Clostridium aerovorum synthesizes 3-hydroxybutyrate

Abstract

The invention discloses a method for constructing an engineered bacterium of Clostridium aeruginosa, comprising the following steps: preparing a desired gene in a biosynthetic pathway of a target product, namely a pathway gene, to form a pathway gene cluster; embedding the pathway gene cluster into a transposase recognition site of a mariner transposon to form a pathway gene expression cassette; selecting a constitutive strong promoter commonly used in Clostridium jundal for driving transposase expression, loading the pathway gene expression cassette into a multiple cloning site of a plasmid, and inserting an inducible repressor element upstream of the replicon to obtain a transposition plasmid; transforming the transposition plasmid into Clostridium aeruginosa to obtain an engineered bacterium of Clostridium aeruginosa. The yield of 3-hydroxybutyric acid expressed by the engineered bacterium constructed by the above method is as high as 3g / L.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to a method for constructing an engineered bacterium of Clostridium aerovorum, and in particular to a method for constructing an engineered bacterium of Clostridium jundal capable of synthesizing 3-hydroxybutyric acid. Background Art

[0002] At present, the fermentation raw materials used to produce fine chemicals by microorganisms through biosynthesis routes are mainly various sugar-based raw materials. A development trend is to use cheaper waste raw materials with efficient recombinant microorganisms as production strains to create new technical systems and process routes, thereby reducing the production cost of target products or target compounds and improving the supply capacity to the market. One-carbon gases represented by CO and CO2 are cheap carbon resources with huge reserves. They come from a wide range of sources, including corporate waste gas and synthesis gas prepared by gasification of carbon-containing materials such as coal, oil, natural gas and biomass. These one-carbon resources can be converted into various bulk chemicals and liquid fuels through autotrophic microbial fermentation. The market prospects are huge and have attracted widespread attention. Compared with the chemical catalytic route, the one-carbon gas bioconversion route has unique advantages in synthesizing long carbon chain high-value products. Therefore, whether from the perspective of energy conservation and emission reduction or the bioconversion of cheap one-carbon resources and the establishment of a new generation of green biomanufacturing technology, it is of great economic value to carry out basic and applied research on the bioconversion of one-carbon gases.

[0003] Known autotrophic microorganisms that can utilize one-carbon gas can be divided into three types according to the type of energy demand: chemical energy, light energy, and electrical energy. Among them, chemoautotrophic microorganisms use hydrogen or other organic compounds as energy donors, which are more suitable for the high-density fermentation mode of the existing traditional fermentation industry. Clostridium aerogenes is a major type of chemoautotrophic microorganism that can convert one-carbon gas into alcohol compounds, and has good prospects for development and utilization. For example, the technology of producing ethanol by fermentation with Clostridium aerogenes as the base bacteria and industrial tail gas as the raw material has been industrialized and has shown good commercial prospects. The main natural products of Clostridium aerogenes are acetic acid and ethanol, and the ability to synthesize other medium and long carbon chain chemicals is insufficient, and the industrial production of these high-value products has not yet been realized. In addition, as a type of strict anaerobic bacteria, the molecular genetic modification of Clostridium aerogenes itself is relatively difficult, resulting in slow progress in the development of Clostridium aerogenes cell factories for the efficient synthesis of new products in recent years. This key technical bottleneck needs to be broken through urgently, so as to establish my country's technological advantages in this field and provide technical support for future large-scale industrial promotion. Summary of the invention

[0004] Clostridium ljungdahlii is a species that has been studied more among Clostridium aerogenes. In view of the defects of large-fragment DNA integration technology on the chromosome of Clostridium ljungdahlii, such as cumbersome operation, long cycle and low integration efficiency, we envisioned that it might be possible to construct a large-fragment DNA rapid integration system based on mariner transposon, and use this system to integrate the target product, such as 3-hydroxybutyric acid (3-HB) synthesis pathway genes, into the genome of Clostridium ljungdahlii, and construct a stable and high-yield Clostridium aerogenes cell factory for target products such as 3-hydroxybutyric acid. Since the insertion of mariner transposon is random and immune, strains with pathway genes inserted at different sites in the genome will be obtained. Affected by factors such as secondary structure and intracellular distribution density of RNA polymerase, when pathway genes are integrated at different sites in the genome, their expression levels will be different, which will in turn affect the yield of the strain. Therefore, after obtaining a large number of integrated strains, through appropriate screening processes, integrated engineering strains with high expression of pathway genes can be obtained.

