTGGCAAGGTGTGCGCGGCTATGTGCACGACGCGGCCAG
GCAGCATTTCGATGGCCGCTTCAACGGTCACAAGGCCTACCT'GTGCGGCCCGCCA
CCCATGATCGATGCGGCCATCACCTGCCTGATGCAAGGCCGCCTGTTCGAACGCG
ACATC7TCATGGAGCGCTTCTACAGCGCTGCCGATGGTACTGCCGAAGGCCAGCG
TTCGGGGCTGTTCAAACGCATCTGA
SEQ ID NO. 29: - Escherichia coll - cseB
MALNIPFRNAYYRFASSYSELFFISWSLWWSLYAIWLKGHLG1..TGTELGTLYSVNQF
TSILFMMFYGIVQDKLGLKKPLIWCMSFILVLTGPFMIYVYEPLLQSNFSVGLILGALF
FGL-GYLAGCGLLDSFTEKMARNFHFEYGTARAWGSFGYAIGAFFAGIFFSISPHINFW
LVSLFGAVFMMFNMRFKDKDHQCIAADAGGVKKEDPiAVFKDRNFWVFViFIVGTW
SFYNIFDQQLFPVFYAGLFE
SEQ ID MO. 30: ■ Escherichia cols - escB
ATGGCACTGAATATTCCATTCAGAAATGCGTACTATCGTTTTGCATCCAGTTACT
CAlTTGTCTTTTTTATTTCCTGGTCGCTGTGGTGGTCGTTATACGCTATTrGGCTGA
AAGGACATCTAGGATTAACAGGGACGGAAITAGGTACACTTTATTCGGTCAACC
AGTTTACCAGCATTCTATTTATGATGTTC'TACGGCATCG'ITCAGGATAAAC'TCGGT CTGAAGAAACCGCTCATCTOGTGTATGAGTTTCATTCTGGTCTTGAGCGGACCGT
TTATCATTTACGTTTATGAACCGTTACTGCAAAGCAATTTTTCTGTAGGTCTAATT
CTGGGGGCGCTCTTrTTTGGCCTGGGGTATCTGGCGGGATGCGGTTTGCTTGACA
GCTTCACCGAAAAAATGGCGCGAAATTTTCATTTCGAATATGGAACAGCGCGCG
CCTGGGGATCTTTTGGCTATGCTATTGGCGCG’TTCTTTGCCGGTATATTTTTTAGT'
ATCAGTCCCCATATCAACTTCTGGTTGGTCTCGCTATnTTGGCGCTGTATTTATGAT
GATCAACATGCGTTTTAAAGATAAGGATCACCAGTGCATAGCGGCGGATGCGGG
AGGGGTAAAAAAAGAGGATTTTATCGCAGTTTTCAAGGATCGAAACTTCTGGGTT
TTCGTGATATrTATTGTGGGGAGGTGGTCTrrCTATAACATTTTTGATCAACAACT
CITTCCI’GT'CTTITATGCAGGTTTATTCGAATCACACGATGTAGGAACGCGCCTGT
ATGGTTATGTCAAGTCATTGCAGGTCGTAGTCGAAGCGCTGTGCATGGCGA'n'AT
TCCTTTCTTTGTGAATCGGGTAGGGCCAAAAAATGCATTACTrATCGGTGTTGTG
ATTATGGCGTIOCGTATCCTTTCCTGCGCGTTGTTCGTTAACCCCTGGATTATTTC
ATTAGTGAAGCTGITACATGCCATTGAGGTTCCACITTGTGTCATATCCGTCTTCA
AATACAGCGTGGCAAACTTTGATAAGCGCCTGT'CGTCGACGATCTTTCTGA'ITGG
TTTiCAAATTGCCAGTTCGCTTGGGAITGTGCTGCTTTCAACGCCGACTGGGATA
CTCITTGACCACGCAGGCTACCAGACAGTTTTCTTCGCAATTTCGGGTATTGTCTG
GGTGATGTTGGTArnXjGCATTTTCTTGGTGAGTAAAAAACGCGAGCAAATAGTT
ATGGAAACGCCTGTACCTTCAGCAATATAG
SEQ ID NO. 31 : - Synechoeoccus - sps
MVAAQNLYILB'IQTHGLLRGQNLELGRDADTGGQTKYVLELAQAQAKSPQVQQVDI
ITRQITDPRVSVGYSQAIEPFAPKGRIVRLPFGPKRYLRKELLWPHLYTFADAILQYLA
QQKRTPTW!QAHYADAGQVGSLLSRWLNVPLIFTGHSLGRIKLKKLLEQDWPLEEIE
AQFNiQQRIDAEEMTLTHADWIVASTQQEVREQYRVYDRYNPERKLVIPPOVDTDRF
RFQPLGDRGVVLQQELSRFLRDPEKPQILCLCRPAPRKNVPALVRAFGEHPWLRKKA
NLVLVLGSRQDINQMDROSRQVFQEIFHLVDRYDLYGSVAYPKQHQADDVPEFYRL
AAHSGGVFVNPALTEPFGLTILEAGSCGVPVVATHDGGPQEILKHCDFGTLVDVSRP
AMLATALATLLSDRDLWQCYHRNGIEKVPAHYSWDQHVNl'LFERMETVALPRRRA
VSFVRSRKRLIDAKRLVVSDIDNTLLGDRQGLENLMTYLDQYRDHFAFGf ATGRRLD
SAQEVLKEWGVPSPNFWVTSVGSEiHYGTDAEPD!SWEKHFNRNWNPQRlRAVMAQ
LPFLELQPEEDQTPFKVSFFVRDRHETVLREVRQHLRRHRLRL.KSIYSHQEFLD1LPLA
ASKGDAiRHLSERWRlPLENitVAGDSGNDEEMLKGHNLGVVVGNYSPELEPLRSYE
RVYFAEGHYANGILEALKHYRFFEAIA
SEQ ID NO, 32: - Synechoeoccus - sps
ATGGTGGCAGCTCAAAATCTCTACATTCTGCACATTCAGACCCATGGTCTGCTGC
GAGGGCAGAACTPGGAACTGGGGCGAGATGCCGACACCGGCGGGCAGACCAAG
TAGGTG7TAGAAGTGGCTCAAGCCCAAGCTAAATCGCCAGAAGTCCAAGAAGTC
GACATCATCACCCGCCAAATCACCGACCCCCGCGTCAGTGTTGGTTACAGTCAGG
CGATCGAACCCTTTGCGCCCAAAGGTCGGATFGTCCGTTTGCCTTTTGGCCCCAA
ACGCTACCTCCGTAAAGAGCTGCTITGGCCCCATCTCTACACCITTGCGGATGCA
ATFGTCGAATATGTGGCTCAGCAAAAGCGCACCCCGAGTTGGATFCAGGCCGACT ATGCTGATGCTGGCCAAGTGGGATCACTGCTGAGTCGCTGGTTGAATGTACCGCT
AATTITCACAGGGCATTCTCTGGGGCGGATCAAGCTAAAAAAGCTGTTGGAGCA
AGACTGGCCGCTTGAGGAAATTGAAGCGCAATTCAATATTCAACAGCGAATTGA
TGCGGAGGAGATGACGCTCACTCATGCTGACTGGATTGTCGCCAGCACTCAGCA
GGAAGTGGAGGAGCAATACCGCGTTTACGATCGCTACAACCCAGAGCGCAAACT
TGTCATTCCACCGGGTGTCGATACCGATCGCTTGAGGTTTCAGCCCrrGGGGGAT
CGCGGTGTTGTTCTCCAACAGGAACTGAGCCGCTTTCTGCGCGACCCAGAAAAAC
CTCAAATT'CTCTGCCTCTGTCGCCCCGCACCTCGCAAAAATG'T'ACCGGCGCTGGT
GCGAGCCTTIGGCGAACATCCTTGGCTGCGCAAAAAAGCCAACCTTGTCTTAGTA
CTGGGCAGCCGCCAAGACATCAACCAGAl GGATCGCGGCAGTCGGCAGGTGTTC
GAAGAGATTTTCCATCTGGTCGATCGGTACGACGTGTACGGCAGCGTCGCCTATC
CCAAACAGCATCAGGCTGATGATGTGCCGGAGTI CTATCGCCTAGCGGCTCATTC
CGGCGGGGTATTCGTCAATCCGGCGCTGACCGAACCTTTTGGTTTGACAATTTTG
GAGGCAGGAAGCTGCGGCGTGCCGGTGGTGGCAACCCAI'GATGGCGGCCCCCAG
GAAATTCTCAAACACTGTGATIYCGGCACTTTAGTTGATGTCAGCCGACCCGCTA
ATATCGCGACTGCACTCGCCACCCTGCTGAGCGATCGCGATCTTTGGCAGTGCTA
IGACCGCAATGGCATTGAAAAAGTTCCCGCCCATTACAGCTGGGATCAACATGTC
AATACCCTGTTTGAGCGCATGGAAAGGGTGGCTTTGCCTCGTGGTCGTGCTGTCA
GTITCGTACGGAGTCGCAAACGCTTGATTGATGCCAAACGCCTTGTCGTTAG'T'GA
CATCGACAAGACACTGTTGGGGGATCGTCAAGGAGTCGAGAATTTAATGACCTAT
CTCGATCAGTATCGCGATCATITTGCCTTTGGAATTGCCACGGGGCGTCGCCTAG
ACTCTGCCCAAGAAGTCTTGAAAGAGTGGGGCGTTCCTTGGCCAAACITGTGGGT
GACTTCCGTCGGCAGCGAGATTCACTATGGCACCGATGCTGAACCGGATATCAG
GTGGGAAAAGCATATGAATCGCAACTGGAATGGTCAGCGAATTCGGGCAGTAAT
GGCACAACTACCCTTTCTTGAACTGCAGCCGGAAGAGGATCAAACACCCTTCAA
AGTCAGCTTCTTTGTCCGCGATCGCCACGAGACTGTGCTGCGAGAAGTACGGCAA
CATCTTCGCCGCCATCGCCTGCGGGTGAAGTCAATCTATTCCCATCAGGAGTTTC
TTGACATTCTGCCGCTAGCTGCCTCGAAAGGGGATGCGATTCGCCACCTCTCACT
CCGCTGGCGGATTCCTCTTGAGAACATTTTGGTGGCAGGCGATTCTGGTAACGAT
GAGGAAATGCTCAAGGGCCATAATCTCGGCGTTGTAGTTGGCAATTACTCACCG
GAATTGGAGCCACTGCGCAGCTACGAGCGCGTCTATTTTGCTGAGGGCCACTATG
CTAATGGCATTCTGGAAGCCTT'AAAACACTATGGCTTTTTTGAGGGGATCGCTTA
A
SEQ !D NO. 33: -Synechocystis ■■ g!gC
MCCWQSRGLLVKRVLAHLGGGAGTRLYPLTKLRAKPAVPLAOKYRLIDIPVSNCiNS
EiVKIYVLTQi-'NSASLNRHISRAYNFSGFQEGFVEVLAAQQTKDNPDWFQGTADAVR
QYLWLFREWDVDEYLILSGDHLYRMDYAQFVKRHRETNADiTLSVVPVDDRKAPEL
GLMKiDAQGRiTDFSEKPQGEALRAMQVDTSVLGLSAEKAKLNPYiASMGIYVFKKE
VLHNLLEKYEOATDFGKEnPDSASDHNLQAYLFDDYWEDIGTIEAFYEANLALTKQP
SPDFSFYNEKAFIYTRGRYLPPTKMLNSTYTESMIGEGCMIKQCR!HHSVLGIRSRIES
DCTIEDTLVMGNDFYESSSERDTLKARGEIAAGIGSGTnRRAnDKNARIGKNVMlVN
KENVQEANREELGFYlRNGIVVViKNVTIADGTVI SEQ !D NO. 34: - Synechocystis - glgC
GTGTGTTGTTGGCAATCGAGAGGTCTGCTTGTGAAACGTGTCTTAGCGATTATCC
TGGGCGGTGGGGCCGGGACCCGCCTCTATCCTTTAACCAAACTCAGAGCCAAAC
CC'GCAGTrCCCTTGGCCGGAAAGTATCGCCTCATCGATATTCCCGTCAGTAATTG
CATCAACTCAGAAATCGTTAAAATTTACGTCCTTACCCAGTTTAATTCCGCCTCCC
