TWI811184B - Genetically engineered bacterium comprising energy-generating fermentation pathway - Google Patents

Abstract

The invention relates to a genetically engineered bacterium comprising an energy-generating fermentation pathway and methods related thereto. In particular, the invention provides a bacterium comprising a phosphate butyryltransferase (Ptb) and a butyrate kinase (Buk) (Ptb-Buk) that act on non-native substrates to produce a wide variety of products and intermediates. In certain embodiments, the invention relates to the introduction of Ptb-Buk into a C1-fixing microoorgansim capable of producing products from a gaseous substrate.

Description

Genetically engineered bacteria including energy-producing fermentation pathways

With the current development of fermentation and metabolic engineering, fermentation pathways for various products have been identified and developed (Clomburg, " Appl Microbiol Biotechnol " , 86: 419-434, 2010; Peralta-Yahya , " Biotechnol J ", 5: 147-162, 2010; Cho, " Biotechnol Adv ", pii: S0734-9750(14)00181-5, 2014). However, all such fermentation pathways are energy-consuming (ATP) or maximal-energy (ATP) balanced, thereby limiting product yield in energy-limited systems and separating product production from microbial growth. The present invention provides a capacity (ATP) pathway to overcome these limitations by providing novel fermentation pathways and pathways to obtain a variety of products, including acids, olefins, aldehydes, alcohols and glycols. These pathways directly couple to microbial growth and provide high product yields.

Specifically, the present invention relates to a fermentation pathway involving Ptb-Buk. Phosphate butyltransferase (Ptb) (EC 2.3.1.19) naturally catalyzes the reaction of butyl-CoA and phosphate to form CoA and butyl-phosphate. Butyrate kinase (Buk) (EC 2.7.2.7) naturally catalyzes the reaction of butyryl phosphate and ADP to form butyrate/butanoate and ATP. Thus, these enzymes together (Ptb-Buk) naturally catalyze the conversion of butyryl-CoA to butyrate and generate an ATP via substrate-level phosphorylation (SLP).

The inventors have discovered that Ptb is hybrid and can accept a variety of acyl-CoA and enyl-CoA as acceptors, so that Ptb-Buk can be used to convert a variety of acyl-CoA and enyl-CoA into their respective counterparts. acid or enoate, and simultaneously generates ATP through phosphorylation at the substrate level.

Furthermore, in combination with aldehyde:ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase, the acid formed via the Ptb-Buk system can be further converted into its respective aldehyde, alcohol or glycol. AOR (EC 1.2.7.5) catalyzes the reaction of acid and reduced ferric redox protein (which can be produced, for example, by oxidation of CO or hydrogen) to form aldehydes and ferric redox protein. Alcohol dehydrogenase (EC 1.1.1.1 and EC 1.1.1.2) converts aldehydes and NAD(P)H into alcohols and NAD(P).

Therefore, the introduction of Ptb-Buk and/or AOR into heterologous materials provides a novel alternative route to form natural and unnatural products (such as acids, alkenes, ketones, aldehydes, alcohols, and glycols) in high yields, thus overcoming the current state of the art. Technical limitations.

The invention provides a genetically engineered bacterium, which includes exogenous phosphobutyltransferase (Ptb) and exogenous butyrate kinase (Buk) (Ptb-Buk). Generally speaking, Ptb-Buk acts on non-natural substrates, such as substrates other than butyl-CoA and/or butyl phosphate, and produces unnatural products, such as products other than butyl phosphate or butyrate. In certain embodiments, Ptb-Buk converts acetyl acetyl-CoA to acetyl acetate, 3-hydroxyisovaleryl-CoA to 3-hydroxyisovalerate, and 3-hydroxyisovalerate. Butyryl-CoA is converted to 3-hydroxybutyrate, or 2-hydroxyisobutyryl-CoA is converted to 2-hydroxyisobutyrate.

The bacteria may produce one or more of acids, alkenes, ketones, aldehydes, alcohols or glycols. More specifically, the bacteria can produce acetone or its precursor, isopropanol or its precursor, isobutylene or its precursor, 3-hydroxybutyrate or its precursor, 1,3-butanediol or its precursor. Precursor, 2-hydroxyisobutyrate or its precursor, adipic acid or its precursor, 1,3-hexanediol or its precursor, 3-methyl-2-butanol or its precursor, 2 - one or more of buten-1-ol or its precursor, isovalerate or its precursor, or isoamyl alcohol or its precursor. The bacteria generally do not produce butanol.

The bacteria may further include disruptive mutations in phosphotransacetylase (Pta) and acetate kinase (Ack). The bacterium may further comprise a disrupting mutation of the thioesterase enzyme. In another embodiment, the present invention provides a genetically engineered bacterium that includes exogenous Ptb-Buk and exogenous or endogenous aldehyde:ferredoxin oxidoreductase.

The present invention further provides a method of producing a product, which comprises culturing the bacterium of any of the above embodiments in the presence of a substrate. The product may be, for example, acetone or its precursor, isopropyl alcohol or its precursor, isobutylene or its precursor, 3-hydroxybutyrate or its precursor, 1,3-butanediol or its precursor, 2-hydroxyl Isobutyrate or its precursor, adipic acid or its precursor, 1,3-hexanediol or its precursor, 3-methyl-2-butanol or its precursor, 2-butene-1- Alcohol or its precursor, isovalerate or its precursor or isoamyl alcohol or its precursor. Typically, the substrate is a gaseous substrate, including, for example, one or more of CO, _CO2 , and _H2 . In one embodiment, the gas substrate is syngas. In another embodiment, the gas substrate is industrial waste gas.

Figure 1 is a diagram of the metabolic pathways that produce various products (including acetone, isopropyl alcohol, isobutylene, 3-hydroxybutyrate, 1,3-butanediol and 2-hydroxyisobutyrate) from acetyl-CoA. . Acetyl-CoA can be produced from any suitable substrate, such as a carbohydrate (eg, sugar) substrate or a gaseous substrate. In the present invention, acetyl-CoA is typically produced from a gaseous substrate. Bold arrows indicate steps that can be catalyzed by Ptb-Buk.

Figure 2 is a diagram showing the reaction naturally catalyzed by Ptb-Buk (ie, converting butyl-CoA to butyrate and producing an ATP).

Figure 3 is a graph comparing the activities of CoA-transferase, thioesterase and Ptb-Buk.

Figure 4 is a graph showing average acetone production in E. coli BL21(D3) modified with plastids including foreign genes. This data demonstrates the ability of Ptb-Buk to convert acetyl acetyl-CoA to acetyl acetate in E. coli in vivo.

Figure 5 is a graph showing the effect of inducing E. coli BL21 (DE3) carrying pACYC-ptb-buk and pCOLA-thlA-adc plasmids (expressing thiolase, Ptb-Buk and acetoacetate decarboxylase).

Figure 6 is a diagram of a pathway designed to produce acetone using Ptb-Buk while recycling the reducing equivalent produced in the production of (R)-3-hydroxybutyryl-CoA and produced from Ptb-Buk of ATP.

Figure 7 is a graph showing the role of aldehyde: ferredoxin oxidoreductase (AOR), ferredoxin and Adh in the production of 1,3-butanediol in C. autoethanogenum . More generally, AOR can be used to catalyze the conversion of acids to aldehydes, and Adh can be used to catalyze the conversion of aldehydes to alcohols/diols.

Figure 8 is a graph showing the stereospecificity of Ptb-Buk for the production of (R)-3-hydroxybutyrate and 2-hydroxyisobutyrate. The term "native" in Figure 8 refers to native thioesterases.

Figure 9 is a graph showing the production of isobutylene via the Ptb-Buk conversion of 3-hydroxyisovaleryl-CoA and 3-hydroxyisovalerate using alternative pathway 1.

Figure 10 is a graph showing the production of isobutylene via the Ptb-Buk conversion of 3-hydroxyisovaleryl-CoA and 3-hydroxyisovalerate using alternative route 2.

Figure 11 is a diagram showing the production of 1,3-butanediol via 3-butyraldehyde dehydrogenase (Bld).

Figure 12 is a graph showing isopropanol production in Clostridium autoethanogenogenum using the Ptb-Buk system relative to a control. ○pMTL85147-thlA-adc, ●pMTL85147-thlA-ptb-buk-adc.

Figures 13A-F are graphs showing the production of 3-hydroxybutyrate, acetate, ethanol and acetone using modular plastids in E. coli in the presence of different concentrations of the inducer IPTG (0, 50, 100 μM). Figure 13A: pACYC-ptb-buk, pCOLA-thlA-adc, pCDF-phaB. Figure 13B: pACYC-ptb-buk, pCOLA-thlA-adc, pCDF-phaB-bdh1. Figure 13C: pCOLA-thlA-adc, pCDF-phaB-bdh1. Figure 13D: pCOLA-thlA-adc. Figure 13E: pCDF-phaB-bdh1. Figure 13F: pCDF-phaB.

Figure 14 is a plasmid diagram of pMTL8225-budA::thlA-phaB.

Figure 15 shows the use of thiolase ( thlA ) and 3-hydroxybutyryl-CoA dehydrogenase ( phaB ) in four pure strains of Clostridium autoethanologenum (1, 4, 7, and 9) compared with the wild type (W). ) Gel image of PCR verification of gene replacement acetyl lactate synthase ( budA ) gene. All clones were positive as seen by the larger PCR fragment size compared to wild type.

Figure 16 is a diagram showing the fermentation profile of the batch fermentation of Clostridium autoethanologenum budA::thlAphaB strain and showing the formation of 3-hydroxybutyrate and 1,3-butanediol from gas.

Figure 17A is a graph showing the production of 1,3-BDO via thiolase, 3-hydroxybutyryl-CoA dehydrogenase (Bld) and butyraldehyde dehydrogenase. Figure 17B is a graph showing the effect of bld expression on growth.

Figure 18A is a graph showing the formation of 3-hydroxybutyrate and 1,3-butanediol from gas acceptors in Clostridium autoethanogenogenum pMTL8315-Pfdx-hbd1-thlA. Figure 18B is a graph showing the reduction of acetate to ethanol in the same culture.

Figure 19 is a graph showing the fermentation profile of the strain Clostridium ethanologenum pMTL8315-Pfdx-hbd1-thlA, which was displayed in a continuous culture (the medium was continuously replenished with an established dilution rate D when indicated) from a gas receiving The substance forms 3-hydroxybutyrate and 1,3-butanediol.

Figures 20A and 20B show the response to a series of acyl-CoA (acetyl-acetyl-CoA) in Clostridium autoethanologenum expressing the Ptb-Buk system from plastid pMTL82256-Ptb-Buk compared to the wild type (WT). -CoA, 3-hydroxybutyl-CoA and 2-hydroxyisobutyl-CoA) graph showing increased CoA hydrolysis activity.

Figures 21A and 21B show a C. autoethanogenum strain with an inactivated thioesterase (CT2640 = thioesterase 1, CT) compared to the acyl-CoA hydrolytic activity seen in C. autoethanogenum LZ1560 or LZ1561 1524 = thioesterase 2, CT1780 = thioesterase 3) activity reduction graph.

Figure 22 is a graph showing increased production of specific isopropanol in a C. autoethanogenum strain with cleaved thioesterase 3 CAETHG_1780 compared to wild-type C. autoethanogenum.

Figures 23A-D show the growth of wild-type Clostridium autoethanogenogenum and a strain with cleaved thioesterase 3 (CAETHG_1780) compared to wild-type Clostridium autoethanogenogenum (Figure 23A) and isopropyl alcohol (Figure 23B), Acetate (Figure 23C) and ethanol (Figure 23D) produced overview plots.

Figure 24 is a plasmid diagram of pMTL8225-pta-ack::ptb-buk.

Figure 25 is a gel image indicating the replacement of the pta and ack genes with the ptb and buk genes and the ermB cassette.

Figure 26 is a graph showing increased conversion of 3-hydroxybutyrate to 1,3-BDO by overexpression of the aldehyde:ferredoxin oxidoreductase gene aor1 .

Figure 27 shows the effects of thioesterase TesB, Pta-Ack and Ptb-Buk systems on acetyl acetyl-CoA, 3-hydroxybutyl-CoA and 2-hydroxyisobutyl-CoA compared to the control (BL21 strain). Plot of CoA hydrolysis activity. Ptb-Buk showed the highest activity, while Pta-Ack showed no activity.

Figures 28A and 28B illustrate the production of 3- by Ptb-Buk and (S)-specific (Hbd) (Figure 28A) or (R)-specific 3-hydroxybutyrate (PhaB) (Figure 28B) dehydrogenases. Diagram of hydroxybutyrate.

Figures 29A-D are graphs showing LC-MS/MS detection of 2-hydroxyisobutyric acid (2-HIB) and 2-hydroxybutyrate (2-HB). Figure 29A: 1mM 2-HIB standard. Figure 29B: 1mM 2-HB standard. Figure 29C: 0.5mM 2-HB and 2-HIB standards. Figure 29D: Duplicates of C. autoethanogenum samples showing production of 2-HIB and 2-HB from gases.

Figure 30 is a set of graphs showing GC-MS confirmation of the production of 2-hydroxyisobutyric acid (8.91 minutes). First panel: Clostridium autoethanogenogenum+pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB+pMTL82256-tesB. Second picture: Clostridium autoethanogenogenum+pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB+pMTL82256-ptb-buk (spectrum). Third panel: E. coli+pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB+pMTL82256-tesB. Figure 4: E. coli+pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB+pMTL82256-ptb-buk.

Figure 31 is a set of real-time PCR diagrams showing the genes of the 2-HIBA pathway in E. coli, Clostridium autoethanogenogenum LZ1561 (at 30°C) and Clostridium autoethanogenogenum LZ1561 (at 37°C) (from pta-ack The expression of thlA , hba , meaBhcmA , hcmB ) of the promoter and corresponding Wood-Ljungdahl operon promoter.

Figure 32 is a graph showing the production of various products in microorganisms including Ptb-Buk, AOR and Adh.

Figure 33 is a diagram showing coupling of firefly luciferase (Luc) to the Ptb-Buk system to characterize Ptb-Buk variants.

Figure 34 is a diagram of metabolic pathways that produce various products, including adipic acid. Bold arrows indicate steps that can be catalyzed by Ptb-Buk.

Figure 35 is a diagram of metabolic pathways that produce various products including 1,3-hexanediol, 2-methyl-2-butanol, and 2-buten-1-ol. Bold arrows indicate steps that can be catalyzed by Ptb-Buk.

Figure 36 is a diagram of metabolic pathways that produce various products, including isovalerate and isoamyl alcohol. Bold arrows indicate steps that can be catalyzed by Ptb-Buk.

Figure 37 is a graph showing 3-HB production in Clostridium autoethanogenans containing plasmid pMTL82256-thlA-ctfAB at various growth points.

Figure 38A is a graph showing the growth and ethanol and 2,3-butanediol production profile of the strain Clostridium autoethanologenum pta-ack::ptb-buk+pMTL85147-thlA-ptb-buk-adc. Figure 38B is a graph showing the isopropyl alcohol and 3-HB production profile of the strain Clostridium ethanologenum pta-ack::ptb-buk+pMTL85147-thlA-ptb-buk-adc.

Figure 39 shows a series of C ₄ , C ₆ , C ₈ , C ₁₀ , C ₁₂ , C14 generated by combining known chain elongation paths (Hbd, Crt, Bcd-EtfAB, Thl) with Ptb-Buk+AOR/Adc-Adh Diagram of the path flow of an alcohol, ketone, enol or diol.

Figure 40 is a graph showing the production of 3-HB and 1,3-BDO by C. autoethanogenans transformed with plasmid pMTL83159-phaB-thlA at various growth points.

Figure 41 is a graph showing the production of 3-HB and 1,3-BDO by C. autoethanogenans at various growth points including budA knockout and pMTL-HBD-ThlA.

Figure 42A is a graph showing the production of 3-HB in fermentation of C. ethanologenum pMTL83159-phaB-thlA+pMTL82256. Figure 42B is a graph showing the production of 3-HB in fermentation of Clostridium autoethanologenum pMTL83159-phaB-thlA+pMTL82256-buk-ptb.

Figure 43 shows 3-HB in Clostridium autoethanogenum strains showing thioesterase gene knockout (ΔCAETHG_1524), expression plasmid pMTL83156-phaB-thlA and with and without Ptb-Buk expression plasmid pMTL82256-buk-ptb. The resulting graph.

Figure 44 is a graph showing ethanol and 1,3-BDO production in a C. autoethanogenum strain expressing plasmid pMTL82256-hbd-thlA(2pf) with and without AOR overexpression plasmid pMTL83159-aor1(+aor1) .

Metabolic pathways in Figure 1 and Figures 34-36

Figures 1 and 34-36 are diagrams of metabolic pathways that produce the following various acids, alkenes, ketones, aldehydes, alcohols and diol products from substrates, including acetone, isopropanol, isobutylene, 3-hydroxybutyrate (R and S-isomer), 1,3-butanediol, 2-hydroxyisobutyrate, adipic acid, 1,3-hexanediol, 2-methyl-2-butanol, 2-butene -1-ol, isovalerate and isopentyl alcohol. Bold arrows indicate steps that can be catalyzed by Ptb-Buk. Exemplary enzymes are provided for each of the steps and enzymatic pathways detailed in Figure 1 and Figures 34-36. However, other suitable enzymes will be known to the person skilled in the art.

Step 1 demonstrates the conversion of acetyl-CoA to acetyl-acetyl-CoA. This step can be catalyzed by the enzyme thiolase, acetyl-CoA acetyltransferase (EC 2.3.1.9). The thiolase may be, for example, ThlA (WP_010966157.1) (SEQ ID NO: 1) from Clostridium acetobutylicum , PhaA (WP_013956452.1) (SEQ ID) from Cupriavidus necator NO: 2), BktB (WP_011615089.1) from Cupria hookworm (SEQ ID NO: 3) or AtoB (NP_416728.1) from Escherichia coli (SEQ ID NO: 4). Clostridium autoethanogenum, Clostridium ljungdahlii and Clostridium ragsdalei have no known natural activity for this step. E. coli is naturally active for this step.

Step 2 demonstrates the conversion of acetyl acetyl-CoA to acetyl acetate. This step can be catalyzed by CoA-transferase (i.e. acetyl-CoA: acetyl-acetyl-CoA transferase) (EC 2.8.3.9). The CoA-transferase may be, for example, CtfAB, a heterodimer including the subunits CtfA and CtfB from Clostridium beijerinckii (CtfA, WP_012059996.1) (SEQ ID NO: 5) (CtfB, WP_012059997 .1) (SEQ ID NO: 6). This step can also be catalyzed by thioesterases (EC 3.1.2.20). The thioesterase may be, for example, TesB (NP_414986.1) from E. coli (SEQ ID NO: 7). This step can also be catalyzed by putative thioesterases from, for example, Clostridium autoethanogenogenum or Clostridium yongdalae. Specifically, three putative thioesterases have been identified in C. autoethanologenum: (1) "Thioesterase 1"(AGY74947.1; annotated as palmitoyl-CoA hydrolase; SEQ ID NO: 8), (2) "Thioesterase 2"(AGY75747.1; labeled as 4-hydroxybenzyl-CoA thioesterase; SEQ ID NO: 9), and (3) "Thioesterase 3"(AGY75999.1; Annotated as putative thioesterase; SEQ ID NO: 10). Three putative thioesterases have also been identified in C. yongdahl: (1) "Thioesterase 1"(ADK15695.1; annotated as predicted acyl-CoA thioesterase 1; SEQ ID NO: 11), (2 ) "Thioesterase 2"(ADK16655.1; annotated as a predicted thioesterase; SEQ ID NO: 12), and (3) "Thioesterase 3"(ADK16959.1; annotated as a predicted thioesterase; SEQ ID NO: 13). This step can also be catalyzed by phosphate butyryl transferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7). Exemplary sources of phosphobutyltransferase and butyrate kinase are described elsewhere in this application. Natural enzymes in Clostridium autoethanogenogenum, Clostridium yongdahlii, and Clostridium larsonii (or E. coli), such as thioesterases from Clostridium autoethanogenogenum, can catalyze this step and produce certain amounts of downstream products. However, it may be necessary to introduce exogenous enzymes or overexpress endogenous enzymes to produce desired levels of downstream products. Additionally, in certain embodiments, it may be necessary to introduce disruptive mutations into endogenous enzymes (such as endogenous thioesterases) to reduce or eliminate competition with the introduced Ptb-Buk.

Step 3 shows the conversion of acetyl acetate to acetone. This step can be catalyzed by acetoacetate decarboxylase (EC 4.1.1.4). The acetoacetate decarboxylase can be, for example, Adc (WP_012059998.1) from Clostridium beijerinckii (SEQ ID NO: 14). This step can also be catalyzed by α-ketoisovalerate decarboxylase (EC 4.1.1.74). The alpha-ketoisovalerate decarboxylase may be, for example, KivD from Lactococcus lactis (SEQ ID NO: 15). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. Additionally, E. coli has no known natural activity for this step. Rarely, the conversion of acetoacetate to acetone can occur spontaneously. However, spontaneous transformation is highly inefficient and unlikely to produce desired levels of downstream products.

Step 4 shows the conversion of acetone to isopropyl alcohol. This step can be catalyzed by primary:secondary alcohol dehydrogenase (EC 1.1.1.2). Primary: Secondary alcohol dehydrogenase can be, for example, SecAdh (AGY74782.1) (SEQ ID NO: 16) from Clostridium autoethanogenogenum, SecAdh (ADK15544.1) (SEQ ID NO: 17) from Clostridium yungdalae ), SecAdh from Clostridium lascheri (WP_013239134.1) (SEQ ID NO: 18) or SecAdh from Clostridium beijerinckii (WP_026889046.1) (SEQ ID NO: 19). This step can also be catalyzed by a primary:secondary alcohol dehydrogenase (EC 1.1.1.80), such as SecAdh (3FSR_A) from Thermoanaerobacter brokii (SEQ ID NO: 20). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active in this step (Köpke, Appl Environ Microbiol , 80: 3394-3403, 2014). However, E. coli has no known natural activity for this step. Down-regulating or genetically deleting the gene content of this enzyme in Clostridium autoethanogenogenum, Clostridium yondahlii or Clostridium larsonii causes the production and accumulation of acetone instead of isopropyl alcohol (WO 2015/085015).

Step 5 shows the conversion of acetone to 3-hydroxyisovalerate. This step can be catalyzed by a hydroxyisovalerate synthase, such as hydroxymethylglutaryl-CoA synthetase (HMG-CoA synthetase) from Mus musculus (EC 2.3.3.10) (SEQ ID NO: 21) (US 2012/0110001). Hydroxymethylglutaryl-CoA synthetase can be engineered to improve activity. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 6 shows the conversion of 3-hydroxyisovalerate to isobutylene/isobutene. This step can be catalyzed by hydroxyisovalerate phosphorylase/decarboxylase. This step can also be catalyzed by mevalonate diphosphate decarboxylase (hydroxyisovalerate decarboxylase) (EC 4.1.1.33). The mevalonate diphosphate decarboxylase may be, for example, Mdd (CAA96324.1) (SEQ ID NO: 22) from Saccharomyces cerevisiae or Mdd (WP_011178157.1) from Picrophilus torridus ) (SEQ ID NO: 23) (US 2011/0165644; van Leeuwen, Appl Microbiol Biotechnol , 93: 1377-1387, 2012). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 7 shows the conversion of acetone to 3-hydroxyisovaleryl-CoA. This step can be catalyzed by 3-hydroxyisovaleryl-CoA synthetase. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 8 demonstrates the conversion of 3-hydroxyisovaleryl-CoA to 3-hydroxyisovalerate. This step can be catalyzed by CoA-transferase (i.e. acetyl-CoA: acetyl-acetyl-CoA transferase) (EC 2.8.3.9). The CoA-transferase can be, for example, CtfAB, a heterodimer including the subunits CtfA and CtfB from Clostridium beijerinckii (CtfA, WP_012059996.1) (SEQ ID NO: 5) (CtfB, WP_012059997.1) ( SEQ ID NO: 6). This step can also be catalyzed by thioesterases (EC 3.1.2.20). The thioesterase may be, for example, TesB (NP_414986.1) from E. coli (SEQ ID NO: 7). This step can also be catalyzed by putative thioesterases from, for example, Clostridium autoethanogenogenum or Clostridium yongdalae. Specifically, three putative thioesterases have been identified in C. autoethanologenum: (1) "Thioesterase 1" (AGY74947.1; annotated as palmitoyl-CoA hydrolase; SEQ ID NO: 8), (2) "Thioesterase 2" (AGY75747.1; labeled as 4-hydroxybenzyl-CoA thioesterase; SEQ ID NO: 9), and (3) "Thioesterase 3" (AGY75999.1 ; Annotated as putative thioesterase; SEQ ID NO: 10). Three putative thioesterases have also been identified in C. yongdahl: (1) "Thioesterase 1" (ADK15695.1; annotated as predicted acyl-CoA thioesterase 1; SEQ ID NO: 11), (2 ) "Thioesterase 2" (ADK16655.1; annotated as a predicted thioesterase; SEQ ID NO: 12), and (3) "Thioesterase 3" (ADK16959.1; annotated as a predicted thioesterase; SEQ ID NO: 13). This step can also be catalyzed by phosphate butyryl transferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7). Exemplary sources of phosphobutyltransferase and butyrate kinase are described elsewhere in this application. Natural enzymes in Clostridium autoethanogenogenum, Clostridium yongdahlii, and Clostridium larsonii (or E. coli), such as thioesterases from Clostridium autoethanogenogenum, can catalyze this step and produce certain amounts of downstream products. However, it may be necessary to introduce exogenous enzymes or overexpress endogenous enzymes to produce desired levels of downstream products. Additionally, in certain embodiments, it may be necessary to introduce disruptive mutations into endogenous enzymes (such as endogenous thioesterases) to reduce or eliminate competition with the introduced Ptb-Buk.

Step 9 shows the conversion of acetyl-CoA to 3-methyl-2-pentoxyvalerate. This step encompasses multiple enzymatic reactions involved in the isoleucine biosynthetic pathway, which occurs naturally in a variety of bacteria, including Clostridium autoethanologenum, Clostridium yongdahlii, and Clostridium larsonii (and E. coli). Enzymes involved in the conversion of acetyl-CoA to 3-methyl-2-pentoxyvalerate may include methylmalate synthase (EC 2.3.1.182), 3-isopropylmalate dehydratase ( EC 4.2.1.35), 3-isopropylmalate dehydrogenase (EC 1.1.1.85), acetyl lactate synthase (EC 2.2.1.6), ketol acid reductoisomerase (EC 1.1.1.86) and/ or dihydroxyacid dehydratase (EC 4.2.1.9). The methylmalate synthase may be, for example, CimA (AGY76958.1) (SEQ ID NO: 24) from Clostridium autoethanogenogenum or CimA (NP_248395.1) (SEQ ID NO. :25). The 3-isopropylmalate dehydratase can be, for example, LeuCD (WP_023162955.1, LeuC; AGY77204.1, LeuD) from Clostridium autoethanogenogenum (SEQ ID NO: 26 and 27, respectively) or from E. coli LeuCD (NP_414614.1, LeuC; NP_414613.1, LeuD) (SEQ ID NO: 28 and 29, respectively). The 3-isopropylmalate dehydrogenase may be, for example, LeuB from Clostridium autoethanogenogenum (WP_023162957.1) (SEQ ID NO: 30) or LeuB from E. coli (NP_414615.4) (SEQ ID NO: 31 ). The acetyl lactate synthase can be, for example, IlvBN (AGY74359.1, IlvB; AGY74635.1, IlvB; AGY74360.1, IlvN) from Clostridium autoethanogenogens (SEQ ID NOs: 32, 33 and 34, respectively) or from IlvBN of E. coli (NP_418127.1, IlvB; NP_418126.1, IlvN) (SEQ ID NO: 35 and 36, respectively). The ketol acid reductoisomerase may be, for example, IlvC (WP_013238693.1) from Clostridium autoethanogenogenes (SEQ ID NO:37) or IlvC (NP_418222.1) (SEQ ID NO:38) from E. coli. The dihydroxyacid dehydratase may be, for example, IlvD (WP_013238694.1) from Clostridium autoethanogenogenes (SEQ ID NO: 39) or IlvD (YP_026248.1) from E. coli (SEQ ID NO: 40). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active for this step.