[0005] Therefore, we designed and constructed the transposase plasmid. First, we determined the optimal 3-HB synthesis pathway in Clostridium jundal, optimized the synthesis pathway, inserted the chloramphenicol resistance gene in the middle of the pathway as a resistance screening tag, and embedded the entire pathway gene cluster inside the transposase recognition site. Then, we selected the constitutive strong promoter P commonly used in Clostridium jundal. 1339 The transposase Himar1C9 was expressed. The above pathway was reproduced in the multiple cloning site of the plasmid pMTL83151, and an "inducible repressor" element Pfac / Pptb::lacI was inserted upstream of its replicon repH. This module can inhibit the work of the plasmid replicon after adding the inducer IPTG, thereby blocking the plasmid replication and successfully constructing a transposable plasmid. The transposable plasmid was transformed into Clostridium jundal, and a transformant capable of producing 3-HB using syngas was obtained. Based on the above research results, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a method for constructing an engineered bacterium of Clostridium aerovorum, which comprises the following steps:

[0007] A. Genes required in the biosynthetic pathway for preparing a target product or target compound, i.e., pathway genes, to form a pathway gene cluster, wherein the preparation method of the pathway gene includes but is not limited to whole gene synthesis or PCR amplification;

[0008] B. embedding the pathway gene cluster obtained in step A into the transposase recognition site of mariner transposon to form a pathway gene expression cassette;

[0009] C. Select a constitutive strong promoter commonly used in Clostridium jundal to drive the expression of the transposase, load the pathway gene expression cassette obtained in step B into the multiple cloning site of a plasmid suitable for expression in Clostridium, and insert an inducible repressor element upstream of the plasmid replicon, which can inhibit the work of the plasmid replicon after adding the inducer IPTG, thereby blocking the plasmid replication and obtaining a transposase plasmid;

[0010] D. The transposition plasmid obtained in step C is transformed into Clostridium aerovorum, and positive clones in which the pathway gene cluster is integrated into the chromosome of Clostridium aerovorum are screened to obtain engineered Clostridium aerovorum bacteria expressing the target product, i.e., the target compound.

[0011] The above-mentioned Clostridium aerovorans may be Clostridium ljungdahlii, for example, Clostridium ljungdahlii DSM 13528.

[0012] Preferably, the pathway gene cluster is integrated into the following site of the Clostridium aerovorum genome: the non-coding region between the CLJU_c05590 and CLJU_c05600 genes. When pathway genes, especially 3-hydroxybutyrate biosynthesis pathway genes, are integrated into this site of the Clostridium aerovorum genome, the expression level of the target product will increase significantly, reflecting the "position effect" of the expression level of the pathway genes.

[0013] In one embodiment, step A in the above method further comprises inserting a resistance gene in the middle of the pathway gene cluster for use as a resistance screening tag, and the resistance gene is, for example, a chloramphenicol resistance gene.

[0014] Preferably, the mariner transposon is the transposase Himar1C9.

[0015] The plasmid suitable for expression in Clostridium can be selected from the group consisting of pMTL 82151, pMTL 83151, pMTL84151, pMTL 85151, pMTL 82251, pMTL 82254, pMTL 83251, pMTL 83254, pMTL 84251, pMTL84254, pMTL 85251, pMTL 85254 and pIMP1. Preferably, the plasmid is pMTL83151, which comprises the replicon repH (pCB102).

[0016] In one embodiment, the above-mentioned strong constitutive promoter is P 1339 .

[0017] The above-mentioned inducible repressor element can be Pfac / Pptb::lacI.

[0018] In a specific embodiment, the target product is 3-hydroxybutyric acid; accordingly, the biosynthetic pathway is a 3-hydroxybutyric acid biosynthetic pathway; the pathway genes include genes required for the metabolic pathway for synthesizing 3-hydroxybutyric acid from one-carbon gas in Clostridium aerovorum, especially some genes in the 3-hydroxybutyric acid biosynthetic pathway that are lacking in wild-type Clostridium aerovorum, and these genes include at least one of the following groups: thiolase (Thl), such as Thl from Clostridium acetobutylicum (Gen / bank No. CA_P0079), used to achieve the conversion of acetyl-CoA to acetoacetyl-CoA; CoA-transferase complex AB (CtfAB), such as CtfAB from Clostridium acetobutylicum (Gen / bank No. CA_P0163, CA_P0164); 3-hydroxybutyrate dehydrogenase (3-hydroxybutyratedehydrogenase, BdhA), such as Clostridium difficile ( R20291) derived from BdhA (GenBank No. CDR20291_2567), used to catalyze acetoacetate to produce 3-HB; acetoacetyl-CoA reductase (Acetoacetyl-CoA reductase, PhaB) such as PhaB derived from Cupriavidus necator H16 (GenBank No. H16_A1439); 3-hydroxybutyryl-CoA dehydrogenase (3-hydroxybutyryldehydrogenase, Hbd) such as Hbd derived from Clostridium kluyveri (GenBank No. CKL_0458) or 3-hydroxybutyryl-CoA dehydrogenase (Hbd2, GenBank No. CLJU_c37300) derived from Clostridium ljungdahlii DSM 13528, used to catalyze acetoacetyl-CoA to produce 3-hydroxybutyryl-CoA.