TFAACCGTCACATCAGCCGGGCCTATAAT!TTTCCGGCTTCCAAGAAGGATTTGT
GGAAGTCCTCGCCGCCCAACAAACCAAAGATAATCCTGATTGGITI'CAGGGCAC
TGCTGATGGGGTACGGCAATAGCTCTGGTTGTTTAGGGAATGGGAGGTAGATGAA
TATCTIATTCTGTCCGGCGACCATCTCTACCGCATGGATTACGCCCAATTTGTTAA
AAGACACCGGGAAACCAATGCCGACATAACCCTFrCCGTTGTGCCCGTGGATGA
CAGAAAGGCACCCGAQCTGGGCTTAATGA.^AATCGACGCCCAGGGCAGAATTAC
TGACTTTTCTGAAAAGCCCCAGGGGGAAGCCCTCCGGGCCATGCAGGTGGACAC
CAGCGITTTGGGCCTAAGTGCGGAGAAGGCTAAGCTT'AATCCTTACATTGCCTCC
ATGGGCATTiACGTTTTCAAGAAGGAAGTATTGCACAACCTCCTGGAAAAATATG
AAGGGGCAACGGACTTTGGGAAAGAAATGArrCGTGATTCAGCCAGTGATGACA
ATCTGGAAGCCTATCTCTTTGATGACTAITGGGAAGACATTGGTACCAITGAAGC
CTTCTATGAGGCTAATFTAGCCCTGACCAAACAACCTAG'T'CCCGACTTTAGTTTTT
ATAACGAAAAAGCCCCCATCTATACCAGGGGTCGTYATCTTCCCCCCACCAAAAT
GTTGAATTCCACCGTGACGGAATCCATGA'T'CGGGGAAGGTTGCATGATTAAGCA
ATGTCGCATCCACCACTCAGTTTTAGGCATTCGCAGTCGCATTGAATCTGATTGC
ACCATTGAGGATACTTTGGTGATGGGCAATGATTTCTACGAATCTTGATCAGAAC
GAGACACCCTCAAAGCCCGGGGGGAAATTGCCGCTGGCATAGGTTCCGGCACCA
CTATCCGCCGAGCCATCATCGACAAAAATGCCCGCATCGGCAAAAACGTCATGA
TTGTCAACAAGGAAAATGTCCAGGAGGCTAACCGGGAAGAGTTAGGTTTTTACA
TCCGCAATGGCATCGTAGTAGTGA'TTAAAAATGTCACGATCGCCGACGGCACGG
TAATCTAG
SEQ ID NO. 35: - Synechocystis - spp
MRQLLL!SDLDNTWVG DQQALEHLQEYLGDRRGN FYXAY ATGRSYHS ARELQKQV
GLMEPDYWLTAVGSEIYHFEGLDQHWADYLSEHWQRDILQAIADGFEALKPQSFLE
QNPWKISYHLDFQACPTVIDQL'rEMLKETGIPVQVIFSSGKDVDLLPQRSNKGNATQ
YLQQHLAMEPSQTLVCGDSGNDIGLFETSARGVlVRNAQPELLHWYDQWGDSRI-rY'
RAQSSH AGAILE A f AHF DELS
SEQ ID NO. 36: - Synechocystis - spp
ATGCGACAGTTATTGCTAATTTCTGACCTGGACAATACCTGGGTCGGAGATCAAC
AAGCCCTGGAACATTTGCAAGAATATCTAGGCGATCGCCGGGGAAATTTTTATTT
GGCCTATGCCACGGGGCGTTCCTACCATTCCGCGAGGGAGTTGCAAAAACAGGT
GGGACTCATGGAACCGGACTATTGGCTCACCGCGGTGGGGAGTGAAATTTACCA
TCCAGAAGGCCTGGACCAACATTGGGCT'GATTACCTCTCTGAGCATTGGCAACGG
GATATCCTCGAGGCGATCGCCGATGGTTTTGAGGCCTTAAAAGCCGAATCTCCCT
TGGAACAAAACCCATGGAAAATTAGCTATCATCTCGATCCCCAGGCTTGCCCCAC
CGTCATCGACCAATTAACGGAGATG'ITGAAGGAAACCGGCATCCCGGTGCAGGT GATTTTCAGCAGTGGCAAAGATGTGGATTTATYGCCCCAACGGAGTAACAAAGG
TAACGCCAGCCAATATCTGCAACAACATTTAGCCATGGAGCCGTCTCAAACCCTG
GTGYGTGGGGACTCCGGCAATGATATTGGCTTATTTGAAACTTCCGCTCGGGGTG
TCATTGTCCGTAATGCCCAGCCGGAATTATTGCACTGGTATGACCAATGGGGGGA
TTCTCGTCATTATCGGGCCCAATCGAGCCATGCTGGCGCTATCCTAGAGGCGATC
GCCCATITCGATTTTTTGAGCTGA
SEQ ID NO, 37: - Synechococcus ■ glgP
MSDSTAQLSYDPTTSY LEFSGLVCEDERTSVTPKTLKRA YEAHLYY SQGKTSA iATLR
DI-!YMALAYMVRORLLQRWLASLSTYQQQHVKVVCYLSAEFLMGRHLENCLINLHL
HDRVQQVLDELGLDFEQLLEKEEEPGLGNGGLGRLAACFLDSMATLDiPAVGYGIR
YEFG!EHQELHNGWQIEiPDNWLREGNPWELERREQAVEIKLGGMTEAYMDARGRY
CVS WIPDRVIRAiPYDTPVPG YiYFNNV SMLRLWKAEGTTELNLEAFN SGNYDDA VA
DKMSSETISKVLYPNDNTPQGRELRLEQQYFFVSASE-QDIIRRHLMNilGHLERiJriEAI
AVQlJNDTHPSVAVPELMRLLIDiiliHLTWDNAWTITQRTFAYTNHTLLPEALERWPV
GMFQRTLPRLMEIIYEINWRFLANVRAWYPGDDTRARRLSLIBEGAEPQVRMAHLA
CVGSRAINGVAALHTQIXKQETLRDFYELWPEKFFNMTNGVTPRRWLLQSNPRLAN
LISDRIGNDWiHDLRQLRRLEDSVNDREFLQRWAEVKHONKVDLSRYiYQQTRiEVD
PHSLFDVQVKRiHEYKRQLLAVMHIVTLYNWLKHNPQLNLVPRTFIFAGKAAPGYY
RAKQiVKUNAVGSi!NHDPDVQGRLKVVFLPMFNVSLGQRIYPAADLSEQ!STAGKE
ASGTGNMKFTMNGALTIGTYDGAN1E1REEVGPENFFLFGLRAED1ARROSRGYRPVE
FWSSNAELRAVLDRFSSGHFT'PDQPMLFQDLVSDLLQRBEYMLMADYQSYiDCQR.E
AAAAYRDSDRWWRMSLLNTARSGKFSSDRTIADYSEQ1WEVKPVPVSLSTSF
SEQ ID NO, 38: - Synechococcus sp, - gigp
ATGAGTGATTCCACCGCCCAACTCAGCTACGACCCCACCACGAGCTACCTCGAGC
CCAGTGGCTTGGTCTGTGAGGATGAACGGAGTTCTGTGACTCCGGAGACCTTGAA
ACGGGCTTACGAGGCCCATCTCTACTACAGCCAGGGCAAAACCTCAGCGATCGC
CACCCTGCGTGATCACTACATGGCACTGGCCTACATGGTCCGCGATCGCCTCCTG
CAACGGTGGCTAGCITCACTGTCGACCTATCAACAACAGCACGTCAAAGTGGTCT
GTTACCTGTCCGCTGAGTYTTTGATGGGTCGGCACCTCGAAAACTGCCTGATCAA
CCTGCATCTTCACGACCGCGTl'CAGCAAGTTTTGGATGAACTGGGTCTCGATTTT
GAGCAACTGCTAGAGAAAGAGGAAGAACCCGGGCTAGGCAACGGTGGCCTCGG
TCGCCTCGCAGCTTGTTTCCTCGACTCCATGGCTACCCTCGACATTCCTGCCGTCG
GCTATGGCATTCGCTATGAGTTCGGTATCTTCCACCAAGAACTCCACAACGGCTG
GCAGATCGAAATCCCCGATAACTGGCTGCGCTTTGGCAACCCTTGGGAGCTAGA
GCGGCGCGAACAGGCCGTGGAAATTAAGTTGGGCGGCCACACGGAGGCCTACCA
CGATGCGCGAGGCCGCTACTGCGTCTCTTGGATCCCCGATCGCGTCATTCGCGCC
ATCCCCTACGACACCCCCGTACCGGGCTACGACACCAATAACGTCAGCATGTTGC
GGCTCTGGAAGGCTGAGGGCACCAGGGAACTCAACCTTGAGGCTTrCAACTCAG
GCAACTACGACGATGCGGTTGCCGACAAAATGTCGTCGGAAACGATCTCGAAGG
TGCTCTATCCCAACGACAACACCGCGCAAGGGCGGGAACTGGGGCTCGAGCAGC
AGTATTTCTTCGTCT'CGGCTTCGCTCCAAGACATCATCCGTCGCCACTTGATGAAC CACGGTGATCTTGAGCGGCTGCATGAGGCGATCGCAGTCCAGCTTAACGACACC
CATCCCAGCGTGGCGGTGCCGGAGTTGATGCGCC'IGCTGATCGATGAGCATCACC
TGACTTGGGACAATGCTTGGACGATTACACAGCGCACCTTCGCCTACACCAACCA
CACGCTGCTACCTGAAGCCTTGGAACGCTGGCCCGTGGGCATGTTCCAGCGCACT
TTACCGCGCTTGATGGAGATTAT'CTACGAAATCAACTGGCGCTTCTTGGCCAATG
TGCGGGCCTGGTATCCCGGT'GACGACACGAGAGCTCGCCGCCTCTCCCTGATTGA
GGAAGGAGCTGAGCCCCAGGTGCGCATGGCTCACCTCGCCTGCGTGGGCAGTCA
TGCCATCAACGGTGTGGCAGCCCTGCATACGCAACTGCTCAAGCAAGAAACCCT
GCGAGATn’CTACGAGCTTTGGCCCGAGAA/vTTCTTCAACATGACCAACGGTGTG
ACGCCCCGCCGCTGGCTGCTGCAAAGTAATCCTC GCCTAGCCAACCTGATCAGCG
ATCGCAITGGCAATGACTGGATTCATGATCTCAO 3CAACTGCGACGGCTGGAAG
ACAGCGTGAACGATCGCGAGTTTTTACAGCGCTGGGCAGAGGTCAAGCACCAAA
ATAAGGTCGATCTGAGCCGCTACATCTACCAGCAGACTCGCATAGAAGl'CGATC
CGCACTCTCTCTTTGATGTGCAAGTCAAACG 3A1TCACGAATACAAACGCCAGCT
CCTCGCTGTCATGCATATCGTGACGCTCTACAACTGGCTGAAGCACAATCCCCAG
CTCAACCTGGTGCCGCGCACTnTATCTTTGCGGGCAAAGCGGCCCCGGGTTACT
ACCGTGCCAAGCAAATCGTCAAACTGATCAATGCGGTCGGGAGCATCATCAACC
ATGATCCCGATGTCCAAGGGCGACTGAAGGTCGTCTTCCTACCTAACTTCAACGT
TTCCTTGGGGCAGCGCATTTATCCAGCTGCCGATTTGTCGGAGCAAATCTGAACT
GCAGGGAAAGAAGCGTCCGGCACCGGCAACATGAAGTTCACCATGAATGGCGCG
CTGACAATCGGAACCTACGATGGTGCCAACATCGAGATCCGCGAGGAAGTCGGC
CCCGAAAACTTCTTCCTGTTTGGCCTGCGAGCCGAAGATATCGCXCGACGCCAAA
GTCGGGGCTATCGACCTGTGGAGTTCTGGAGCAGCAATGCGGAACTGCGGGCAG
TCCTCGATCGCTrTAGCAGTGGTCACTYCACACCGGATGAGCCCAACCTCTTCCA
AGACTTGGTCAGCGATCTGCTGCAGCGGGATGAGTACATGTTfOATGGGGGACTA
TCAGTCCTACATCGACTGCCAGCGCGAAGCTGCTGCTGCCTACCGCGATTCCGAT