Step 10 shows the conversion of 3-methyl-2-pentoxyvalerate to 2-methylbutyl-CoA. This step is catalyzed by ketoisovalerate oxidoreductase (EC 1.2.7.7). The ketoisovalerate oxidoreductase may be, for example, VorABCD from Methanothermobacter thermautotrophicus (WP_010876344.1, VorA; WP_010876343.1, VorB; WP_010876342.1, VorC; WP_010876341.1, VorD) ( SEQ ID NO: 41-44, respectively) or VorABCD from Pyrococcus furiosus (WP_011012106.1, VorA; WP_011012105.1, VorB; WP_011012108.1, VorC; WP_011012107.1, VorD) (respectively SEQ ID NO: 45-48). VorABCD is a 4-unit enzyme. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 11 demonstrates the conversion of 2-methylbutyl-CoA to 2-methylcrotonyl-CoA. This step is catalyzed by 2-methylbutyl-CoA dehydrogenase (EC 1.3.99.12). The 2-methylbutyl-CoA dehydrogenase may be, for example, AcdH (AAD44196.1 or BAB69160.1) (SEQ ID NO: 49) from Streptomyces avermitilis or from Streptomyces coelicolor AcdH (AAD44195.1) (SEQ ID NO: 50). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 12 demonstrates the conversion of 2-methylcrotonyl-CoA to 3-hydroxyisovaleryl-CoA. This step can be catalyzed by crotonase/3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55). The crotonase/3-hydroxybutyryl-CoA dehydratase may be, for example, Crt (ABR34202.1) from Clostridium beijerinckii (SEQ ID NO: 51), Crt (NP_349318.1) from Clostridium acetobutylicum (NP_349318.1) SEQ ID NO: 52) or LiuC (WP_011553770.1) from Myxococcus xanthus . This step can also be catalyzed by crotonyl-CoA carboxylase-reductase (EC 1.3.1.86). The crotonyl-CoA carboxylase-reductase may be, for example, Ccr (NP_971211.1) from Treponema denticola (SEQ ID NO: 53). This step can also be catalyzed by crotonyl-CoA reductase (EC 1.3.1.44). The crotonyl-CoA reductase may be, for example, Ter (AAW66853.1) from Euglena gracilis (SEQ ID NO: 54). This step can also be catalyzed by 3-hydroxypropyl-CoA dehydratase (EC 4.2.1.116). The 3-hydroxypropyl-CoA dehydratase may be, for example, Msed_2001 (WP_012021928.1) from Metallosphaera sedula . This step can also be catalyzed by enyl-CoA hydratase. This enyl-CoA hydratase (4.2.1.17) can be, for example, YngF (WP_000787371.1) from Bacillus anthracis . Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 13 demonstrates the conversion of acetyl acetyl-CoA to 3-hydroxybutyl-CoA. This step is catalyzed by 3-hydroxybutyl-CoA dehydrogenase (EC 1.1.1.157). The 3-hydroxybutyryl-CoA dehydrogenase may be, for example, Hbd (WP_011967675.1) from Clostridium beijerinckii (SEQ ID NO: 55), Hbd (NP_349314.1) from Clostridium acetobutylicum (SEQ ID NO: 56) or Hbd1 (WP_011989027.1) from Clostridium kluyveri (SEQ ID NO: 57). This step can also be catalyzed by acetyl-CoA reductase (EC 4.2.1.36). The acetoacetyl-CoA reductase can be, for example, PhaB (WP_010810131.1) (SEQ ID NO: 58) from Cupriaphila ancylostoma. This step can also be catalyzed by acetyl-CoA hydratase (EC 4.2.1.119). Notably, PhaB is R-specific and Hbd is S-specific. In addition, Hbd1 derived from Clostridium cruzi is NADPH-dependent, and Hbd derived from Clostridium acetobutylicum and Clostridium beijerinckii is NADH-dependent. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 14 demonstrates the conversion of 3-hydroxybutyl-CoA to 3-hydroxybutyrate. This step can be catalyzed by thioesterase (EC 3.1.2.20). The thioesterase may be, for example, TesB (NP_414986.1) from E. coli (SEQ ID NO: 7). This step can also be catalyzed by putative thioesterases from, for example, Clostridium autoethanogenogenum or Clostridium yongdalae. Specifically, three putative thioesterases have been identified in C. autoethanologenum: (1) "Thioesterase 1" (AGY74947.1; annotated as palmitoyl-CoA hydrolase; SEQ ID NO: 8), (2) "Thioesterase 2" (AGY75747.1; labeled as 4-hydroxybenzyl-CoA thioesterase; SEQ ID NO: 9), and (3) "Thioesterase 3" (AGY75999.1 ; Annotated as putative thioesterase; SEQ ID NO: 10). Three putative thioesterases have also been identified in C. yongdahl: (1) "Thioesterase 1" (ADK15695.1; annotated as predicted acyl-CoA thioesterase 1; SEQ ID NO: 11), (2 ) "Thioesterase 2" (ADK16655.1; annotated as a predicted thioesterase; SEQ ID NO: 12), and (3) "Thioesterase 3" (ADK16959.1; annotated as a predicted thioesterase; SEQ ID NO: 13). This step can also be catalyzed by phosphate butyryl transferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7). Exemplary sources of phosphobutyltransferase and butyrate kinase are described elsewhere in this application. Natural enzymes in Clostridium autoethanogenogenum, Clostridium yongdahlii, and Clostridium larsonii (or E. coli), such as thioesterases from Clostridium autoethanogenogenum, can catalyze this step and produce certain amounts of downstream products. However, it may be necessary to introduce exogenous enzymes or overexpress endogenous enzymes to produce desired levels of downstream products. Additionally, in certain embodiments, it may be necessary to introduce disruptive mutations into endogenous enzymes (such as endogenous thioesterases) to reduce or eliminate competition with the introduced Ptb-Buk.

Step 15 demonstrates the conversion of 3-hydroxybutyrate to acetyl acetate. This step is catalyzed by 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30). The 3-hydroxybutyrate dehydrogenase may be, for example, Bdh1 from Ralstonia pickettii (BAE72684.1) (SEQ ID NO: 60) or Bdh2 from Ralstonia pickettii (BAE72685 .1) (SEQ ID NO: 61). The reverse reaction, the conversion of acetoacetate to 3-hydroxybutyrate, can be catalyzed by different 3-hydroxybutyrate dehydrogenases (EC 1.1.1.30). For example, the conversion of acetoacetate to 3-hydroxybutyrate can be catalyzed by Bdh (AGY75962) (SEQ ID NO: 62) from Clostridium autoethanogenogenum. Clostridium yongdahlii and Clostridium larsonii likely contain enzymes with similar activity. E. coli has no known natural activity for this step.

Step 16 demonstrates the conversion of 3-hydroxybutyrate to 3-hydroxybutyraldehyde. This step is catalyzed by aldehyde:ferredoxin oxidoreductase (EC 1.2.7.5). The aldehyde:ferredoxin oxidoreductase (AOR) may be, for example, the AOR from Clostridium autoethanogenogenum (WP_013238665.1; WP_013238675.1) (SEQ ID NO: 63 and 64, respectively) or from Clostridium yungdalae AOR (ADK15073.1; ADK15083.1) (SEQ ID NO: 65 and 66, respectively). In other embodiments, the aldehyde:ferredoxin oxidoreductase may be from or may be derived from, for example, any of the following sources, the sequences of which are publicly available:

AOR catalyzes the reaction of acid and reduced ferric redox protein to form aldehyde and oxidized ferric redox protein. In acetogens, this reaction can be combined with either oxidized CO (via CO dehydrogenase, EC 1.2.7.4) or hydrogen (via ferredoxin-dependent hydrogenase, EC 1.12.7.2) to produce reduced ferredoxin. or 1.12.1.4) coupling (Köpke, "Curr Opin Biotechnol", 22: 320-325, 2011; Köpke, " Proceedings of the National Academy of Sciences ( PNAS USA )", 107: 13087-13092, 2010) . Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active for this step. However, overexpression of endogenous AOR or introduction of exogenous AOR may be required to increase product yield in C. autoethanologenum, C. yongdahlii, or C. larsonii. Alternatively, exogenous AORs can be introduced into microorganisms that do not naturally include AORs (eg, E. coli). In particular, co-expression of Ptb-Buk with AOR (and optionally Adh) may enable such microorganisms to produce novel unnatural products.

Step 17 demonstrates the conversion of 3-hydroxybutyraldehyde to 1,3-butanediol. This step can be catalyzed by alcohol dehydrogenase (EC 1.1.1.1. or 1.1.1.2). Alcohol dehydrogenase converts aldehydes and NAD(P)H into alcohols and NAD(P). The alcohol dehydrogenase may be, for example, Adh from Clostridium autoethanogenogenum (AGY76060.1) (SEQ ID NO: 67), Clostridium yongdahl (ADK17019.1) (SEQ ID NO: 68), or Clostridium lashanii; BdhB from Clostridium acetobutylicum (NP_349891.1) (SEQ ID NO: 69); Bdh from Clostridium beijerinckii (WP_041897187.1) (SEQ ID NO: 70); Bdh1 from Clostridium yongdahl (YP_003780648 .1) (SEQ ID NO: 71); Bdh1 (AGY76060.1) from Clostridium autoethanogenogenum (SEQ ID NO: 72); Bdh2 (YP_003782121.1) (SEQ ID NO: 73) from Clostridium yungdalae ), Bdh2 (AGY74784.1) (SEQ ID NO: 74) from Clostridium autoethanogenogenum, AdhE1 (NP_149325.1) (SEQ ID NO: 75) from Clostridium acetobutylicum, Clostridium acetobutylicum AdhE2 (NP_149199.1) (SEQ ID NO: 76), AdhE (WP_041893626.1) (SEQ ID NO: 77) from Clostridium beijerinckii, AdhE1 (WP_023163372.1) from Clostridium autoethanogenogenum (SEQ ID NO: 78) or AdhE2 (WP_023163373.1) from Clostridium autoethanogenogenum (SEQ ID NO: 79). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active for this step. However, overexpression of endogenous alcohol dehydrogenases or introduction of exogenous alcohol dehydrogenases may be required to increase product yield in C. autoethanologenum, C. jungdahl, or C. larsonii. E. coli is most likely not naturally active for this step.

Step 18 demonstrates the conversion of 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde. This step is catalyzed by butyraldehyde dehydrogenase (EC 1.2.1.57). The butyraldehyde dehydrogenase may be, for example, Bld (AAP42563.1) from Clostridium saccharoperbutylacetonicum (SEQ ID NO: 80). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 19 demonstrates the conversion of 3-hydroxybutyl-CoA to 2-hydroxyisobutyl-CoA. This step can be catalyzed by 2-hydroxyisobutyl-CoA mutase (EC 5.4.99.-). The 2-hydroxyisobutyl-CoA mutase may be, for example, HcmAB (AFK77668.1, major unit; AFK77665.1, minor unit) from Aquincola tertiaricarbonis (SEQ ID NO: 81 and AFK77665.1, respectively) 82) or HcmAB (WP_013074530.1, major subunit; WP_013074531.1, minor subunit) from Kyrpidia tusciae (SEQ ID NO: 83 and 84, respectively). Although the chaperone MeaB (AFK77667.1, three-carbon Proteobacteria; WP_013074529.1, Kyrpidia tusciae ) (SEQ ID NOs: 85 and 86, respectively) is not required for HcmAB function, MeaB has been described to modify HcmAB by reactivating HcmAB. Activity (Yaueva, J Biol Chem , 287: 15502-15511, 2012). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 20 demonstrates the conversion of 2-hydroxyisobutyryl-CoA to 2-hydroxyisobutyrate. This step can be catalyzed by phosphate butyltransferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7). Exemplary sources of phosphobutyltransferase and butyrate kinase are described elsewhere in this application. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 21 shows the conversion of acetyl-CoA to succinyl-CoA. This step encompasses multiple enzymatic reactions involved in the TCA-reducing pathway that occurs naturally in a variety of bacteria, including Clostridium autoethanologenum, Clostridium yongdalae, and Clostridium larsonii (and E. coli) (Brown, " Biotechnol Biofuels , 7: 40, 2014; U.S. Patent 9,297,026). Enzymes involved in the conversion of acetyl-CoA to succinyl-CoA may include pyruvate: ferredoxin oxidoreductase (PFOR) (EC 1.2.7.1), pyruvate carboxylase (PYC) (EC 6.4 .1.1), malic enzyme/malate dehydrogenase (EC 1.1.1.38, EC 1.1.1.40), pyruvate phosphate dikinase (PPDK) (EC: 2.7.9.1), PEP carboxykinase (PCK) (EC 4.1 .1.49), fumarate hydratase/fumarase (EC 4.2.1.2), fumarate reductase (EC 1.3.5.1)/succinate dehydrogenase (EC 1.3.5.4 ) and succinyl-CoA synthetase (EC 6.2.1.5). The pyruvate:ferredoxin oxidoreductase can be from, for example, Clostridium autoethanogenans (AGY75153, AGY77232) or E. coli (NP_415896). Pyruvate carboxylase may, for example, be from Clostridium autoethanogenogenum (AGY75817). Malic enzyme/malate dehydrogenase may be derived, for example, from Clostridium autoethanologenum (AGY76687) or E. coli (NP_416714, NP_417703). Pyruvate phosphodikinase (PPDK) can be derived, for example, from Clostridium autoethanogenogenum (AGY76274, AGY77114). PEP carboxykinase (PCK) can be derived, for example, from Clostridium autoethanogenogenum (AGY76928) or E. coli (NP_417862). The fumarate hydratase/fumarase may for example be from Clostridium autoethanogenans (AGY76121, AGY76122) or E. coli (NP_416128, NP_416129, NP_418546). Fumarate reductase/succinate dehydrogenase may be derived, for example, from Clostridium autoethanogenans (AGY74573, AGY74575, AGY75257, AGY77166) or E. coli (NP_415249, NP_415250, NP_415251, NP_415252, NP_418575, NP_418576, NP_418577, NP_418578 ). Succinyl-CoA synthetase may, for example, be from E. coli (NP_415256, NP_415257).

Step 22 shows the conversion of acetyl-CoA and succinyl-CoA to 3-side oxy-adipyl-CoA. This step can be catalyzed by β-ketoadipyl-CoA thiolase (EC 2.3.1.16). The ketoisovalerate oxidoreductase may be, for example, PaaJ from E. coli (WP_001206190.1). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 23 demonstrates the conversion of 3-side oxy-adipyl-CoA to 3-hydroxyadipyl-CoA. This step can be catalyzed by 3-hydroxybutyl-CoA dehydrogenase (EC 1.1.1.157) or acetyl-CoA hydratase (EC 4.2.1.119). The 3-hydroxybutyryl-CoA dehydrogenase or acetoacetyl-CoA hydratase may be, for example, Hbd from Clostridium beijerinckii (WP_011967675.1) (SEQ ID NO: 55), Hbd from Clostridium acetobutylicum (NP_349314.1) (SEQ ID NO: 56), Hbd1 (WP_011989027.1) (SEQ ID NO: 57) from Clostridium cruzi or PaaH1 (WP_010814882.1) from Cupriaphila hookworm. Notably, PhaB is R-specific and Hbd is S-specific. In addition, Hbd1 derived from Clostridium cruzi is NADPH-dependent and Hbd derived from Clostridium acetobutylicum and Clostridium beijerinckii is NADH-dependent. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 24 shows the conversion of hydroxyadipate-CoA to 2,3-dehydroadipate-CoA. This step can be catalyzed by enyl-CoA hydratase (EC: 4.2.1.17) or enyl-CoA reductase (EC: 1.3.1.38). The enyl-CoA hydratase or enyl-CoA reductase may be, for example, Crt (NP_349318.1) from Clostridium acetobutylicum or PhaJ (032472) from Aeromonas caviae (Seq. ID No.52). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 25 demonstrates the conversion of 2,3-dehydrohexanediyl-CoA to hexadecyl-CoA. This step can be catalyzed by trans-2-enyl-CoA reductase (EC 1.3.8.1, EC 1.3.1.86, EC 1.3.1.85, EC 1.3.1.44). Trans-2-enyl-CoA reductase can be, for example, Bcd (NP_349317.1) from Clostridium acetobutylicum, which forms a complex with the electron flavoprotein EtfAB (NP_349315, NP_349316); Ccr (AAA92890) from Streptomyces collinus; Ccr (YP_354044.1) from Rhodobacter sphaeroides; Ter (NP_971211.1) from Treponema denticola or Ter (AY741582.1) from Euglena gracilis . Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 26 shows the conversion of adipyl-CoA to adipic acid. This step can be catalyzed by phosphate butyltransferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7). Exemplary sources of phosphobutyltransferase and butyrate kinase are described elsewhere in this application. Natural enzymes in Clostridium autoethanogenogenum, Clostridium yongdahlii, and Clostridium larsonii (or E. coli), such as thioesterases from Clostridium autoethanogenogenum, can catalyze this step and produce certain amounts of downstream products. However, it may be necessary to introduce exogenous enzymes or overexpress endogenous enzymes to produce desired levels of downstream products. Additionally, in certain embodiments, it may be necessary to introduce disruptive mutations into endogenous enzymes (such as endogenous thioesterases) to reduce or eliminate competition with the introduced Ptb-Buk.

Step 27 demonstrates the conversion of 3-hydroxybutyl-CoA to crotonyl-CoA. This step can be catalyzed by crotonyl-CoA hydratase (crotonase) (EC 4.2.1.17) or crotonyl-CoA reductase (EC 1.3.1.38). The crotonyl-CoA hydratase (crotonase) or crotonyl-CoA reductase may be, for example, Crt (NP_349318.1) (SEQ ID NO: 52) from Clostridium acetobutylicum or from Aeromonas caviae of PhaJ(O32472). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 28 demonstrates the conversion of crotonyl-CoA to crotonate. This step can be catalyzed by phosphate butyltransferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7). Exemplary sources of phosphobutyltransferase and butyrate kinase are described elsewhere in this application. Natural enzymes in Clostridium autoethanogenogenum, Clostridium yongdahlii, and Clostridium larsonii (or E. coli), such as thioesterases from Clostridium autoethanogenogenum, can catalyze this step and produce certain amounts of downstream products. However, it may be necessary to introduce exogenous enzymes or overexpress endogenous enzymes to produce desired levels of downstream products. Additionally, in certain embodiments, it may be necessary to introduce disruptive mutations into endogenous enzymes (such as endogenous thioesterases) to reduce or eliminate competition with the introduced Ptb-Buk.

Step 29 shows the conversion of crotonate to crotonaldehyde. This step is catalyzed by aldehyde:ferredoxin oxidoreductase (EC 1.2.7.5). Aldehydes: Exemplary sources of ferredoxin oxidoreductases are described elsewhere in this application. AOR catalyzes the reaction of acid and reduced ferric redox protein to form aldehyde and oxidized ferric redox protein. In acetogens, this reaction can be combined with either oxidized CO (via CO dehydrogenase, EC 1.2.7.4) or hydrogen (via ferredoxin-dependent hydrogenase, EC 1.12.7.2) to produce reduced ferredoxin. or 1.12.1.4) coupling (Köpke, "New Opinions in Biotechnology", 22: 320-325, 2011; Köpke, " Proceedings of the National Academy of Sciences , 107: 13087-13092, 2010"). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active for this step. However, overexpression of endogenous AOR or introduction of exogenous AOR may be required to increase product yield in C. autoethanologenum, C. yongdahlii, or C. larsonii. The AOR of Pyrococcus furiosus has demonstrated activity in converting crotonaldehyde and crotonate (Loes, J Bacteriol , 187: 7056-7061, 2005). Alternatively, exogenous AORs can be introduced into microorganisms that do not naturally include AORs (eg, E. coli). In particular, co-expression of Ptb-Buk with AOR (and optionally Adh) may enable such microorganisms to produce novel unnatural products.

Step 30 shows the conversion of crotonaldehyde to 2-buten-1-ol. This step can be catalyzed by alcohol dehydrogenase (EC 1.1.1.1. or 1.1.1.2). Alcohol dehydrogenase converts aldehydes and NAD(P)H into alcohols and NAD(P). The alcohol dehydrogenase may be, for example, Adh from Clostridium autoethanogenogenum (AGY76060.1) (SEQ ID NO: 67), Clostridium yongdahl (ADK17019.1) (SEQ ID NO: 68), or Clostridium lashanii; BdhB from Clostridium acetobutylicum (NP_349891.1) (SEQ ID NO: 69); Bdh from Clostridium beijerinckii (WP_041897187.1) (SEQ ID NO: 70); Bdh1 from Clostridium yongdahl (YP_003780648 .1) (SEQ ID NO: 71); Bdh1 (AGY76060.1) from Clostridium autoethanogenogenum (SEQ ID NO: 72); Bdh2 (YP_003782121.1) (SEQ ID NO: 73) from Clostridium yungdalae ), Bdh2 (AGY74784.1) (SEQ ID NO: 74) from Clostridium autoethanogenogenum, AdhE1 (NP_149325.1) (SEQ ID NO: 75) from Clostridium acetobutylicum, Clostridium acetobutylicum AdhE2 (NP_149199.1) (SEQ ID NO: 76), AdhE (WP_041893626.1) (SEQ ID NO: 77) from Clostridium beijerinckii, AdhE1 (WP_023163372.1) from Clostridium autoethanogenogenum (SEQ ID NO: 78) or AdhE2 (WP_023163373.1) from Clostridium autoethanogenogenum (SEQ ID NO: 79). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active for this step. However, overexpression of endogenous alcohol dehydrogenases or introduction of exogenous alcohol dehydrogenases may be required to increase product yield in C. autoethanologenum, C. jungdahl, or C. larsonii. E. coli is most likely not naturally active for this step.

Step 31 shows the conversion of crotonyl-CoA to butyl-CoA. This step can be catalyzed by butyl-CoA dehydrogenase or trans-2-enyl-CoA reductase (EC 1.3.8.1, EC 1.3.1.86, EC 1.3.1.85, EC 1.3.1.44). Butyl-CoA dehydrogenase or trans-2-enyl-CoA reductase can be, for example, Bcd from Clostridium acetobutylicum (NP_349317.1), which forms a complex with the electron flavoprotein EtfAB (NP_349315, NP_349316) ; Ccr from Streptomyces capilans (AAA92890); Ccr from Rhodobacter sphaeroides (YP_354044.1); Ter (NP_971211.1) from Treponema denticola; or Ter (AY741582.1) from Euglena gracilis . Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 32 shows the conversion of butyl-CoA to acetyl-butyl-CoA. This step can be catalyzed by thiolase or acyl-CoA acetyltransferase (EC 2.3.1.9). The thiolase may be, for example, ThlA from Clostridium acetobutylicum (WP_010966157.1) (SEQ ID NO: 1), ThlA1 from Clostridium kludii (EDK35681), ThlA2 from Clostridium kludii (EDK35682), ThlA3 (EDK35683) from Clostridium cruzi, PhaA (WP_013956452.1) from Cupria ancylostoma (SEQ ID NO: 2), BktB (WP_011615089.1) (SEQ ID NO: 3) from Cupria ancytosoma or AtoB (NP_416728.1) from E. coli (SEQ ID NO: 4). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli is naturally active for this step.

Step 33 shows the conversion of acetylbutyryl-CoA to acetylbutyrate. This step can be catalyzed by phosphate butyltransferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7). Exemplary sources of phosphobutyltransferase and butyrate kinase are described elsewhere in this application. Natural enzymes in Clostridium autoethanogenogenum, Clostridium yongdahlii, and Clostridium larsonii (or E. coli), such as thioesterases from Clostridium autoethanogenogenum, can catalyze this step and produce certain amounts of downstream products. However, it may be necessary to introduce exogenous enzymes or overexpress endogenous enzymes to produce desired levels of downstream products. Additionally, in certain embodiments, it may be necessary to introduce disruptive mutations into endogenous enzymes (such as endogenous thioesterases) to reduce or eliminate competition with the introduced Ptb-Buk.

Step 34 shows the conversion of acetylbutyrate to acetylacetone. This step can be catalyzed by acetoacetate decarboxylase (EC 4.1.1.4). The acetoacetate decarboxylase can be, for example, Adc (WP_012059998.1) from Clostridium beijerinckii (SEQ ID NO: 14). This step can also be catalyzed by α-ketoisovalerate decarboxylase (EC 4.1.1.74). The alpha-ketoisovalerate decarboxylase may be, for example, KivD from Lactococcus reuteri (SEQ ID NO: 15). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. Furthermore, E. coli has no known natural activity for this step. Rarely, the conversion of acetoacetate to acetone can occur spontaneously. However, spontaneous transformation is highly inefficient and unlikely to produce desired levels of downstream products.

Step 35 shows the conversion of acetylacetone to 3-methyl-2-butanol. This step can be catalyzed by primary:secondary alcohol dehydrogenase (EC 1.1.1.2). Primary: Secondary alcohol dehydrogenase can be, for example, SecAdh (AGY74782.1) (SEQ ID NO: 16) from Clostridium autoethanogenogenum, SecAdh (ADK15544.1) (SEQ ID NO: 17) from Clostridium yungdalae ), SecAdh from Clostridium lascheri (WP_013239134.1) (SEQ ID NO: 18) or SecAdh from Clostridium beijerinckii (WP_026889046.1) (SEQ ID NO: 19). This step can also be catalyzed by a primary:secondary alcohol dehydrogenase (EC 1.1.1.80), such as SecAdh (3FSR_A) from Thermoanaerobacter brucei (SEQ ID NO: 20). Clostridium autoethanologenum, Clostridium yongdahl and Clostridium larsonii are naturally active in this step (Köpke, Appl Environ Microbiol , 80: 3394-3403, 2014). However, E. coli has no known natural activity for this step. Down-regulating or genetically deleting the gene content of this enzyme in Clostridium autoethanogenogenum, Clostridium yongdalae or Clostridium larsonii causes the production and accumulation of acetylacetone instead of 3-methyl-2-butanol (WO 2015/085015 ).

Step 36 shows the conversion of acetylbutyl-CoA to 3-hydroxyhexyl-CoA. This step can be catalyzed by 3-hydroxybutyl-CoA dehydrogenase (EC 1.1.1.157) or acetyl-CoA hydratase (EC 4.2.1.119). 3-Hydroxybutyl-CoA dehydrogenase or acetoacetyl-CoA can be, for example, Hbd from Clostridium beijerinckii (WP_011967675.1) (SEQ ID NO: 55), Hbd from Clostridium acetobutylicum (NP_349314 .1) (SEQ ID NO: 56), Hbd1 from Clostridium cruzi (WP_011989027.1) (SEQ ID NO: 57), Hbd2 from Clostridium cruzi (EDK34807) or PaaH1 ( WP_010814882.1). Notably, PhaB is R-specific and Hbd is S-specific. In addition, Hbd1 derived from Clostridium cruzi is NADPH-dependent and Hbd derived from Clostridium acetobutylicum and Clostridium beijerinckii is NADH-dependent. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 37 shows the conversion of 3-hydroxybutyl-CoA to 3-hydroxycaproate. This step can be catalyzed by phosphate butyltransferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7). Exemplary sources of phosphobutyltransferase and butyrate kinase are described elsewhere in this application. Natural enzymes in Clostridium autoethanogenogenum, Clostridium yongdahlii, and Clostridium larsonii (or E. coli), such as thioesterases from Clostridium autoethanogenogenum, can catalyze this step and produce certain amounts of downstream products. However, it may be necessary to introduce exogenous enzymes or overexpress endogenous enzymes to produce desired levels of downstream products. Additionally, in certain embodiments, it may be necessary to introduce disruptive mutations into endogenous enzymes (such as endogenous thioesterases) to reduce or eliminate competition with the introduced Ptb-Buk.

Step 38 shows the conversion of 3-hydroxycaproate to 1,3-hexanal. This step is catalyzed by aldehyde:ferredoxin oxidoreductase (EC 1.2.7.5). Aldehydes: Exemplary sources of ferredoxin oxidoreductases are described elsewhere in this application. AOR catalyzes the reaction of acid and reduced ferric redox protein to form aldehyde and oxidized ferric redox protein. In acetogens, this reaction can be combined with either oxidized CO (via CO dehydrogenase, EC 1.2.7.4) or hydrogen (via ferredoxin-dependent hydrogenase, EC 1.12.7.2) to produce reduced ferredoxin. or 1.12.1.4) coupling (Köpke, "New Opinions in Biotechnology", 22: 320-325, 2011; Köpke, " Proceedings of the National Academy of Sciences , 107: 13087-13092, 2010"). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active for this step. However, overexpression of endogenous AOR or introduction of exogenous AOR may be required to increase product yield in C. autoethanologenum, C. yongdahlii, or C. larsonii. Alternatively, exogenous AORs can be introduced into microorganisms that do not naturally include AORs (eg, E. coli). In particular, co-expression of Ptb-Buk with AOR (and optionally Adh) may enable such microorganisms to produce novel unnatural products.

Step 39 demonstrates the conversion of 1,3-hexanal to 1,3-hexanediol. This step can be catalyzed by alcohol dehydrogenase (EC 1.1.1.1 or 1.1.1.2). Alcohol dehydrogenase converts aldehydes and NAD(P)H into alcohols and NAD(P). The alcohol dehydrogenase may be, for example, Adh from Clostridium autoethanogenogenum (AGY76060.1) (SEQ ID NO: 67), Clostridium yongdahl (ADK17019.1) (SEQ ID NO: 68), or Clostridium lashanii; BdhB from Clostridium acetobutylicum (NP_349891.1) (SEQ ID NO: 69); Bdh from Clostridium beijerinckii (WP_041897187.1) (SEQ ID NO: 70); Bdh1 from Clostridium yongdahl (YP_003780648 .1) (SEQ ID NO: 71); Bdh1 (AGY76060.1) from Clostridium autoethanogenogenum (SEQ ID NO: 72); Bdh2 (YP_003782121.1) (SEQ ID NO: 73) from Clostridium yungdalae ), Bdh2 (AGY74784.1) (SEQ ID NO: 74) from Clostridium autoethanogenogenum, AdhE1 (NP_149325.1) (SEQ ID NO: 75) from Clostridium acetobutylicum, Clostridium acetobutylicum AdhE2 (NP_149199.1) (SEQ ID NO: 76), AdhE (WP_041893626.1) (SEQ ID NO: 77) from Clostridium beijerinckii, AdhE1 (WP_023163372.1) from Clostridium autoethanogenogenum (SEQ ID NO: 78) or AdhE2 (WP_023163373.1) from Clostridium autoethanogenogenum (SEQ ID NO: 79). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active for this step. However, overexpression of endogenous alcohol dehydrogenases or introduction of exogenous alcohol dehydrogenases may be required to increase product yield in C. autoethanologenum, C. jungdahl, or C. larsonii. E. coli is most likely not naturally active for this step.