[0019] Accordingly, the transposition plasmid obtained in step C above is used to express the 3-hydroxybutyrate biosynthetic pathway genes, and the nucleotide sequence of the transposition plasmid can be SEQ ID NO: 1, and is named plasmid pCYG4-P herein. 1339 -3HB; preferably also includes a plasmid overexpressing 3-hydroxybutyryl-CoA dehydrogenase (Hbd2, GenBank No. CLJU_c37300) from Clostridium ljungdahlii DSM 13528, whose nucleotide sequence can be SEQ ID NO: 2, and is named plasmid pMTL83251-P in this article pta -hbd2.

[0020] In the method for constructing 3-hydroxybutyrate-producing bacteria, it is preferred to enhance the expression of the 3-hydroxybutyryl-CoA dehydrogenase (Hbd2) gene hbd2 from C. ljungdahlii DSM 13528 in the 3-hydroxybutyrate pathway gene. The 3-hydroxybutyryl-CoA dehydrogenase (Hbd2) is used to catalyze 3-hydroxybutyryl-CoA to produce acetoacetyl-CoA.

[0021] The second aspect of the present invention provides an engineered bacterium of Clostridium aerovorum, which is constructed according to the above method.

[0022] The third aspect of the present invention provides the use of the engineered Clostridium aerovorum in producing a target product using a carbon-containing gas.

[0023] Preferably, the engineered Clostridium aerovorum bacteria can convert a carbon-containing gas such as syngas into 3-hydroxybutyric acid, a fine chemical with high added value, through fermentation.

[0024] The present invention verifies the function of mariner transposon as a tool for rapid integration of large DNA fragments in Clostridium jundal, optimizes the high-level expression scheme of target products such as 3-hydroxybutyric acid biosynthesis pathway genes in Clostridium aerogenes, and the 3-hydroxybutyric acid production of the constructed engineered bacteria is as high as 3 g / L, demonstrating the great application potential of the mariner transposon integration method in the construction of Clostridium engineered bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The construction and comparison of three 3-hydroxybutyrate biosynthetic pathways are shown. Among them, Figure A is a schematic diagram of three 3-hydroxybutyrate biosynthetic pathways, where: Th1, thiolase; CtfAB, CoA transferase complex; PhaB, acetoacetyl-CoA reductase; Hbd, 3-hydroxybutyryl-CoA dehydrogenase; BdhA, 3-hydroxybutyrate dehydrogenase; TesB, thioesterase; cac, Clostridium acetobutylicum; cne, Ralstonia; ckl, Clostridium kluyveri. Figure B is a simplified diagram of the three 3-hydroxybutyrate biosynthetic pathways introduced into Clostridium jundal, pathway ① is abbreviated as clj-3hb-i, introducing CtfAB and Th1; pathway ② is abbreviated as clj-3hb-ii, introducing PhaB and Th1; pathway ③ is abbreviated as clj-3hb-iii, introducing Hbd and Th1. Figure C shows a comparative curve of the growth of strains introduced with three synthetic pathways in syngas fermentation. Figure D shows a bar chart comparing the 3-hydroxybutyrate production of syngas fermentation by strains into which three synthetic pathways were introduced.

[0026] Figure 2 Three plasmids are shown: pMTL83151-P pta-phaB-thl, pMTL83151-P pta -HBD-THL and pMTL83151-P pta -map of thl-ctfAB.

[0027] Figure 3 The entire process flow chart of transposase plasmid construction, Clostridium Yongdal engineered bacteria construction, and fermentation verification is shown.

[0028] Figure 4 Comparison of integration site analysis, growth phenotype, target product yield, and pathway gene expression levels of 64 strains integrated into the 3-HB biosynthesis pathway are shown. Among them, Figure A is the distribution of 3-hydroxybutyrate synthesis pathway gene insertion sites in 64 single bacteria on the genome of Clostridium Yongdal; Figure B is a statistical bar chart of 3-hydroxybutyrate production of 64 engineered bacteria with pathway genes integrated at different sites; Figure C is a growth comparison curve of 3-hydroxybutyrate integrated strains clj-2, clj-13, clj-48, clj-45, clj-51, and clj-64 in syngas fermentation; Figure D is a bar chart comparing 3-hydroxybutyrate production of 3-hydroxybutyrate integrated strains clj-2, clj-13, clj-48, clj-45, clj-51, and clj-64 after 96 hours of syngas fermentation; Figure E is a bar chart comparing the relative expression levels of pathway genes in 3-hydroxybutyrate integrated strains clj-2, clj-13, and clj-45 at 36 hours of syngas fermentation. CtfAB, CoA transferase complex; BdhA, 3-hydroxybutyrate dehydrogenase; Th1, thiolase.