CGCTGGTGGCGGATGTCGCTACTCAACACCGCGAGAT'CGGGCAAGTTCTCCTCCG
ATCGCACGATCGCTGACTACAGCGAACAGATCTGGGAGGTCAAACCAGTCCCCG
TCAGCCTAAGCACTAGCTTTTAG
SEQ ID NO. 39; - Escherichia coli ■■ ga!U
MAAiNTKVKKAVlPVAGLGTRMLPATKAIPKEMLPLVDKPLIQYVVNECIAAGITEiV
LVTHSSKNSiENHFDTSFELEAMLEKRVKRQLLDEVQSiCPPHVT!MQVRQGLAKGL
GHAVLCAHPVVGEffiPVAVILPDVlLDBYESDLSQDNLAEMIRRFDETGHSQtMVEPV
ADVTAYGWDCKGVELAPGESVPMVGVVEK.PKADVAPSNLAIVGRYVLSAD1WPL
LAKTPPGAGDEiQLTDAlDMLIEKETVEAYHMKGKSHDCGNKLGYMQAFVEYGIRl-f
NTLGTEFKA WLEEEMGIKK
SEQ ID NO, 40; - Escherichia coli - gaiti
ATGGCTGCCATTAATACGAAAGTCAAAAAAGCCGTTATCCCCGTTGCGGGATTA
GGAACCAGGATGTTGCCGGCGACGAAAGCCATCCCGAAAGAGATGCTGCCACTT
GTGGATAAGCCATTAATTGAATACGTCGTGAATGAATGTATTGGGGCTGGCATTA
CTGAAATTGTGCTGGTTACACACTCATCTAAAAACTCTATTGAAAACCACTTTGA TACCAGTTTTGAACTGGAAGCAATGCTGGAAAAACGTGTAAAACGTCAACTGCT
TGATGAAGTGGAGTCTATTTGTCCACCGCACGTGACTATTATGCAAGTTCGTCAG
GGTCTGGGGAAAGGCCTGGGACACGGGGTATFGTGTGCTCACCCGGTAGTGGGT
GATGAACCGGTAGCTGTTATTTTGCCTGATG'IYATTCTGGATGAATATGAAT'CCG
ATTTGTCACAGGATAACCTGGCAGAGATGATCCGCCGCriTGATGAAACGGGTC
ATAGCCAGATCATGGTTGAACCGGTTGCTGATGTGACCGCATATGGCGTTGTGGA
ITGCAAAGGCGTTGAATTAGCGCCGGGTGAAAGCGTACCGATGGTTGGTGTGGT
AGAAAAACCGAAAGCGGATGTTGCGCCGTCTAATCTCGCTATTGTGGGTCG1TAC
GTACTTAGCGCGGATATTTGGCCGTTGCTGGCAAAAACCCCTCCGGGAGCTGGTG
ATGAAATTCAGCTCACCGACGCAATTGATATGCTGATCGAAAAAGAAACGGTGG
AAGCCTATCATATGAAAGGGAAGAGCCATGACTGCGGTAATAAATTAGGTTACA
TGCAGGCCTTCGTTGAATACi tGTATTCGTCATAACACCCTFGGCACGGAATTTAA
AGCCTGGCTTGAAGAAGAGATGGGCATTAAGAAGTAA
SEQ ID NO. 41 : - Synechocystis ·■ Invertase
MKSPQAQQILDQARRLLYEKAMVKINGQYVGTVAAIPQSDHHDLNYTEVFIRDNVP
VMlFLLLQNETEIVQNFLHICLTLQSKOFPTYOiFFTSFVETENHELKADYGQRAKIRV
CSVDASLWWPILAYYYVQRTGNEAWARQTHVQLGLQKFLNLILHPVFRDAPTLFVP
DGAFMiDRPMDVWGAPLElQTlX-YGALKSAAGLLLIDLKAKGYCSNKDH'PFDSFTM
EQSHQFNLSVDWLKKLRTYLLKHYWINCNiVQALRRRPTEQYGEFASNEHNVHTETi
PNWLQDWLGDRGGYUGNIRTGRPDFRFFSLGNCLGAIFDVTSLAQQRSFFRLYLNN
QRELCAQMPLR!CHPPLKDDDWRSKTGFDRKNLPWCYHNAGHWPCLFWFLVVAVL
RHSCHSNYGTVEYAEMGNLIRNNYEVLLRRLPKHKWAEYFDGPTGFWVGQQSRSY
QTWTIVGLLLVHHiTEVNPDDAl..MFDl..PSLKSLHQALH
SEQ ID NO. 42: - Synechocystis ·· invertase
ATG A A ATCCCCCC AGGGTC A AC A A ATCCTAG ACC AGGCCCGCCGTTTGCTCT ACG
AAAAAGCCATGGTCAAAATCAATGGGCAATACGTGGGGACGGTGGCGGGCATTC
CCGAATCGGATCACCATGATTTGAACTATACGGAAGTTTTCATTGGGGACAATGT
GCCGGTGATGATCTTCTrGTFACTGCAAAATGAAACGGAAATTGTCCAAAACTTT
ITGGAAATTTGCCTCACCCTCCAAAGTAAGGGCTTTCCCACCTACGGCATTTTTCC
CACTAGTTTTGTGGAAACGGAAAACCATGAACTCAAGGCAGACTATGGCCAACG
GGCGATCGGTCGAGTTTGCTCGGTGGATGCGTCCCTCTGGTGGCCTATTn’GGCC
TATTACTACGTGCAAAGAACCGGCAATGAAGCCTGGGCTAGACAAACCCATGTG
CAATTGGGGCTACAAAAGTTTTTAAACCTCAITCTCCATCCAGTCTTTCGGGATG
CACGGAC/mGTTTGTGCCCGACGGGGCCTTTATGATTGAGCGCGCCATGGATGT
GTGGGGAGCGCCGTTGGAAATCCAAACCCTGCTCTACGGAGCCCTGAAAAGTGC
GGCGGGGTTACTGTTAATCGACCTCAAGGCGAAGGGTTATTGCAGCAATAAAGA
CCATCCTTTTGACAGCTrCAGGATGGAGCAGAGTCATCAATTTAACCTGAGTGTG
GATTGGCTCAAAAAACTGCGCACCTATCTGCTCAAGCATTATFGGATTAATTGCA
A'T'ATTGTCCAAGCTCTCCGCCGCCGTCCCACGGAACAGTACGGTGAAGAAGCCA
GCAACGAACATAATGTCCACACAGAAACCATTCCCAACTGGCTCCAGGATTGGC
TCGGCGATCGGGGAGGCTATTFAATCGGCAATATCCGCACGGGTCGCCCCGATTT TCGCTTTTI'CTCCCTGGGTAATTGCTT'GGGGGCAATTTTCGATGTCACTAGCTTGG
CCCAGCAACGTI'CCITTTTCCGITTGGTATTAAATAATCAGCGGGAGTTATGTGC
CCAAATGCCCCTGAGGATTFGCCATCCCCCCCTCAAAGATGACGA1TGGCGCAGT
AAAACCGGCTTTGACCGCAAAAATTTACCCTGGTGCTACGACAACGCCGGCCATT
GGCCCTGTTTAlTTTGGnTCTGGTGGTGGCGGTGCTCCGCCATAGCTGCCATTCC
AACTACGGCACGGTGGAGTATGCGGAAATGGGGAACCTAATTCGCAATAACTAT
GAGGTGCTTTTGCGCCGTTTGCCCAAGCATAAATGGGCTGAATATTTTGATGGCC
CCACGGGCTTFI'GGGTCGGGCAAGAATCCCGTTCCTACGAAAGCTGGAGCATTGT
GGGCCTATTGCTAGTACACCATTTCACAGAAGTI'AACCCCGACGATGCTTTGATG
'ITCGA'ITTGCCTAGTTTGAAAAGTTTGCATCAAGCGCTGCATTAA
SEQ ID NO. 43: - Synechoeystis - ggpS
MNSSLVILYHJREPYDEVRENGKTVYREKKSPNGILPTLKSFFADAEQSTWVAWKQV
SPKQKDDFQADMSffiGLGDRCTVRRVPLTAEQVKNFYHITSKEAFWPILHSFPWQFT
YDSSDWDNFQHINRLFAEAACADADDNALFWVHDYNLWLAPLYIRQLKPNAKIAFF
HHTPFPSVDIFNILPWREAIVESLLACDLCGFHIPRYVENFVAVARSLKPVEITRRVVV
DQAFTPYGTALAEPELTTQLRYGDRLiNLDAFPVGTNPANiRAiVAKESVQQKVAEIK
QDLGGKRUVSAGRVDYVKGTKEMLMCYERLLERRPELQGEISLVVPVAKAAEGMR iYRNAQNEIERLAGKINGRFAKLSWTPVMLFTSPLAYEELiALFCAADIAWITPLRDG
LNLVAKEYVVAKNGEEGVLILSEFAGCAVELPDAVLTNPYASSRMDESIDQALAMD
KDEQKKRMGRMYAAIKRYDVQQWANHLXREAYADVVL.GEPPQM
SEQ ID NO. 44: --Synechoeystis -■ ggpS
ATGAATTCATCCCTTGTGATCCTTTACCACCGTGAGCCCTACGACGAAGTTAGGG
A A A ATGGC A A A AGGGTGTATCG AG AG A A A A A G AGTCCC AACGGG ATTTTGCCC A
CCCTCAAAAGTTTTTTTGCCGATGCGGAAGAGAGCAGCTGGGTCGCATGGAAAC
AGGTTTCGCCGAAGCAAAAGGATGATTn'CAGGCGGATATGTCCATTGAAGGCC
TTGGCGATCGTTGTACGGTGCGCCGGGTGCCCCTGACGGCGGAGCAGGTAAAAA
ACTTCTATC AC ATG AG'ITCC A AGG A AGCCTTTTGGCiCC ATTCTCC ACTCTTTCCCC
TGGCAGTFCACCTACGATTCTTCTGATTGGGATAATTTTCAGCACATTAACCGCTT
ATTTGCCGAGGCGGCCTGTGCCGATGCCGATGACAATGCATTGTTTTGGGTCCAC
GACTATAACCT’CTGGITAGCGCCCCTTTACATTCGTCAGCTCAAGCCCAACGCCA
AGATFGCCiTTTTCCACCACACCCCCTTCCCCAGCGlTGATATITTCAATATTTTG
CCCTGGCGGGAGGCGATCGTAGAAAGCTTGCTGGCCTGTGATCTCTGTGGTTTTC
AT'ATTCCCCGCTACGTAGAAAATTTTGTCGCCGTGGCCCGTAGTCTCAAGCCGGT
GGAAATCACCAGACGGGTTGTGGTAGACCAAGCCTTTACCCCCTACGGTACGGC
CGTGGCGGAACCGGAACTCACCACCCAGTTGGGTTATGGCGATGGGCTCATTAAG
CTCGATGCG7TTGCCGTGOGCACCAATCCGGCAAATATCCGGGCGATCGTGGCCA
AAGAAAG'I'GTGCAACAAAAAGTTGCTGAAATTAAACAAGA'ITTAGGCGGTAAGA
GGCTAATTGT1TCCGCTGGGCGGGTGGATTACGTGAAGGGCACCAAGGAAATGT
TGATGTGCTATGAACGTCTACTGGAGCGTCGCCCCGAATTGCAGGGGGAAATTA
GCCTGGTAGTCCCCGT'AGCCAAGGCCGCTGAGGGAATGCGTATTTATCGCAACG
CGCAAAACGAAATTGAACGACTGGCAGGGAAAATTAACGGTCGCTTTGCCAAAC TGTCCTGGACACCAGTGATGCTGTTCAGCTCTCCTITAGCGTATGAGGAGCTCATT
GCCCTGTTCTGTGCCGCCGACATTGCCTGGATCACTCCCCTGCGGGATGGGCTAA
AiXTGGTGGCTAAGGAGTATGTGGTGGCTAAAAATGGCGAAGAAGGAGTTCTGA
TCCTCTCGGAATTTGCCGG'TTGTGCGGTGGAACTACCCGATGCQGTGTFGACTAA
CCCCTAGGC'TTCCAGCCGTATGGACGAATCCATTGACCAGGCCCTGGCCATGGAC