Step 40 shows the conversion of acetyl acetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA. This step can be catalyzed by hydroxymethylglutaryl-CoA synthase (HMG-CoA synthase) (EC 2.3.3.10). HMG-CoA synthases broadly include multiple genera and biological kingdoms and include, for example, MvaS from Staphylococcus aureus (WP_053014863.1), ERG13 from Saccharomyces cerevisiae (NP_013580.1), HMGCS2 from Mus musculus (NP_032282.2) and various other members of the EC 2.3.3.10 enzyme class. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 41 shows the conversion of 3-hydroxy-3-methylglutaryl-CoA to 3-methylglucosyl-CoA. This step can be catalyzed by 3-hydroxybutyl-CoA dehydratase (EC 4.2.1.55). The 3-hydroxybutyl-CoA dehydratase may be, for example, LiuC from Myxococcus xanthus (WP_011553770.1). This step can also be catalyzed by short-chain enyl-CoA hydratase (EC 4.2.1.150) or enyl-CoA hydratase (EC 4.2.1.17). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 42 shows the conversion of 3-methylglucosyl-CoA to 2-methylcrotonyl-CoA. This step can be accomplished by methylcrotonyl-CoA decarboxylase with high structural similarity to glutaconate-CoA transferase (EC 2.8.3.12) (e.g. aibAB from Myxococcus xanthus (WP_011554267.1) and WP_011554268.1)) catalysis. This step can also be catalyzed by methylcrotonyl-CoA carboxylase (EC 6.4.1.4), such as LiuDB (NP_250702.1 and NP_250704.1) from Pseudomonas aeruginosa or from Arabidopsis ( thaliana ) MCCA and MCCB (NP_563674.1 and NP_567950.1). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 43 shows the conversion of methylcrotonyl-CoA to isopentyl-CoA. This step can be catalyzed by oxidoreductase (zinc-binding dehydrogenase). This oxidoreductase (zinc-binding dehydrogenase) can be, for example, AibC from Myxococcus xanthus (WP_011554269.1). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii have no known natural activity for this step. E. coli has no known natural activity for this step.

Step 44 shows the conversion of isopentyl-CoA to isovalerate. This step can be catalyzed by CoA-transferase (i.e. acetyl-CoA: acetyl-acetyl-CoA transferase) (EC 2.8.3.9). The CoA-transferase can be, for example, CtfAB, a heterodimer including the subunits CtfA and CtfB from Clostridium beijerinckii (CtfA, WP_012059996.1) (SEQ ID NO: 5) (CtfB, WP_012059997.1) ( SEQ ID NO: 6). This step can also be catalyzed by thioesterases (EC 3.1.2.20). The thioesterase may be, for example, TesB (NP_414986.1) from E. coli (SEQ ID NO: 7). This step can also be catalyzed by putative thioesterases from, for example, Clostridium autoethanogenogenum or Clostridium yongdalae. Specifically, three putative thioesterases have been identified in C. autoethanologenum: (1) "Thioesterase 1" (AGY74947.1; annotated as palmitoyl-CoA hydrolase; SEQ ID NO: 8), (2) "Thioesterase 2" (AGY75747.1; labeled as 4-hydroxybenzyl-CoA thioesterase; SEQ ID NO: 9), and (3) "Thioesterase 3" (AGY75999.1 ; Annotated as putative thioesterase; SEQ ID NO: 10). Three putative thioesterases have also been identified in C. yongdahl: (1) "Thioesterase 1" (ADK15695.1; annotated as predicted acyl-CoA thioesterase 1; SEQ ID NO: 11), (2 ) "Thioesterase 2" (ADK16655.1; annotated as a predicted thioesterase; SEQ ID NO: 12), and (3) "Thioesterase 3" (ADK16959.1; annotated as a predicted thioesterase; SEQ ID NO: 13). This step can also be catalyzed by phosphate butyryl transferase (EC 2.3.1.19) + butyrate kinase (EC 2.7.2.7). Exemplary sources of phosphobutyltransferase and butyrate kinase are described elsewhere in this application. Natural enzymes in Clostridium autoethanogenogenum, Clostridium yongdahlii, and Clostridium larsonii (or E. coli), such as thioesterases from Clostridium autoethanogenogenum, can catalyze this step and produce certain amounts of downstream products. However, it may be necessary to introduce exogenous enzymes or overexpress endogenous enzymes to produce desired levels of downstream products. Additionally, in certain embodiments, it may be necessary to introduce disruptive mutations into endogenous enzymes (such as endogenous thioesterases) to reduce or eliminate competition with the introduced Ptb-Buk.

Step 45 shows the conversion of isovalerate to isovaleraldehyde. This step is catalyzed by aldehyde:ferredoxin oxidoreductase (EC 1.2.7.5). The aldehyde:ferredoxin oxidoreductase (AOR) may be, for example, the AOR from Clostridium autoethanogenogenum (WP_013238665.1; WP_013238675.1) (SEQ ID NO: 63 and 64, respectively) or from Clostridium yungdalae AOR (ADK15073.1; ADK15083.1) (SEQ ID NO: 65 and 66, respectively). Aldehydes: Other exemplary sources of ferredoxin oxidoreductases are described elsewhere in this application. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active for this step. However, overexpression of endogenous AOR or introduction of exogenous AOR may be required to increase product yield in C. autoethanologenum, C. yongdahlii, or C. larsonii. Alternatively, exogenous AORs can be introduced into microorganisms that do not naturally include AORs (eg, E. coli). In particular, co-expression of Ptb-Buk with AOR (and optionally Adh) may enable such microorganisms to produce novel unnatural products.

Step 46 shows the conversion of isovaleraldehyde to isopentyl alcohol. This step can be catalyzed by alcohol dehydrogenase (EC 1.1.1.1. or 1.1.1.2). Alcohol dehydrogenase converts aldehydes and NAD(P)H into alcohols and NAD(P). The alcohol dehydrogenase may be, for example, Adh from Clostridium autoethanogenogenum (AGY76060.1) (SEQ ID NO: 67), Clostridium yongdahl (ADK17019.1) (SEQ ID NO: 68), or Clostridium lashanii; BdhB from Clostridium acetobutylicum (NP_349891.1) (SEQ ID NO: 69); Bdh from Clostridium beijerinckii (WP_041897187.1) (SEQ ID NO: 70); Bdh1 from Clostridium yongdahl (YP_003780648 .1) (SEQ ID NO: 71); Bdh1 (AGY76060.1) from Clostridium autoethanogenogenum (SEQ ID NO: 72); Bdh2 (YP_003782121.1) (SEQ ID NO: 73) from Clostridium yungdalae ), Bdh2 (AGY74784.1) (SEQ ID NO: 74) from Clostridium autoethanogenogenum, AdhE1 (NP_149325.1) (SEQ ID NO: 75) from Clostridium acetobutylicum, Clostridium acetobutylicum AdhE2 (NP_149199.1) (SEQ ID NO: 76), AdhE (WP_041893626.1) (SEQ ID NO: 77) from Clostridium beijerinckii, AdhE1 (WP_023163372.1) from Clostridium autoethanogenogenum (SEQ ID NO: 78) or AdhE2 (WP_023163373.1) from Clostridium autoethanogenogenum (SEQ ID NO: 79). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are naturally active for this step. However, overexpression of endogenous alcohol dehydrogenases or introduction of exogenous alcohol dehydrogenases may be required to increase product yield in C. autoethanologenum, C. jungdahl, or C. larsonii. E. coli is most likely not naturally active for this step.

Step 47 demonstrates the conversion of isopentyl-CoA to isovaleraldehyde. This step is catalyzed by butyraldehyde dehydrogenase (EC 1.2.1.57). The butyraldehyde dehydrogenase may be, for example, Bld (AAP42563.1) from Clostridium glycoacetate (SEQ ID NO: 80). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium larsonii are not naturally active for this step. E. coli has no known natural activity for this step.

Overview of Ptb-Buk

The present invention provides novel pathways utilizing the Ptb-Buk enzyme system. In nature, this enzyme system is found in a range of butyrate-producing microorganisms, such as butyrate-producing Clostridia or Butyrivibrio . Specifically, phosphate butyltransferase (Ptb) (EC 2.3.1.19) naturally catalyzes the reaction of butyl-CoA+ phosphate to form CoA+ butyl phosphate, and butyrate kinase (Buk) (EC 2.7.2.7) naturally catalyzes the reaction of butyl-butyl phosphate. The reaction of ester and ADP forms butyrate and ATP. Therefore, these enzymes together (Ptb-Buk) naturally catalyze the conversion of butyryl-CoA to butyrate and generate an ATP via phosphorylation at the substrate level (Figure 2). However, the inventors have discovered that Ptb is hybrid and can accept a variety of acyl-CoA and enyl-CoA as acceptors, so that Ptb-Buk can be used to convert a variety of acyl-CoA and enyl-CoA into Its corresponding acid or enoate ester produces ATP at the same time. Ptb has been reported to be active in vitro against a range of acyl-CoA, including acetyl-acetyl-CoA (Thompson, Applied & Environmental Microbiology , 56: 607-613, 1990). Acetoacetyl-phosphate has not previously been shown to serve as an acceptor for Buk. Although Buk is known to accept a wide substrate range (Liu, Appl Microbiol Biotechnol , 53:545-552, 2000), it does not exhibit activity in vivo.

In addition, the inventors have discovered that the introduction of exogenous Ptb-Buk enables specific microorganisms to produce useful products, including acetone, isopropanol, isobutylene, 3-hydroxybutyrate, 1,3-butanediol and 2-hydroxyisobutyrate. acid esters, as well as other products such as propionate, caproate, and octanoate.

The novel pathway that relies on Ptb-Buk is compared to other known and existing pathways that rely on CoA-transferase to produce products (such as in the typical Clostridial acetone-butanol-ethanol (ABE) fermentation pathway) or that rely on sulfur. Other known and existing routes to product production from esterases offer several advantages (Jones, Microbiol Rev , 50: 484-524, 1986; Matsumoto, Applied Microbiology and Biotechnology , 97: 205 -210, 2013; May, " Metabol Eng ", 15: 218-225, 2013) (Figure 3). In particular, these novel pathways (1) do not rely on the presence or production of specific molecules required for the CoA-transferase reaction (such as organic acids, such as butyrate or acetate), and (2) enable Phosphorylation at the plasma membrane produces ATP that is not conserved in thioesterase or CoA-transferase reactions. The same advantages apply when using the Ptb-Buk system for other reactions such as the conversion of 3-hydroxybutyl-CoA to 3-hydroxybutyrate. Therefore, these novel pathways can produce much higher production titers and rates by generating additional energy and producing target products without co-producing undesirable by-products such as acetate.

Specifically, it is undesirable for microorganisms to produce acetate (or other organic acids required for the CoA transferase reaction) as a by-product at commercial scales because acetate transfers carbon from the target product and therefore affects the target. Product efficiency and yield. Additionally, acetate may be toxic to microorganisms and/or may serve as a substrate for the growth of contaminating microorganisms. In addition, the presence of acetate makes it difficult to recover and isolate the target product and control fermentation conditions to promote the production of the target product.

The generation of ATP through phosphorylation at the substrate level can be used as a driving force for product synthesis, especially in ATP-limited systems. In detail, acetogenic bacteria are known to survive on the edge of thermodynamic life (Schuchmann, Nat Rev Microbiol , 12:809-821, 2014). Thus, all acetogenic microorganisms isolated to date have been described to produce acetate (Drake, "Acetogenic Prokaryotes", in The Prokaryotes , 3rd ed., pp. 354-420, New York, NY, Springer, 2006), because the production of acetate provides microorganisms with phosphate from the substrate via Pta (phosphotransacetylase) (EC 2.3.1.8) and Ack (acetate kinase) (EC 2.7.2.1) ation directly produces ATP. Although mechanisms such as membrane gradients and electrobifurcating enzymes coupled to ion or proton translocation systems (e.g., Rnf complexes) (Schuchmann, Nature Reviews: Microbiology , 12:809-821, 2014) make these microorganisms ATP is conserved, but direct ATP production is still critical for its survival. Thus, when introducing a heterologous pathway that does not allow ATP production, acetate is produced as a by-product (Schiel-Bengelsdorf, FEBS Lett , 586: 2191-2198, 2012). However, the Ptb-Buk pathway described herein provides microorganisms with an alternative mechanism to generate ATP via phosphorylation at the substrate level, thus avoiding acetate production. Specifically, an otherwise essential acetate-forming enzyme such as Pta-Ack (Nagarajan, Microb Cell Factories , 12:118, 2013) can be replaced with Ptb-Buk for ATP production alternative way. Since microorganisms can then rely on ATP production via Ptb-Buk, this system provides the power to ensure maximum flux through novel pathways using Ptb-Buk. The production of ATP may also be critical for downstream pathways that require ATP. For example, the production of isobutylene from acetone fermentation requires ATP. The entire path from acetyl-CoA to isobutylene consumes ATP when using CoA-transferase or thioesterase, whereas the path is energy balanced when using Ptb-Buk.

Provides illustrative sources for Ptb and Buk. However, it is understood that other suitable sources of Ptb and Buk may be used. In addition, Ptb and Buk can be engineered to improve activity and/or the gene encoding Ptb-Buk can be codon-optimized for expression in a specific host microorganism.

The phosphobutyltransferase enzyme may be or may be derived from, for example, the following sources, the sequences of which are publicly available:

In a preferred embodiment, the phosphate butyltransferase is from Clostridium acetobutylicum (WP_010966357; SEQ ID NO: 87) or Clostridium beijerinckii (WP_026886639; SEQ ID NO: 88) (WP_041893500; SEQ ID NO: 89 ) of Ptb. Clostridium autoethanologenum, Clostridium yongdalae and Clostridium lashoni naturally do not contain phosphate butyl transferase.

Butyrate kinase may be derived from or may be derived from sources such as:

In a preferred embodiment, butyrate kinase is derived from Clostridium acetobutylicum (WP_010966356; SEQ ID NO: 90) or Clostridium beijerinckii (WP_011967556; SEQ ID NO: 91) (WP_017209677; SEQ ID NO: 92) (WP_026886638; SEQ ID NO:93) Buk of (WP_041893502; SEQ ID NO:94). Clostridium autoethanologenum, Clostridium yongdalae and Clostridium lashanii naturally do not contain butyrate kinase.

Since Ptb-Buk has been shown to act on a large number of substrates, it is reasonable to assume that if Ptb-Buk does not exhibit any activity on a desired substrate, it can be engineered to achieve activity on that substrate. One strategy could be, but is not limited to, rational design based on available crystal structures of Ptb and Buk with or without substrates, where the binding pocket is altered to accommodate novel substrates, or via saturation mutagenesis. Once activity is achieved, it can be further improved through repeated enzyme engineering cycles. These engineering operations will be combined with assays to test enzyme activity. These types of strategies have previously proven effective (see, e.g., Huang, Nature , 537:320-327, 2016 ; Khoury, Trends Biotechnol , 32:99-109, 2014; Packer , "Nature Rev Genetics ", 16: 379-394, 2015; Privett, " Proceedings of the National Academy of Sciences , 109: 3790-3795, 2012").

To improve the substrate specificity of Ptb-Buk for a specific acyl-CoA substrate, high-throughput analysis of Ptb-Buk variants from public databases or generated Ptb-Buk mutants (e.g., by directed evolution) Screening, that is, overexpressing the Ptb-Buk enzyme pair in E. coli, adding test substrates and screening for ATP production using bioluminescence assays. The assay may use accepted practices of correlating ATP concentration with firefly luciferase bioluminescence. The adaptability of this assay to a porous disk format will facilitate better efficient screening of substrates across novel Ptb-Buk combinations (Figure 33).

The assay avoids the measurement of spontaneous hydrolysis of CoA groups by screening for ATP production rather than substrate elimination or product accumulation. However, an alternative approach described in the literature is the use of free CoA using established assays using Ellman's reagent (5,5'-disulfobis-(2-nitrobenzoic acid) or DTNB) (Thompson, Applied and Environmental Microbiology, 56: 607-613, 1990.) were measured to evaluate the coupling efficiency of the Ptb-Buk reaction (Figure 33). Cyl-CoA and the corresponding free acid and phosphorylated intermediate can also be measured during the validation phase using LC-MS/MS.

In high-throughput screening methods, it is difficult to collect kinetic data due to the labor involved in protein quantification. Alternatively, for each preparation of an E. coli lysate containing the Ptb-Buk enzyme, the activity of each relevant substrate (measured as luminescence per unit time) can be compared with that of a positive control substrate (butyl-CoA) and a comparison of activity against acetyl-CoA, a physiological substrate that is likely to provide the greatest competition for the enzyme active site of the target acetyl-CoA.

To ensure that the assay is not biased by native phosphotransacetylase (Pta) and/or acetate kinase (Ack) activity, the assay can also be evaluated in E. coli strains in which the pta and/or ack genes have been deleted.

Produce acetone and isopropyl alcohol

Acetone and isopropyl alcohol are important industrial solvents, with a combined market size of 8 million tons and a total market value of $8.5-11 billion. In addition, acetone and isopropyl alcohol are precursors to the following valuable downstream products, including polymethylmethacrylate (PMMA), with a total market value of $7 billion; isobutylene, with a total market value of $25-29 billion; and Propylene has a total market value of $125 billion. In addition, fuel ejection pathways from acetone have been reported in recent years. Currently, industrial acetone production is directly linked to petrochemical phenol production as it is a by-product of the cumene process. Approximately 92% by volume of the acetone output is a by-product of phenol production from cumene. This has significant impacts on both the environment and the market. During the cumene production process, one mole of sodium sulfite accumulates for each mole of phenol, causing serious waste disposal problems and challenges to the natural environment and human health. World market demand for phenol is expected to decrease or decline, while demand for acetone is forecast to increase. An alternative route to phenol production by direct oxidation of benzene is being developed and is expected to be commercialized soon; this would allow the complete elimination of acetone production.

Acetone has been produced on an industrial scale for about 100 years as a by-product of butanol in ABE fermentation. Although industrial ABE fermentation declined in the second half of the 20th century due to low oil prices and high sugar costs, it has recovered in recent years, with several commercial plants built in recent years. Acetone production from sugars has also been demonstrated by several academic groups in heterologous hosts using metabolic engineering and synthetic biology approaches to express acetone from ABE fermentation organisms, especially E. coli and yeast. corresponding enzyme. However, the low yields and high costs associated with the pretreatment required to release the polysaccharide component of biomass make the production of acetone via standard fermentation uneconomical because current biochemical conversion technologies do not utilize the lignin component of biomass, lignin. Components may constitute up to 40% of this substance.

The present invention provides a microorganism capable of producing acetone or its precursor from a substrate. The present invention further provides a method for producing acetone or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of acetone may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

Obtaining acetone via steps 1, 2 and 3: In one embodiment, the present invention provides a microorganism comprising the enzymes of steps 1, 2 and 3, by which the microorganism is able to obtain acetone from a substrate (such as a gas substrate). substance) to produce acetone or its precursors. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 2 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 2, and 3 are described elsewhere in this application. If the microorganism is derived from a parent microorganism that naturally contains a primary: secondary alcohol dehydrogenase capable of converting acetone to isopropyl alcohol (step 4) (e.g. Clostridium autoethanologenum, Clostridium yongdahl or Clostridium larsonii), The microorganism can then be modified to block or eliminate the expression of primary:secondary alcohol dehydrogenase (e.g., by disrupting the gene encoding primary:secondary alcohol dehydrogenase), allowing the microorganism to produce acetone without converting it is isopropyl alcohol (WO 2015/085015).

Obtaining acetone through steps 1, 13, 14, 15 and 3: In one embodiment, the invention provides a microorganism comprising steps 1, 13, 14, 15 and 3 exogenous enzymes, by which the The microorganism is capable of producing acetone or its precursors from a substrate such as a gaseous substrate. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 14 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 13, 14, 15, and 3 are described elsewhere in this application. If the microorganism is derived from a parent microorganism that naturally contains a primary: secondary alcohol dehydrogenase capable of converting acetone to isopropyl alcohol (step 4) (e.g. Clostridium autoethanologenum, Clostridium yongdahl or Clostridium larsonii), The microorganism can then be modified to block or eliminate the expression of primary:secondary alcohol dehydrogenase (e.g., by disrupting the gene encoding primary:secondary alcohol dehydrogenase), allowing the microorganism to produce acetone without converting it is isopropyl alcohol (WO 2015/085015).

In one embodiment, the microorganism may include more than one pathway for producing acetone.

The present invention provides a microorganism capable of producing isopropyl alcohol or its precursor from a substrate. The present invention further provides a method for producing isopropanol or a precursor thereof by culturing such microorganisms in the presence of a substrate. In preferred embodiments, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of isopropanol may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

Obtaining isopropyl alcohol through steps 1, 2, 3 and 4: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 1, 2, 3 and 4, by which the microorganism can self- A substrate, such as a gaseous substrate, produces isopropanol or a precursor thereof. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 2 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 2, 3, and 4 are described elsewhere in this application. If the microorganism is derived from a parent microorganism that naturally contains a primary: secondary alcohol dehydrogenase capable of converting acetone to isopropyl alcohol (step 4) (e.g. Clostridium autoethanologenum, Clostridium yongdahl or Clostridium larsonii), Then step 4 does not require the introduction of exogenous enzymes to produce isopropanol. However, conditioning microorganisms such as overexpression of natural primary:secondary alcohol dehydrogenases can increase isopropanol production.

Obtaining isopropyl alcohol through steps 1, 13, 14, 15, 3 and 4: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 1, 13, 14, 15, 3 and 4, by Described enzyme, described microorganism can produce isopropanol or its precursor from substrate (such as gaseous substrate). Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 14 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 13, 14, 15, 3, and 4 are described elsewhere in this application. If the microorganism is derived from a parent microorganism that naturally contains a primary: secondary alcohol dehydrogenase capable of converting acetone to isopropyl alcohol (step 4) (e.g. Clostridium autoethanologenum, Clostridium yongdahl or Clostridium larsonii), Then step 4 does not require the introduction of exogenous enzymes to produce isopropanol. However, modulating microorganisms, such as by overexpressing natural primary:secondary alcohol dehydrogenases, can increase isopropanol production.

In one embodiment, the microorganism may include more than one pathway for producing isopropanol.

produces isobutylene

Isobutylene is an important chemical building block, with a market size of more than 15 million tons and a total market value of $25-29 billion. In addition to isobutylene's use in chemical processes and as a fuel additive (15 Mt/yr), it can also be converted into isooctane, a drop-in, high-efficiency fuel for gasoline vehicles. Global Bioenergies has submitted a patent application for the production of isobutylene from acetone fermentation, but the disclosed pathways do not include Ptb-Buk (WO 2010/001078; EP 2295593; WO 2011/076691; van Leeuwen, " Applications " Microbiology and Biotechnology , 93: 1377-1387, 2012).

The present invention provides a microorganism capable of producing isobutylene or its precursor from a substrate. The present invention further provides a method for producing isobutylene or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of isobutylene may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

Figure 1 shows two alternative routes to isobutylene. The first pathway involves the production of isobutylene via steps 1, 2, 3, 5 and 6. The second pathway includes the production of isobutylene via steps 1, 2, 3, 7, 8 and 6. Steps 2 and 8 can be catalyzed by Ptb-Buk. Therefore, each pathway may involve Ptb-Buk.

Obtaining isobutylene through steps 1, 2, 3, 5 and 6: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 1, 2, 3, 5 and 6, by which the microorganism Isobutylene or its precursors can be produced from a substrate such as a gaseous substrate. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 2 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 2, 3, 5, and 6 are described elsewhere in this application. If the microorganism is derived from a parent microorganism that naturally contains a primary: secondary alcohol dehydrogenase capable of converting acetone to isopropyl alcohol (step 4) (e.g. Clostridium autoethanologenum, Clostridium yongdahl or Clostridium larsonii), The microorganism can then be modified to block or eliminate the expression of primary:secondary alcohol dehydrogenase (e.g., by disrupting the gene encoding primary:secondary alcohol dehydrogenase) to prevent the conversion of acetone to isopropyl alcohol and allow The conversion of acetone to isobutylene reaches the maximum.

Obtaining isobutylene via steps 1, 2, 3, 7, 8 and 6: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 1, 2, 3, 7, 8 and 6, by which the enzyme , the microorganism is capable of producing isobutylene or its precursor from a substrate (such as a gas substrate). Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 2 and/or step 8 are catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 2, 3, 7, 8, and 6 are described elsewhere in this application. If the microorganism is derived from a parent microorganism that naturally contains a primary: secondary alcohol dehydrogenase capable of converting acetone to isopropyl alcohol (step 4) (e.g. Clostridium autoethanologenum, Clostridium yongdahl or Clostridium larsonii), The microorganism can then be modified to block or eliminate the expression of primary:secondary alcohol dehydrogenase (e.g., by disrupting the gene encoding primary:secondary alcohol dehydrogenase) to prevent the conversion of acetone to isopropyl alcohol and allow The conversion of acetone to isobutylene reaches the maximum.

Produces 3-hydroxybutyrate

3-Hydroxybutyrate (3-HB) is a four-carbon carboxylic acid in the β-hydroxy acid family. 3-Hydroxybutyrate is a cosmetic ingredient for oily skin cleansing, an intermediate in anti-aging cream formulations, an intermediate in polyhydroxybutyrate (PHB), a biodegradable polymer resin, and in novel Co-monomer of bioplastics with other polyhydroxy acids. In addition, 3-hydroxybutyrate is particularly used in biocompatible and biodegradable nanocomposites, especially in medical implants, intermediates of C3/C4 chemicals, chiral building blocks and fine chemicals. Taste. Although (R)- and (S)-3-hydroxybutyrate are produced by recombinant E. coli grown on glucose, the production of 3-hydroxybutyrate from microorganisms grown on gaseous substrates has not been demonstrated (Tseng, " Applied and Environmental Microbiology , 75: 3137-3145, 2009). Notably, the system previously demonstrated in E. coli cannot be directly transferred to acetogens (including Clostridium autoethanogenans) due to the presence of native thioesterases in acetogens. Although E. coli also has a thioesterase TesB that can act on 3-HB-CoA, Tseng shows very little background activity (<0.1g/L). Although the production of stereopure isomers has been reported in E. coli , the inventors unexpectedly discovered that a mixture of isomers can be produced in Clostridium autoethanogenogenum. Without being bound by this theory, this is most likely due to natural isomerase activity. This enables the combination of (S)-specific 3-hydroxybutyl-CoA dehydrogenase (Hbd) with (R)-specific Ptb-Buk to optimize yields. To produce stereopure isomers, this activity can be eliminated. Taken together, the present invention enables the production of several grams/liter of 3-HB compared to the low yields in E. coli and the use of Ptb-Buk with (R) or (S)-specific 3-hydroxybutyryl-CoA. Any combination of hydrogenase and natural autoethanogenic Clostridial thioesterase.

The present invention provides a microorganism capable of producing 3-hydroxybutyrate or its precursor from a substrate. The present invention further provides a method for producing 3-hydroxybutyrate or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of 3-hydroxybutyrate may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

Figure 1 shows two alternative routes to 3-hydroxybutyrate. The first pathway involves the production of 3-hydroxybutyrate via steps 1, 2 and 15. The second route involves the production of 3-hydroxybutyrate via steps 1, 13 and 14. Steps 2 and 14 can be catalyzed by Ptb-Buk. Therefore, each pathway may involve Ptb-Buk. In one embodiment, the microorganism may include more than one pathway to produce 3-hydroxybutyrate, where Ptb-Buk may catalyze more than one step (eg, steps 2 and 14).

Obtaining 3-hydroxybutyrate through steps 1, 2 and 15: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 1, 2 and 15, by which the microorganism is capable of being subjected to A substrate (such as a gaseous substrate) produces 3-hydroxybutyrate or a precursor thereof. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 2 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 2, and 15 are described elsewhere in this application.

Obtaining 3-hydroxybutyrate through steps 1, 13 and 14: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 1, 13 and 14, by which the microorganism is able to undergo A substrate (such as a gaseous substrate) produces 3-hydroxybutyrate or a precursor thereof. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 14 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 13, and 14 are described elsewhere in this application.

Produce 1,3-butanediol

1,3-Butanediol (1,3-BDO) is commonly used as a solvent in food flavorings and is a comonomer used in certain polyurethane and polyester resins. More importantly, 1,3-butanediol can be catalytically converted to 1,3-butadiene (Makshina, Chem Soc Rev , 43:7917-7953, 2014). Butadiene is used in the manufacture of rubber, plastics, lubricants, latex and other products. Although a large amount of butadiene is produced today for use in rubber in automobile tires, it is also used to produce adiponitrile, which is used to make nylon 6,6. Overall demand for butadiene is rising. In 2011, it was estimated that 10.5 million tons would be needed, worth $40 billion.

The present invention provides a microorganism capable of producing 1,3-butanediol or its precursor from a substrate. The present invention further provides a method for producing 1,3-butanediol or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of 1,3-butanediol may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

In certain embodiments, the microorganism can produce 1,3-butanediol without co-producing ethanol (or only producing a small amount of ethanol, such as less than 0.1-1.0 g/L ethanol or less than 1-10 g/L ethanol) .

Figure 1 shows three alternative routes to 1,3-butanediol. The first pathway includes the production of 1,3-butanediol via steps 1, 2, 15, 16 and 17. The second pathway includes the production of 1,3-butanediol via steps 1, 13, 14, 16 and 17. The third pathway includes the production of 1,3-butanediol via steps 1, 13, 18 and 17. Steps 2 and 14 can be catalyzed by Ptb-Buk. Thus, at least the first and second pathways may involve Ptb-Buk. In one embodiment, the microorganism may include more than one pathway for producing 1,3-butanediol. In a related embodiment, Ptb-Buk can catalyze more than one step (eg, steps 2 and 14).