[0029] Figure 5 The phenotype and 3-HB production of the strain clj-2-hbd2 overexpressing the hbd2 gene (GenBank No. CLJU_c37300) and the control strain clj-2 (control) not expressing hbd2 in gas fermentation are shown. Among them, Figure A is a curve chart comparing the growth of the two strains in syngas fermentation; Figure B is a bar chart comparing the 3-HB production of the two strains in syngas fermentation. DETAILED DESCRIPTION

[0030] This study established a technology for rapidly constructing engineered Clostridium aerovorum, which can be used as a cell factory to achieve efficient bioconversion of syngas (rich in CO and CO2) into important target compounds such as the four-carbon compound 3-hydroxybutyrate (3-HB) through fermentation.

[0031] Clostridium aeruginosa is an important chemoautotrophic anaerobic bacterium. It is known that it can convert CO2 / CO into simple organic compounds such as acetic acid and ethanol. The technology of manufacturing ethanol from industrial carbon-containing tail gas based on Clostridium aeruginosa has been industrialized in recent years. However, as a type of autotrophic anaerobic bacteria, Clostridium aeruginosa is difficult to genetically modify and has low efficiency, which seriously restricts the development of new products such as four-carbon or longer carbon chain organic compounds. Based on the advantages of high loading capacity and high transposition efficiency of transposons, the inventors carried the target product synthesis gene cluster on the chromosome of Clostridium aeruginosa through transposons to achieve random integration, obtained a large number of engineered bacteria that can synthesize the target product at one time, and quickly discovered the high-yield strains through subsequent phenotypic analysis. The strain with the best performance obtained by screening and modification has better ability to produce 3-hydroxybutyric acid using syngas than similar strains currently reported. This research result has laid an important technical foundation for the development of a new type of Clostridium aeruginosa one-carbon cell factory.

[0032] In subsequent studies, we selected strains with integrated 3-HB pathway genes to determine the integration sites of their pathway genes on chromosomes, and found that the integration of pathway genes driven by mariner transposon on the chromosomes of Clostridium ljungdahlii was random. Subsequently, the 3-HB yield under fructose fermentation conditions was determined by high-performance liquid chromatography, and the 3-HB synthesis ability of the integrated engineering strains was screened. It was found that the expression of 3-hydroxybutyrate pathway genes in the integrated strains had a "position effect". When the pathway genes were integrated at different sites in the genome, there were large differences in the expression of each gene in the 3-hydroxybutyrate pathway. Further selection of differentially expressed strains for gas fermentation found that differential expression still existed under gas fermentation conditions. In order to improve the expression level of 3-HB in the engineered bacteria, we optimized the metabolic network of 3-hydroxybutyrate synthesis and found that 3-hydroxybutyryl-CoA can be derived from the pyruvate pathway, and two reducing forces will be generated in the process of generating acetoacetyl-CoA catalyzed by endogenous (C. ljungdahlii DSM 13528 source) 3-hydroxybutyryl-CoA dehydrogenase Hbd2. By enhancing the expression of the gene hbd2, the production of 3-hydroxybutyrate was further increased to 3g / L.

[0033] The mariner transposon integration strategy of the present invention can effectively and rapidly integrate large DNA fragments, such as biosynthetic pathway gene clusters, into the chromosomes of Clostridium, so that Clostridium aerogenes can convert one-carbon gases, such as syngas, which pollute the environment, into high-value-added organic compounds, such as 3-hydroxybutyric acid, thereby achieving the environmental significance and economic benefits of "turning waste into treasure".

[0034] When investigating the genetic engineering strategy of the present invention, we used simulated steel mill tail gas (with a composition of CO:CO2:H2:N2=56%:20%:9%:15%) as a syngas (also known as C1 gas) carbon source to carry out syngas fermentation of engineered bacteria to produce 3-hydroxybutyric acid (3-HB).

[0035] After obtaining a large number of integrated strains, the integration sites of large DNA fragments in the genome are optimized through appropriate screening processes, which can further increase the production of target compounds, that is, the expression level of pathway genes.

[0036] The terms "(target compound production / pathway gene expression) increase", "elevate" or "enhance" can mean an increase of at least 10% compared to a reference level (such as a starting strain), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including an increase of 100%, or any increase between 10%-100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level.