AAAGACGAACAGAAAAAAGGCATGGGGAGAATGTACGCCGCCATTAAGCGTTA
CGACGITCAACAATGGGCCAATCACCTACTGCGGGAAGCCTACGCCGATGTGGT
ACTGGGAGAGCCCCCCCAAATGTAG
SEQ ID NO. 45: ~ Synechocystis - g!gA
MKILFVAAEVSPLAKVGGMGDVVGSLPKVLHQLGHDVRVFMPYYGFIGDKfDVPKE
PVWKGEAMFQQFAYYQSYLPDTKIPLYLFGHPAFDSRRIYGGDDEAWRFFFFSNGA
AEFAWNHWKPE11HCHDWMTGMIPVWMHQSPDIATVFTIHNI..AYQGPWRGLLETMT
WCPWYMQGDNVMAAAiQFANRVTTVSPTYAQQIQTPAYGEKLEGLLSYLSGNLYGl
LNGIDTEIYNPAEDRFiSMVFDADSLDKRVKNKIAIQEETGLEiNRNAMVVGIVARLV
EQKGI DE' VTQILDRFM S YTDSQL ilLGTGDRH YETQL WQM A SRFPGRM A VQLLHNDA
LSRRVYAGADVFLMPSRFEPCGLSQLMAMRYGCIPlVRRTGGLVDTVSFYDPiNEAG
TGYCFDRYEPLDCFTAMVRAWEGFRFKADWQKLQQRAMRADPSWYRSAGEYIKV
YKG V VG KP EELS PM E E E K i AEL'TA S Y R
SEQ ID NO. 46; - Synechocystis sp. - g!gA
ATGAAGATTTTATTTGTGGCGGCGGAAGTATCCCCCCTAGCAAAGGTAGGTGGC
ATGGGGGATGTGGTGGGTTCCCTGCCTAAAGTTCTGCATCAGTTGGGCGATGATG
TCCGTGTCTTCATGCCCTACTACGGTTrCATGGGCGACAAGATTGATGTGCCGAA
GGAGCCGGTCTGGAAAGGGGAAGCCATGTTCCAGCAGTTTGCTGTTTACCAGTCC
TATCTACCGGACACCAAAATTCCTCTCTACTTGTTCGGCCATCCAGCTTTCGACTC
CCGAAGGATCTATGGCGGAGATGACGAGGCGTGGCGGTTCACTTTTTTTTCTAAC
GGGGCAGCTGAATTTGCCTGGAACCATTGGAAGCCGGAAATTATCCATTGCCAT
GATTGGCACACTGGCATGATCCCTGTTTGGATGCATCAGTCCCCAGACATCGCCA
CCGTTTTCACCATCCATAATCTTGCTTACCAAGGGCCCTGGCGGGGCTTGCTTGA
AACTATGACITGGTGTCCTTGGTACATGCAGGGAGACAATGTGATGGCGGCGGC
GATTCAAITT’GCCAATCGGGTGACTACCGTTFCTCCCACCTATGCCCAACAGATC
CAAACCCCGGGGTATGGGGAAAAGCTGGAAGGGTTATTGTCCTACCTGAGTCJGT
AATTTAGTCGGTATTCTCAACGGTATTGATACGGAGATTTAGAACCCGGCGGAAG
ACCGCTTTATCAGCAATGTTTTCGATGCGGACAGTTTGGACAAGCGGGTGAAAA
ATAAAATTGCCATCCAGGAGGAAACGGGGTTAGAAATTAATCGTAATGCCATGG
TGGTGGGTATAGTGGCTCGCTTGGTGGAACAAAAGGGGATTGATTTGGTGATTCA
GATCCTTGACGGC7TCATGTCCTACACGGATFCCCAGTTAATTATCCTCGGCACTG
GCGATCGCCATTACGAAACCCAACTTrGGCAGATGGCTTCCCGATITCCTGGGCG
GATGGCGGTGCAATTACTCCACAACGATGCCCTTIXX'CGTCGAGTCTATGCCGGG
GCGGATGTGTFTTTAATGCCTTCTCGCTTTGAGCCCTGTGGGCTGAGTCAATTGAT
GGCGATGCGTTATGGGTGTATCCCCATTGTGCGGCGGACAGGGGGTTTGGTGGAT
ACGGTATCCTTCTACGATCC'T'ATCAATGAAGCCGGCACCGGCTATTGCTTTGACC GTTATGAACCCCTGGATTGCTTTACGGCCATGGTGCGGGCCTGGGAGGGTTTCCG
TTTCAAGGCAGATTGGCAAAAATTACAGCAACGGGCCATGCGGGCAGACTTTAG
TTGGTACCGTTCCGCCGGGGAATATATCAAAGTTTATAAGGGCGTGGTGGGGAA
ACCGGAGGAATTAAGCCCCATGGAAGAGGAAAAAATCGCTGAGTTAACTGCTTC
CTATCGCTAA
SEQ ID NO. 47: -- Deha!ococcoides - TceAB
ATGAGTGAAAAATACCAC'T'CCACCGTGACCCGTCGCGATTTTATGAAACGCCTGG
GCCTCGCCGGTGCCGGTGCCGGTGCCCTGGGTGCCGCCGTGCTCGCCOAAAACA
ATCTGCCCCACGAGTTTAAAGATGTGGATGATCTGCTCAGCGCCGGCAAAGCCCT
GGAGGGTGATCACGCCAATAAGGTGAACAATCACCCCTGGTGGGTGACCACCCG
CGATCACGAAGATCCCACCTGCAACATTGATTGGAGCCTGATTAAGCGCTACAGT
GGCTGGAACAATCAAGGTGCCi'ATTTTCTCCCCGAAGATTATCTCAGCCCCACCT
ATACCGGCCGCCGCCACACCATTGTGGATAGCAAACTGGAAATTGAACTGCAAG
GCAAAAAGTATCGCGATAGCGCCTTTATTGAAAGTGGTATTGATTGGATGAAGG
AGAATATTGATCCCGATTACGATCCCGGCGAACTCGGCTATGGTGATCGTCGCGA
GGATGCCCTGATTTACGCCGCCACCAACGGTTCCCACAATTGCTGGGAAAACCCC
CTCTACGGCCGCTATGAGGGTTCCCGCCCCTATCTGAGCATGCGCACCATGAACG
GCATrAATGGTCTCCACGAGTTTGGCCACGCCGATATTAAAACCACCAATTACCC
CAAGTGGGAAGGCACCCCCGAAGAGAACCTGCTCATTATGCGCACCGCCGCCCG
CTATTTTGGTGCCTCCAGCGTGGGTGCCATCAAGATCACGGATAAGGTGAAGAAA
ATTTTCTACGCCAAGGCCCAACCCTITTGCCTGGGCCCCTGGTATACCATTACCA
ATATGGCCGAATACATTGAGTATCCCGTGCCCGTGGATAACTACGCCATTCCCAT
TGTG'TTTGAGGATATI'CCCGCCGATCAAGGCCACTACAGCTAT'A AACGCTTTGGC
GGTGATGATAAGATTGCCGTGCCCAATGCCCTGGATAACAITTTTACCTACACCA
TTATGCTGCCCGAAAAACGCTTTAAGTATGCCCAC'T'CCATTCCTATGGACCCCTG
CAGCTGCATTGCCTACCCCCTGTTTACCGAAGTGGAGGCCCGCATTCAACAATTT
ATTGCCGGCCTGGGITATAATAGCATGGGGGGCGGCGTGGAAGCCTGGGGTGCC
GGTT'CCGCCTITGGTAACCTGAGCGGCCTCGGTGAACAAAGTCGCGTGAGTTCCA
CCATTGAGCCCCGCTACGGCAGTAACACCAAAGGTl’CCCTGCGCATGCTCACCGA
TCTGCCCCTCGCCCCCACCAAACCCATTGATGCCGGCATTCGCGAATTTTGCAAG
AGCTGCGGTATTTGCGCCGAGCACTGGCCCACGCAAGCCATTAGTCACGAAGGCC
CCCGCTATGATAGTCCCCACTGGGATTGCGTGTCCGGCTACGAGGGTTGGCACCT
GGATTATCACAAGTGCACCAATTGCACCAWTGCGAAGCCGTGTGCCCCTTTTTC
ACCATGTCCAACAATAGCTGGGTGCAGAACCTCGTGAAATGCACCGTGGCCACC
ACCCGCGTGTTTAATGGCTTTTTGAAGAACATGGAAGAGGCCTTTGGCTAGGGTG
CCCGCTATAGCCCCAGTCGCGATGAATGGTGGGCCAGCGAGAACCCCATTCGGG
GTGCCAGTGTGGATATTriYTGAGAGAAAGGATGGAATAGATTATTATGGGCGGT
GCCCTCT'ACTA'ITTTCTGGTGGGCATGCTCATTGGCGGTGCCGCCATn'GGTTCAT
G ACCT AC ACCC A ATTC AAG A AGA TTAGTiTT A AGTGGT GGG AATGG AGGCTC ATG
GGCCTGAGTGTGCTCCiGGTGTGGAGCATTTTTCAACACATGTATAGTTCCATGTC
CGTGGAAATGGAGTACCAAAGCGCCTTTATGTATCTGGGCGTGTTTGGCACCCTG
GCCGTGATTCTCAATCTGATTGTGTGGCGCACCTACAGCGGTCGCAAAGAGTAG SEQ ID NO. 48: - BamHl GGATCC
SEQ ID NO. 49 - Hindi!! AAGCTT
SEQ ID NO, 50 - Kpni GOT ACC
SEQ ID NO, 51 - T7 promoter TAAT.4CG ACTC ACTAT AGG
SEQ ID NO. 52 - T7 terminator GCTAGTTATTOCTCAGCGG
SEQ ID NO. 53 - His tag HHHHHH
SEQ ID NO. 54 - psbA promoter
ATTTAGCGTCTTCTAATCCAGTGTAGACAGTAGTTTTGGCTCCGTTGAGCACTGTA
GCCTTGGGCGATCGCTCTAAACATTACATAAATTCACAAAGTTTTCGTTACATAA
AAATAGTGTCrACTTAGCTAAAAATTAAGGGTTTTTTACACCTTTTTGACAGTTAA
TCTCCTAGCCTAAAAAGCAAGAGTTTTTAACTAAGACTCTTGCCCTTTACAACCT
C
SEQ ID NO. 55 ~ rrnB terminator
TGCCTGGCGGCAGTAGCGCGGTGGTCCGACCTGACCCCATGCCGAACTGAGAAG
TGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCGATGCGAGAGTAGGGA
ACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTT
SEQ ID NO. 56 ···· Synecbococcus - NS 1 integration
CAATGCCITCTCCAAGGGCGGCATTCCCCTGACTGTTGAAGGCGTTGCCAATATC
AAGATTGCTGGGGAAGAACCGACCATCCACAACGCGATCGAGCGGCTGCTTGGC AAAAACCGTAAGGAAATCGAGCAAATTGCCAAGGAGACCCTCGAAGGCAACTTG
CGTGGTGTnTAGCCAGCCTCACGCCGGAGCAGATCAACGAGGACAAAATTGCC
TTTGCCAAAAGTCTGCTGGAAGAGGCGGAGGATGACCTTGAGCAGCTGGGTCAA
GTCCTCGATACGCTGCAAGTCCAGAACATTTCCGATGAGGTCGGTTATCTCTCGG
CTAGTGGACGGAAGCAGCGGGCTGATCTGCAGGGAGATGCCCGAATTGCTGAAG
CCGATGCCCAGGCTGCCTCTGCGATCCAAACGGCCGAAAATGACAAGA'I'CACGG
CCCTGCGTCGGATCGATCGCGATGTAGCGATCGCCCAAGCCGAGGCCGAGCGCC
GGATTCAGGATGCGTTGACGCGGCGCGAAGCGGTGGTGGCCGAAGCTGAAGCGG