Obtain 1,3-butanediol through steps 1, 2, 15, 16 and 17: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 1, 2, 15, 16 and 17, by The enzyme is capable of producing 1,3-butanediol or a precursor thereof from a substrate such as a gaseous substrate. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 2 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 2, 15, 16, and 17 are described elsewhere in this application.

Obtain 1,3-butanediol through steps 1, 13, 14, 16 and 17: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 1, 13, 14, 16 and 17, by The enzyme is capable of producing 1,3-butanediol or a precursor thereof from a substrate such as a gaseous substrate. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 14 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 13, 14, 16, and 17 are described elsewhere in this application.

Obtain 1,3-butanediol through steps 1, 13, 18 and 17: In one embodiment, the present invention provides a microorganism comprising the enzymes of steps 1, 13, 18 and 17, by which the enzyme The microorganisms are capable of producing 1,3-butanediol or its precursors from substrates such as gaseous substrates (Figure 11). Typically, at least one enzyme in this pathway is exogenous to the microorganism. Exemplary types and sources of enzymes for steps 1, 13, 18, and 17 are described elsewhere in this application. Similar pathways are shown in E. coli but not in acetogens such as Clostridium autoethanologenum, Clostridium yongdallii and Clostridium larsonii (Kataoka, J Biosci Bioeng ) , 115:475-480, 2013). Although Ptb-Buk can be used to produce (R)-1,3-butanediol, this pathway does not require the use of Ptb-Buk to produce (S)-1,3-butanediol.

produces 2-hydroxyisobutyrate

2-Hydroxyisobutyrate (2-HIB) is a four-carbon carboxylic acid that can serve as a building block in a variety of polymers. Methyl methacrylate (which can be synthesized by dehydration of 2-hydroxyisobutyrate or via the corresponding amide) is polymerized to form polymethyl methacrylate (PMMA) to produce acrylic glass, durable coatings and inks. For this compound alone, the total market exceeds 3 million tons. Other branched C4 carboxylic acids (such as the chlorine and amine derivatives of 2-hydroxyisobutyrate) and isobutylene glycol and its oxides are also used in polymers and in many other applications.

The stereospecificity of the Ptb-Buk system is particularly suitable for overcoming the limitations of current advanced technology for the preparation of 2-hydroxyisobutyrate. Both Ptb-Buk and thioesterases are promiscuous, allowing lateral activity on 3-hydroxybutyl-CoA to divert resources from the target pathway to produce 2-hydroxyisobutyl-CoA (see, eg, Figures 1 and 8). However, Ptb-Buk is able to distinguish between stereoisomers and acts on (R)-3-hydroxybutyl-CoA but not (S)-3-hydroxybutyl-CoA. In contrast, thioesterases cannot differentiate between 3-hydroxybutyryl-CoA stereoisomers. In a preferred embodiment, (S)-specific acetoacetyl-CoA hydratase (EC 4.2.1.119) (step 13) is selected to be combined with Ptb-Buk (step 20) to avoid the generation of 3-hydroxyl The loss caused by butyrate resulted in the maximum production of 2-hydroxyisobutyrate (Figure 8). The (S)-specific form of 3-hydroxybutyl-CoA is also a preferred substrate for 2-hydroxyisobutyl-CoA mutase (EC 5.4.99.-) (step 19) (Yaneva, Journal of Biochemistry , 287:15502-15511, 2012).

The present invention provides a microorganism capable of producing 2-hydroxyisobutyrate or a precursor thereof from a substrate. The present invention further provides a method for producing 2-hydroxyisobutyrate or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of 2-hydroxyisobutyrate may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

Obtaining 2-hydroxybutyrate through steps 1, 13, 19 and 20: In one embodiment, the present invention provides a microorganism comprising the enzyme of steps 1, 13, 19 and 20, by which the enzyme, Microorganisms are capable of producing 2-hydroxybutyrate or precursors thereof from substrates such as gaseous substrates. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 20 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 13, 19, and 20 are described elsewhere in this application.

In certain embodiments, the invention also provides a microorganism capable of producing 2-hydroxybutyrate (2-HB) or a precursor thereof from a substrate. The present invention further provides a method for producing 2-hydroxybutyrate or a precursor thereof by culturing such microorganisms in the presence of a substrate. Without wishing to be bound by any particular theory, the inventors believe that the observed production of 2-hydroxybutyrate is attributable to microorganisms such as Clostridium autoethanologenum, Clostridium yundalis and Clostridium larvae non-specific mutase activity in bacteria).

produce adipic acid

Adipic acid is the most important dicarboxylic acid, with an estimated market greater than US$4.5 billion, of which approximately 2.5 billion kilograms are produced annually. More than 60% of the adipic acid produced is being used as a monomer precursor for the production of nylon, and by 2019, the total market for adipic acid is expected to reach US$7.5 billion. Currently, adipic acid is produced almost exclusively by petrochemical processes, for example by carbonylation of butadiene.

The present invention provides a microorganism capable of producing adipic acid or its precursor from a substrate (Fig. 34). The present invention further provides a method for producing adipic acid or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of adipic acid may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

Obtaining adipic acid through steps 22, 23, 24, 25 and 26: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 22, 23, 24, 25 and 26, by which the enzyme The microorganism is capable of producing adipic acid or its precursors from a substrate, such as a gaseous substrate. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 26 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 22, 23, 24, 25, and 26 are described elsewhere in this application.

Obtaining adipic acid through steps 21, 22, 23, 24, 25 and 26: In one embodiment, the invention provides a microorganism comprising the enzymes of steps 21, 22, 23, 24, 25 and 26, by The enzyme is capable of producing adipic acid or a precursor thereof from a substrate, such as a gaseous substrate. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 26 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 21, 22, 23, 24, 25, and 26 are described elsewhere in this application.

In one embodiment, the microorganism may include more than one pathway for producing adipic acid.

Produce 1,3-hexanediol

The present invention provides a microorganism capable of producing 1,3-hexanediol or its precursor from a substrate (Fig. 35). The present invention further provides a method for producing 1,3-hexanediol or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of 1,3-hexanediol may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

The pathway depicted in Figure 35 begins with 3-hydroxybutyl-CoA, which can be generated via steps 1 and 13 as depicted in Figure 1 .

Obtain 1,3-hexanediol via steps 1, 13, 27, 31, 32, 36, 37, 38 and 39: In one embodiment, the invention provides a microorganism comprising steps 1, 13, 27, 31 , 32, 36, 37, 38 and 39 enzymes, by which the microorganism can produce 1,3-hexanediol or its precursor from a substrate (such as a gas substrate). Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 37 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 13, 27, 31, 32, 36, 37, 38, and 39 are described elsewhere in this application.

Produces 3-methyl-2-butanol

The present invention provides a microorganism capable of producing 3-methyl-2-butanol or its precursor from a substrate (Fig. 35). The present invention further provides a method for producing 3-methyl-2-butanol or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of 3-methyl-2-butanol may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

The pathway depicted in Figure 35 begins with 3-hydroxybutyl-CoA, which can be generated via steps 1 and 13 as depicted in Figure 1 .

Obtain 3-methyl-2-butanol via steps 1, 13, 27, 31, 32, 33, 34 and 35: In one embodiment, the invention provides a microorganism comprising steps 1, 13, 27, 31 , 32, 33, 34 and 35 enzymes, by which the microorganism can produce 3-methyl-2-butanol or its precursor from a substrate (such as a gas substrate). Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 33 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 13, 27, 31, 32, 33, 34, and 35 are described elsewhere in this application.

Produces 2-buten-1-ol

The present invention provides a microorganism capable of producing 2-buten-1-ol or a precursor thereof from a substrate (Fig. 35). The present invention further provides a method for producing 2-buten-1-ol or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of 2-buten-1-ol may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

The pathway depicted in Figure 35 begins with 3-hydroxybutyl-CoA, which can be generated via steps 1 and 13 as depicted in Figure 1 .

Obtain 2-buten-1-ol through steps 1, 13, 27, 28, 29 and 30: In one embodiment, the invention provides a microorganism comprising steps 1, 13, 27, 28, 29 and 30 An enzyme by which the microorganism is capable of producing 2-buten-1-ol or a precursor thereof from a substrate, such as a gaseous substrate. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 28 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 13, 27, 28, 29, and 30 are described elsewhere in this application.

produces isovalerate

The present invention provides a microorganism capable of producing isovalerate or its precursor from a substrate (Fig. 36). The present invention further provides a method for producing isovalerate or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of isovalerate may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

Obtaining isovalerate through steps 1, 40, 41, 42, 43 and 44: In one embodiment, the present invention provides a microorganism comprising the microorganisms of steps 1, 40, 41, 42, 43 and 44, by The enzyme, the microorganism is capable of producing isovalerate or a precursor thereof from a substrate, such as a gaseous substrate. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 44 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 40, 41, 42, 43, and 44 are described elsewhere in this application.

produces isoamyl alcohol

The present invention provides a microorganism capable of producing isoamyl alcohol or its precursor from a substrate (Fig. 36). The present invention further provides a method for producing isoamyl alcohol or a precursor thereof by culturing such microorganisms in the presence of a substrate. In a preferred embodiment, the microorganism is derived from a parent microorganism selected from the group consisting of Clostridium ethanologenum, Clostridium yongdahlii, or Clostridium larsonii. However, the microorganisms can also originate from completely different microorganisms, such as E. coli. The enzymatic pathways described for the production of isoamyl alcohol may include endogenous enzymes and, in the case where endogenous enzyme activity is absent or low, exogenous enzymes.

Isoamyl alcohol is obtained via steps 1, 40, 41, 42, 43, 44, 45 and 46: In one embodiment, the invention provides a microorganism comprising steps 1, 40, 41, 42, 43, 44, 45 and 46. A microorganism capable of producing isoamyl alcohol or a precursor thereof from a substrate (such as a gaseous substrate) via the enzyme. Typically, at least one enzyme in this pathway is exogenous to the microorganism. In a preferred embodiment, step 44 is catalyzed by Ptb-Buk. Exemplary types and sources of enzymes for steps 1, 40, 41, 42, 43, 44, 45, and 46 are described elsewhere in this application.

Obtaining isoamyl alcohol through steps 1, 40, 41, 42, 43, 47 and 46: In one embodiment, the invention provides a microorganism comprising the microorganisms of steps 1, 40, 41, 42, 43, 47 and 46 , by means of the enzyme, the microorganism is able to produce isoamyl alcohol or its precursor from a substrate (such as a gas substrate). Typically, at least one enzyme in this pathway is exogenous to the microorganism. Exemplary types and sources of enzymes for steps 1, 40, 41, 42, 43, 47, and 46 are described elsewhere in this application.

In one embodiment, the microorganism may include more than one pathway for producing isoamyl alcohol.

produce other products

The present invention provides a microorganism, which includes exogenous Ptb-Buk and exogenous or endogenous aldehyde: ferredoxin oxidoreductase (AOR). Such microorganisms can produce, for example, 1-propanol, 1-butanol, 1-hexanol, and 1-octanol or precursors thereof from acetyl-CoA produced, for example, from gaseous substrates (Figure 32). The present invention further provides a method for producing 1-propanol, 1-butanol, 1-hexanol and 1-octanol or precursors thereof by cultivating such microorganisms in the presence of gaseous substrates. Clostridium autoethanologenum, Clostridium yongdahlii, and Clostridium larsonii naturally include AOR. However, AOR can be overexpressed in such microorganisms in combination with expression of exogenous Ptb-Buk. Alternatively, exogenous AOR and exogenous Ptb-Buk may be expressed in microorganisms other than Clostridium autoethanologenum, Clostridium yongdahlii, and Clostridium larsonii, such as E. coli.

Produce precursors and intermediates

The pathways depicted in Figures 1, 34, 35 and 36 can be modified to produce precursors or intermediates to the products described above. In particular, part of the enzymatic pathway of any of the pathways described herein can be inserted into a host microorganism to obtain the production of precursors or intermediates.

Definition and background

The term "genetic modification" or "genetic engineering" generally refers to the manipulation of the genome or nucleic acid of microorganisms. Likewise, the term "genetically engineered" refers to the microorganism including the genome or nucleic acid upon which it has been manipulated. Genetic modification methods include, for example, heterologous gene expression, gene or promoter insertion or deletion, nucleic acid mutation, altered gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutation induction methods, gene shuffling, and Codon optimization.

"Recombinant" indicates that nucleic acids, proteins or microorganisms are the product of genetic modification, engineering or recombination. Generally speaking, the term "recombinant" refers to a nucleic acid, protein or microorganism that contains genetic material derived from multiple sources or consists of genetic material derived from multiple sources, such as two or more different strains or species of microorganisms. Encoding. As used herein, the term "recombinant" may also be used to describe a microorganism that includes mutated nucleic acids or proteins, including mutated forms of endogenous nucleic acids or proteins.

"Endogenous" means that a nucleic acid or protein is present or expressed in the wild-type or parent microorganism from which the microorganism of the invention originates. By way of example, endogenous genes are genes naturally present in the wild-type or parent microorganism from which the microorganism of the invention arises. In one embodiment, expression of endogenous genes can be controlled by exogenous regulatory elements, such as exogenous promoters.

"Foreign" refers to a nucleic acid or protein that is not present in the wild-type or parent microorganism from which the microorganism of the invention arises. In one embodiment, foreign genes or enzymes may be derived from a heterologous (ie, different) strain or species and introduced or expressed in the microorganism of the invention. In another embodiment, exogenous genes or enzymes may be formed artificially or recombinantly and introduced or expressed in the microorganism of the invention. The exogenous nucleic acid can be adapted to be integrated into the genome of the microorganism of the invention or to be maintained in an additional chromosomal state (eg, a plastid) in the microorganism of the invention.

"Enzymatic activity" or simply "activity" refers broadly to enzyme activity, including (but not limited to) the activity of an enzyme, the amount of enzyme, or the availability of enzyme-catalyzed reactions. Therefore, "increasing" enzyme activity includes increasing enzyme activity, increasing enzyme amount, or increasing the availability of enzyme-catalyzed reactions. Similarly, "reducing" enzyme activity includes reducing the activity of the enzyme, reducing the amount of the enzyme, or reducing the availability of the enzyme for a reaction catalyzed by the enzyme.

Regarding enzyme activity, the "substrate" is the molecule the enzyme acts on and the "product" is the molecule produced by the action of the enzyme. Therefore, a "natural substrate" is a molecule on which an enzyme in a wild-type microorganism naturally acts, and a "natural product" is a molecule on which an enzyme in a wild-type microorganism naturally acts. For example, butyl-CoA is a natural acceptor for Ptb and butyl phosphate and is a natural acceptor for Buk. In addition, butyryl phosphate is a natural product of Ptb and butyrate is a natural product of Buk. Likewise, an "unnatural substrate" is a molecule on which an enzyme does not naturally act in a wild-type microorganism, and an "unnatural product" is a molecule on which an enzyme does not naturally occur in a wild-type microorganism. Enzymes, whether natural or non-natural, that act on a variety of different substrates are often referred to as "hybrid" enzymes. The inventors have discovered that Ptb is hybrid and can accept a variety of acyl-CoA and enyl-CoA as acceptors, so that Ptb-Buk can be used to convert multiple acyl-CoA and enyl-CoA into their respective counterparts. acid or enoate, while producing ATP. Therefore, in a preferred embodiment, the Ptb-Buk of the present invention acts on a non-natural substrate (i.e., a substrate other than butyl-CoA and/or butyl phosphate) to produce a non-natural product (i.e., except butyl phosphate). products other than esters and/or butyrate esters).

The term "butyryl-CoA" may be used interchangeably herein with "butanoyl-CoA".

The term "capacity" or similar terms may be used interchangeably herein with "energy conservation" or similar terms. These terms are commonly used in the literature.

"Mutation" refers to a modification of a nucleic acid or protein in a microorganism of the invention compared to the wild-type or parent microorganism from which the microorganism of the invention arises. In one embodiment, the mutation may be a deletion, insertion, or substitution of the gene encoding the enzyme. In another embodiment, the mutation may be a deletion, insertion or substitution of one or more amino acids in the enzyme.

Specifically, "disruptive mutations" are mutations that reduce or eliminate (i.e., "interfere with") the expression or activity of a gene or enzyme. Breaking mutations can partially inactivate a gene or enzyme, completely inactivate it, or delete it. Breaking mutations can be knockout (KO) mutations. A disruptive mutation may be any mutation that reduces, prevents, or blocks the biosynthesis of a product produced by an enzyme. Disruptive mutations may include, for example, mutations in a gene encoding an enzyme, mutations in a genetic regulatory element involved in the expression of a gene encoding an enzyme, introduction of a nucleic acid that produces a protein that reduces or inhibits the activity of an enzyme, or introduction of a nucleic acid that inhibits the expression of an enzyme (e.g., reverse sense RNA, siRNA, CRISPR) or protein. Breaking mutations can be introduced using any method known in the art.

Introduction of disruptive mutations produces microorganisms of the invention that produce no target product, substantially no target product, or a reduced amount of the target product compared to the parent microorganism that produced the microorganism of the invention. For example, the microorganism of the invention may produce no target product or at least about 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50% less than the target product produced by the parent microorganism. , 60%, 70%, 80%, 90% or 95%. For example, the microorganisms of the invention can produce less than about 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0 g/L of the target product.

"Codon optimization" refers to the mutation of a nucleic acid, such as a gene, to optimize or improve translation of a particular strain or species of nucleic acid. Codon optimization can result in faster translation rates or higher translation accuracy. In a preferred embodiment, the gene of the present invention is codon-optimized for expression in Clostridium autoethanogenans, Clostridium autoethanologenum, Clostridium yongdahlii or Clostridium larsonii . In another preferred embodiment, the gene of the present invention is codon-optimized for performance in Clostridium autoethanologenum LZ1561, which is registered with DSMZ registration number DSM23693.

"Overexpression" refers to increased expression of nucleic acids or proteins in a microorganism of the invention compared to the wild-type or parent microorganism from which the microorganism of the invention arises. Overexpression can be achieved by any means known in the art, including altering gene copy number, gene transcription rate, gene translation rate, or enzyme degradation rate.

The term "variant" includes nucleic acids and proteins whose sequences differ from those of reference nucleic acids and proteins, such as those disclosed in the prior art or exemplified herein. The invention can be practiced using variant nucleic acids or proteins that function substantially the same as a reference nucleic acid or protein. For example, a variant protein may perform substantially the same function or catalyze substantially the same reaction as the reference protein. A variant gene may encode the same or substantially the same protein as the reference gene. A variant promoter can have substantially the same ability to drive expression of one or more genes as a reference promoter.

Such nucleic acids or proteins may be referred to herein as "functionally equivalent variants." For example, functionally equivalent variants of nucleic acids may include allelogenic variants, gene fragments, mutant genes, polymorphisms, and the like. Homologous genes from other microorganisms are also examples of functionally equivalent variants. It contains homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii or Clostridium yongdahl, details of which are publicly available on websites such as GenBank or NCBI. Functionally equivalent variants also include nucleic acids that result in sequence changes due to codon optimization for a particular microorganism. Functionally equivalent variants of nucleic acids preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or greater nucleic acid sequence identity (percent homology) with the reference nucleic acid. ). Functionally equivalent variants of a protein preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or greater amino acid identity (homology) with the reference protein. percentage). The functional equivalence of variant nucleic acids or proteins can be assessed using any method known in the art.

Nucleic acids can be delivered to the microorganisms of the invention using any method known in the art. For example, the nucleic acid may be delivered as naked nucleic acid, or may be formulated with one or more reagents, such as liposomes. The nucleic acid may be DNA, RNA, cDNA or a combination thereof, as appropriate. Limiting inhibitors may be used in certain embodiments. Other vectors may include plastids, viruses, phages, myxoplasts, and artificial chromosomes. In a preferred embodiment, plasmids are used to deliver nucleic acids to the microorganisms of the invention. For example, transformation (including transduction or transfection) can be achieved by electroporation, sonication, polyethylene glycol-mediated transformation, chemical or natural competence, protoplast transformation, prophage induction or conjugation. In certain embodiments with active restriction enzyme systems, it may be necessary to methylate the nucleic acid before introducing it into the microorganism.

In addition, nucleic acids can be designed to include regulatory elements, such as promoters, to enhance or otherwise control the expression of a particular nucleic acid. A promoter can be a constitutive promoter or an inducible promoter. Ideally, the promoter is a Wood-Ljungdahl pathway promoter, a ferredoxin promoter, a pyruvate:ferredoxin oxidoreductase promoter, a promoter of the Rnf complex operon, a promoter of the ATP synthase operon or the promoter of the phosphotransacetylase/acetate kinase operon.

"Microorganisms" are microscopic organisms, especially bacteria, archaea, viruses or fungi. The microorganisms of the present invention are usually bacteria. As used herein, the reference to "microorganisms" should be understood to include "bacteria."

A "parent microorganism" is a microorganism used to produce the microorganism of the invention. The parent microorganism can be a naturally occurring microorganism (ie, a wild-type microorganism) or a previously modified microorganism (ie, a mutant or recombinant microorganism). The bacteria of the invention can be modified to express or overexpress one or more enzymes that are not expressed or overexpressed in the parent microorganism. Similarly, microorganisms of the invention may be modified to contain one or more genes that are not present in the parent microorganism. Microorganisms of the invention may also be modified to express none or a lower amount of one or more of the enzymes expressed in the parent microorganism. In one embodiment, the parent microorganism is Clostridium autoethanogenogenum, Clostridium yongdahlii, or Clostridium larsonii. In a preferred embodiment, the parent microorganism is Clostridium autoethanogenogenum LZ1561, which is registered under DSMZ registration number DSM23693.

The term "derived from" indicates that a nucleic acid, protein or microorganism is modified or adapted from a different (eg, parent or wild-type) nucleic acid, protein or microorganism in order to produce a novel nucleic acid, protein or microorganism. Such modifications or adaptations typically include insertion, deletion, mutation or substitution of nucleic acids or genes. Generally speaking, the microorganisms of the invention are derived from a parent microorganism. In one embodiment, the microorganism of the present invention is derived from Clostridium autoethanogenogenum, Clostridium yongdahlii or Clostridium larsonii. In a preferred embodiment, the microorganism of the present invention is derived from Clostridium autoethanogenogenum LZ1561 registered under DSMZ registration number DSM23693.

The microorganisms of the present invention can be further classified based on functional characteristics. For example, the microorganisms of the present invention may be or may be derived from C1-fixing microorganisms, anaerobic bacteria, acetogens, ethanologens, carboxyl-oxidizing bacteria and/or methanotrophic bacteria. Table 1 provides a representative list of microorganisms and identifies their functional characteristics.

"C1" refers to a one-carbon molecule, such as CO, CO ₂ , CH ₄ or CH ₃ OH. "C1-oxygenate" refers to a carbon molecule that also includes at least one oxygen atom, such as CO, _CO2 , or _CH3OH . "C1--carbon source" refers to a carbon molecule that serves as part or the only carbon source for the microorganism of the present invention. For example, the C1 carbon source may include _one or more of CO, _CO2 , _CH4 , _CH3OH , or _CH2O2 . Preferably, the C1-carbon source includes one or both of CO and _CO2 . A "C1-fixing microorganism" is a microorganism capable of producing one or more products from a C1-carbon source. Typically, the microorganisms of the present invention are C1-fixing bacteria. In a preferred embodiment, the microorganism of the present invention is derived from the C1-fixed microorganism identified in Table 1.

"Anaerobic bacteria" are microorganisms that do not require oxygen to grow. If oxygen is present above a certain threshold, anaerobic bacteria can react negatively or even die. Typically, the microorganisms of the present invention are anaerobic bacteria. In a preferred embodiment, the microorganism of the present invention is derived from the anaerobic bacteria identified in Table 1.

"Acetogens" are microorganisms that produce or are capable of producing acetate (or acetic acid) as a product of anaerobic respiration. Typically, acetogens are absolutely anaerobic bacteria that use the Wood-Ljungdahl pathway as their primary mechanism for energy conservation and synthesis of acetyl-CoA and acetyl-CoA-derived products such as acetate (Ragsdale, " Biochemistry and Biophysics" Acta Sinica Sinica , 1784: 1873-1898, 2008). Acetogens use the acetyl-CoA pathway as (1) the mechanism to synthesize acetyl-CoA from CO ₂ reduction, (2) the final process of electron acceptance and energy conservation, and (3) fixation (assimilation) of synthesized cellular carbon The mechanism of CO ₂ (Drake, "Acetogenic Prokaryotes", in "Prokaryotes", 3rd edition, p. 354, New York, NY, 2006). All naturally occurring acetogens are C1-fixed, anaerobic, autotrophic and non-methanotrophic. Typically, the microorganisms of the present invention are acetogenic bacteria. In a preferred embodiment, the microorganism of the present invention is derived from the acetogenic bacteria identified in Table 1.

"Ethanologenic bacteria" are microorganisms that produce or are capable of producing ethanol. Typically, the microorganisms of the present invention are ethanologenic bacteria. In a preferred embodiment, the microorganism of the present invention is derived from the ethanologenic bacteria identified in Table 1.

"Autotrophs" are microorganisms that can grow in the absence of organic carbon. Instead, autotrophs use inorganic carbon sources such as CO and/or _CO2 . Typically, the microorganisms of the present invention are autotrophs. In a preferred embodiment, the microorganism of the present invention is derived from the autotrophic organism identified in Table 1.

"Carbon monoxide oxidizing bacteria" are microorganisms that can use CO as the sole carbon source. Typically, the microorganisms of the present invention are carbon monoxide oxidizing bacteria. In a preferred embodiment, the microorganism of the present invention is derived from the carboxy-oxidizing bacteria identified in Table 1.

"Methanotrophs" are microorganisms that can use methane as their sole source of carbon and energy. In certain embodiments, the microorganisms of the invention are derived from methanotrophs.

More broadly, the microorganisms of the invention may be derived from any genus or species identified in Table 1.

In a preferred embodiment, the microorganism of the present invention is derived from a cluster of Clostridium species including Clostridium autoethanologenum, Clostridium yongdahlii and Clostridium larsonii. These species were first identified by Abrini, Arch Microbiol, 161: 345-351, 1994 (Clostridium autoethanologenum), Tanner, Int J System Bacteriol , 43: 232-236, 1993 (Clostridium jungdalae) and Huhnke, WO 2008/028055 (Closobacter larsonii) reported and characterized.

These three species have many similarities. Specifically, these species are C1-fixed, anaerobic, acetogenic, ethanologenic, and carbon monoxide trophic members of the genus Clostridium. These species have similar genotypes, phenotypes, energy conservation and fermentation metabolism patterns. In addition, these species cluster in Clostridium rRNA homology group I with greater than 99% identity in 16S rRNA DNA, have a DNA G+C content of approximately 22-30 mol%, are Gram-positive, and have similar morphology and size (logarithmically growing cells between 0.5-0.7 × 3-5 μm), are mesophilic (optimally grown at 30-37°C), have a similar pH range of approximately 4-7.5 (where the optimal pH is approximately 5.5-6), does not possess cytochromes and conserves energy via the Rnf complex. Additionally, reduction of carboxylic acids to their corresponding alcohols has been demonstrated in these species (Perez, Biotechnol Bioeng , 110:1066-1077, 2012). Importantly, these species also all display strong autotrophic growth under CO-containing gases, produce ethanol and acetate (or acetic acid) as the main fermentation products, and produce small amounts of 2,3-butyrate under certain conditions. Glycol and lactic acid.

However, these three species also have many differences. These species were isolated from different sources: Clostridium autoethanologenum was isolated from rabbit intestines, Clostridium yongdahl was isolated from chicken house waste, and Clostridium lashanii was isolated from freshwater sediments. These species are present in various sugars (e.g. rhamnose, arabinose), acids (e.g. gluconate, citrate), amino acids (e.g. arginine, histidine) and other substrates (e.g. betaine , butanol) are different in their utilization. In addition, these species differ in their auxotrophy of certain vitamins (eg, thiamine, biotin). The species differ in the nucleic acid and amino acid sequences of the Wood-Ljungdahl pathway genes and proteins, but the general organization and number of these genes and proteins have been found to be the same in all species (Köpke, " New Insights in Biotechnology " , 22: 320-325, 2011).

Thus, in general, many of the characteristics of C. autoethanologenum, C. jungdahl, or C. larsonii are not specific to the species in question, but are C1-fixed, anaerobic, acetogenic, General characteristics of ethanologenic and carbonotrophic members of this cluster. However, because these species are actually distinct, genetic modifications or manipulations in one of these species may have different effects in another of these species. For example, differences in growth, potency, or product production may be observed.

The microorganisms of the present invention may also be derived from isolates or mutants of Clostridium ethanologenum, Clostridium yongdahlii or Clostridium larsonii. Isolates and mutants of C. autoethanogenum include JA1-1 (DSM10061) (Abrini, Archives of Microbiology, 161:345-351, 1994), LBS1560 (DSM19630) (WO 2009/064200) and LZ1561 (DSM23693 ). Isolates and mutants of Clostridium yongdahl include ATCC 49587 (Tanner, " International Journal of Systematic Bacteriology ", 43: 232-236, 1993), PETCT (DSM13528, ATCC 55383), ERI-2 (ATCC 55380) (US 5,593,886), C-01 (ATCC 55988) (US 6,368,819), O-52 (ATCC 55989) (US 6,368,819) and OTA-1 (Tirado-Acevedo, "Production of bioethanol from self-synthesis gas using Clostridium yungdalae" (Production of bioethanol from synthesis gas using Clostridium ljungdahlii)", "PhD thesis", North Carolina State University, 2010). Isolates and mutants of Clostridium larsonii include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008/028055).