[0037] In the description of the technical solutions of the present invention, the term "and / or" used in terms such as "A and / or B", "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0038] The terms "engineered Clostridium aerovorum", "engineered Clostridium bacterium" and "engineered bacteria" herein have the same meaning, and all refer to strains of wild-type Clostridium aerovorum that have been genetically modified to express the target product. Wild-type Clostridium aerovorum may also be referred to as the "original strain" or "starting strain".

[0039] In this article, for the sake of simplicity, a certain protein, such as 3-hydroxybutyryl-CoA dehydrogenase (Hbd2) from C. ljungdahlii DSM13528, and its encoding gene (DNA) name hbd2 are sometimes used interchangeably. Those skilled in the art should understand that they represent different types of substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context. For example, for Hbd2, when used to describe the function or category of 3-hydroxybutyryl-CoA dehydrogenase, it refers to the protein; when described as a gene, it refers to the encoding gene hbd2 of the protein.

[0040] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0041] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0042] Example

[0043] Materials and methods

[0044] The molecular biology experiments in this article, including plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., are mainly carried out with reference to "A Guide to Molecular Cloning Experiments" (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). For example, the competent cell transformation method and the competent cell preparation method are all carried out with reference to "A Guide to Molecular Cloning Experiments" (3rd edition), Chapter 1, page 96. If necessary, the specific experimental conditions can be determined by simple experiments.

[0045] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0046] Main culture medium:

[0047] LB medium: 5g / L yeast extract, 10g / L tryptone, 10g / L sodium chloride. (LB solid medium plus 20g / L agar powder.)

[0048] YTF (yeast extract, tryptone, fructose) medium: 10g / L yeast extract, 16g / L tryptone, 10g / L fructose, 0.2g / L sodium chloride, 0.3g / L cysteine, 500μL / L resazurin storage solution, 2mL / L trace element storage solution, 1mL / L vitamin storage solution, dissolved in double distilled water, and adjusted to pH 6.0 with hydrochloric acid. Among them,

[0049] Trace element storage solution: 10.0g / L nitrilotriacetic acid, 5.0g / L manganese sulfate, 4.0g / L ferrous ammonium sulfate hexahydrate, 1.0g / L cobalt chloride hexahydrate, 1.0g / L zinc sulfate heptahydrate, 0.1g / L cupric chloride dihydrate, 0.1g / L nickel chloride hexahydrate, 0.1g / L sodium molybdate dihydrate, 0.1g / L sodium stannate, 0.1g / L sodium tungstate dihydrate, dissolved in double distilled water.

[0050] Vitamin storage solution: 100 mg / L vitamin B6, 50.0 mg / L thiamine, 50.0 mg / L vitamin B2, 50.0 mg / L calcium pantothenate, 50.0 mg / L lipoic acid, 50.0 mg / L p-aminobenzoic acid, 50.0 mg / L nicotinic acid, 50.0 mg / L vitamin B12, 20.0 mg / L biotin, 20.0 mg / L folic acid, dissolved in double distilled water.

[0051] Resazurin stock solution: 1g / L Resazurin, dissolved in double distilled water.

[0052] PETC 1754 Medium:

[0053] 30mL / L mineral element stock solution, 1mL / L vitamin stock solution, 2mL / L trace element stock solution, 0.5g / L yeast extract, 5g / L 2-morpholineethanesulfonic acid, 5mL / L reducing agent stock solution, 500μL / L resazurin stock solution, dissolved in double distilled water, and adjusted to pH 6.0 with NaOH.

[0054] Mineral element storage solution: 80g / L sodium chloride, 100g / L ammonium chloride, 10g / L potassium chloride, 10g / L dipotassium hydrogen phosphate, 20g / L magnesium sulfate heptahydrate, 4g / L calcium chloride dihydrate, dissolved in double distilled water.

[0055] Reducing agent stock solution: 90 g / L sodium hydroxide, 40 g / L cysteine ​​hydrochloride, 40 g / L sodium sulfide nonahydrate, dissolved in double distilled water.

[0056] SMP buffer: 270 mM sucrose, 4.4 mM NaH2PO4, 0.6 mM Na2HPO4, 1 mM MgCl2·6H2O.

[0057] C. ljungdahlii DSM 13528 was purchased from the German National Culture Collection (DMSZ).

[0058] The plasmids expressing 3-HB pathway genes in the embodiments, such as pCYG4 and pMTL83251-aor-fadk, are constructed and preserved by the research group of Jiang Weihong of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. Any unit or individual may obtain the plasmid for verification of the present invention, but may not be used for other purposes, including development and utilization, scientific research and teaching, without the permission of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences.