ACATTGCTACCGAAGTCGCTCGTAGCCAAGCAGAACTCCCTGTGCAGCAGGAGC
GGATCAAACAGGTGCAGCAGCAACTTGAAGCGGATGTGATCGCCCCAGGTGAGG
CAGCTTGTAAACGGGCGATCGCGGAAGCGCGGGGGGCCGCCGCCCGTATCGTCG
AAGATGGAAAAGCTCAAGCGGAAGGGACCCAACGGGTGGGGGAGGCTTGGCAG
ACCGCTGGTGCTAATGCCGGCGACATCITCCTGCTCGAGAAGCTCGAGTCCCTGC
TCGTCACGCTTTCAGGCACCGTGCCAGATATCGACGTGGAGTCGATCACTGT'GAT
TGGCGAAGGGGAAGGCAGCGCTACCCAAATCGCTAGCTTGCTGGAGAAGGTGAA
ACAAACCACGGGCATTGATCTGGCGAAATCCCTACCGGGTCAATCCGACTCGCC
CGCTGCGAAGTCCTAAGAGATAGCGATGTGACCGCGATCGCTTGTCAAGAATCC
CAGTGATCCCGAACCATAGGAAGGCAAGCTCAATGCTTGCCTCGTCTTGAGGACT
ATCTAGATGTCTGTGGAACGCACATiTATTGCCATCAAGCCCGATGGCGTTCAGC
GGGGTTTGGTCGGTACGATCATCGGCCGCTTYGAGCAAAAAGGCTTCAAACTGGT
GGGCCTAAAGGAGCTGAAGCCCAGTCGGGAGCTGGCGGAACAGGACTATGCTGT
GCAGGGGGAGCGCCCGTTGTTCAATGGCCTCGTGGAGrrCATCAGGTCTGGGCGG
ATCGTGGCGATCGTCTTGGAAGGCGAAGGCGTT'GTGGCGGCTGCTCGCAAGTTG
ATCGGCGCTACCAATCCGCTGACGGCAGAACCGGGCACCATCCGTGGTGATT1TG
GTGTCAATATTGGCCGCAACATCATCCATGGCTCGGATGCAATCGAAACAGCAC
AACAGGAAATTGCTGTCTGGTTTAGCCCAGCAGAGCTAAGTGAITGG-AGCCCCAC
GATTCAACCCTGGCTGTACGAATAAGGTCTGCATi'CCTTCAGAGAGACATTGCCA
T
7

Claims

1. A vinyl chloride monomer (VCM) producing recombinant microorganism having a VCM improved production ability, wherein the VCM producing recombinant microorganism expresses at least one VCM producing enzyme by i) expressing at least one non-native VCM producing enzyme nucleotide sequence and / or it) overexpressing at least one native VCM producing enzyme nucleotide sequence, wherein an amount of the VCM producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native VCM producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native VCM producing enzyme nucleotide sequence, wherein the VCM producing recombinant microorganism is capable of utilizing a chlorinated hydrocarbon source to produce VCM.
2. The VCM producing recombinant microorganism of Claim 1, wherein the chlorinated hydrocarbon source is a chlorinated alkane or a chlorinated aikene,
3. The VCM producing recombinant microorganism of Claim 1 or 2, wherein the chlorinated hydrocarbon source Is a C\.:> hydrocarbon.
4. The VCM producing recombinant microorganism of any one of Claims 1 to 3, wherein the chlorinated hydrocarbon source is trichloroethylene, dichloroetbylene or chloroform.
5. The VCM producing recombinant microorganism of any one of Claims 1 to 4, wherein the at least one VCM producing enzyme comprises TCE reductive dehalogenase (TceA), TceA anchor protein (TeeB), tetrachloroethyiene reductive dehalogenase (pceA), vinyl chloride reductase (vcrA), 1 ,2-dich!oropropane-to-propene reductive dehalogenase (depA), 1 ,2-trans-dishloroethene reductive dehalogenase catalytic A (TdrA), or a combination thereof.
6. The VCM producing recombinant microorganism of any one of Claims 1 to 5, wherein VCM producing recombinant microorganism expresses TceA having an amino acid sequence at least 95% identical to SEQ ID NO: I by expressing a native or non-native TceA nucleotide sequence at least 95% identical to SEQ ID NO: 2; expresses TeeB having an amino acid sequence at least 95% identical to SEQ ID NO: 3 by expressing a native or non- native TceB nucleotide sequence at least 95% identical to SEQ ID NO: 4; expresses pceA having an amino acid sequence at least 95% identical to SEQ ID NO: 5 by expressing a native or non-native pceA nucleotide sequence at least 95% identical to SEQ ID NO: 6; expresses vcrA having an amino acid sequence at least 95% identical to SEQ ID NO: 7 by expressing a native or non-native ver A nucleotide sequence at least 95% identical to SEQ ID NO: 8; expresses dcpA having an amino acid sequence at least 95% identical to SEQ ID NO: 9 by expressing a native or non-native depA nuc.eotide sequence at least 95% identical to SEQ ID NO: 10; expresses TdrA having an arr ino acid sequence 95% identical to SEQ ID NO: 11 by expressing a native or non-native Ί irA nucleotide sequence at least 95% Identical to SEQ ID NO: 12; or a combination thereof
7. The VCM producing recombinant microorganism of any one of Claims 1 to 6, wherein the VCM producing recombinant microorganism expresses at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or is) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the VCM producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme nucleotide sequence, wherein the VCM producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE,
8. The VCM producing recombinant microorganism of any one of Claims 1 to 7, wherein the VCM producing recombinant microorganism is a microorganism of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehalococcoid.es strain FL2, Dehalococcoides mccarty e.g. strains KS, RC, JNA, MB, I ia or GY50. Dehalococcoides ethenogenes e.g, strain 195, Dehalococcoides strain BAVI, Dehalococcoides strain VS, Dehalococcoides strain CBDB1, Dehalococcoides strain GT) Escherichia (optionally Escherichia coil), Synechococcus (optionally Synechococcus elongaius), Sulfurospirilium (optionally Sulfurospirilium multi varans or Sulfurospirilium harnesn), Dehalobacter (optionally Dehalobacter restrictus), Desulfuromonas (optionally strain BB1 or Desulfitromonas chloroethenica), Desulfitobacterium (optionally Desulfitobacterium hqfhieme), Geobacier (optionally Geobacter bemidjiensis, Geobacter lovleyi, Geobacler psychrophilus, Geobacter sp. FRC-32, Geobacter sp. M21, Geobacter sulfurreducens, or Geobacter uraniireducens), Pelobacter (optionally Pelobacter propionicus) Dehalogenimonas (optionally sp. Strain WBC-2) Pseudomonas, Flavobacterium,
Comamonas, Cytophaga, Acidovorax, Sphingomonas, Bacillus, Acimtobacter or a combination thereof.