However, in some embodiments, the microorganism of the present invention is a microorganism other than Clostridium autoethanologenum, Clostridium yongdahlii, or Clostridium larsonii. For example, the microorganism may be selected from the group consisting of: Escherichia coli, Saccharomyces cerevisiae, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharbutyricum , Clostridium saccharbutylicum, butyricum Clostridium difficile, Clostridium thetmocellum, Clostridium cellulolyticum, Clostridium cellulolyticum , Clostridium cellulolytica, Clostridium phytofermentans, Lactococcus reuteri, Bacillus subtilis, Bacillus licheniformis, Zymomonas mobilis , Klebsiella oxytoca , Klebsiella pneumoniae Klebsiella pneumonia , Corynebacterium glutamicum , Trichoderma reesei , Cupria hookworm, Pseudomonas putida , Lactobacillus plantarum and Methylobacterium extorquens .

"Substrate" refers to the carbon source and/or energy source of the microorganism of the present invention. Typically, the substrate is a gaseous substrate and includes a C1-carbon source, such as CO, _CO2 and/or _CH4 . Preferably, the substrate includes a C1-carbon source containing CO or CO+ _CO2 . The acceptor may further include other non-carbon components such as H ₂ , N ₂ or electrons.

The substrate generally includes at least some amount of CO, such as about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mol% CO. The substrate may include a range of CO, such as about 20-80, 30-70, or 40-60 mol% CO. Preferably, the substrate includes about 40-70 mol% CO (such as steel plant or blast furnace gas), about 20-30 mol% CO (such as basic oxygen converter gas), or about 15-45 mol% CO (such as syngas). In some embodiments, the substrate may include relatively low amounts of CO, such as about 1-10 or 1-20 mol% CO. The microorganisms of the present invention typically convert at least a portion of the CO in the substrate into products. In some embodiments, the substrate does not include or does not include substantially CO.

The substrate may include a certain amount of H ₂ . For example, the substrate may include about 1, 2, 5, 10, 15, 20, or 30 mol% _H2 . In some embodiments, the substrate may include relatively high amounts of _H2 , such as about 60, 70, 80, or 90 mol% _H2 . In other embodiments, the substrate does not include or does not include substantially _H2 .

The substrate may include a certain amount of CO ₂ . For example, the substrate may include about 1-80 or 1-30 mol% _CO2 . In some embodiments, the substrate may include less than about 20, 15, 10, or 5 mol% _CO2 . In another embodiment, the substrate does not include or does not include substantially _CO2 .

Although substrates are usually in the gaseous state, substrates may be provided in alternative forms. For example, the substrate can be dissolved in a CO gas-saturated liquid using a microbubble dispersion generator. For another example, the substrate can be adsorbed onto a solid support.

The substrate and/or C1-carbon source may be exhaust gas obtained as a by-product of an industrial process or from some other source such as smoke from automobile exhaust or biomass gasification. In certain embodiments, the industrial process is selected from the group consisting of: ferrous metal product manufacturing (such as steel plant manufacturing), non-ferrous product manufacturing, petroleum refining processes, coal gasification, electricity production, carbon black production, Ammonia production, methanol production and coke manufacturing. In these embodiments, the substrate and/or C1-carbon source may be captured from an industrial process using any convenient method before being emitted to the atmosphere.

The substrate and/or C1-carbon source may be a syngas, such as that obtained by gasification of coal or refinery residues, gasification of biomass or lignocellulosic materials, or reforming of natural gas. In another embodiment, syngas may be obtained from the gasification of municipal solid waste or industrial solid waste.

The composition of the substrate can have a significant impact on reaction efficiency and/or cost. For example, the presence of oxygen (O ₂ ) can reduce the efficiency of anaerobic fermentation processes. Depending on the composition of the substrate, it may be necessary to treat, scrub or filter the substrate to remove any undesirable impurities, such as toxins, undesirable components or dust particles and/or to increase the concentration of the desired components.

Microorganisms of the invention can be cultured to produce one or more products. For example, Clostridium ethanologenum produces or can be engineered to produce ethanol (WO 2007/117157), acetate (WO 2007/117157), butanol (WO 2008/115080 and WO 2012/053905), butanol (WO 2008/115080 and WO 2012/053905), butanol acid ester (WO 2008/115080), 2,3-butanediol (WO 2009/151342), lactic acid ester (WO 2011/112103), butene (WO 2012/024522), butadiene (WO 2012/024522 ), methyl ethyl ketone (2-butanone) (WO 2012/024522 and WO 2013/185123), ethylene (WO 2012/026833), acetone (WO 2012/115527), isopropyl alcohol (WO 2012/115527) , lipid (WO 2013/036147), 3-hydroxypropionate (3-HP) (WO 2013/180581), isoprene (WO 2013/180584), fatty acid (WO 2013/191567), 2-butanol (WO 2013/185123), 1,2-propanediol (WO 2014/0369152) and 1-propanol (WO 2014/0369152). In addition to one or more target products, the microorganisms of the invention can also produce ethanol, acetic acid and/or 2,3-butanediol. In certain embodiments, the microbial biomass itself can be considered the product.

"Natural products" are products produced by microorganisms that have not been genetically modified. , for example, ethanol, acetate, and 2,3-butanediol are natural products of Clostridium ethanologenum, Clostridium yongdahlii, and Clostridium larsonii. "Non-natural products" are products produced by a genetically modified microorganism other than the non-genetically modified microorganism that produced the genetically modified microorganism.

The terms "intermediate" and "precursor" as used interchangeably herein refer to molecular entities in the enzymatic pathway upstream of the observed or target product.

"Selectivity" refers to the ratio of the yield of the target product to the yield of all fermentation products produced by the microorganism. Microorganisms of the present invention can be engineered to produce products with specific selectivity or minimal selectivity. In one embodiment, the target product accounts for at least about 5%, 10%, 15%, 20%, 30%, 50% or 75% of all fermentation products produced by the microorganism of the invention. In one embodiment, the target product accounts for at least 10% of all fermentation products produced by the microorganism of the present invention, such that the selectivity of the microorganism of the present invention to the target product is at least 10%. In another embodiment, the target product accounts for at least 30% of all fermentation products produced by the microorganism of the present invention, such that the selectivity of the microorganism of the present invention to the target product is at least 30%.

The terms "increased efficiency" and the like include, but are not limited to, increasing growth rate, product production rate or volume, product volume per unit volume of substrate consumed, or product selectivity. Efficiency can be measured relative to the efficacy of the parent microorganism that produced the microorganism of the invention.

Typically, cultivation is performed in bioreactors. The term "bioreactor" includes culture/fermentation units consisting of one or more vessels, towers or pipe configurations, such as continuously stirred tank reactors (CSTR), immobilized cell reactors (ICR), trickle bed reactors (TBR), bubble column, air lift fermentation tank, static mixer or other containers or other devices suitable for gas-liquid contact. In some embodiments, a bioreactor may include a first growth reactor and a second culture/fermentation reactor. The substrate can be provided to one or both of these reactors. As used herein, the terms "culture" and "fermentation" are used interchangeably. These terms cover the growth phase and product biosynthesis phase of the culture/fermentation process.

Cultures are typically maintained in an aqueous medium containing sufficient nutrients, vitamins and/or minerals to allow growth of the microorganisms. Preferably, the aqueous medium is an anaerobic microbial growth medium, such as a minimal anaerobic microbial growth medium. Suitable media are well known in the art.

Culture/fermentation should advantageously be carried out under conditions suitable for the production of the target product. Typically, culture/fermentation is performed under anaerobic conditions. Reaction conditions that should be considered include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, medium pH, medium redox potential, stirring rate (if using a continuously stirred tank reactor), inoculum level, ensuring liquid phase neutralization. The maximum gas substrate concentration at which the gas does not become the limiting factor and the maximum product concentration at which product inhibition is avoided. In particular, the rate of substrate introduction can be controlled to ensure that the concentration of gas in the liquid phase does not become a limiting factor, since under gas-limiting conditions the culture may consume product.

Operating the bioreactor at high pressure allows for increased gas mass transfer rates from the gas phase to the liquid phase. Therefore, it is usually better to perform culture/fermentation at a pressure higher than atmospheric pressure. Furthermore, because a given gas conversion rate is in part a function of substrate residence time, and residence time dictates the required bioreactor volume, the use of a pressurized system can significantly reduce the required bioreactor volume, thus Reduce the capital cost of culture/fermentation equipment. This in turn means that the residence time (defined as the volume of liquid in the bioreactor divided by the input gas flow rate) can be reduced when the bioreactor is maintained at high pressure rather than atmospheric pressure. Optimal reaction conditions will depend in part on the specific microorganism used. Generally speaking, however, it is better to operate fermentation at a pressure above atmospheric pressure. In addition, since the given gas conversion rate is partly a function of the substrate residence time, and obtaining the required residence time also stipulates the required volume of the bioreactor, the use of a pressurized system can greatly reduce the required volume of the bioreactor. Therefore, the capital cost of fermentation equipment is reduced.

The target product may be isolated or purified from the fermentation broth using any method or combination of methods known in the art, including, for example, fractionation, evaporation, pervaporation, gas stripping, phase separation, and extractive fermentation, including, for example, liquid- liquid extraction. In certain embodiments, the target product is recovered from the fermentation broth by continuously removing a portion of the culture broth from the bioreactor, separating the microbial cells from the culture broth (conveniently performed by filtration), and self-cultivation Liquid recovery of one or more target products. The alcohol and/or acetone can be recovered, for example, by distillation. The acid can be recovered, for example, by adsorption onto activated carbon. The separated microbial cells are preferably returned to the bioreactor. It is also preferred to return the cell-free permeate remaining after removal of the target product to the bioreactor. Other nutrients, such as B vitamins, can be added to the cell-free permeate to replenish the culture medium before returning to the bioreactor.

Example

The following examples further illustrate the invention, but of course should not be construed as limiting its scope in any way.

Example 1

This example demonstrates the ability of Ptb-Buk to convert acetyl acetyl-CoA to acetyl acetate in vivo in E. coli and its use to produce acetone, isopropanol, 3-hydroxybutyrate and isobutylene.

The design and construction relied on the Ptb-Buk system's pathway to generate acetoacetate from acetoacetyl-CoA. This was performed in a modular fashion using the pDUET vector system (Novagen). One module contains the ptb-buk gene from C. beijerinckii NCIMB8052 (GenBank NC_009617, positions 232027..234147; Cbei_0203-204; NCBI-GeneID 5291437-38) on plastid pACYC. Another module contains the thiolase gene thlA of Clostridium acetobutylicum (GenBank NC_001988, position 82040..83218; CA_P0078; NCBI-GeneID 1116083) and the acetoacetic acid removal gene of Clostridium beijerinckii NCIMB8052 on plastid pCOLA. Carboxylase gene adc (GenBank NC_009617, positions 4401916..4402656; Cbei_3835; NCBI-GeneID 5294996). The ptb and buk genes were amplified from the genomic DNA of Clostridium beijerinckii NCIMB8052 and thlA and adc were amplified from the existing acetone plasmid pMTL85147-thlA-ctfAB-adc (WO 2012/115527), and were initiated by T7 present in the pDUET vector Selection was carried out via restriction-independent selection under sub-control using the Circular Polymerase Extension Selection (CPEC) method (Quan, PLoS One , 4: e6441, 2009).

Oligonucleotides used to amplify ptb and buk genes:

Oligonucleotides used to amplify thlA and adc genes:

After constructing plastids pACYC-ptb-buk (SEQ ID NO: 105) and pCOLA-thlA-adc (SEQ ID NO: 106), they were individually transformed together into E. coli BL21 (DE3) (Novagen), and in M9 minimal medium containing glucose was used in 1.5 mL cultures in 12-well dishes at 28°C with gyroscopic shaking at 160 rpm (Sambrook, Molecular Cloning: A Laboratory Manual, 3 Volume, Cold Spring Harbor Press, 1989) Growth experiments were performed in quadruplicate (Fig. 4). Cultures were inoculated at an OD600nm of 0.1 and induced with different concentrations of IPTG (0, 50, 100 μM) after 2 hours of growth. The plates were sealed with plate tape and the wells were pierced with a green-tipped needle to provide microaerobic conditions. Growth was allowed to proceed for an additional 64 hours of induction. The experiment was repeated three times.

Gas chromatography (GC) analysis was performed using Supelco polyethylene glycol (PEG) 60μm solid-phase microextraction fiber, Restek Rtx-1 (30m × 0.32μm × 5μm) column and flame ionization detector (FID). ), the Agilent 6890N headspace GC measures acetone concentration and the concentration of other metabolites (such as isobutylene). The sample (4 mL) was transferred to a 20-ml headspace vial, and the fibers were incubated (exposed) at 50°C for 10 minutes. The sample was allowed to desorb in a syringe at 250°C for 9 minutes. Chromatography was performed using the following oven program: 40°C (hold 5 min), and 10°C/min up to 200°C, followed by 5 min hold at 220°C. The column flow rate was 1 mL/min, and hydrogen was used as the carrier gas. The FID was maintained at 250°C with 40 ml/min hydrogen, 450 ml/min air and 15 ml/min nitrogen as supplementary gases.

The production of acetone in the strain harboring pACYC-ptb-buk and pCOLA-thlA-adc plastids (expressing thiolase, Ptb-Buk and acetoacetate decarboxylase) was immediately apparent. The average final acetone production was measured to be 0.19g/L, while in the plastid-free control, medium control and single plastid control pACYC-ptb-buk (expressing Ptb-Buk) or pCOLA-thlA-adc plastid (expressing thiolysis enzyme and acetoacetate decarboxylase), no acetone is produced (below the reliable detection limit). Uninduced cultures of strains harboring pACYC-ptb-buk and pCOLA-thlA-adc plasmids (expressing thiolase, Ptb-Buk and acetoacetate decarboxylase) did not produce significant amounts of acetone.

Average acetone production in E. coli BL21(DE3):

This experiment clearly demonstrates that Ptb-Buk can convert acetoacetyl-CoA to acetoacetate and can replace CoA-transferase or thioesterase for the production of acetone. Its use includes steps 1 and 2 in Figure 1. and 3 examples of pathways.

It is well known that isopropanol can be produced from acetone by adding primary:secondary alcohol dehydrogenases (Köpke, Applied and Environmental Microbiology , 80: 3394-3403, 2014) (step 4 in Figure 1), and isobutylene can be produced from acetone. Prepared by adding hydroxyisovalerate synthase (step 5 in Figure 1) and decarboxylase (step 6 in Figure 1) (van Leeuwen, Applied Microbiology and Biotechnology , 93: 1377-1387, 2012) . The following path can be constructed, which includes the genes thlA , ptb-buk and adc shown above and the first-level: secondary alcohol dehydrogenase gene (for example, GenBank accession number NC_022592, pos.609711..610766; CAETHG_0553; NCBI-GeneID : 17333984) an acetone pathway via Ptb-Buk, which pathway enables the production of isopropanol in E. coli via the Ptb-Buk system, which includes steps 1, 2, 3 and 4 of Figure 1 . Similarly, a pathway can be constructed that includes the genes shown above with genes thlA , ptb-buk and adc and hydroxyisovalerate synthase and decarboxylase to convert acetyl acetyl-CoA via Ptb-Buk. The acetone pathway of acetoacetate, which allows the production of isobutene in E. coli via the Ptb-Buk system, includes steps 1, 2, 3, 5 and 6 of Figure 1 . Acetoacetate can also be converted to 3-hydroxybutyrate via 3-hydroxybutyrate dehydrogenase Bdh. It can be used in combination with Ptb-Buk to convert acetyl acetyl-CoA to acetyl acetate to produce 3-hydroxybutyrate in strains expressing the genes thlA , ptb-buk and bdh , resulting in products including Figure 1 The path of steps 1, 2 and 15.

Example 2

This example demonstrates the ability of Ptb-Buk to convert acetoacetyl-CoA into acetoacetate in vivo in Clostridium autoethanologenum and the ability of Ptb-Buk to produce acetone, isopropanol, 3- Uses in hydroxybutyrate and isobutylene.

In order to demonstrate that the Ptb-Buk system can also synthesize acetone, isopropanol or isobutylene from gas substrates, the following plasmid was constructed, which contained the same gene as in Example 1 on a shuttle vector under the control of a Clostridial promoter, thl + ptb- buk + adc , such that the plastids may be expressed in acetogens such as Clostridium autoethanologenum, Clostridium yongdahlii or Clostridium larsonii .

The pMTL plasmid is a shuttle plastid system used to introduce circular DNA into Clostridium via E. coli conjugation (Heap, J Microbiol Methods , 78: 79-85, 2009). The genes of interest (i.e., hbd, phaB, thlA, ptb, buk, and aor1) were selected in the lacZ region of the plastids using common techniques in molecular biology, including DNA restriction digestion followed by conjugation, and in Golden gate DNA assembly technology is used when a DNA fragment is simultaneously selected and colonized in the plastid. The constructed plasmids were verified by DNA sequencing.

Production of acetone and isopropanol was previously demonstrated using the plasmid pMTL85147-thlA-ctfAB-adc (WO 2012/115527) encoding thl + ctfAB + adc under the control of a Clostridial promoter from the Wood-Ljungdahl gene cluster. in Clostridium ethanolum. In this plastid, the ctfAB gene encoding CoA transferase is directly replaced with the ptb -buk gene encoding the Ptb-Buk system. This was performed as described in Example 1 using the CPEC method. The resulting plasmid was pMTL85147-thlA-ptb-buk-adc.

The oligonucleotides used to amplify ptb-buk and select for colonization in pMTL8317-thl-ptb-buk-adc are described below.

Clostridium autoethanogenogens DSM10061 and DSM23693 (derivatives of DSM10061) were obtained from DSMZ (The German Collection of Microorganisms and Cell Cultures), Inhoffenstraße 7 B, 38124 Braunschweig, Germany.

strains using standard anaerobic techniques (Hungate, Meth Microbiol , 3B: 117-132, 1969; Wolfe, Adv Microb Physiol , 6: 107-146, 1971) Grow in PETC medium pH 5.6 at 37°C. 30 psi of steel plant gas containing CO (collected from New Zealand Steel site (Glenbrook, NZ)) or the same composition with 44% CO, 32% N ₂ , 22% CO ₂ , 2% H ₂ The synthetic gas blend serves as a substrate for autotrophic growth. For solid media, add 1.2% bacterial agar (BD, Franklin Lakes, NJ 07417, USA).

The construct is synthesized and subsequently transformed via conjugation into self-produced ethanol. To do this, the expression vector was first introduced into the conjugate donor strain E. coli HB101+R702 (CA434) using standard heat shock transformation (Williams, J Gen Microbiol , 1136:819-826, 1990) (donor). Donor cells were recovered in SOC medium (Sambrook, Molecular Selection: A Laboratory Manual, Vol. 3, Cold Spring Harbor Press, 1989) for 1 hour at 37°C and subsequently plated on plates containing 100 μg/ml Spectidium LB medium containing spectinomycin and 25 μg/ml chloramphenicol (Sambrook, "Molecular Selection: Laboratory Manual," Vol. 3, Cold Spring Harbor Press, 1989) was placed on the plate. Incubate LB plates at 37 °C overnight. The next day, inoculate several donor colonies with 5 ml aliquots of LB containing 100 μg/ml spectinomycin and 25 μg/ml chloramphenicol and incubate at 37°C, shaking for approximately 4 hours or until dense cultures are visible But it has not yet entered the stationary phase. Collect 1.5 ml of donor culture in a microcentrifuge tube by centrifugation at 4000 rpm for 2 minutes at room temperature and discard the supernatant. Gently resuspend the donor cells in 500 μl of sterile PBS buffer (Sambrook, Molecular Selection: A Laboratory Manual, Vol. 3, Cold Spring Harbor Press, 1989), centrifuge at 4000 rpm for 2 min and discard the PBS Clear liquid. The aggregate pellets were introduced into the anaerobic chamber and gently resuspended in 200 μl of late exponential phase C. autoethanogenum culture (recipient). The binding mixture (mixture of donor and recipient cells) was spotted on PETC-MES+fructose agar plates and allowed to dry. When moisture is no longer visible at the spots, the culture plates are introduced into a pressure tank pressurized with syngas to 25-30 psi and incubated at 37°C for approximately 24 hours. After 24 hours of incubation, remove the binding mixture from the culture plate by gently scraping with a 10 μl inoculation loop. The removed mixture was suspended in 200-300 μl PETC medium. Aliquots of 100 μl of the binding mixture were spread on agar plates of PETC medium supplemented with 15 μg/ml thiamphenicol to select for transformants with plasmids expressing chloramphenicol acetyl Transferase confers resistance to tetracycline.

Three unique colonies of Clostridium autoethanogenogens with pMTL85147-thlA-ptb-buk-adc plasmids were inoculated into 2 mL of PETC-MES medium containing 15 μg/ml of capricillin and shaken at 100 rpm at 37°C. Grow autotrophically for three days. The culture was diluted to OD _600nm = 0.05 in a serum-based bottle using 10 mL of PETC-MES containing 15 μg/ml formamycin, and grown autotrophically for five days at 37°C with gyroscopic shaking at 100 rpm. Samples were taken every day to measure biomass. and metabolites. At the same time, a control strain was examined, in which the expressoplast encoded only thl and adc under the control of the Wood-Ljungdahl cluster promoter and did not contain the ctfAB or ptb-buk genes that catalyze the formation of acetyl acetate from acetyl acetyl-CoA ( pMTL85147-thlA-adc). Cultures were sampled over five days to monitor metabolite and biomass accumulation.

Isopropyl alcohol concentration and the concentrations of ethanol, acetic acid, 2,3-butanediol and lactic acid were measured by high performance liquid chromatography (HPLC) on an Agilent LC at 35°C with refractive index (RI) detection. Prepare samples by diluting 400 μL of 5-sulfosalicylic acid solution (1 w/v% in 1 M sulfuric acid) with 100 μL of 5-sulfosalicylic acid solution (1 w/v% in 1 M sulfuric acid), followed by centrifugation at 14,000 rpm for 3 minutes; transfer the supernatant to a glass bottle for analysis . Separation was performed by injecting 10 μL onto an Alltech IOA-2000 column (150 mm × 6.5 mm × 8 μm) at 0.7 mL/min and 65°C under isocratic conditions using 5 mM sulfuric acid mobile phase.

In some cases, longer HPLC methods were used to improve peak separation. In this method, the concentrations of isopropanol, ethanol, acetate, 2,3-butanediol, and 3-hydroxybutyrate (which are not separated using the shorter method) are determined by high performance liquid chromatography (HPLC) in Measured on an Agilent 1260 Infinity LC at 35°C with refractive index (RI) detection. Prepare samples by diluting 400 μL of 5-sulfosalicylic acid solution (1 w/v% in 1 M sulfuric acid) with 100 μL of 5-sulfosalicylic acid solution (1 w/v% in 1 M sulfuric acid), followed by centrifugation at 14,000 rpm for 3 minutes; transfer the supernatant to a glass bottle for analysis . Separation was performed by injecting 10 μL onto an Aminex HPX-87H column (300 mm × 7.8 mm × 9 μm) at 0.6 mL/min and at 35°C under isocratic conditions using 5 mM sulfuric acid mobile phase.

C. autoethanogenum with pMTL85147-thlA-ptb-buk-adc produced up to 0.804 g of isopropanol IPA per gram of biomass, whereas the control C. autoethanogenum with pMTL85147-thlA-adc and no Ptb-Buk No IPA was produced (Fig. 12).

This experiment clearly demonstrates that Ptb-Buk is capable of performing the conversion of acetyl acetyl-CoA to acetyl acetate in the isopropanol pathway when using a gaseous substrate. Ptb-Buk can be used in place of CoA transferase or thioesterase in gas fermentation acetogens such as Clostridium autoethanogenogens, exemplified using a pathway including steps 1, 2, 3 and 4 of Figure 1 .

Clostridium autoethanogenum contains natural primary:secondary alcohol dehydrogenase enzymes that convert acetone to isopropyl alcohol (Köpke, Applied & Environmental Microbiology , 80: 3394-3403, 2014). It has been shown that deletion of this gene eliminates the conversion of acetone to isopropanol in Clostridium ethanologenum (WO 2015/085015). In the context of this gene knockout, the same genes including steps 1, 2 and 3 of Figure 1 can be used to generate acetone (instead of isopropanol) from the gas feed via the Ptb-Buk system. Adding hydroxyisovalerate synthase and decarboxylase genes to this strain (van Leeuwen, Applied Microbiology and Biotechnology , 93:1377-1387, 2012) would allow C. autoethanologenum or similar bacteria to Isobutylene is produced, which includes steps 1, 2, 3, 5 and 6 of Figure 1.

Acetoacetate can also be converted to 3-hydroxybutyrate via 3-hydroxybutyrate dehydrogenase Bdh. 3-Hydroxybutyrate dehydrogenase was identified in the genomes of Clostridium autoethanologenum (AGY75962) and other acetogens such as Clostridium yongdahl (ADK16920.1). This activity can be combined with Ptb-Buk (or CoA transferase) conversion of acetoacetyl-CoA to acetoacetate to produce 3- in strains expressing the genes thlA , ptb-buk (or ctfAB ), and bdh Hydroxybutyrate, resulting in a route including steps 1, 2 and 15 of Figure 1. Clostridium autoethanogenum has been shown to form low levels of 3-hydroxybutyrate (up to 2 g/L) via this pathway. These levels can be increased by overexpression of the Bdh gene, which is naturally expressed only at low levels.

In one experiment, C. autoethanogenum was transformed with plasmid pMTL82256-thlA-ctfAB as described in Example 2. Generation was monitored for 10 days from six biological replicates under autotrophic conditions as described in Example 2. The average value of 3-HB after 10 days was 1.86±0.14g/L. On day 10, 1,3-butanediol (from 3-HB) was produced with an average titer of 0.38 ± 0.05 g/L (Figure 37). No acetone or isopropyl alcohol is formed. This demonstrates that 3-HB can be efficiently produced with natural enzymes via acetoacetate.

In certain embodiments, it may be necessary to knock out or block the performance of 3-hydroxybutyrate dehydrogenase (such as Bdh) to prevent the consumption of carbon to obtain 3-HB, thereby increasing the product (such as acetone, isopropanol and isobutylene) Yield.

Example 3

This example demonstrates the in vivo conversion of (R)-3-hydroxybutyryl-CoA to (R)-3-hydroxybutyrate in E. coli by Ptb-Buk to prepare (R)-hydroxybutyrate, acetone, isopropyl alcohol or isobutylene.

The following pathway was designed and constructed to generate (R)-3-hydroxybutyrate from (R)-3-hydroxybutyryl-CoA relying on the Ptb-Buk system. Additionally, 3-hydroxybutyrate dehydrogenase (Bdh) can be used to convert (R)-3-HB to acetoacetate. Ralstonia picketii has been reported to have two 3-hydroxybutyrate dehydrogenases, Bdh1 and Bdh2, which can convert 3-hydroxybutyrate into acetyl acetate in vitro (Takanashi, " Biology and Biology" Journal of Engineering , 101: 501-507, 2006). Design a pathway that utilizes this enzyme to produce acetone (steps 1, 13, 14, 15, 3 in Figure 1), while simultaneously producing the reducing equivalents produced in (R)-3-hydroxybutyl-CoA and the production of Ptb -Recycling of ATP produced by Buk (Figure 6).

Pathways were constructed using the pDUET vector system (Novagen) in a modular manner. The two modules described in the above examples (pACYC-ptb-buk expressing Ptb-Buk and pCOLA-thlA-adc expressing thiolase and acetoacetate decarboxylase) were combined with two separate modules containing the hookworm Copper. An additional module (pCDF-phaB) for any (R)-specific 3-hydroxybutyrate dehydrogenase phaB (WP_010810131.1) and a 3-hydroxybutyrate dehydrogenase with Ralstonia picketii The module (pCDF-phaB-bdh1) of the enzyme bdh1 gene (BAE72684.1) is used together in the vector pCDF. Both phaB and bdh1 genes were synthesized from GeneArt and cloned under the control of the T7 promoter via restriction-independent selection using the circular polymerase extension selection (CPEC) method (Quan, PLoS 4: e6441, 2009).

Oligonucleotides used to amplify the bdh1 gene:

Oligonucleotides used to amplify the phaB gene:

Construct plasmids pACYC-ptb-buk (SEQ ID NO: 105), pCOLA-thlA-adc (SEQ ID NO: 106), pCDF-phaB (SEQ ID NO: 119) and pCDF-phaB-bdh1 (SEQ ID NO: 120), they were individually transformed and combined in Escherichia coli BL21 (DE3) (Novagen), and at 28°C, under gyroscopic shaking at 160 rpm, glucose-containing solution was used in a culture in a 1.5 mL 12-well plate. Growth experiments were performed in quadruplicate in M9 minimal medium. Cultures were inoculated at an OD600nm of 0.1 and induced with different concentrations of IPTG (0, 50, 100 μM) after 2 hours of growth. The plates were sealed with BioRad plate tape and each well was pierced with a green-tipped needle to provide microaerobic conditions. Growth was allowed to proceed for an additional 64 hours of induction. The experiment was repeated three times. Metabolites were measured as described in the previous example.

Cultures containing the combination of plastid pACYC-ptb-buk, pCOLA-thlA-adc and pCDF-phaB produced 1.65-2.4g/L (R)-3-hydroxybutyrate (depending on inducer level) and only Minimal amounts of by-products (Fig. 13A-F), demonstrating the efficiency of the Ptb-Buk system in converting (R)-3-hydroxybutyryl-CoA to (R)-3-hydroxybutyrate and supporting growth (Fig. 13A-F). In cultures that also expressed bdh1 (a combination containing plasmids pACYC-ptb-buk, pCOLA-thlA-adc, and pCDF-phaB-bdh1), only small amounts of (R)-3-hydroxybutyrate were found in the culture medium, 0.89-1.16g/L acetone (depending on inducer level) was also found, indicating that the bdh1 gene efficiently converts (R)-3-hydroxybutyrate to acetoacetate and further to acetone. No 3-hydroxybutyrate or acetone was found in all plastid combinations lacking Ptb-Buk (Figure 13A-F). Acetate levels were significantly higher in these cultures.