[0059] The PCR primers used in the examples are listed in Table 1 and were synthesized by Boshang Biotechnology (Shanghai) Co., Ltd.

[0060] Table 1. Primers used in constructing the recombinant plasmids in the examples

[0061]

[0062]

[0063] In Table 1, "-f" in the name stands for forward direction; "-r" stands for reverse direction.

[0064] The provider of the simulated steel plant exhaust gas (gas composition is CO:CO2:H2:N2=56%:20%:9%:15%) is Shanghai Haoqi Gas Co., Ltd.

[0065] Detection method of 3-hydroxybutyric acid (3-HB) in fermentation broth:

[0066] The content of 3-hydroxybutyric acid in the fermentation broth was determined by gas chromatography. The gas chromatograph machine and detection method are as follows: Agilent technology 1100 series, column: Bio-Rad Aminex HPX–87H (300×7.8mm), mobile phase: 5mM H2SO4, flow rate: 0.6mL / min, column temperature: 60℃, differential refractive index detector. The sample was centrifuged at 4℃12,000rpm for 10min, and the supernatant of the fermentation broth was taken for the determination of 3-HB content. The 3-HB content was calculated by the external standard method.

[0067] It should be noted that, for the sake of convenience of description, in the embodiments, the plasmid number and the corresponding strain number may share the same number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0068] Example 1: Design of 3-HB Synthesis Pathway

[0069] According to genome annotation and previous reports, Clostridium jundal contains some genes of the 3-HB synthesis pathway, including 3-hydroxybutyrate dehydrogenase (3-hydroxybutyrate dehydrogenase, BdhA) that catalyzes acetoacetate to produce 3-HB, 3-hydroxybutyryl-CoA dehydrogenase (3-hydroxybutyryldehydrogenase, Hbd) that catalyzes acetoacetyl-CoA to produce 3-hydroxybutyryl-CoA, and thioesterase (thioesterase, TesB) that catalyzes 3-hydroxybutyryl-CoA to produce 3-HB. Therefore, only the introduction of partially deleted genes can achieve the production of 3-HB (Tokiwa & Ugwu, 2007; Wei et al., 2021).

[0070] To this end, we decided to first introduce thiolase (Th1) from Clostridium acetobutylicum (GenBank accession number CA_P0079) into Clostridium jundal to achieve the conversion of acetyl-CoA to acetoacetyl-CoA (see Figure 1 Then, using acetoacetyl-CoA as the metabolic starting point, three 3-HB synthesis pathways were constructed and introduced into the cells of Clostridium jundal using the same plasmid vector (see Figure 1 B). Pathway ① (abbreviated as clj-3hb-i) introduced CoA-transferase complex AB (CtfAB) from Clostridium acetobutylicum (GenBank No. CA_P0163, CA_P0164) and BdhA (GenBank No. CDR20291_2567) from Clostridium difficile R20291. Pathway ② (abbreviated as Clj-3hb-ii) introduced Acetoacetyl-CoA reductase (PhaB) (GenBank No. H16_A1439) from Cupriavidus necator H16. Pathway ③ (abbreviated as clj-3hb-iii) introduced Hbd (GenBank No. CKL_0458) from Clostridium kluyveri.

[0071] Example 2: Optimization of 3-HB Synthesis Pathway

[0072] 2.1 The 3-HB pathway genes of the three designed pathways ①, ② and ③ were introduced into Clostridium cells through plasmid transformation. The plasmids to be constructed were pMTL83151-Ppta-phaB-thl, pMTL83151-Ppta-hbd-thl and pMTL83151-Ppta-thl-ctfAB (see Figure 2 ).

[0073] Plasmid construction: The starting plasmid was pMTL83151, which was digested with restriction endonucleases BamHI and XhoI to obtain the plasmid backbone. pta First, the C. ljungdahlii genome was used as a template and primers (BamHI-Ppta-f / Ppta-r) were used to amplify the pta promoter sequence P pta .

[0074] PCR reaction program: 98°C pre-denaturation for 5 min; 98°C denaturation for 30 s, 55°C annealing for 30 s, 68°C extension for 2 min, 30 cycles; 68°C extension for 10 min, and storage at 16°C. High-fidelity DNA polymerase was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., product number: KFX-101, and the PCR reaction system was prepared according to the instructions.

[0075] The amplified product was recovered by 1.5% (w / v) agarose gel electrophoresis. The PCR product recovery kit was purchased from AXYGEN (Shanghai) Company, product number: AP-GX-250.