9. The VCM producing recombinant microorganism of any one of Claims I to 8, wherein the chloride source is a chloride salt {e.g. NaCl, KCl, MgCh, CaCfe or combinations thereof) and / or HC!.
10. A method of producing vinyl chloride monomer (VCM) comprising: providing a reaction medium comprising a VCM producing enzyme and a chlorinated hydrocarbon; maintaining the reaction medium under conditions which permit the production of VCM from the chlorinated hydrocarbon by the VCM producing enzyme; and collecting VCM from the reaction medium.
1 1. The method of Claim 10. wherein the VCM producing enzyme is produced by a VCM producing microorganism which is native.
12. The method of Claim 10, wherein the VCM producing enzyme is produced by a VCM producing microorganism is recombinant.
13. The method of Claim 12. wherein the recombinant VCM producing organism is a vinyl chloride monomer (VCM) producing recombinant microorganism as defined by any one of Claims l to 8.
14. The method of any one of Claims 10 to 13, wherein the VCM producing enzyme is produced by a VCM producing microorganism which VCM producing microorganism is provided in the reaction medium.
15. The method of any one of Claims 11 to 14, wherein the VCM producing microorganism is a microorganism of the phylum Cyanobacteria or the genus Dekalococeoides (optionally Dehalococcoides strain FL2, Dehalococcoides mccarty e.g. strains KS5 RC, JNA, MR, 1 1a or GY5Q. Dehalococcoides ethenogenes e g. strain 195. Dehalococcoides strain BAVl , Dehalococcoides strain VSs Dehalococcoides strain CBDBl, Dehalococcoides strain GT) Escherichia (optionally Escherichia coi'i ), Synechococcus (optionally Synechococcus elongatus), Sulfurospirillum (optionally Sulfurospirillum multivoram or Sui/urospirilium barmsii), Dehalobacter (optionally Dehalobacter restrictus), Desu!furomonas (optionally strain BR] or Desulfuromonas chloroethenica). Desulfitobacterium (optionally Desulfitobacterium hafniense), Geobacter (optionally Geobacter bemidjiensis, Geobacter lovleyi, Geobacter ps ychrophilus, Geobacter sp. FRC-32, Geohacter sp. M21, Geobacier sulfurreducem, or Geobacter uraniireducem), Pelohacier (optionally Pelobacter propionicus) Dehalogenimonas (optionally sp. Strain WBC-2) Pseudomonas, Flavohacterium, Comamoms, Cyiophaga. Acidovorax, Sphingomonas, Bacillus, Acinetobacter or a combination thereof
16. The method of any one of Claims 11 to 15, wherein the VCM producing microorganism comprises a consortium of bacteria,
17. The method of any one of Clai. is 11 to 16, wherein the VCM producing microorganism comprises a plurality of strains i -elonging to the genus Dehalococcoid.es.
18. The method of any one of Claims i 1 to 17, wherein the VCM producing microorganism expresses Tee A having an amino a< id sequence at least 95% identical to SEQ ID NO: 1 by expressing a TceA nucleotide sequence at least 95% identical to SEQ ID NO: 2; expresses TceB having an amino acid sequence at least 95% identical to SEQ ID NO: 3 by expressing a TceB nucleotide sequence at least 95% identical to SEQ ID NO: 4; expresses peeA having an amino acid sequence at least 95% identical to SEQ ID NO: 5 by expressing a pee A nucleotide sequence at least 95% identical to SEQ ID NO: 6; expresses vcrA having an amino acid sequence at least 95% identical to SEQ ID NO: 7 by expressing a vcrA nucleotide sequence at least 95% identical to SEQ ID NO: 8; expresses dcpA having an amino acid sequence at least 95% identical to SEQ ID NO: 9 by expressing a dcpA nucleotide sequence at least 95% identical to SEQ ID NO: 10; and I or expresses TdrA having an amino acid sequence 95% identical to SEQ ID NO: 11 by expressing a TdrA nucleotide sequence at least 95% identical to SEQ ID NO: 12.
19. A trichloroethylene (TCE) producing recombinant microorganism having an improved TCE producing ability, wherein the TCE producing recombinant microorganism expresses at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or ii) overexpressing at feast one native TCE producing enzyme nucleotide sequence, wherein an amount of TCE producing enzyme produced by the TCE producing recombinant microorganism is greater than that produced relative to a control microorganism lacking the at least one non-native TCE producing enzyme nucleotide sequence and / or which is not engineered to overexpress the at least one native TCE producing enzyme nucleotide sequence, wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE.
20. The TCE producing recombinant microorganism of Claim 19, wherein the at least one TCE producing enzyme comprises phenol hydrase (PH), particulate methane monooxygenase (pMMO), benzene (and/or toluene) dioxygenase (BDO/ToD), toluene o- xyiene monooxygenase oxygenase subunit (TouA), toluene-4-monooxygenase system hydroxylase component subunit alpha, chlorobenzene dioxygenase, cis-chlorobenzene dihydrodiol dehydrogenase, toluene 2~monooxygenase, or a combination thereof.
21. The TCE producing recombinant microorganism of Claim 39 or 20, wherein the TCE producing recombinant microorganism expresses PH having an amino acid sequence at least 95% identical to SEQ ID NO: 13 by expressing a native or non-native PH nucleotide sequence at least 95% identical to SEQ ID NO: 14; expresses pMMO having an amino acid sequence at least 95% identical to SEQ ID NO: 15 by expressing a native or non-native pMMO nucleotide sequence at least 95% identical to SEQ ID NO: 16; expresses ToD having an amino acid sequence at least 95% identical to SEQ ID NO: 1 ? by expressing a native or non-native ToD nucleotide sequence at least 95% identical to SEQ ID NO: 18; expresses TouA having an amino add sequence at least 95% identical to SEQ ID NO: 19 by expressing a native or non-native TouA nucleotide sequence at least 95% identical to SEQ ID NO: 20; expresses toluene-4-monooxygenase system hydroxylase component subunit alpha having an amino add sequence at least 95% identical to SEQ ID NO: 2! by expressing a native or nonnative toiuene-4-monoGxygenase system hydroxylase component subunit alpha nucleotide sequence at least 95% identical to SEQ ID NO: 22; expresses chlorobenzene dioxygenase having an amino acid sequence 95% identical to SEQ ID NO: 23 by expressing a native or non-native chlorobenzene dioxygenase nucleotide sequence at least 95% identical to SEQ ID NO: 24; expresses cis-chlorobenzene dihydrodiol dehydrogenase having an amino acid sequence at least 95% identical to SEQ ID NO: 25 by expressing a native or non-native cis- chlorobenzene dihydrodiol dehydrogenase nucleotide sequence at least 95% identical to SEQ ID NO: 26; or expresses toluene 2-monooxygenase having an amino acid sequence at least 95% identical to SEQ ID NO: 27 by expressing a native or non-native toluene 2- monooxygenase nucleotide sequence at least 95% identical to SEQ ID NO: 28; or a combination thereof.
22. The TCE producing recombinant microorganism of any one of Claims 19 to 21, wherein the TCE producing recombinant microorganism is a microorganism of the genus Rhizobiim (optionally Rhizobium meliloti e.g, Rhizobium meliloti strain Dangeard), Porphyridiurn (optionally Porphyriaium purpureum), Emiliania (optionally Emiliana huxleyi), Sinorhizobium (optionally Sinorhizobium meliloti), Calcidiscus (optionally Calcidiscus leploporus), Phaeodaetylum (optionally Phaeodactylum tricomutum), Chaetoceros (optionally Chaetoceros neogracilis), Dumliella (optionally DunalieUa tertiolecta), Meristiella (optionally Meristiella gelidium), Ulva (optionally Ulva laciuca or Ulva rigida , e.g. Ulva rigida Agardh), Enieromorpha (optionally Enieromorphia intestinalis , Ciadophora (optionally Cladophora mpesiris), Fucus (optionally Fucus serratus), Laminaria (optionally Laminaria saccarina, e.g. Laminaria sacearina (L) Lamour or Laminaria digitaia, e.g. Laminaria digitata (Huds) Lamour), Desmarestia (optionally Desmarestia aculeata, e.g. Desmarestia aculeata (L) Lamour), Chorda (optionally Chorda filum, e.g. Chorda filum (L) Stackh), Chondrus (optionally Chondrus crispus, e.g. Ckondrus crispus Stackh), Phyllophora (optionally Phyllophora pseudoeeranoides, e.g. Phyllophora pseudoceranoi'des (Gmelin)), Porphyra (optionally Porphyra umbilicalis, e.g. Porphyra
Figure imgf000085_0001
(optionally Polysiphonia nigrsscem, e.g. Polysiphonia nigrescens (Huds.) Greviiie), Furcellaria (optionally Furcellaria lumbricalis, e.g.
Furcellaria lumbricalis (Huds.) Lamour). Ceramium rubrurn (optionally Ceramium rubrum, e.g. Ceramium rubrum (Huds.) Agardh), Ahnfeltia (optionally Ahnfeltia plicata, e.g.