This experiment clearly demonstrates that Ptb-Buk can perform the conversion of (R)-3-hydroxybutyrate-CoA to 3-hydroxybutyrate, and that Bdh1 can be recycled to produce (R)-3-hydroxybutyryl-CoA. The resulting reducing equivalent further converts 3-hydroxybutyrate to acetyl acetate in vivo. The experiments also highlighted the ability of Ptb-Buk to support growth such that acetate production became unnecessary. The production of (R)-3-hydroxybutyrate formation is exemplified in the strains comprising steps 1, 13 and 14 of Figure 1. Acetone production is exemplified via a pathway including steps 1, 13, 14, 15 and 3 of Figure 1.

It is well known that isopropyl alcohol can be produced from acetone by adding primary:secondary alcohol dehydrogenases (step 4 in Figure 1) (Köpke, Applied and Environmental Microbiology , 80: 3394-3403, 2014), and that isobutylene can be produced from acetone. Prepared by adding hydroxyisovalerate synthase (step 5 in Figure 1) and decarboxylase (step 6 in Figure 1) (van Leeuwen, Appl Microbiol Biotechnology , 93: 1377-1387, 2012). The following path can be constructed, which includes the genes thlA , ptb-buk and adc shown above and the first-level: secondary alcohol dehydrogenase gene (for example, GenBank accession number NC_022592, pos.609711..610766; CAETHG_0553; NCBI-GeneID : 17333984) acetone pathway via Ptb-Buk, which pathway enables the production of isopropanol via the Ptb-Buk system in E. coli (steps 1, 13, 14, 15, 3 and 4 of Figure 1). Similarly, a pathway can be constructed that includes the acetone pathway shown above with the genes thlA, ptb-buk and adc and the genes for hydroxyisovalerate synthase and decarboxylase via Ptb-Buk, which pathway allows for Isobutylene is produced via the Ptb-Buk system in E. coli (steps 1, 13, 14, 15, 3, 5 and 6 of Figure 1).

Example 4

This example demonstrates the production of (R)-3-hydroxybutyrate and 1,3-butanediol from Clostridium ethanologenum. It also demonstrates the production of 1,3-butanediol in the absence of 2,3-butanediol.

A strain from Clostridium ethanologenum was constructed in which the natural pathway for 2,3-butanediol production was inactivated and the gene for (R)-3-hydroxybutyryl-CoA formation was replaced. This is achieved by using a thiolase ( thlA of Clostridium acetobutylicum; GenBank NC_001988, positions 82040..83218; CA_P0078; NCBI-GeneID 1116083) and (R)-specific 3-hydroxybutyrate dehydrogenase (Ancylostoma spp. The gene replacement of the acetyl lactate decarboxylase gene ( budA ) on the genome of Clostridium ethanologenum was achieved, and the strain budA::thlAphaB of Clostridium ethanologenum was obtained .

To replace the budA gene with the thlA and phaB genes, the plasmid pMTL8225-budA::thlA-phaB (Fig. 14) containing the E. coli toxin gene mazF (for counter selection) under the tet3n0 tetracycline-inducible promoter, and the budA gene The approximately 1 kb upstream homology arm, the thlA , phaB , ermB cassette flanked by the loxP site, and the approximately 1 kb downstream homology arm of the budA gene were assembled on plastid pMTL-tet3no.

Approximately 1 kb of the upstream and downstream homology arms of budA were PCR amplified from Clostridium autoethanogenogens using primers SN01/SN02 and SN07/SN08. The thlA and phaB genes were PCR amplified from the genomic DNA of Cupriaphila hookworm using primers SN03/SN04mod. The ermB cassette flanked by loxP sites was PCR amplified using primers SN05mod/SN06. The tet3no promoter flanked by FseI and Pmel was synthesized and treated with restriction enzymes FseI and Pmel and washed. The PCR product and digested vector were assembled using the GeneArt Seamless Selection Kit (from Life Technologies), and the plasmid pMTL8225-budA::thlA-phaB (SEQ ID NO: 121) without mutations in the insert was used as previously described The combined transformation of Clostridium ethanologenum is described in the Examples.

After combination and selection on trimethoprim and clarithromycin, 9 colonies were streaked twice on PETC-MES agar plates, in which clarithromycin and anhydrotetracycline were used to induce mazF gene expression. The budA gene from the clarithromycin and anhydrotetracycline community should have been replaced by thlA and phaB genes and the ermB cassette. This was tested by PCR using primers Og31f/Og32r flanked by homology arms and KAPA polymerase (Fig. 15).

Approximately 3.3 kb bands were amplified from the wild-type strain, while approximately 5.7 kb bands were amplified from communities 1, 4, 7, and 9, indicating that the budA gene was replaced by thlA , phaB , and ermB cassettes. The above events were further confirmed by sequencing the PCR products of all four pure lines. Under the obtained modification, the expression of th1A and phaB genes is driven through the promoter upstream of the budA gene.

Fermentation was performed with Clostridium autoethanologenum budA::thlA-phaB strain. Cultures were grown at 37°C under synthesis gas (50% CO, 18% CO ₂ , 2% H ₂ and 30% N ₂ ), which was continuously fed into the bioreactor. The gas flow rate was initially set at 50 ml/min and increased to 400 ml/min during the experiment, while stirring was increased from 200 to 500 rpm. Fermentation is carried out for approximately 5 days. Metabolites were measured as described in the examples above.

The concentration of 1,3-butanediol and other metabolites (such as 2-hydroxyisobutyric acid) was analyzed using gas chromatography (GC) using an Agilent CP-SIL 5CB-MS (50m×0.25μm×0.25μm). Agilent 6890N GC measurement of column, autosampler and flame ionization detector (FID). Samples were prepared by diluting 400 μL of sample with 400 μL of acetonitrile and centrifuging at 14,000 rpm for 3 minutes; transferring the supernatant to a glass bottle and drying the sample in a Thermo SpeedVac. After drying, the sample was then suspended in 400 μL of a solution of N,O-bistrifluoroacetamide (BSTFA) and pyridine (3:1 ratio) and heated in a sealed glass bottle at 60°C for 60 minutes. Samples were transferred to the autosampler for analysis using a 1 μL injection volume, a separation ratio of 30 to 1, and an inlet temperature of 250°C. Chromatography was performed using the following oven program: 70°C (no hold) to 3°C/min ramp to 110°C to 15°C/min ramp to 230°C, then 40°C/min final ramp to 310°C, hold for 3 minutes . The column flow rate was 1.8 mL/min, and helium was used as the carrier gas. The FID was maintained at 320°C with 40 ml/min hydrogen, 400 ml/min air and 20 ml/min helium as supplementary gases.

Unexpectedly, C. autoethanogenum budA::thlA-phaB strain expressing thlA and phaB produced as much as 1.55g/L 3-hydroxybutyrate from gas (Fig. 16). Natural thioesterase can convert the formed 3-hydroxybutyryl-CoA into 3-hydroxybutyrate. In the genome sequence, three putative thioesterases were identified.

Even more surprisingly, it was also found that along with 3-hydroxybutyrate formation, up to 150 mg/L of 1,3-butanediol was formed (Figure 16). This can be attributed to natural aldehydes: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase activities. Two AOR genes and several alcohol dehydrogenases are present in the genome of Clostridium autoethanogenogenum (Mock, Journal of Bacteriology , 197: 2965-2980, 2015). This reduction of 3-hydroxybutyrate is powered by reduced ferredoxin and can therefore be directly coupled to CO oxidation (CO+Fd _ox □CO ₂ +Fd _red ) providing reduced ferredoxin (Fig. 7 ).

It has also been shown that 1,3-BDO is produced from gas via an alternative pathway using butyraldehyde dehydrogenase Bld (AAP42563.1) (SEQ ID NO:80) from Clostridium glycoacetate. The bld gene was synthesized and colonized in plastid pMTL8315-Pfdx together with the same thiolase ( thlA from Clostridium acetobutylicum) and (R)-specific 3-hydroxybutyrate dehydrogenase ( phaB from Cupriaphila ancytoides). -thlA-phaB-bld (SEQ ID NO: 132). The Bld and phaB genes were amplified from the above plasmid using the primers in the table below and cloned into the existing plasmid pMTL85147-thlA (WO 2012/115527).

The resulting constructs were transformed into C. autoethanogenum as described above and cultured in 50-mL PETC medium at 30 psi with CO-containing steel plant gas (collected from the New Zealand Steel Station (Glenbrook, NZ)) or with 44% CO The growth experiment was carried out in a serum bottle pressurized by a synthetic gas mixture of the same composition, 32% N ₂ , 22% CO ₂ and 2% H ₂ .

Demonstrated production of 1,3-BDO from gases via this pathway (Figure 17A), but the yield was lower (up to 67 mg/L 1,3-BDO) than via the AOR pathway, and compared to the AOR pathway, when the bld gene was expressed Growth of C. autoethanogenogenum was affected compared to wild type (Fig. 17B).

In another experiment, C. autoethanogenum transformed with plasmid pMTL83159-phaB-thlA as described in Example 2 produced 0.33 and 0.46, respectively, in bottle experiments performed under autotrophic conditions as described in Example 2. g/L 3-HB (Figure 40).

Example 5

This example demonstrates the production of (S)-3-hydroxybutyrate and 1,3-butanediol from Clostridium ethanologenum.

Plasmids were constructed in which the ferredoxin promoter ( _Pfdx isolated from C. autoethanogenum; SEQ ID NO: 138) or the pyruvate-ferredoxin oxidoreductase promoter (P fdx isolated from C. autoethanogenum) was constructed. The bacterial isolate P _pfor ; SEQ ID NO: 139) exhibits thiolase ( thlA from Clostridium acetobutylicum; SEQ ID NO: 136) and (S)-specific 3-hydroxybutyrate dehydrogenase (from hbd1 of Clostridium cruzi; SEQ ID NO: 137). The following plasmid was constructed: P-hbd1-rbs2-thlA was assembled together and cloned into the pMTL83151 vector by the following conventional methods in molecular selection (Heap, Journal of Microbiological Methods , 78: 79-85, 2009) , including restriction enzyme digestion followed by ligation, overlap extension polymerase chain reaction, seamless cloning (Thermo Fisher Scientific), and GeneArt IIs (Thermo Fisher Scientific). The operon P-hbd1-rbs2-thlA was selected between the restriction sites NotI and XhoI found in multiple selection regions of the plastids. P is a constitutive promoter, which contains a complete ribosome binding site (rbs). rbs2 (SEQ ID NO: 140) is the ribosome binding site expressing thlA. The step-by-step procedure is to amplify P, hbd1 and thlA from existing templates with the primers listed below.

Polymerase chain reaction was performed using the Kapa Taq PCR kit (Kapa Biosystems) as follows. The annealing temperature was set at 56°C and the extension was 1 minute. Repeat the PCR reaction for 30 cycles. Afterwards, the PCR products were desalted using the DNA Clean & Concentrator Kit (Zymo Research Corporation).

pMTL83151 was prepared by NotI/XhoI double digestion using FastDigest NotI and FastDigest XhoI (Thermo Fisher Scientific) following the protocol provided, followed by alkaline phosphate treatment using FastAP alkaline phosphatase (Thermo Fisher Scientific) and the protocol provided Plastid backbone. Subsequently, the digested backbone was desalted using a DNA Clean & Concentrator Kit (Zymo Research Corporation).

GeneArt IIs type kit (Thermo Fisher Scientific) was used for assembly of PCR products and plastid backbones. The resulting plastids were then isolated from the E. coli plastid expression host using the QIAprep Spin Miniprep Kit (Qiagen).

To introduce the assembled plasmids pMTL8315-Pfdx-hbd1-thlA and pMTL8315-Ppfor-hbd1-thlA composed of the operon, the plastids were first introduced into E. coli CA434 strain by chemical transformation. Thereafter, by mixing the transformed CA434 strain with the Clostridium autoethanogenogen production host on solid LB-agar medium and in an anaerobic environment under pressure in the presence of a mixture consisting of carbon monoxide and hydrogen as described in Example 2 Cultivation is carried out in combination. C. autoethanogenans were selected after conjugation to remove remaining E. coli CA434 strains by continuous growth under anaerobic conditions on solid media containing appropriate antibiotics and trimethroprim.

The Clostridium autoethanogenum strain carrying the introduced pMTL8315-Pfdx-hbd1-thlA or pMTL8315-Ppfor-hbd1-thlA plasmid consisting of the operon P-hbd1-rbs2-thlA was tightly sealed with a rubber septum and sealed Lid and synthesized at 30 psi with CO containing steelworks gas (collected from New Zealand Steel Station (Glenbrook, NZ)) or the same composition with 44% CO, 32% N ₂ , 22% CO ₂ , 2% H ₂ The gas blend was grown in 10-mL PETC medium in a pressurized 250-mL Schott bottle. Metabolites were measured as described in the previous example.

Unexpectedly, 3-hydroxybutyrate was produced from gas in C. autoethanogenum cultures expressing thlA and hbd1 (Figure 18A). Natural thioesterase can convert the formed 3-hydroxybutyryl-CoA into 3-hydroxybutyrate. In the genome sequence, three putative thioesterases were identified. In the strain harboring pMTL8315-Pfdx-hbd1-thlA, up to 2.55 g/L 3-hydroxybutyrate was found (Figure 18A).

Even more strikingly, 3-hydroxybutyrate was also found to be converted to 1,3-butanediol over time, producing as much as 1.1g/ L 1,3-butanediol (Figure 18A). This can be attributed to natural aldehydes: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase activities. Two AOR genes and several alcohol dehydrogenases are present in the genome of Clostridium autoethanogenogenum (Mock, Journal of Bacteriology , 197: 2965-2980, 2015). This reduction of 3-hydroxybutyrate (and the reduction of acetate to ethanol; Figure 18B) is powered by reduced ferredoxin and thus can be coupled with the CO oxidation (CO+ _Fdox) that provides reduced ferredoxin. □CO ₂ +Fd _red ) direct coupling (Figure 7).

The same strain of C. autoethanogenans harboring plasmid pMTL8315-Pfdx-hbd1-thlA was also tested in continuous fermentation. Fermentation was performed as described in the previous example, but the culture was continuously varied with dilution rates with fresh medium, approximately 0.05 on day 2, and subsequently increased to 1.0 on day 3. High 3-hydroxybutyrate production of up to 7 g/L was observed, and 1,3-BDO production of 0.5 g/L.

In order to improve the production of (S)-3-hydroxybutyrate and 1,3-butanediol and avoid the synthesis of another form of butanediol (2,3-butanediol), plasmid pMTL-HBD-ThlA A strain with an inactivated 2,3-butanediol pathway in which the acetyl lactate decarboxylase gene BudA was deleted (U.S. 9,297,026) was introduced. This budA deletion removes the main pathway to obtain 2,3-BDO, thereby improving the specificity for 3-HB and 1,3-BDO production. When pMTL-HBD-ThlA was expressed in the budA deletion strain, a total of 15 mol% C was obtained for 3-HB and 1,3-BDO (Figure 41).

For comparison, in a strain expressing the same plasmid pMTL83159-hbd-thlA but without deletion of budA, the overall specificity for the production of 3-HB and 1,3-BDO at steady state was only 6.9%.

Example 6

This example shows that the Ptb-Buk system in Clostridium autoethanologenum is effective for a series of acyl-CoA, including acetoacetyl-CoA, 3-hydroxybutyryl-CoA and 2-hydroxyisobutyryl-CoA.

The Ptb-Buk system was expressed from plastids in Clostridium autoethanogenogenum and its activity was measured using a CoA hydrolysis assay. For this, the ptb-buk gene from C. beijerinckii NCIMB8052 (GenBank NC_009617, positions 232027..234147; Cbei_0203-204; NCBI-GeneID 5291437-38) was amplified from the genomic DNA of C. beijerinckii NCIMB8052, and By the following conventional method in molecular selection, the _pMTL83151 vector (Heap , Journal of Methods in Microbiology , 78:79-85, 2009), including restriction enzyme digestion followed by ligation, overlap extension polymerase chain reaction, seamless selection (Thermo Fisher Scientific) and GeneArt type IIs (Thermo Fisher Scientific). Oligonucleotides are described below.

The resulting plasmid pMTL82256-ptb-buk (SEQ ID NO: 153) was introduced into C. autoethanogenans as described in the previous example.

Carboxyl-CoA hydrolysis analysis was performed as follows. OD 2 (late exponential phase) C. autoethanogenic cells were collected by centrifugation (14,000 rpm, 1 min at 4°C). Cells were resuspended in 500 μl of lysis buffer (potassium phosphate buffer, pH 8). Lyse cells by sonicating on ice for 6 × 30 seconds at an amplitude of 20 using a freeze-thaw cycle (optional). The sample was centrifuged at 14,000 rpm for 10 minutes at 4°C and the supernatant with soluble protein was removed. Measure protein concentration, e.g. using Bradford assay.

The assay mixture contained: 484 μl potassium phosphate buffer (pH 8.0), 1 μl DTNB (final concentration 0.1 mM), 10 μl cell lysate, and 5 μl CoA (final concentration 500 μM). All components, except protein, were mixed in a quartz colorimetric tube (1 ml colorimetric tube, 1 cm reading length). The analysis was started by adding cell lysates, followed by reaction in a spectrophotometer at 405 nm for 3 minutes at 30°C. A control without dissolved matter was run to measure the autodecomposition of acyl-CoA.

To determine activity, the slope of the linear portion of the curve (usually the first 30 seconds) is calculated. Normalize the protein amount and divide the slope by the protein amount. Specific activity (M/s/mg) was calculated using the extinction coefficient (14,150M ^-1 cm ^-1 ). Subtract the activity of the negative control.

The analysis was performed with acetoacetyl-CoA, a racemic mixture of 3-hydroxybutyryl-CoA (3-HB-CoA) and 2-hydroxyisobutyryl-CoA (2-HB-CoA). The possibility of artificially low hydrolysis rates of 3-HB-CoA and 2-HIB-CoA due to potential substrate limitations was investigated by using different concentrations (500 μM and 200 μM) of acyl-CoA in autoethanogenic Clostridium lysates. Repeat analysis to resolve.

The analysis results show that for a series of acyl-CoA (including acetyl-acetyl-CoA, 3-hydroxybutyl-CoA and 2-hydroxyisobutyl-CoA), the pMTL82256-ptb-bearing plasmid representing the Ptb-Buk system CoA hydrolysis was significantly increased in lysates of C. autoethanogenogenum buk (Figure 20A-B). It is worth noting that CoA hydrolysis also occurs for acyl-CoA in the form of 2-hydroxyisobutyl-CoA, which is not hydrolyzed by wild-type C. autoethanologenum. Some native CoA hydrolytic activity was observed for acetoacetyl-CoA and 3-hydroxybutyryl-CoA.

Example 7

This example demonstrates that disruption of the identified native thioesterase gene improves Ptb by increasing the pool of available acyl-CoA, such as acetyl-acetyl-CoA, 3-hydroxybutyl-CoA, or 2-hydroxyisobutyl-CoA. -Efficiency of Buk and CoA transferase systems.

In contrast to the Ptb-Buk system where energy is conserved in the form of ATP during the conversion of acyl-CoA to its respective acid, energy is not conserved if CoA is simply hydrolyzed.

In the hydrolase analysis, it was found that Clostridium autoethanologenum has natural hydrolytic activity for acetoacetyl-CoA and 3-hydroxybutyryl-CoA.

Hydrolysis of Acetyl-CoA with Acetyl-CoA, a racemic mixture of 3-hydroxybutyl-CoA (3-HB-CoA) and 2-hydroxyisobutyl-CoA (2-HB-CoA) The analysis was performed as described in the previous example. The analysis showed cleavage of acetoacetyl-CoA and 3-HB-CoA, but not 2-HIB-CoA, and confirmed that the native activity is present in C. autoethanologenum (Figure 11).

Analysis of the genome of Clostridium autoethanologenum identified three putative CoA-thioesterases (thioester-hydrolases) that can cause cleavage of acetoacetyl-CoA or 3-hydroxybutyryl-CoA thioester bonds. It is also present in other acetogenic bacteria, such as Clostridium yungdalae.

Inactivation of these three putative CoA thioesterases resulted in higher product titers, thereby improving the efficiency of the Ptb-Buk system. Three putative thioesterases were inactivated using ClosTron technology. Briefly, the targeting domain of type II Ltr was reprogrammed using the ClosTron website and the retargeting ClosTron plasmid was ordered from DNA 2.0. The ClosTron knockout vector pMTL007C-E2-Cau-2640-571s (CAETHG_0718) targeting thioesterase 1, pMTL007C-E2-PBor3782-166s (CAETHG_1524) targeting thioesterase 2 and thioesterase 3 were used. pMTL007C-E2-PBor4039-199s (CAETHG_1780) was introduced into Clostridium autoethanogenogenum.

Selection for integration was performed by selecting PETC supplemented with 5 μg/ml clarithromycin and successful inactivation by integrating the type II intron was confirmed by PCR across the insertion site.

The assay described above was used to measure CoA hydrolase activity of acetyl-CoA against wild-type C. autoethanogenogenum and C. autoethanogenogenum with one of the putative genes inactivated. It was shown that all three strains with inactivated putative thioesterases exhibited lower hydrolytic activity towards acetyl acetyl-CoA and 3-hydroxybutyl-CoA (Figure 21A-B).

To demonstrate that reduced CoA hydrolase activity and thus increased acetyl-CoA pooling is beneficial for the production of acetyl-CoA derivatives, the isopropanol plasmid pMTL85147-thlA- encoding thl + ctfAB + adc ctfAB-adc (WO 2012/115527) was introduced into a wild-type C. autoethanogenum strain and a strain with inactivated thioesterase 1. Growth experiments were performed in technical triplicate with Co gas in 40 ml PETC medium in 1 L Schott bottles at 37 °C with shaking at 110 rpm. Synthesis gas (50% CO, 18% CO ₂ , 2% H ₂ and 30% N ₂ ) is used as the sole energy and carbon source. A headspace exchange was performed and aerated to 21 psi (1.5 bar) at 37°C under synthesis gas (50% CO, 18% _CO2 , 2% _H2 and 30% _N2 ). Samples for OD and analysis were collected twice a day.

The strain with inactivated thioesterase 3 CAETHG_1780 produced significantly higher levels of isopropyl alcohol compared to the wild type (Figure 22 and Figure 23A-D).

Similarly, deletion of thioesterase in Clostridium autoethanogenum will increase the recruitment of 3-hydroxybutyl-CoA, allowing Ptb-Buk to more efficiently utilize 3-hydroxybutyl-CoA and induce acetone, isopropanol, isobutylene , higher production of (R)-3-hydroxybutyrate, 1,3-butanediol and/or 2-hydroxyisobutyric acid. When pMTL8315-Pfdx-hbd1-thlA of Example 5 was introduced into a C. autoethanogenum strain with disrupted thioesterase 2CAETHG_1524, 3-hydroxybutyrate synthesis was eliminated (compared to when in C. autoethanogenum wild-type As much as 2.55g/L 3-hydroxybutyrate) was found in strains expressing this plasmid. There is no competitive activity for 3-hydroxybutyl-CoA in this strain.

These results demonstrate that by reducing thioesterase activity, higher CoA pooling of the Ptb-Buk system and product synthesis can be obtained.

In addition, the production of 3-HB and 1,3-BDO can be increased by overexpression of Ptb-Buk. In a control experiment, C. autoethanologenum was transformed with plasmid pMTL83159-phaB-thlA from Example 4 plus pMTL82256 as described in Example 2 (Heap, Journal of Methods in Microbiology , 78: 79-85, 2009), in which pMTL82256 is an empty plasmid used as a background control. Fermentation of this strain resulted in the production of 1.68g/L 3-HB at the highest efficiency on day 10 (Figure 42A). When pMTL82256-buk-ptb replaced empty plasmid pMTL82256 and pMTL83159-phaB-thlA was co-expressed in Clostridium autoethanogenogenum, the fermentation produced a higher titer of 3-HB of 4.76g/L at an earlier time - day 4 (Figure 42B).

Deletion of the native thioesterase improves the efficiency of the Ptb-Buk system, which prefers (R)-3-HB-CoA. The locus of the thioesterase gene in the genome was deleted and replaced with a buk-ptb DNA fragment via a common molecular biology technique called homologous recombination. Replacement of the thioesterase gene by buk-ptb was confirmed by PCR, followed by agarose gel electrophoresis and DNA sequencing.

In bottle experiments, when expressing pMTL83156-phaB-thlA but not ptb-buk in the above thioesterase deletion mutant, the average maximum titer of 3-HB produced was 0.50±0.05g/L, similar to Titers obtained using unmodified C. ethanologenum strains. When pMTL82256-buk-ptb and pMTL83156-phaB-thlA plasmids were co-expressed in the thioesterase gene knockout strain, the production of 3-HB increased to 1.29±0.10g/L (Figure 43).

Example 8

This example demonstrates that the Ptb-buk system can be used to remove the acetogenic bacteria Clostridium ethanologenum from the acetate-producing system.

All acetogenic microorganisms have been described to produce acetate (Drake, "Acetogenic Prokaryotes," in Prokaryotes , 3rd ed., pp. 354-420, New York, NY, Springer, 2006) because The production of acetate provides microorganisms with the option of directly producing ATP from phosphorylation at the substrate level via Pta (phosphotransacetylase) and Ack (phosphotransacetylase-acetate kinase). Therefore, natural acetate-forming enzymes such as Pta-Ack are believed to be essential in acetogens (Nagarajan, Microbial Cell Factory , 12:118, 2013). Since Ptb-Buk provides an alternative way to generate energy, Ptb-Buk can be used to replace the natural Pta-Ack system.

The pta and ack genes in Clostridium autoethanogenogens are in one operon. In order to replace the pta and ack genes with the ptb and buk genes, the plasmid pMTL8225-pta-ack::ptb-buk (Fig. 24) containing the mazF counter-selection marker under the tetracycline-inducible promoter and about 1 kb of the upstream homology arm was , loxP site flanked by ptb , buk , ermB cassette and about 1kb downstream homology arm assembly (SEQ ID NO: 160).

Approximately 1 kb of upstream and downstream homology arms were PCR amplified from Clostridium autoethanogenogens using primers SN22f/SN23r and SN28f/SN29r. The ptb and buk genes were PCR amplified from pIPA_16 plasmid using primers SN24f/SN25r. The ermB cassette with loxP site was PCR amplified using primers SN26f/SN27r. The plastid backbone was PCR amplified using primers SN30f/SN31r. KAPA polymerase was used for all PCR amplifications. PCR products were assembled using the GeneArt seamless cloning kit (from Life Technologies), and C. ethanologenum was transformed by conjugation as previously described using plasmids without mutations in the insert.

After combination and selection on trimethoprim and clarithromycin, 7 colonies were streaked twice on PETC-MES agar plates, in which clarithromycin and anhydrotetracycline were used to induce mazF gene expression. The pta and ack genes from the clarithromycin and anhydrotetracycline communities should have been replaced by the ptb and buk genes and the ermB cassette. This was tested by PCR using primers Og29f/Og30r flanked by homology arms and KAPA polymerase (Figure 25). The approximately 4.6 kb band was amplified from the wild-type strain, while the approximately 5.7 kb band was amplified from communities 1 and 4-7, indicating that the pta and ack genes were replaced by the ptb and buk genes and the ermB cassette. The above events were further confirmed by sequencing the PCR products from pure lines 4-7.

Under the resulting modification, the expression of ptb and buk genes is driven by the promoter upstream of the pta gene.

The strain obtained by transforming the isopropanol-producing plasmid pMTL85147-thlA-adc of Example 2 was pta-ack::ptb-buk from Clostridium ethanologenum as described above, in which the pta-ack operon was replaced by the ptb-buk operon. Growth studies were performed under autotrophic conditions and metabolic end products were analyzed. No acetate production was observed, but in addition to ethanol and 2,3-butanediol, isopropanol (up to 0.355g/L) and 3-HB (up to 0.29g/L) were still produced (Figure 39A and 39B). This demonstrates the use of a Ptb-Buk system to produce isopropanol and 3-HB from the gaseous substrates CO and/or _CO and _H without the production of acetate.

If acetone instead of isopropanol is the target product, the primary:secondary alcohol dehydrogenase gene (SEQ ID NO: 17) can be produced from this strain using the method described above and detailed in WO 2015/085015 Clostridium ethanolum pta-ack::ptb-buk was further eliminated. Introduction of plasmid pMTL85147-thlA-adc into this strain resulted in the production of acetone at similar levels as described above for isopropanol, without co-production of acetate. Ethanol, 2,3-butanediol and 3-HB can be further products.

These products can also be eliminated by further deletion, for example, deleting the acetolactate decarboxylase gene BudA to obtain a strain that cannot produce 2,3-butanediol (U.S. 9,297,026). 3-HB production can be reduced or eliminated by deleting the 3-hydroxybutyrate dehydrogenase gene Bdh (SEQ ID NO: 62).

Example 9

This example demonstrates improved conversion of 3-hydroxybutyrate to 1,3-BDO by overexpression of the aldehyde:ferredoxin oxidoreductase gene aor1 .