[0076] Using plasmid pTrc99aABC (for a description of this plasmid, see the literature: Wei Xiangju, Wu Ju, Guo Pengye, et al. Effect of short-chain thioesterase deficiency on the synthesis of polyhydroxybutyrate lactate in Escherichia coli [J]. Journal of Biotechnology, 2021, 37(1):11.) as a template, primers Ppta-phaB-f / phaB-RBS-thl-r were used to amplify the phaB gene fragment (GenBank number H16_A1439). Using the genome of Clostridium acetobutylicum as a template, primers phaB-thl-f / thl-XhoI-R were used to amplify the thl gene fragment (GenBank number CA_P0079). Using the phaB gene fragment and promoter P pta The fragment was used as a template and the fragment P was obtained by overlapping PCR. pta -phaB. pta -phaB and thl were used as templates, and overlap PCR amplification was performed again to obtain fragment P pta -phaB-thl. The fragment P was assembled using the ClonExpress MultiS One Step cloning kit. pta -phaB-thl and the plasmid backbone to obtain the plasmid pMTL83151-P pta -phaB-thl.

[0077] Plasmid pMTL83151-P pta The construction method of -hbd-thl is basically the same as the above steps, except for the amplification of the fragment hbd. The fragment hbd was amplified using the genome of Clostridium kluyveri as a template and primers Ppta-hbd-f / hbd-thl-r.

[0078] Plasmid pMTL83151-P ptaThe construction method of -thl-ctfAB is basically the same as the above steps, except for the amplification of the fragment ctfAB. The fragment ctfAB was amplified using the genome of Clostridium acetobutylicum as a template and primers thl-ctfA-f / ctfB-XhoI-r.

[0079] 2.2 Preparation of competent culture medium of Clostridium Yongdal: Clostridium Yongdal is a strict anaerobic bacterium. All reagents and culture media need to be prepared in advance and placed in an anaerobic box for deoxygenation for more than 12 hours. All operations except centrifugation need to be completed in a high-efficiency anaerobic workstation.

[0080] The reagents that need to be prepared in advance are: SMP buffer, YTF medium, 9.375% glycine solution, 2M sucrose solution, 100% dimethyl sulfoxide (DMSO). SMP buffer and YTF medium are sterilized by high temperature and high pressure at 121°C for 20 minutes. Glycine solution and sucrose solution are sterilized by filtration with a 0.22μm filter membrane in a clean bench. After the above reagents are prepared, they are placed in an anaerobic box for deoxygenation at least 12 hours in advance.

[0081] 2.2.1 Preparation of bacterial solution: Take a frozen Clostridium jundal glycerol stock from a -80℃ refrigerator and inoculate it into 5mL YTF liquid culture medium for activation and culture, usually at 37℃ for 24h. Use a pipette to draw 20μL of activated Clostridium jundal into the YTF solid plate placed in advance, take a clean and sterile inoculation loop and evenly streak it using the three-line method. After a single colony grows on the streaked plate (usually 2-3 days), pick a single bacterium and inoculate it into 5mL YTF liquid culture medium for culture. Culture at 37℃ for 24h to obtain the seed solution for preparing Clostridium jundal competent cells.

[0082] 2.2.2 Inoculate 1 / 1000 of the inoculum into 200 mL YTF expansion system. After culturing for about 16 hours, take 1 mL of the mixed system and measure the OD in a visible spectrophotometer. 600 , when OD 600 When the pH is between 0.4 and 0.6, add 40 mL of 9.375% glycine and 60 mL of 2 M sucrose to the 200 mL culture system, mix well and leave it to stand for 2 hours.

[0083] 2.2.3 Dispense the above culture system into sterile 50mL centrifuge tubes, tighten the tube caps and precool on ice for 10 minutes. After precooling, centrifuge at 4℃ and 5,000rpm for 10 minutes. After centrifugation, pour out the supernatant in an anaerobic box. This process is called bacterial collection. Repeat the above bacterial collection steps as needed until all strains in the expansion culture system are collected.

[0084] 2.2.4 After resuspending the cells with a small amount of SMP buffer, add SMP to 40 mL and centrifuge at 4°C and 5000 rpm for 10 min. After centrifugation, pour out the supernatant in an anaerobic box and repeat the above steps once.

[0085] 2.2.5 Add DMSO and SMP to the remaining bacteria, resuspend with 100 μL DMSO and 900 μL SMP for every 100 mL of bacterial solution, and dispense into cryopreservation tubes. The prepared competent cells can be frozen in a -80°C refrigerator for later use.