Ahnfeltia plicaia (Hudson) Fries), Laurencia (optionally Laurencia pinnatifida, e.g. Laurencia pinnatifida (Huds.) Lamour, or Laurencia obtuse, e g. Laurencia obtusa (Huds.) Lamour), Caulerpa, Hypnea (optionally Hypnea musciformis, e.g. Hypnea musciformis (Wulfen) Lamouroux), Asparagopsis (optionally A, sparagopsis taxiformis, e.g. Asparagopsis taxiformis (Deliie) Trev), Gelidium (optionally Gelidium camriensis), Falkenbergia (optionally Falkenbergia hillebrandii, e.g. Falkenbergia hillebrandii (Born) Falkenb), Corallina (optionally Corallina officinalis), Gracilariopsis (optionally Gracilariopsis lemaneiformis), Gracilaria (optionally Gracilaria cornea, e.g. Gracilaria cornea J. Agardh), Methylosm' us (optionally Methylosinus trichosponum, e.g. Methylosims trichosporium OB 3b), Desuljitobacterium (optionally Desulfitobacterium frappieri, e.g. Desulfitohacterium frappieri TCE1, or Desulfitobacterium metaUireducens), Melhylomicrobium (optionally Methyiomicrobium album, e.g. Methylomicrobium album BG8), Methylococcus (optionally Methylococcus capsulatus, e.g. Methylococcus capsulatus (Bath)), Ralstonia (e.g. Ralstonia sp. KN 1-1 OA)t Pseudomonas (optionally Pseudomonas putida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4), Rhodobacier (optionally Rhodobacter sphaeroides or Rhodobacter capsulatus), Burkholderia (optionally Burkholder ia cepacian, e.g. Burkholderia cepacian G4) or a combination thereof.
23. The TCE producing recombinant microorganism of any one of Claims 19 to 22, wherein the chloride source Is a chloride salt (e.g. NaCi, KC!, MgCh, CaCh or combinations thereof) and / or HCL
24. A method of producing trichloroethylene (TCE) comprising: providing a reaction medium comprising a TCE producing enzyme and a TCE feedstock; maintaining the reaction medium under conditions which permit the production of TCE from the TCE feedstock by the TCE producing enzyme; and collecting TCE from the reaction medium.
25. The method of Claim 24, wherein the TCE producing enzyme is produced by a TCE producing microorganism which is native.
26. The method of Claim 24, wherein the TCE producing enzyme is produced by a TCE producing microorganism which is recombinant
27. The method of Claim 26, wherein the recombinant TCE producing organism is a trichloroethylene (TCE) producing recombinant microorganism as defined by arty one of Claims 17 to 20.
2k, The method of any one of Claims 24 to 27, wherein the TCE producing enzyme is produced by a TCE producing microorganism which TCE producing microorganism is provided in the reaction medium.
29. The method of any one of Claims 24 to 28, wherein the TCE feedstock comprises a carbohydrate, a chlorinated hydrocarbon and / or a polyol
30. The method of Claim 29, wherein the carbohydrate is a monosaccharide, optionally xylose, giyceraldehyde, fructose, galactose, mannose and / or glucose, or a disaccharide, optionally lactose, maltose, sucrose, cellobiose and / or trehalose.
31. The method of Claim 29, wherein the chlorinated hydrocarbon is a Cj-2 chlorinated alkane, optionally chloroform and / or carbon tetrachloride.
32. The method of Claim 29, wherein the polyol comprises glycerol, mannitol, sorbitol, ma!titoi and / or xylito!.
33. The method of any one of Claims 25 to 28, wherein the TCE producing microorganism is a microorganism of the genus Rhizobium (optionally Rhizobium meliloti e g Rhizobium meliloti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emiliana kuxleyi), Sinorhizobium (optionally Sinorhizobium meliloti), Calcidlscus (optionally Calddiscus leptoporus), Phaeodactylum (optionally Phaeodactylum tricornutum), Ckaeioceros (optionally Chaetoceros neogracilis),
§5 Dunaliella (optionally Dumliella iertiolecia ), Meristiella (optionally Meristiella gelidium), Ulva (optionally Ulva lactuca or Ulva rigida, e.g. Ulva rigida Agardh), Enteromorpha (optionally Enieromorphia intestimlis, Cladophora (optionally Cladophora rupestris), Fucus (optionally Fucus serratus), Laminaria (optionally Laminaria saccarina, e.g. Laminaria saccarim (L) Lamour or Laminaria digitata, e.g. Laminaria digiiata (Huds) Ixonour), Desmarestia (optionally Desmarestia acuieata, e.g. Desmaresiia aculeata (L) Lamour), Chorda (optionally Chorda fihm, e.g. Chorda filum (L) Stackh), Chondrus (optionally Chondrm crispus, e.g. Chondrus crispus Stackh), Phyllophora (optionally Phytlophora pseudoceranoides , e.g. Phyllophora pseudoceranoi'des (Gmelin)), Porphyra (optionally Porphyra umbilicalis, e.g. Porphyra umbilicalis, (L) J. Ag.), Polysiphonia (optionally Poiysiphonia nigrescem, e.g. Polysiphonia nigrescens (Huds.) Greville), Furcellaria (optionally Furcellaria lumhricalis, e.g. Furcellaria Itmbrlealis (Huds) Lamour), Ceramium rubrum (optionally Ceramium rubrum, e.g Ceramium rubrum (Huds.) Agardh), Ahnfeltia (optionally Ahnfeltia plicata, e.g. Ahnfeltia plicata (Hudson) Fries), Laurencia (optionally Laurencia pimatifida, e.g. Laurencia pinnatifida (Huds.) Lamour, or Laurencia obtuse, e.g. Laurencia obiusa (Huds.) Lamour), Caulerpa, Hypnea (optionally Hypnea musdformis, e.g. Hypnea musdformis (Wulfen) Lamouroux), Asparagopsis (optionally Asparagopsis taxiformis, e.g. Asparagopsis taxiformis (Delile) Trev), Gelidium (optionally Gelidium canariensis), Falkenbergia (optionally Falkenbergia hillebrandii, e.g. Falkenbergia hillebrandii (Bom.) Falkenb), Coralline (optionally Corallina officinalis), Gracilariopsis (optionally Gracilariopsis lemaneiformis), Gracilaria (optionally Gracilaria cornea, e.g. Gracilaria cornea J. Agardh), Methylosinus (optionally Methylosinus trichosporium, e.g. Methylosinus trichosporium OB 3b). Desulfitobacterium (optionally Desulfttobacterium frappieri, e.g. Desulfitobacterium frappieri TCE1, or Desulfitobacterium metallireducens), Methylomicrobium (optionally Methylomicrobium album, e.g. Methylomicrobium album BG8), Meihylococcus (optionally Meihylococcus capsulatus, e.g. Methylococcus capsulaius (Bath)), Ralstonia (e.g. Ralstonia $p. KNJ-IQA), Pseudomonas (optionally Pseudomonas piitida, e.g. Pseudomonas putida FI, or Pseudomonas sp. M4), Rhodobacter (optionally Rhodobacter sphaeroides or Rhodobacter capsulatus), Burkholderia (optionally Burkholderia cepaclan, e.g. Burkholderia cepacian G4) or a combination thereof
34. The method of any one of Claims 25 to 28, wherein the TCE producing microorganism expresses at least one TCE producing enzyme selected from phenol hydrase (PH), particulate methane monooxygenase (pMMO), benzene (and/or toluene) dioxygenase (ToD), toluene o-xyiene monooxygenase oxygenase subunit (TouA), toluene-4- monooxygenase system hydroxylase component subunit alpha, chlorobenzene dioxygenase, cis-ch!orobenzene dihydrodiol dehydrogenase, toluene 2-monooxygenase, or a combination thereof.
35. The method of any one of Claims 25 to 28, wherein the TCE producing microorganism expresses PH having an amino acid sequence at least 95% identical to SEQ ID NO: 13 by expressing a PH nucleotide sequence at least 95% identical to SEQ ID NO: 14: expresses pMMO having an amino acid sequence at least 95% identical to SEQ ID NO: 15 by expressing a pMMO nucleotide sequence at least 95% identical to SEQ ID NO: 16; expresses ToD having an amino acid sequence at least 95% identical to SEQ ID NO: 17 by expressing a ToD nucleotide sequence at least 95% identical to SEQ ID NO: 18; expresses TouA having an amino acid sequence at least 95% identical to SEQ ID NO: 19 by expressing a TouA nucleotide sequence at least 95% identical to SEQ I'D NO: 20; expresses toluene-4- monooxygenase system hydroxylase component subunit alpha having an amino acid sequence at least 95% identical to SEQ ID NO: 21 by expressing a toluene-4-monooxygenase system hydroxylase component subunit alpha nucleotide sequence at least 95% identical to SEQ ID NO: 22; expresses chlorobenzene dioxygenase having an amino acid sequence 95% identical to SEQ ID NO: 23 by expressing a chlorobenzene dioxygenase nucleotide sequence at least 95% identical to SEQ ID NO: 24; expresses cis-chlorobenzene dihydrodiol dehydrogenase having an amino acid sequence at least 95% identical to SEQ ID NO: 25 by- expressing a cis-chlorobenzene dihydrodiol dehydrogenase nucleotide sequence at least 95% identical to SEQ ID NO: 26; or expresses toluene 2-monooxygenase having an amino acid sequence at least 95% identical to SEQ ID NO: 27 by expressing a toluene 2-monooxygenase nucleotide sequence at least 95% identical to SEQ ID NO: 28; or a combination thereof.
36. The method of any one of Claims 24 to 35, wherein the reaction medium comprises a chloride source,
37. The method of any one of Claims 24 to 36, wherein the chloride source is a chloride salt (e.g. NaCi, KCI, MgCfe, CaCl? or combinations thereof) and / or HC!.
38. A method of producing trichloroethylene (TCE) comprising: producing TCE by reacting glucose with a chloride source in the presence of a TCE producing catalytic element, wherein the TCE producing catalytic element comprises a TCE bioreactor culture containing a TCE producing recombinant microorganism having ars improved TCE producing ability, wherein the TCE producing recombinant microorganism expresses the at least one TCE producing enzyme by i) expressing at least one non-native TCE producing enzyme nucleotide sequence and / or it) overexpressing at least one native TCE producing enzyme nucleotide sequence, wherein the TCE producing recombinant microorganism is capable of utilizing a TCE feedstock and a chloride source to produce TCE,
39. The method of Claim 38, farther comprising: producing glucose by reacting a carbon source with a hydrogen source in the presence of a glucose producing catalytic element comprising at least one photosynthesis enzyme comprising Rubisco, wherein the glucose producing catalytic element comprises a glucose bioreactor culture containing a glucose producing microorganism, wherein the glucose producing microorganism expresses the at least one glucose producing enzyme, wherein the glucose producing microorganism is capable of utilizing the carbon source and the hydrogen source to produce glucose.
40. The method of Claim 38 or Claim 39, further comprising a VCM producing catalytic element, wherein the VCM producing catalytic element comprises a VCM bioreactor culture containing a VCM producing recombinant microorganism having an improved VCM producing ability, wherein the VCM producing recombinant microorganism expresses the at least one VCM producing enzyme by i) expressing at least one non-native VCM producing enzyme nueieotide sequence and / or ii) overexpressing at least one native VCM producing enzyme nucleotide sequence, wherein the VCM producing recombinant microorganism is capable of utilizing TCE to produce VCM; or wherein the carbon source comprises a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof; or wherein the hydrogen source comprises water; or wherein the chloride source comprises sodium chloride, HC!, or a combination thereof.