Overexpression of the C. autoethanogenogenum aor1 gene using the pMTL82251 plastid backbone. The pMTL82251 plasmid was chosen because it has a different origin of replication and antibiotic markers and can be co-expressed with the plasmid used in Example 5 containing hbd1 and thlA . The preparation and assembly reaction of the plastid backbone followed the above procedures. First, the Clostridium autoethanogenum ferredoxin promoter was introduced into plastid pMTL82251 to generate plastid pMTL82256, and then the aor1 gene was added to form plastid pMTL82256- aor1. Use the following primer.

After the resulting plasmid pMTL82256-aor1 was transformed into E. coli CA434 strain, conjugation was performed to the previous C. ethanologenum 1,3-BDO production host. Thus, under different replication sources and selection markers, the resulting C. autoethanogenogens strains harbored two plasmids, one for overexpression of hbd1 and thlA and the other for overexpression of aor1 . Follow the above procedure to characterize and quantify the production of 1,3-BDO.

The results clearly demonstrate that 1,3-BDO production can be improved by overexpression of aor1 . Likewise, other aldehyde:ferredoxin oxidoreductase genes may be expressed in C. autoethanogenum to promote the conversion of 3-hydroxybutyrate to 1,3-butanediol.

To improve 1,3-BDO production, AOR is overexpressed to improve the conversion of 3-HB to 3-HB-aldehyde. To do this, pMTL82256-hbd-thlA and pMTL83159-aor1 were co-expressed in C. autoethanogenogenum. Compared with the strain carrying only pMTL82256-hbd-thlA, the aor1 co-expressing strain produced more ethanol and 1,3-BDO (Figure 44).

Example 10

This example demonstrates the stereospecificity of Ptb-Buk that allows the production of 2-hydroxyisobutyric acid without generating undesired by-products.

2-Hydroxyisobutyric acid can be converted into Escherichia coli and Clostridium autoethanologenum by introducing thiolase and 3-hydroxybutyryl-CoA dehydrogenase that convert acetyl-CoA into 3-hydroxybutyryl-CoA. 2-Hydroxyisobutyl-CoA mutase converts 3-hydroxybutyl-CoA into 2-hydroxyisobutyl-CoA and an enzyme that hydrolyzes CoA to form 2-hydroxyisobutyric acid. The 3-hydroxybutyryl-CoA dehydrogenase may be (R) or (S)-specific, and the enzyme converts 2-hydroxyisobutyl-CoA to 2 according to steps 1, 13, 19 and 20 of Figure 1 -Hydroxybutyrate. This last step can be performed via thioesterase or Ptb-Buk systems.

Three potential candidate genes, Escherichia coli thioesterase type II TesB, Clostridium autoethanologenum phosphoacetyltransferase/acetate kinase pair, and Clostridium beijerinckii butyryltransferase/butyrate kinase pair, were cloned using the above method and the following primers. in the E. coli pDUET T7 expression vector.

The obtained plasmids pDUET-pta-ack (SEQ ID NO: 185), pDUET-ptb-buk (SEQ ID NO: 186), and pDUET-tesB (SEQ ID NO: 187) were introduced into E. coli BL21 (DE3) for expression. , and then measured its activity towards acetyl acetyl-CoA, 3-hydroxybutyl-CoA and 2-hydroxyisobutyl-CoA. The results are shown in Figure 27. E. coli BL21 has small but measurable amounts of activity on all three substrates. Pta-Ack produced no activity above background, while thioesterases TesB and Ptb-Buk showed high activity against all three substrates (including 2-hydroxyisobutyl-CoA).

Thioesterases TesB and Ptb-Buk are more active towards linear acetyl acetyl-CoA and 3-hydroxybutyl-CoA than branched chain 2-hydroxyisobutyl-CoA. This creates a problem in the pathway as it causes the pathway to end prematurely at 3-hydroxybutyl-CoA, especially when the activity is higher than the mutase activity towards 2-hydroxyisobutyl-CoA.

However, in contrast to thioesterases, Ptb-Buk is able to distinguish between stereoisomers and acts only (or preferentially) on (R)-3-hydroxybutyl-CoA rather than (S)-3-hydroxybutyl-CoA. CoA. This was demonstrated by expressing the Ptb-Buk system in E. coli and ThlA and (S)-specific Hbd (Figure 28A) or (R)-specific phaB (Figure 28B) in the pDuet system. Constructs were constructed as described in Examples 1 and 3. Growth studies confirmed the formation of significant amounts of 3-hydroxybutyrate only when Ptb-Buk was expressed in combination with (S)-specific Hbd but not (R)-specific phaB.

Therefore, the pathway via (S)-specific 3-hydroxybutyl-CoA dehydrogenase and Ptb-Buk offers significant advantages because the Ptb-Buk system (unlike thioesterases) CoA is inactive, but (S)-3-hydroxybutyl-CoA is also the preferred isomer of 2-hydroxyisobutyl-CoA mutase (Yaueva, Journal of Biochemistry , 287: 15502-15511, 2012) . The produced 2-hydroxyisobutyl-CoA can then be used via Ptb-Buk to produce 2-hydroxyisobutyric acid and (unlike thioesterases) hydrolysis of 2-hydroxyisobutyl-CoA provides additional energy (Figure 8).

Modular constructs are designed to compare the performance of paths. A gene cassette containing the Wood-Ljnngdahl promoter in front of the genes meaB , hcmA and hcmB was codon optimized and synthesized (SEQ ID NO: 188). HcmA and hcmB encode 2-hydroxyisobutyl-CoA mutase and meaB encodes a chaperone protein from three-carbon Proteobacteria. In the construct, the hcmA and meaB genes are in the form of one protein (SEQ ID NO: 189) fused together (Yaneva, Journal of Biochemistry , 287:15502-15511, 2012). The gene cassette was selected using the restriction enzymes KpnI and NcoI to contain thiolase ( thlA from Clostridium acetobutylicum; SEQ ID NO: 136) and (S)-specific 3-hydroxybutyrate dehydrogenase (from hbd of Clostridium acetobutylicum; SEQ ID NO: 190) plasmid (pMTL83155-thlA-hbd) or containing (R)-specific 3-hydroxybutyric acid (from R. eutropha ) of phaB ) in the plasmid (pMTL83155-thlA-phaB), pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB (SEQ ID NO: 191) and pMTL83155-thlA-phaB-Pwl-meaBhcmA-hcmB were formed respectively. (SEQ ID NO: 192). Colonization of the codon-optimized 2-hydroxyisobutyl-CoA mutase cassette into E. coli Top-10 was successful only after some initial colonization complications; discovery of the 2-hydroxyisobutyl-CoA mutase cassette The enzyme cassette can only be colonized in plastids at a lower temperature (28°C).

By first amplifying the promoter region of phosphoacetyltransferase (SEQ ID NO: 193) from Clostridium ethanologenum, and using NotI and NdeI restriction sites to select and colonize the vector pMTL83151 (FJ797647.1; Heap, " Journal of Microbiological Methods , 78: 79-85, 2009), the genes thlA and hbd or correspondingly the phaB generation vectors pMTL83155-thlA-hbd and pMTL83155-thlA-phaB were subsequently introduced via NdeI and KpnI in a double ligation reaction.

In addition, construct compatible plasmid modules for representing ptb-buk or tesB . For this, the respective genes were amplified from genomic DNA and introduced into the plastid pMTL82256 as described in Example 9, followed by introduction of ptb-buk or phaB using NdeI and NcoI and the Seamless Selection Kit (Life technologies) to form plastids pMTL82256-ptb-buk (SEQ ID NO: 194) and pMTL82256-tesB (SEQ ID NO: 195).

Plasmids pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB, pMTL83155-thlA-phaB-Pwl-meaBhcmA-hcmB, pMTL82256-ptb-buk and pMTL82256-tesB were introduced into the large intestine by transformation in the following combinations as described in the previous examples Bacillus Top-10 (all steps at 28°C) and Clostridium autoethanogenum: pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB+pMTL82256-ptb-buk, pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB+ pMTL82256-tesB, pMTL83155-thlA-phaB-Pwl-meaBhcmA-hcmB+pMTL82256-ptb-buk and pMTL83155-thlA-phaB-Pwl-meaBhcmA-hcmB+pMTL82256-tesB.

Growth experiments were performed with E. coli in LB medium at 30°C for 4 days and in the presence of 30 psi of CO-containing steelworks gas (collected from the New Zealand Steel Station (Glenbrook, NZ)) at 30 and 37°C. Clostridium ethanologenum was grown in PETC medium for 6 days. Metabolites were measured as described above. In addition to measurement by GC-MS, liquid chromatography tandem mass spectrometry (LC-MS/MS) and ¹ H nuclear magnetic resonance (NMR) spectroscopy were also used to confirm the production of 2-hydroxyisobutyric acid.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) data were acquired on a Dionex UltiMate 3000 liquid chromatography system (Dionex, California, USA) coupled to an ABSciex 4000 QTRAP mass spectrometer (ABSciex, Concord, Canada). The liquid chromatography system was controlled by Chromeleon software (Dionex), and the chromatography separation was performed by a Gemini-NX C18 150mm×2mm I.D., 3μm 110Å particle column equipped with a pre-column Security Guard Gemini-NX C18 4mm×2mm I.D. column. (Phenomenex, Aschaffenburg, Germany) was achieved by injecting 10 μL. Control the column oven temperature and maintain it at 55°C during the entire collection, and the mobile phase is as follows: 7.5mM tributylamine aqueous solution (eluent A) adjusted to pH 4.95 (±0.05) with glacial acetic acid and acetonitrile (eluent B) ). The mobile phase flow rate was maintained at 300 μL/min throughout the gradient profile and introduced directly into the mass spectrometer without separation. The mass spectrometer was controlled by Analyst 1.5.2 software (ABSciex) and was equipped with a TurboV electrospray source operating in negative ionization mode. Predetermined multiple reaction monitoring (MRM) data were collected using the following previously optimized (and therefore universal) parameters: ion spray voltage -4500V, spray (GS1), auxiliary (GS2), curtain (CUR) gas and collision (CAD ) gases are 60, 60, 20 and the average (arbitrary unit), respectively, generated by an N300DR nitrogen generator (Peak Scientific, Massachusetts, USA). The additive gas temperature is maintained at 350°C. The entrance potential (EP) is -10 volts. This method can also detect and separate 2-hydroxybutyric acid.

The field strength of ¹ H nuclear magnetic resonance (NMR) spectroscopy is 400MHz. Samples were prepared by diluting 400 μL of sample with 400 μL of 20 mM phosphate buffer prepared with D ₂ O and containing trimethylsilylpropionic acid (TMSP) as internal standard (pH 7). The samples were then transferred to glass NMR tubes (5 mm x 8 inches) and analyzed by ¹ H NMR at 27°C with a 30° excitation pulse, 15 sec relaxation delay and 64 scans using presaturation for water suppression. analyze. After acquisition, the spectra were converted, flattened and integrated using Agilent VnmrJ software. Quantification of 2-hydroxyisobutyric acid was performed using TMSP of known concentration using resonance at 1.36 ppm (single peak).

In heterotrophically growing E. coli and in autotrophically growing C. autoethanogenum, the construct pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB+pMTL82256-tesB (in C. autoethanogenum, in LC- 1.5 mg/L in the MS/MS method and 8 mg/L in the GC-MS; in E. coli 0.5 mg/L in the LC-MS/MS method and 2 mg/L in the GC-MS) and pMTL83155 -thlA-phaB-Pwl-meaBhcmA-hcmB+pMTL82256-ptb-buk (in Clostridium autoethanogenogenum, 15 mg/L in the LC-MS/MS method, and 75 mg/L in the GC-MS; in E. coli 2-Hydroxyisobutyric acid was detected in 1.1 mg/L in the LC-MS/MS method and 8.5 mg/L in the GC-MS) but not in all other constructs including controls arrive. The highest yields so far occurred in the strain harboring the plasmid pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB+pMTL82256-ptb-buk (10-fold higher than all other pathways), which had the optimal pathway, which Contains thiolase, (S)-specific (S)-specific 3-hydroxybutyl-CoA dehydrogenase, 2-hydroxyisobutyl-CoA mutase and Ptb-Buk system (Figure 29A-D). Unexpectedly, it was found that 2-hydroxybutyrate (2-HB) was also produced in this strain (up to 64 mg/L by LC-MS/MS and up to 64 mg/L by GC-MS in Clostridium autoethanologenum 50 mg/L; in E. coli , up to 12 mg/L by LC-MS/MS, and up to 9.5 mg/L by GC-MS), indicating non-specific mutase activity (Figure 30). This phenomenon was also found in the tesB strain, but again at significantly lower levels (18 mg/L in LC-MS-MS and 9 mg/L in GC-MS in C. autoethanogenogenum). The production of 2-hydroxyisobutyric acid was also confirmed by NMR.

In addition, qRT-PCR was also performed to confirm the expression of genes thlA , hbd , meaBhcmA and hcmB (Fig. 31).

The RT-PCR plot shows that under the P _pta-ack promoter, thlA gene product expression level is slightly higher than hbd (as expected, with a second gene in the operon), and hmcB shows slightly lower expression level than meaBhcmA . The performance of Clostridium autoethanologenum at 30°C was also lower than that at 37°C and E. coli at 30°C. For specific cycle numbers, see below.

The ratio of (S)-3-hydroxybutyric acid to (R)-3-hydroxybutyric acid was measured by high performance liquid chromatography (HPLC) on an Agilent 1260 Infinity LC with UV detection at 210 nm. Samples were prepared by centrifugation at 14,000 rpm for 3 minutes, followed by evaporation of 200 μL of supernatant to dryness. The aggregated pellets were then resuspended in 100% isopropyl alcohol and sonicated with heat for 1 hour. Centrifugation was repeated and the supernatant transferred to HPLC vials for analysis. By injecting 5 μL into a TCI Chiral MB-S column (250 mm×4.6mm×3μm).

Stereospecific analysis of the production of 3-HB has been performed. It was unexpectedly found that a mixture of isomers is produced in Clostridium autoethanogenum. The enzymes Hbd and PhaB are described to be stereospecific, PhaB to be R-specific and Hbd to be S-specific, and when these enzymes are expressed in E. coli , a stereopure product is observed (Tseng, " Applications and Environmental Microbiology 1, 75: 3137-3145, 2009).

The table below indicates the distribution of the (R) and (S)-forms of 3-HB at equilibrium produced by three different pathways in C. autoethanogenogenum. These data indicate the presence of an isomerase in C. autoethanogenogenum.

Eliminating the natural isomerase prevents the interconversion of the (R) and (S) forms of 3-HB. Alternatively, expression or overexpression of isomerase could generate novel PTB-Buk pathways. For example, Hbd can be used to produce (S)-3-HB, an isomerase can convert (S)-3-HB to (R)-3-HB, and ptb-buk can act on (R)-3 -HB to produce related products.

Example 11

This example demonstrates the conversion of 3-hydroxyisovaleryl-CoA and 3-hydroxyisovalerate via Ptb-Buk to produce isobutylene.

Different pathways for the production of isobutylene have been described, such as the conversion of acetone to isobutylene via hydroxyisovalerate synthase and decarboxylase (van Leeuwen, Appl Microbiol Biotechnol 93: 1377-1387, 2012). However, the hydroxyisovalerate decarboxylase step is an ATP-requiring step and the kinetics of this enzyme may not be ideal. Two alternative pathways to isobutylene using the Ptb-Buk system have been identified via 3-hydroxyisovaleryl-CoA, which has been shown to be a usable acceptor of the Ptb-Buk system in vitro (Liu, " Applied Microbiology Biotechnology ", 53: 545-552, 2000).

Alternative pathway 1 consists of a synthetase that converts acetone to 3-hydroxyisovaleryl-CoA (Figure 9).

Alternative pathway 2 proceeds via 3-methyl-2-pentoxyvalerate, a known intermediate in isoleucine biosynthesis common in bacteria such as E. coli or Clostridium autoethanogenans (Figure 10).

Example 12

This example describes methods for characterizing Ptb-Buk variants.

In view of the promiscuous nature of Ptb-Buk acceptors, it is likely that Ptb-Buk systems with different amino acid sequences have different preferences for a given acceptor. To identify Ptb-Buk systems favoring the desired acceptor (e.g., acetyl-acetyl-CoA, 3-hydroxybutyl-CoA, 2-hydroxyisobutyl-CoA, acetyl-CoA, and/or butyl-CoA), High-throughput screening is required. Such screening can be accomplished by coupling firefly luciferase (Luc) to the Ptb-Buk system (Figure 33). Luc reacts with D-luciferin to produce oxyluciferin, carbon dioxide and light. In addition to magnesium and molecular oxygen, Luc requires ATP for the reaction to proceed. ATP is the product produced by Ptb-Buk when provided with an appropriate acyl-CoA or enyl-CoA substrate. Therefore, the reaction rate and preference of Ptb-Buk to different substrates can be quantified by measuring the reaction rate and preference of Ptb-Buk, Luc, d-luciferin, magnesium, molecular oxygen, phosphate, ADP and acyl-CoA or enyl group. -Comparison of the amount of light produced by the reaction of CoA.

Example 13

This example uses genome-scale modeling to demonstrate that high selectivity for unnatural products can be achieved using Ptb-Buk. In addition, the use of Ptb-Buk has been shown to allow coupling of cell growth to product production, allowing the construction of stable and high-yielding fermentation strains.

A genome-scale metabolic model similar to that described by Marcellin, Green Chem , 18:3020-3028, 2006, was used. Variants of this model were generated that incorporated additional metabolic reactions, each presenting a different genetically modified microorganism to form unnatural products. Three model versions were generated for each unnatural product pathway, incorporating thioesterase, acetate CoA-transferase or Ptb-Buk reactions.

The maximum selectivity was calculated using flux balance analysis (FBA) using the instruction code of COBRA Toolbox v2.0 from MATLAB R2014a (Mathworks, Inc.), with Gurobi version 6.0.4 as the solver (Gurobi Optimization, Inc.). Exchange reactions are defined to represent chemically defined minimal growth media using CO as carbon source and energy. Evolutionary algorithms were used to search for the presence of strain designs incorporating up to ten genetic knockouts that couple the target's unnatural chemical production to growth.

FBA predicted that pathways using Ptb-Buk or CoA transferases would provide the highest product selectivity due to ATP acquisition via phosphorylation at the substrate level. The results are illustrated in Table 2. However, it should be noted that one limitation of genome-scale models and FBA analyzes is the inability to capture enzyme kinetics. The CoA transferase reaction requires a specific base level of acetate to produce functionality, so the actual maximum selectivity using CoA transferase is less than 100% due to the need for a base level of acetate to be present.

It is necessary to construct strains in which targeted non-natural chemicals must be produced for cell growth. FBA predicts that in most cases it will be difficult to couple target chemical production to growth when using thioesterases or CoA transferases; the natural products acetate and ethanol will facilitate this. However, when using Ptb-Buk, there are a variety of growth-coupling chemical-producing strain designs that often incorporate disruption of the phosphotransacetylase-acetate kinase reaction. Table 3 summarizes the growth coupling ability of each strain.

Although both Ptb-Buk and CoA transferases can support high selectivity, flux balance analysis predicts that in most cases only Ptb-Buk can construct stable, high-yield fermentation strains that couple the production and growth of unnatural chemicals.

Example 14

This example demonstrates the generation of adipic acid from a gas feed via Ptb-Buk.

The production of adipic acid from sugars in E. coli has been described by utilizing the Ptb-Buk pathway (Yu, Biotechnology and Bioengineering , 111: 2580-2586, 2014). But the yield is very low, in the μg/L range. Without wishing to be bound by any particular theory, the inventors believe that this is likely due to the lack of driving force in the formation of reducing power and remaining ATP. This current limitation can be overcome using reduced gas substrates such as CO and _H2 and acetogenic bacteria such as Clostridium autoethanogenans. In contrast to E. coli growing heterotrophically on more oxidized sugars, CO and _H oxidation provide sufficient driving force to convert 3 - Reduction of the pendant oxy-adipadiyl-CoA to 3-hydroxyadipyl-CoA and 2,3-dehydroadipidyl-CoA by enyl-CoA hydrolase or enyl-CoA reductase -CoA is reduced to adipyl-CoA (Figure 34, steps 23 and 25). Compared with E. coli, which grows heterotrophically on sugar and produces excess ATP through glycolysis, acetogenic bacteria rely on high-energy life limits to survive. Therefore, the ATP production reaction of the Ptb-Buk system has a strong driving force to ensure that adipyl- CoA is efficiently converted to adipic acid (Figure 34 step 26).

To produce adipic acid from gas in Clostridium autoethanogenum, genes encoding succinyl-CoA synthetase from Escherichia coli (NP_415256, NP_415257), ketoisovalerate oxidoreductase PaaJ from Escherichia coli (WP_001206190.1), 3-hydroxybutyl-CoA dehydrogenase from Clostridium beijerinckii (WP_011967675.1), trans-2-enyl-CoA reductase Crt from Clostridium acetobutylicum (NP_349318.1 ), trans-2-enyl-CoA reductase Bcd (NP_349317.1) and electronic flavoprotein EtfAB (NP_349315, NP_349316) from Clostridium acetobutylicum were selected and colonized on the expression plasmid, and then transformed as described above In the C. autoethanogenum strain pta-ack::ptb-buk or CAETHG_1524::ptb-buk from the previous example. Adipic acid is produced according to the steps depicted in Figure 34.

Example 15

This example demonstrates the production of various products including 2-buten-1-ol, 3-methyl-2-butanol, 1,3-hexanediol (HDO) via Ptb-Buk and AOR.

As shown in Example 6, Ptb-Buk is highly promiscuous and operates on a variety of CoAs as acceptors or can be engineered to use a range of non-natural CoAs as acceptors. Likewise, AOR enzymes have been shown to act on a variety of substrates. Together, these two enzymes can convert various CoAs via their acids to aldehydes, which can then be further converted to alcohols, ketones or enols via alcohol dehydrogenases, for which various variants exist in nature. Although the reduction of acids to aldehydes via the AOR with ferredoxin is endergonic under standard conditions (Thauer, Bacteriol Rev. 41:100-180, 1977) and therefore not feasible, unexpectedly This is the case in carboxytrophic acetogens such as Clostridium autoethanogenogens that operate at low pH and use CO or H2 as substrate (Mock, Journal of Bacteriology , 197: 2965-2980, 2015 ). A common limitation for acetogens is that they are ATP-limited and survive on the edge of thermodynamic life (Schuchmann, Nature Reviews Microbiology , 12:809-821, 2014), which can be reduced by coupling this acid with via The Ptb-Buk system is overcome by acid formation from CoA in an ATP-related form.

The Ptb-Buk system and the AOR system have been shown in the above examples for obtaining several different products, but can be extended to other products, such as producing 2-buten-1-ol, 3-methyl-2-butanol, 1,3-hexanediol (HDO). 2-Buten-1-ol can be produced from crotonyl-CoA via Ptb-Buk, AOR and alcohol dehydrogenase (Figure 35). 1,3-Hexanediol can be produced from 3-hydroxy-hexanediol-CoA via Ptb-Buk, AOR, and alcohol dehydrogenase (Figure 35). By combining Ptb-Buk, Adc, and an alcohol dehydrogenase (such as a natural primary:secondary alcohol dehydrogenase), 3-methyl-2-butanol can be formed from acetylbutyl-CoA.

All such precursors, i.e. crotonyl-CoA, 3-hydroxy-hexanoyl-CoA or acetylbutyl-CoA, can be synthesized by, for example, C. -381, 1945; Seedorf, Proceedings of the National Academy of Sciences , 105: 2128-2133, 2008) and other Clostridium known fermentation pathways reduce and elongate acetyl-CoA, acetyl-B as described in the previous examples. Formation of acyl-CoA and 3-HB-CoA. The enzymes involved include crotonyl-CoA hydratase (crotonic enzyme) or crotonyl-CoA reductase, butyl-CoA dehydrogenase or trans-2-enyl-CoA reductase, thiolase or thiolase -CoA acetyltransferase and 3-hydroxybutyryl-CoA dehydrogenase or acetyl-CoA hydratase (Figure 35). Individual genes from C. cruzi or other Clostridium species were selected on expressoplasts (US2011/0236941) and subsequently transformed into the C. autoethanogenogenum strain pta-ack::ptb- from the previous example as described above. buk or CAETHG_1524::ptb-buk to produce 2-buten-1-ol, 3-methyl-2-butanol, 1,3-hexanediol (HDO). 2-Buten-1-ol, 3-methyl-2-butanol and 1,3-hexanediol (HDO) can be precursors of other downstream products.

Although this is only a few examples, it should be apparent that this pathway can be further extended to produce a series of C4, C6, C8, C10, C12, C14 using the same enzyme or its engineered variants specific for longer chain lengths Alcohols, ketones, enols or diols (Figure 39). Different types of molecules can also be obtained by using primers or extender units other than acetyl-CoA in the thiolase step as described elsewhere (Cheong, Nature Biotechnol , 34:556 -561, 2016).

All references (including publications, patent applications, and patents) cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and were fully incorporated herein by reference. The exposition is general. Reference to any prior art in this specification is not and shall not be taken as an admission that prior art forms part of the common general knowledge in any country's field of endeavor.

Unless otherwise indicated herein or clearly contradicted by the context, in describing the invention (especially in the context of the following claims), the use of the terms "a", "said" and similar referents shall be understood to encompass Singular and plural. Unless stated otherwise, the terms "includes," "has," "includes," and "contains" are to be understood as open-ended terms (i.e., meaning "including (but not limited to)"). Unless otherwise indicated herein, recitation of numerical ranges herein is intended only to serve as a shorthand method for individually referring to each individual value falling within that range, and each individual value is incorporated into this specification as if individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (eg, "such as") provided herein is intended merely to better illuminate the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Preferred embodiments of the invention are described herein. Variations of the preferred embodiments may become apparent to those of ordinary skill upon reading the above description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter set forth in the appended claims hereto, as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

<110> New Zealand Business Lanser Technology New Zealand Co., Ltd. (LANZATECH NEW ZEALAND LIMITED)

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<221> MISC_FEATURE

<223> IlvD,YP_026248.1

<400> 40

<210> 41

<211> 477

<212> PRT

<213> Methanothermobacter thermoautotrophicum

<220>

<221> MISC_FEATURE

<223> VorA,WP_010876344.1

<400> 41

<210> 42

<211> 352

<212> PRT

<213> Methanothermobacter thermoautotrophicum

<220>

<221> MISC_FEATURE

<223> VorB,WP_010876343.1

<400> 42

<210> 43

<211> 79

<212> PRT

<213> Methanothermobacter thermoautotrophicum

<220>

<221> MISC_FEATURE

<223> VorC,WP_010876342.1

<400> 43

<210> 44

<211> 124

<212> PRT

<213> Methanothermobacter thermoautotrophicum

<220>

<221> MISC_FEATURE

<223> VorD,WP_010876341.1

<400> 44

<210> 45

<211> 394

<212> PRT

<213> Pyrococcus fulminantis

<220>

<221> MISC_FEATURE

<223> VorA,WP_011012106.1

<400> 45

<210> 46

<211> 311

<212> PRT

<213> Pyrococcus fulminantis

<220>

<221> MISC_FEATURE

<223> VorB,WP_011012105.1

<400> 46

<210> 47

<211> 185

<212> PRT

<213> Pyrococcus fulminantis

<220>

<221> MISC_FEATURE

<223> VorC,WP_011012108.1

<400> 47

<210> 48

<211> 105

<212> PRT

<213> Pyrococcus fulminantis

<220>

<221> MISC_FEATURE

<223> VorD,WP_011012107.1

<400> 48

<210> 49

<211> 386

<212> PRT

<213> Streptomyces anthemicidum

<220>

<221> MISC_FEATURE

<223> AcdH,AAD44196.1 or BAB69160.1

<400> 49

<210> 50

<211> 386

<212> PRT

<213> Streptomyces coelicolor

<220>

<221> MISC_FEATURE

<223> AcdH,AAD44195.1

<400> 50

<210> 51

<211> 261

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> Crt,ABR34202.1

<400> 51

<210> 52

<211> 261

<212> PRT

<213> Clostridium acetobutylicum

<220>

<221> MISC_FEATURE

<223> Crt,NP_349318.1

<400> 52

<210> 53

<211> 397

<212> PRT

<213> Treponema denticola

<220>

<221> MISC_FEATURE

<223> Ccr,NP_971211.1

<400> 53

<210> 54

<211> 539

<212> PRT

<213> Euglena gracilis

<220>

<221> MISC_FEATURE

<223> Ter,AAW66853.1

<400> 54

<210> 55

<211> 282

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> Hbd,WP_011967675.1

<400> 55

<210> 56

<211> 282

<212> PRT

<213> Clostridium acetobutylicum

<220>

<221> MISC_FEATURE

<223> Hbd,NP_349314.1

<400> 56

<210> 57

<211> 282

<212> PRT

<213> Clostridium cruzi

<220>

<221> MISC_FEATURE

<223> Hbd1,WP_011989027.1

<400> 57

<210> 58

<211> 246

<212> PRT

<213> Hookworm Cupriaphila

<220>

<221> MISC_FEATURE

<223> PhaB,WP_010810131.1

<400> 58

<210> 59

<211> 134

<212> PRT

<213> Aeromonas guinea pig

<220>

<221> MISC_FEATURE

<223> PhaJ,O32472

<400> 59

<210> 60

<211> 260

<212> PRT

<213> Ralstonia picketii

<220>

<221> MISC_FEATURE

<223> Bdh1,BAE72684.1

<400> 60

<210> 61

<211> 256

<212> PRT

<213> Ralstonia picketii

<220>

<221> MISC_FEATURE

<223> Bdh2,BAE72685.1

<400> 61

<210> 62

<211> 254

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> Bdh,AGY75962

<400> 62

<210> 63

<211> 607

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> AOR,WP_013238665.1

<400> 63

<210> 64

<211> 607

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> AOR,WP_013238675.1

<400> 64

<210> 65

<211> 607

<212> PRT

<213> Clostridium yungdalae

<220>

<221> MISC_FEATURE

<223> AOR,ADK15073.1

<400> 65

<210> 66

<211> 607

<212> PRT

<213> Clostridium yungdalae

<220>

<221> MISC_FEATURE

<223> AOR,ADK15083.1

<400> 66

<210> 67

<211> 405

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> Adh,AGY76060.1

<400> 67

<210> 68

<211> 388

<212> PRT

<213> Clostridium yungdalae

<220>

<221> MISC_FEATURE

<223> Adh,ADK17019.1

<400> 68

<210> 69

<211> 390

<212> PRT

<213> Clostridium acetobutylicum

<220>

<221> MISC_FEATURE

<223> BdhB,NP_349891.1

<400> 69

<210> 70

<211> 387

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> Bdh,WP_041897187.1

<400> 70

<210> 71

<211> 388

<212> PRT

<213> Clostridium yungdalae

<220>

<221> MISC_FEATURE

<223> Bdh1,YP_003780648.1

<400> 71

<210> 72

<211> 405

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> Bdh1,AGY76060.1

<400> 72

<210> 73

<211> 388

<212> PRT

<213> Clostridium yungdalae

<220>

<221> MISC_FEATURE

<223> Bdh2,YP_003782121.1

<400> 73

<210> 74

<211> 388

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> Bdh2,AGY74784.1

<400> 74

<210> 75

<211> 862

<212> PRT

<213> Clostridium acetobutylicum

<220>

<221> MISC_FEATURE

<223> AdhE1,NP_149325.1

<400> 75

<210> 76

<211> 858

<212> PRT

<213> Clostridium acetobutylicum

<220>

<221> MISC_FEATURE

<223> AdhE2,NP_149199.1

<400> 76

<210> 77

<211> 860

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> AdhE,WP_041893626.1

<400> 77

<210> 78

<211> 870

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> AdhE1,WP_023163372.1

<400> 78

<210> 79

<211> 877

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> AdhE2,WP_023163373.1

<400> 79

<210> 80

<211> 468

<212> PRT

<213> Clostridium glycoacetate

<220>

<221> MISC_FEATURE

<223> Bld,AAP42563.1

<400> 80

<210> 81

<211> 562

<212> PRT

<213> Three-carbon Proteobacteria

<220>

<221> MISC_FEATURE

<223> HcmAB, major subunit, AFK77668.1

<400> 81

<210> 82

<211> 136

<212> PRT

<213> Three-carbon Proteobacteria

<220>

<221> MISC_FEATURE

<223> HcmAB, small subunit, AFK77665.1

<400> 82

<210> 83

<211> 563

<212> PRT

<213> Kyrpidia tusciae

<220>

<221> MISC_FEATURE

<223> HcmAB, major subunit, WP_013074530.1

<400> 83

<210> 84

<211> 132

<212> PRT

<213> Kyrpidia tusciae

<220>

<221> MISC_FEATURE

<223> HcmAB, small subunit, WP_013074531.1

<400> 84

<210> 85

<211> 327

<212> PRT

<213> Three-carbon Proteobacteria

<220>

<221> MISC_FEATURE

<223> MeaB,AFK77667.1

<400> 85

<210> 86

<211> 312

<212> PRT

<213> Kyrpidia tusciae

<220>

<221> MISC_FEATURE

<223> MeaB,WP_013074529.1

<400> 86

<210> 87

<211> 301

<212> PRT

<213> Clostridium acetobutylicum

<220>

<221> MISC_FEATURE

<223> Ptb,WP_010966357.1

<400> 87

<210> 88

<211> 302

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> Ptb

<400> 88

<210> 89

<211> 302

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> Ptb,WP_041893500.1

<400> 89

<210> 90

<211> 355

<212> PRT

<213> Clostridium acetobutylicum

<220>

<221> MISC_FEATURE

<223> Buk,WP_010966356.1

<400> 90

<210> 91

<211> 355

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> Buk,WP_011967556

<400> 91

<210> 92

<211> 355

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> Buk,WP_017209677

<400> 92

<210> 93

<211> 355

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> Buk,WP_026886638

<400> 93

<210> 94

<211> 355

<212> PRT

<213> Clostridium beijerinckii

<220>

<221> MISC_FEATURE

<223> Buk,WP_041893502

<400> 94

<210> 95

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pACYCDuet-ptb-buk-pACYC-ptb-R1, reverse

<400> 95

<210> 96

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pACYCDuet-ptb-buk-ptb-pACYC-F1, forward

<400> 96

<210> 97

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pACYCDuet-ptb-buk-buk-pACYC-R1, reverse

<400> 97

<210> 98

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pACYCDuet-ptb-buk-pACYC-buk-F1, forward

<400> 98

<210> 99

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCOLADuet-thlA-adc-thlA-adc-R1, reverse

<400> 99

<210> 100

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCOLADuet-thlA-adc-adc-ThlA-F1, forward

<400> 100

<210> 101

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCOLADuet-thlA-adc-adc-pCOLA-R1, reverse

<400> 101

<210> 102

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCOLADuet-thlA-adc-pCOLA-adc-F1, forward

<400> 102

<210> 103

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCOLADuet-thlA-adc-thlA-pCOLA-F1, forward

<400> 103

<210> 104

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCOLADuet-thlA-adc-pCOLA-thlA-R1, reverse

<400> 104

<210> 105

<211> 5791

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pACYC-ptb-buk, plasmid

<400> 105

<210> 106

<211> 5609

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCOLA-thlA-adc, plasmid

<400> 106

<210> 107

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> thlA-ptb-R1, reverse

<400> 107

<210> 108

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> adc-buk-F1, forward

<400> 108

<210> 109

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> thlA-ptb-F1, forward

<400> 109

<210> 110

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Buk-adc-R1, reverse

<400> 110

<210> 111

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pDuet-insert2-R1, forward

<400> 111

<210> 112

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> insert2-pDuet-F1, forward

<400> 112

<210> 113

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pDuet-insert2-F1, forward

<400> 113

<210> 114

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> insert2-pDuet-R1, forward

<400> 114

<210> 115

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCDF-phaB-pACYC-phaB-R1, forward

<400> 115

<210> 116

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCDF-phaB-phaB-pACYC-F1, forward

<400> 116

<210> 117

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pcdf-phab-pacyc-phab-f1, forward

<400> 117

<210> 118

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCDF-phaB-phaB-pACYC-R1, forward

<400> 118

<210> 119

<211> 4486

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCDF-phaB, plastid

<400> 119

<210> 120

<211> 5221

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pCDF-phaB-bdh1, plastid

<400> 120

<210> 121

<211> 10922

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pMTL8225-budA::thlA-phaB, plastid

<400> 121

<210> 122

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN01

<400> 122

<210> 123

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN02

<400> 123

<210> 124

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN03

<400> 124

<210> 125

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN04mod

<400> 125

<210> 126

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223>SN05mod

<400> 126

<210> 127

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN06

<400> 127

<210> 128

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN07

<400> 128

<210> 129

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN08

<400> 129

<210> 130

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Og31f

<400> 130

<210> 131

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Og32r

<400> 131

<210> 132

<211> 7951

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pMTL8315-Pfdx-thlA-phaB-bld, plastid

<400> 132

<210> 133

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> bld-phaB-F1, forward

<400> 133

<210> 134

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> bld-pMTL-R1, forward

<400> 134

<210> 135

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pMTL-bld-F1, forward

<400> 135

<210> 136

<211> 1179

<212> DNA

<213> Clostridium acetobutylicum

<220>

<221> misc_feature

<223> thlA

<400> 136

<210> 137

<211> 849

<212> DNA

<213> Clostridium cruzi

<220>

<221> misc_feature

<223> hbd1

<400> 137

<210> 138

<211> 176

<212> DNA

<213> Clostridium autoethanologenum

<220>

<221> misc_feature

<223> ferredoxin promoter

<400> 138

<210> 139

<211> 474

<212> DNA

<213> Clostridium autoethanologenum

<220>

<221> misc_feature

<223> Pyruvate-ferredoxin oxidoreductase promoter

<400> 139

<210> 140

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Ribosome binding site rbs2

<400> 140

<210> 141

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Pfdx-F1, forward

<400> 141

<210> 142

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Pfdx-R1, reverse

<400> 142

<210> 143

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Ppfor-F1, forward

<400> 143

<210> 144

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Ppfor-R1, reverse

<400> 144

<210> 145

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> hbd1-F1, forward

<400> 145

<210> 146

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> hbd1-R1, reverse

<400> 146

<210> 147

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> thlA-F1, forward

<400> 147

<210> 148

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> thlA-R1, reverse

<400> 148

<210> 149

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Ppfor-F2, forward

<400> 149

<210> 150

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Ppfor-R2, reverse

<400> 150

<210> 151

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Ptb-Buk-F2, forward

<400> 151

<210> 152

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Ptb-Buk-F2, reverse

<400> 152

<210> 153

<211> 7884

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pMTL82256-ptb-buk, plasmid

<400> 153

<210> 154

<211> 436

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> Thioesterase 1

<400> 154

<210> 155

<211> 60

<212> PRT

<213> Clostridium yungdalae

<220>

<221> MISC_FEATURE

<223> Thioesterase 2

<400> 155

<210> 156

<211> 128

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> Thioesterase 3

<400> 156

<210> 157

<211> 436

<212> PRT

<213> Clostridium yungdalae

<220>

<221> MISC_FEATURE

<223> Thioesterase 1

<400> 157

<210> 158

<211> 137

<212> PRT

<213> Clostridium autoethanologenum

<220>

<221> MISC_FEATURE

<223> Thioesterase 2

<400> 158

<210> 159

<211> 128

<212> PRT

<213> Clostridium yungdalae

<220>

<221> MISC_FEATURE

<223> Thioesterase 3

<400> 159

<210> 160

<211> 11184

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pMTL8225-pta-ack::ptb-buk, plastid

<400> 160

<210> 161

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN22f

<400> 161

<210> 162

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN23r

<400> 162

<210> 163

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN24f

<400> 163

<210> 164

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN25r

<400> 164

<210> 165

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN26f

<400> 165

<210> 166

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN27r

<400> 166

<210> 167

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN28f

<400> 167

<210> 168

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN29r

<400> 168

<210> 169

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN30f

<400> 169

<210> 170

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> SN31r

<400> 170

<210> 171

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Og29f

<400> 171

<210> 172

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Og30r

<400> 172

<210> 173

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Pfdx-F1, forward

<400> 173

<210> 174

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Pfdx-R1, reverse

<400> 174

<210> 175

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> aor1-F1, forward

<400> 175

<210> 176

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> aor1-R1, reverse

<400> 176

<210> 177

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pETDuet-pta-ack-ack-DuetI2-R1

<400> 177

<210> 178

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pETDuet-pta-ack-DuetI2-ack-F1

<400> 178

<210> 179

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pETDuet-pta-ack-DuetI2-pta-R1

<400> 179

<210> 180

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pETDuet-pta-ack-pta-DuetI2-F1

<400> 180

<210> 181

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pETDuet-tesB-DuetI2-tesB-F1

<400> 181

<210> 182

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pETDuet-tesB-DuetI2-tesB-R1

<400> 182

<210> 183

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pETDuet-tesB-tesB-DuetI2-F1

<400> 183

<210> 184

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pETDuet-tesB-testB-DuetI2-R1

<400> 184

<210> 185

<211> 7606

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pDUET-pta-ack, plasmid

<400> 185

<210> 186

<211> 7492

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pDUET-ptb-buk, plasmid

<400> 186

<210> 187

<211> 6233

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pDUET-tesB, plasmid

<400> 187

<210> 188

<211> 3120

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> Codon-optimized gene cassette containing Wood-Ljungdahl promoter before genes meaB, hcmA and hcmB promoter in front of the genes meaB,hcmA and hcmB

<400> 188

<210> 189

<211> 894

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptides

<220>

<221> MISC_FEATURE

<223> hcmA and meaB fusion

<400> 189

<210> 190

<211> 849

<212> DNA

<213> Clostridium acetobutylicum

<220>

<221> misc_feature

<223> hbd

<400> 190

<210> 191

<211> 10647

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pMTL83155-thlA-hbd-Pwl-meaBhcmA-hcmB

<400> 191

<210> 192

<211> 10539

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pMTL83155-thlA-phaB-Pwl-meaBhcmA-hcmB

<400> 192

<210> 193

<211> 487

<212> DNA

<213> Clostridium autoethanologenum

<220>

<221> misc_feature

<223> Promoter region of phosphoacetyltransferase

<400> 193

<210> 194

<211> 7884

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pMTL82256-ptb-buk

<400> 194

<210> 195

<211> 6624

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<220>

<221> misc_feature

<223> pMTL82256-tesB

<400> 195

Claims (41)

A genetically engineered carbonotrophic acetogenic bacterium, which includes exogenous phosphobutyryl transferase (Ptb) and exogenous butyrate kinase (Buk) (Ptb-Buk), wherein the Ptb-Buk can transform non-natural receptors quality to produce unnatural products. The bacterium according to claim 1, wherein the Ptb-Buk acts on substrates other than butyl-CoA and/or butyl-phosphate. The bacterium of claim 1, wherein the Ptb-Buk produces products other than butyl phosphate or butyrate. The bacterium according to claim 1, wherein the bacterium does not produce butanol. The bacterium of claim 1, wherein the bacterium produces one or more of acids, alkenes, ketones, aldehydes, alcohols or glycols. The bacterium according to claim 1, wherein the bacterium produces acetone or its precursor, isopropyl alcohol or its precursor, isobutylene or its precursor, 3-hydroxybutyrate or its precursor, 1,3-butyrate Diol or its precursor, 2-hydroxyisobutyrate or its precursor, adipic acid or its precursor, 1,3-hexanediol or its precursor, 3-methyl-2-butanol or its precursor One or more of precursors, 2-buten-1-ol or its precursors, isovalerate or its precursors, or isoamyl alcohol or its precursors. The bacterium of claim 1, wherein the Ptb-Buk converts acetyl acetyl-CoA into acetyl acetate (step 2), and converts 3-hydroxyisovaleryl-CoA into 3-hydroxyl Isovalerate (step 8), convert 3-hydroxybutyl-CoA to 3-hydroxybutyrate (step 14), convert 2-hydroxyisobutyl-CoA to 2-hydroxyisobutyrate (step 20) , convert crotonyl-CoA to crotonate (step 28), convert acetylbutyryl-CoA to acetylbutyrate (step 33) or convert isopentyl-CoA to isovalerate (step 44). The bacterium of claim 1, wherein the bacterium produces one or more of 1-propanol, 1-hexanol and 1-octanol. The bacterium as described in claim 1, wherein the bacterium is derived from a parent bacterium selected from the group consisting of: Clostridium autoethanogenum , Clostridium ljungdahlii , Clostridium ljungdahlii Clostridium ragsdalei , Escherichia coli , Saccharomyces cerevisiae , Clostridium acetobutylicum , Clostridium beijerinckii, Clostridium saccharbutyricum , glycoacetate poly Clostridium saccharoperbutylacetonicum , Clostridium butyricum , Clostridium diolis , Clostridium kluyveri , Clostridium pasterianium , Clostridium edema ( Clostridium novyi , Clostridium difficile , Clostridium thermocellum, Clostridium cellulolyticum , Clostridium cellulovorans, Clostridium phytofermentans , Lactococcus lactis, Bacillus subtilis , Bacillus licheniformis , Zymomonas mobilis , Klebsiella oxytoca , Klebsiella pneumoniae Klebsiella pneumonia , Corynebacterium glutamicum , Trichoderma reesei , Cupriavidus necator , Pseudomonas putida , Lactobacillus plantarum ( Lactobacillus plantarum ) and Methylobacterium extorquens . The bacterium of claim 1, wherein the bacterium further includes exogenous or endogenous aldehyde: ferredoxin oxidoreductase (AOR). The bacterium according to claim 1, wherein the bacterium further includes disruptive mutations of phosphotransacetylase (Pta) and acetate kinase (Ack). The bacterium of claim 1, wherein the bacterium further includes a disruptive mutation of thioesterase. A genetically engineered carbonotrophic acetogenic bacterium for producing acetone or its precursor, the bacterium comprising: (a) converting acetyl-CoA into acetyl-acetyl-CoA (step 1) enzyme, an enzyme that converts acetyl acetyl-CoA to acetyl acetate (step 2), and An enzyme that converts acetyl acetate to acetone (step 3), wherein the enzyme that converts acetyl acetyl-CoA to acetyl acetate (step 2) is Ptb-Buk, or (b) Enzyme that converts acetyl-CoA to acetyl-acetyl-CoA (step 1), enzyme that converts acetyl-acetyl-CoA to 3-hydroxybutyl-CoA (step 13), enzyme that converts 3-hydroxybutyl-CoA Enzymes that convert CoA to 3-hydroxybutyrate (step 14), 3-hydroxybutyrate to acetyl acetate (step 15), and acetyl acetate to acetone (step 3) The enzyme, wherein the enzyme that converts 3-hydroxybutyryl-CoA into 3-hydroxybutyrate (step 14) is Ptb-Buk. The bacterium as claimed in claim 13, wherein the bacterium produces acetone or its precursor. The bacterium as claimed in claim 13, wherein the bacterium further comprises a primary:secondary alcohol dehydrogenase disruptive mutation. A genetically engineered carbonotrophic acetogenic bacterium for producing isopropanol or a precursor thereof, the bacterium comprising: (a) converting acetyl-CoA into acetyl-acetyl-CoA (step 1), an enzyme that converts acetyl acetyl-CoA to acetyl acetate (step 2), an enzyme that converts acetyl acetate to acetone (step 3), and an enzyme that converts acetone to isopropanol The enzyme of (step 4), wherein the enzyme that converts acetyl acetyl-CoA into acetyl acetate (step 2) is Ptb-Buk, or (b) converts acetyl-CoA into acetyl acetate Enzyme that converts acetyl-CoA (step 1), enzyme that converts acetyl-acetyl-CoA to 3-hydroxybutyryl-CoA (step 13), that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyrate (Step 14) enzyme, an enzyme that converts 3-hydroxybutyrate to acetyl acetate (step 15), an enzyme that converts acetyl acetate to acetone (step 3), and an enzyme that converts acetone to isopropanol (step 4) ), wherein the enzyme that converts 3-hydroxybutyryl-CoA into 3-hydroxybutyrate (step 14) is Ptb-Buk. The bacterium according to claim 16, wherein the bacterium produces isopropyl alcohol or its precursor. A genetically engineered carbonotrophic acetogenic bacterium for producing isobutylene or its precursor, the bacterium comprising: (a) converting acetyl-CoA into acetyl-acetyl-CoA (step 1) enzyme, enzyme that converts acetyl acetyl-CoA to acetyl acetate (step 2), enzyme that converts acetyl acetate to acetone (step 3), enzyme that converts acetone to 3-hydroxyisoamyl Enzymes for converting acetyl acetyl-CoA to acetyl acetate (step 2) and enzymes for converting 3-hydroxyisovalerate to isobutylene (step 6) The enzyme is Ptb-Buk, or (b) an enzyme that converts acetyl-CoA to acetyl acetyl-CoA (step 1), an enzyme that converts acetyl acetyl-CoA to acetyl acetate (step 2) ), an enzyme that converts acetyl acetate to acetone (step 3), an enzyme that converts acetone to 3-hydroxyisovaleryl-CoA (step 7), an enzyme that converts 3-hydroxyisovaleryl-CoA Enzyme for conversion to 3-hydroxyisovalerate (step 8) and enzyme for conversion of 3-hydroxyisovalerate to isobutylene (step 6), wherein acetyl acetyl-CoA is converted to acetyl acetate The enzyme (step 2) and/or the enzyme converting 3-hydroxyisovaleryl-CoA into 3-hydroxyisovalerate (step 8) is Ptb-Buk. The bacterium of claim 18, wherein the bacterium produces isobutylene or its precursor. A genetically engineered carbonotrophic acetogenic bacterium for producing 3-hydroxybutyrate or its precursor, the bacterium comprising: (a) converting acetyl-CoA into acetyl-acetyl- Enzymes for CoA (step 1), enzymes that convert acetyl acetyl-CoA to acetyl acetate (step 2), and enzymes that convert acetyl acetate to 3-hydroxybutyrate (step 15) , wherein the enzyme that converts acetyl acetyl-CoA into acetyl acetate (step 2) is Ptb-Buk, or (b) converts acetyl acetyl-CoA into acetyl acetyl-CoA ( The enzyme of step 1), the enzyme that converts acetyl acetyl-CoA into 3-hydroxybutyryl-CoA (step 13), and the enzyme that converts 3-hydroxybutyryl-CoA into 3-hydroxybutyrate (step 14) , wherein the enzyme that converts 3-hydroxybutyryl-CoA into 3-hydroxybutyrate (step 14) is Ptb-Buk. The bacterium according to claim 20, wherein the bacterium produces 3-hydroxybutyrate or its precursor. A genetically engineered carbonotrophic acetogenic bacterium for producing 1,3-butanediol or its precursor, the bacterium comprising: (a) converting acetyl-CoA into acetyl-acetyl -Enzyme for CoA (step 1), enzyme for converting acetyl acetyl-CoA to acetyl acetate (step 2), enzyme for converting acetyl acetate to 3-hydroxybutyrate (step 15) Enzyme, enzyme that converts 3-hydroxybutyrate to 3-hydroxybutyraldehyde (step 16) and 3-hydroxybutyraldehyde The enzyme that converts acetyl acetyl-CoA to acetyl acetyl-CoA (step 2) is Ptb-Buk, or (b) Enzyme that converts acetyl-CoA to acetyl-acetyl-CoA (step 1), enzyme that converts acetyl-acetyl-CoA to 3-hydroxybutyl-CoA (step 13), enzyme that converts 3-hydroxybutyl-CoA Enzymes that convert CoA to 3-hydroxybutyrate (step 14), 3-hydroxybutyrate to 3-hydroxybutyraldehyde (step 16), and 3-hydroxybutyraldehyde to 1,3-butanal An enzyme for diol (step 17), wherein the enzyme for converting 3-hydroxybutyryl-CoA to 3-hydroxybutyrate (step 14) is Ptb-Buk. The bacterium according to claim 22, wherein the bacterium produces 1,3-butanediol or its precursor. A genetically engineered carbonotrophic acetogenic bacterium for producing 2-hydroxyisobutyrate or its precursor, the bacterium comprising: (a) converting acetyl-CoA into acetyl-acetyl - Enzyme for CoA (step 1), enzyme for converting acetyl acetyl-CoA to 3-hydroxybutyl-CoA (step 13), enzyme for converting 3-hydroxybutyl-CoA to 2-hydroxyisobutyl-CoA (step 13) 19) The enzyme and the enzyme that converts 2-hydroxyisobutyl-CoA into 2-hydroxyisobutyrate (step 20), wherein 2-hydroxyisobutyl-CoA is converted into 2-hydroxyisobutyrate (step 20) ) is Ptb-Buk. The bacterium according to claim 24, wherein the bacterium produces 2-hydroxyisobutyrate or its precursor. A genetically engineered carbonotrophic acetogenic bacterium for producing adipic acid or a precursor thereof, the bacterium comprising: (a) converting acetyl-CoA into succinyl-CoA (step 21 ), an enzyme that converts succinyl-CoA to 3-Pendantoxy-adipacyl-CoA (step 22), an enzyme that converts 3-Pendantoxy-adipacyl-CoA to 3-hydroxy Enzyme for adipidyl-CoA (step 23), enzyme for converting 3-hydroxyadipyl-CoA to 2,3-dehydroadipidyl-CoA (step 24), enzyme for converting 2,3-dehydradipidyl-CoA Enzymes for converting hydrogen adipyl-CoA to adipidyl-CoA (step 25) and enzymes for converting adipidyl-CoA to adipic acid (step 26), wherein adipidyl-CoA is converted The enzyme for adipate (step 26) is Ptb-Buk, or (b) the enzyme for converting acetyl-CoA into 3-side oxy-adipyl-CoA (step 22), converting 3 - Enzyme that converts side oxy-adipadiyl-CoA to 3-hydroxyadipadiyl-CoA (step 23), converts 3-hydroxyadipadiyl-CoA into 2,3-dehydradipidyl-CoA Enzyme for converting 2,3-dehydroadipyl-CoA (step 24), converting 2,3-dehydroadipyl-CoA to adipidyl-CoA (step 25), and converting adipidyl-CoA to adipic acid (step 26), wherein the enzyme that converts adipyl-CoA into adipic acid (step 26) is Ptb-Buk. The bacterium of claim 26, wherein the bacterium produces adipic acid or its precursor. A genetically engineered carbonotrophic acetogenic bacterium for producing 3-methyl-2-butanol or its precursor from 3-hydroxybutyl-CoA, the bacterium includes: (a) An enzyme that converts 3-hydroxybutyryl-CoA into crotonyl-CoA (step 27), an enzyme that converts crotonyl-CoA into butyryl-CoA (step 31), an enzyme that converts butyryl-CoA into ethyl an enzyme that converts acetylbutyryl-CoA (step 32), an enzyme that converts acetylbutyryl-CoA to acetylbutyrate (step 33), an enzyme that converts acetylbutyrate to acetylacetone (step 34), and The enzyme that converts acetyl acetone to 3-methyl-2-butanol (step 35), wherein the enzyme that converts acetyl butyryl-CoA to acetyl butyrate (step 33) is Ptb-Buk. The bacterium according to claim 28, wherein the bacterium produces 3-methyl-2-butanol or its precursor. A genetically engineered carbonotrophic acetogenic bacterium for producing 2-buten-1-ol or its precursor from 3-hydroxybutyryl-CoA, the bacterium comprising: (a) converting 3-hydroxybutyryl-CoA The enzyme that converts -CoA to crotonyl-CoA (step 27), the enzyme that converts crotonyl-CoA to crotonate (step 28), the enzyme that converts crotonate to crotonaldehyde (step 29), and the The enzyme that converts crotonaldehyde to 2-buten-1-ol (step 30), wherein the enzyme that converts crotonyl-CoA to crotonate (step 28) is Ptb-Buk. The bacterium according to claim 30, wherein the bacterium produces 3-methyl-2-butanol or its precursor. A genetically engineered carbonotrophic acetogenic bacterium, which is used In producing isovalerate or its precursor, the bacteria include: (a) an enzyme that converts acetyl-CoA into acetyl-acetyl-CoA (step 1), converting acetyl-acetyl-CoA Enzyme that is 3-hydroxy-3-methylglutaryl-CoA (step 40), converts 3-hydroxy-3-methylglutaryl-CoA into 3-hydroxy-3-methylglucosyl -Enzyme for CoA (step 41), enzyme for converting 3-hydroxy-3-methylglucosyl-CoA into 2-methylcrotonyl-CoA (step 42), enzyme for converting 2-methylcrotonyl-CoA Enzyme that converts CoA to isopentyl-CoA (step 43) and enzyme that converts isopentyl-CoA to isovalerate (step 44), wherein isopentyl-CoA is converted to isovalerate The enzyme in (step 44) is Ptb-Buk. The bacterium of claim 32, wherein the bacterium produces isovalerate or its precursor. A genetically engineered carbonotrophic acetogenic bacterium for producing isoamyl alcohol or a precursor thereof, the bacterium comprising: (a) converting acetyl-CoA into acetyl-acetyl-CoA (step The enzyme of 1), the enzyme that converts acetyl acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA (step 40), the enzyme that converts 3-hydroxy-3-methylglutaryl-CoA Enzyme that converts CoA to 3-hydroxy-3-methylglucosyl-CoA (step 41), converts 3-hydroxy-3-methylglucosyl-CoA to 2-methylcrotonyl-CoA (step 41) 42), an enzyme that converts 2-methylcrotonyl-CoA into isopentyl-CoA (step 43), an enzyme that converts isopentyl-CoA into isovalerate (step 44), Enzymes that convert isopentyl-CoA to isovaleraldehyde (step 45) and enzymes that convert isovaleraldehyde to isopentyl alcohol (step 46), wherein isopentyl-CoA is converted to isovalerate (step 46) The enzyme in step 44) is Ptb-Buk. The bacterium of claim 34, wherein the bacterium produces isoamyl alcohol or a precursor thereof. A method of producing a product, which includes culturing the bacterium of claim 1 in the presence of a substrate, and the bacterium produces the product by the method. The method of claim 36, wherein the bacterium produces acetone or its precursor, isopropyl alcohol or its precursor, isobutylene or its precursor, 3-hydroxybutyrate or its precursor, 1,3-butyrate Diol or its precursor, 2-hydroxyisobutyrate or its precursor, adipic acid or its precursor, 1,3-hexanediol or its precursor, 3-methyl-2-butanol or its precursor One or more of precursors, 2-buten-1-ol or its precursors, isovalerate or its precursors, or isoamyl alcohol or its precursors. The method of claim 36, wherein the Ptb-Buk converts acetyl acetyl-CoA into acetyl acetate (step 2), and converts 3-hydroxyisovaleryl-CoA into 3-hydroxyl Isovalerate (step 8), convert 3-hydroxybutyl-CoA to 3-hydroxybutyrate (step 14), convert 2-hydroxyisobutyl-CoA to 2-hydroxyisobutyrate (step 20) , convert crotonyl-CoA to crotonate (step 28), convert acetylbutyryl-CoA to acetylbutyrate (step 33) or convert isopentyl-CoA to isovalerate (step 44). The method of claim 36, wherein the substrate is a gaseous substrate including one or more of CO, CO ₂ and H ₂ . The method of claim 36, wherein the gas substrate is synthesis gas. The method according to claim 36, wherein the gas substrate is industrial waste gas.

Images