41. The method of any one of Claims 38 to 40, wherein the chloride source is a chloride salt ( e.g , NaCl, KC1, MgCfe, CaCfe or combinations thereof? and / or HCI.
42. A glucose producing recombinant organism wherein the glucose producing microorganism expresses at least one glucose producing enzyme, wherein the glucose producing microorganism is capable of utilizing a carbon source and a hydrogen source to produce glucose.
43. The glucose producing recombinant organism of Claim 42, wherein the glucose producing enzyme comprises sucrose permease (cscB), sucrose-phosphate synthase (sps), glucose- 1 -phosphate adenylyitransferase (glgC), sucrose phosphate phosphatase (spp), glycogen phophory!ase (gigP), UDP-g!ucose pyrophosphorylase (gaiU), invertase, glucosy Iglyceroi-phosphate synthase (ggpS), glycogen synthase(glgA); or combinations thereof,
44. The glucose producing recombinant organism of Claim 43, wherein the glucose producing microorganism expresses cscB having an amino acid sequence at least
95% identical to SEQ ID NO: 29 by expressing a non-native cscB nucleotide sequence at least 95% identical to SEQ ID NO: 30; expresses sps having an amino acid sequence at least 95% identical to SEQ ID NO: 31 by expressing a non-native sps nucleotide sequence at least 95% identical to SEQ ID NO: 32; expresses glgC having an amino acid sequence at least 95% identical to SEQ ID NO: 33 by expressing a non-native glgC nucleotide sequence at least 95% identical to SEQ ID NO: 34; expresses spp having an amino acid sequence at least 95% identical to SEQ ID NO: 35 by expressing a non-native spp nucleotide sequence at least 95% identical to SEQ ID NO: 36; expresses glgP having an amino acid sequence at least 95% identical to SEQ ID NO: 37 by expressing a non-native g!gP nucleotide sequence at least 95% identical to SEQ ID NO: 38; expresses ga!U having an amino acid sequence 95% identical to SEQ ID NO: 39 by expressing a non-native gaiU nucleotide sequence at least 95% identical to SEQ ID NO: 40; expresses invertase having an amino acid sequence at least 95% identical to SEQ ID NO: 41 by expressing a non-native invertase nucleotide sequence at least 95% identical to SEQ ID NO: 42; expresses ggpS having an amino acid sequence at least 95% identical to SEQ ID NO: 43 by expressing a non-native ggpS nucleotide sequence at least 95% identical to SEQ ID NO: 44; expresses glgA having an amino acid sequence at least 95% identical to SEQ ID NO: 45 by expressing a non-native glgA nucleotide sequence at least 95% identical to SEQ ID NO: 46; or a combination thereof.
45. The glucose producing recombinant organism of any one of Claims 42 to 44, wherein the glucose producing recombinant organism comprises a recombinant microorganism, a photosynthetic microorganism, a microorganism belonging to the phylum Cyanobacteria, a microorganism belonging to the genus Synechococcus (optionally Synechococcus elongates oxSynechococcus leopoliensis), Synechocystis, Anabaena, Pseudomonas (optionally Pseudomonas syringae or Pseudomonas savastanoi }, Chlamydomonas (optionally Ck!amydomonas reinhardtii), an algae, a microalgae, an electrosynthesis bacteria, a photosynthetic microorganism, a yeast, a filamentous fungi, or a plant cell
46. The glucose producing recombinant organism of claims 42 to 45, wherein the carbon source comprises a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source, or a combination thereof; and / or
§9 the hydrogen source comprises water; and / or the chloride source comprises a chloride salt (e.g, NaCl, KCi, MgC?2, CaCI2 or combinations thereof) and / or MCI.
47. A method of producing glucose comprising: providing a reaction medium comprising the glucose producing recombinant microorganism of any one of Claims 42 to 46, a hydrogen source and a carbon source; maintaining the reaction medium under conditions which permit the production of glucose by the glucose producing microorganism; and collecting glucose from the reaction medium.
48. The method of Claim 47, wherein the hydrogen source comprises water; and / or the carbon source comprises a carbon dioxide source, a gaseous carbon dioxide source, a bicarbonate carbon source.
49. A method of producing a vinyl chloride monomer (VCM) producing recombinant microorganism having an VCM improved producing ability or a trichloroethylene (TCE) producing recombinant microorganism having an improved TCE producing ability comprising: producing the VCM producing recombinant microorganism by inserting at least one of a non-nat ive VCM expressing nuc leotide sequence, a native VCM expressing nuc leotide sequence, a nucleotide sequence promoting the overexpression of a native VCM expressing nucleotide sequence, a non-native TCE expressing nucleotide sequence, a native TCE expressing nucleotide sequence, and / or a nucleotide sequence promoting the overexpression of a native TCE expressing nucleotide sequence into a bacterial plasmid of a microorganism; wherein the native or non-native VCM expressing nucleotide sequence optionally has a nucleotide sequence at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or a combination thereof; or wherein the native or non-native TCE expressing nucleotide sequence optionally has a nucleotide sequence at least 95% identical to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:
18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO; 26, SEQ ID NO: 28, or a combination thereof,
50. The method of Claim 49, wherein the VCM producing recombinant microorganism is a microorganism of the phylum Cyanobacteria or the genus Dehalococcoides (optionally Dehalococcoides strain FL2, Dehalococcoides mccarty e.g. strains KS, RC, JNA, MB, I la or GY50, Dehalococcoides ethenogenes e.g. strain 195, Dehalococcoides strain BAY i, Dehalococcoides strain VS, Dehalococcoides strain CBDBI, Deha!ococcoides strain GT) Escherichia (optionally Escherichia coil), Synechococcus (optionally Synechococcus elongatus), Suifurospiriihm (optionally Sulfurospiriihm multivorans or Sulfurospirillum barms ii), Dekalobacter (optionally Dehalobacter resir ictus), Desulfitromonas (optionally strain BB! or Desulfuromonas chloroethenica), Desulfitobacterium (optionally Desulfitobacterium kafniense), Geobacler (optionally Geobaeter bemidjiensis, Geobacter lovleyi, Geobacier psychrophilus, Geobacler sp. FRC-32, Geobacter sp. M21, Geobacter sulfurreducens, or Geobacier uraniireducens), Pelobacter (optionally Pelobacter propionicus) Dehalogenimonas (optionally sp. Strain WBC-2) Pseudomonas, Fiavobacterium, Comamonas, Cytophaga, Acidovorax, Sphingomonas, Bacillus, Acineiobacter or a combination thereof.
5 i . The method of Claim 49, wherein the TCE producing microorganism is a microorganism of the genus Rhizobium (optionally Rhizobium meliioti e.g. Rhizobium meliloti strain Dangeard), Porphyridium (optionally Porphyridium purpureum), Emiliania (optionally Emiliana huxleyi), Sinorhizobium (optionally Sinorhizobium meliloti),
Calcidiscus (optionally Calcidiscus leptoporus), Phasodaciylum (optionally Phaeodaciytum tricornutum), Chaetoceros (optionally Chaetoceros neogracilis), Dunalieila (optionally Dunaliella tertiolecta), Meristiella (optionally Meristieila geiidium), Ulva (optionally Ulva laetuca or Viva rigida, e.g. Ulva rigida Agardh), Enteromorpha (optionally Enteromorphia intesiinalis, Cladophora (optionally Cladophora rupestris), Fucus (optionally Fucm serratm), Laminaria (optionally Laminaria saecarina, e.g. Laminaria saccarina (L) humour or Laminaria digiiata, e.g. Laminaria digitaia (Muds) Lamour), Desmaresila (optionally Desmaresiia aculeata, e.g. Desmaresila aculeaia (L) Lamour), Chorda (optionally Chorda filum, e.g. Chorda fllum (L) Stackh), Chondrus (optionally Chondrus crispus, e.g. Chondrus crispus Stackh), Phyllophora (optionally Phyllophora pseudoceranoides, e.g. Phyllophora pseudoceranoi'des (Gmelin)), Porphyra (optionally Porphyra umbilicalis, e.g. Porphyra umbilicalis, (L) J. Ag.), Polys iphonia (optionally Polysiphonia nigrescens, e.g. Polys iphonia nigrescens (Huds.) Greviiie), Furcellaria (optionally Furcellaria lumbricalis, e.g.
Furcellaria lumbricalis (Huds.) Lamour ), Ceramium rubrum (optionally Ceramium rubrum , e.g. Ceramium rubrum (Huds.) Agardh), Ahnfeltia (optionally Ahnfeltia plicata, e.g. AhnfeUia plicata (Hudson) Fries), Laurencia (optionally Laurencia pinnatifida, e.g. Laurencia pinnatifida (Huds) Lamour , or Laurencia obtuse, e.g. Laurencia ohiusa (Huds) Lamour), Caulerpa, Hypnea (optionally Hypnea musciformis, e g Hypnea musciformis (Wulfen) Lamouroux), Asparagopsis (optionally Asparagopsis taxiformis, e g. Asparagopsis taxiformis (Delile) Trev), Geiidium (optionally Geiidium canadensis), Falkenbergia (optionally Falkenbergia hillebrandii, e.g Falkenbergia hillebrandii (Born.) Falkenb), Corallina (optionally Corallina officinalis), Grocilariopsis (optionally Gracilariopsis lemaneiformis), Gracilaria (optionally Gracilaria cornea, e.g. Gracilaria cornea J. Agardh), Meihylosinus (optionally Methyhsinm trichosporium, e.g Methylosinus trichosporium OB 3b), Desulfitobacterium (optionally Desulfitobacterium frappieri, e.g. Desulfitobacterium frappieri TCE l, or Desulfitobacterium metallireducens), Methylomicrohium (optionally Methylomicrobium album, e.g. Methylomicrohium album BG8), Methylococcus (optionally Methyiococcus capstdatus, e.g Methylococcus ca fsulatus (Bath)), Rabionia (e.g. Rabtonia sp. KN 1-lOA), Pseudomonas (optionally Pseudomonas putida, e g. Pseudomonas putida FI, or Pseudomonas sp. M4), Rhodobacter (optionally Rhode bacter sphaeroides or Rhodobacier capsulatus), Burkholderia (optionally Burkholderia cepacian, e.g. Burkholderia cepacian G4) or a combination thereof
52, The method of any one of Claims 49 to 5 !, wherein the VCM producing recombinant microorganism and the TCE producing recombinant microorganism are the same or different,
53. The method of producing TCE of any one of Claims 24 to 37, wherein the TCE feedstock comprises glucose and said glucose is produced by the glucose producing recombinant organism of any one of Claims 42 to 46 and / or by the process of Claim 47 or 48.
54. The method of producing VCM of any one of Claims 10 to 18, wherein the chlorinated alkane Is TCE and said TCE is produced by the TCE producing recombinant microorganism of any one of Claims 19 to 23 and / or the process of any one of Claims 24 to 41.
55. The process of Claim 54, wherein the reaction to produce glucose as the TCE feedstock is not conducted in the same bioreactor as the production of VCM from TCE.
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