[0086] 2.3 Electrotransformation: First, you need to prepare in advance Clostridium jundal efficient electrotransformation competent cells (200 μL of competent cells for each electrotransformation), ice box, electrotransformation plasmid, electrotransformation cup (2 mm in diameter), and electrotransformer. After testing, the amount of electrotransformation plasmid is 4 μg / 10 μL. After the preparation of all materials, first thaw the competent cells on ice, and pre-cool the electrotransformation cup and the plasmid to be electrotransformed on ice. Adjust the electrotransformation parameters of the electrotransformer to 1000V, 200Ω, and 50μF (you can use an empty electrotransformation cup to test the working status of the electrotransformer once. If the electrotransformation reading is 10.3 or 10.4, it means that the working status is normal). After the competent cells are thawed, pipette 200 μL into the EP tube containing the plasmid in the anaerobic box, gently blow and suck a few times with the tip of the gun to mix slightly, and let it stand on ice for 10 minutes. Transfer the mixed system to an electroporation cup, wipe off the condensed water on the cup body, and perform electroporation. Successful electroporation is indicated by a layer of white foam on the upper layer of the competent cells and a pulse reading between 7.5-9.5 milliseconds. After the electroporation is completed, quickly transfer the competent cells to 800 μL YTF medium for recovery for 20 hours. After the recovery is completed, spread them on a YTF plate supplemented with 5 μg / mL thiamphenicol (Tm).

[0087] 2.4 Syngas fermentation of expression pathway gene strains: The fermentation experiment used in this study was syngas fermentation. The fully synthetic culture medium used for fermentation was PETC 1754. The gas used was simulated steel plant tail gas (gas composition was CO: CO2: H2: N2 = 56%: 20%: 9%: 15%). The fermentation was carried out in a 120mL serum bottle, the fermentation system was 30mL, the atmospheric pressure in the fermentation bottle was 0.2Mpa, and the fermentation system was cultured in a 37°C shaker with a shaker speed of 120rpm. All culture media and fermentation serum bottles need to be sterilized in advance at 121°C for 20min. The specific operation steps are as follows:

[0088] 2.4.1 Preparation of primary seed solution: Pick a single bacterium from the plate and inoculate it into 5 mL YTF medium and culture it at 37°C for about 24 hours until the OD of the bacterial solution reaches 600 Greater than 1.0.

[0089] 2.4.2 Preparation of secondary seed solution: Pipette 1 mL of primary seed solution and measure OD 600 , according to OD 600 Calculate the amount of secondary seeds to be transferred, the transfer amount is about 500 / OD 600 μL, incubate at 37°C for 12–16 h.

[0090] 2.4.3 Fermentation: Pipette 0.5 mL of secondary seed solution and measure OD 600 , when OD 600 Fermentation can be carried out when the pH is between 1.0 and 1.2. First, draw 1.5 mL of the secondary seed solution into the serum bottle, use a measuring cylinder to measure 28.5 mL of PETC 1754 medium (add 5 μg / mL Tm) and place it in the serum bottle, press the cap tightly and then inflate. The inflation conditions are to replace the gas in the bottle with synthetic gas at an atmospheric pressure of 0.2 MPa for 2 minutes and inflate for 1 minute and 30 seconds. After the inflation is completed, place it in a 37°C shaker for culture, and the speed of the shaker is 120 rpm.

[0091] 2.4.4 Gas exchange and sampling: Gas fermentation requires gas replacement in the fermentation bottle every 24 hours. The replacement conditions are 0.2MPa atmospheric pressure synthesis gas exchange for 2 minutes and aeration for 1 minute 30 seconds. Before each gas exchange, a 1mL sterile syringe is used to sample, and the sample is measured by a visible spectrophotometer. 600 , a growth curve was drawn, and then the production of 3-hydroxybutyrate was determined by high performance liquid chromatography.

[0092] 2.5 Product determination: The fermentation broth sample was centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was taken for determination of 3-HB content. The 3-HB content was calculated using the external standard method.

[0093] 2.6 Experimental results:

[0094] The constructed plasmid pMTL83151-P pta -phaB-thl, pMTL83151-P pta -HBD-THL and pMTL83151-P pta -thl-ctfAB was electroporated into Clostridium jundal cells and the transformants were subjected to gas fermentation and 3-hydroxybutyrate production assays. It was found that the plasmid pMTL83151-P pta -thl-ctfAB strain biomass OD at 144h of fermentation 600 Up to 1.88 (see Figure 1 C), the yield of 3-HB reached 1.03 g / L (see Figure 1D), compared with the other two strains (pathway ② and ③), the growth was the best and the 3-HB production was the highest. Therefore, we believe that pathway ① is a more suitable pathway for Clostridium jundal to produce 3-HB.

[0095] Example 3: Design and construction of transposable plasmids and screening of high-yield strains

[0096] 3.1 Plasmid construction: The transposition plasmid constructed in this study is pCYG4-P 1339 -3HB. The starting plasmid was the plasmid pCYG4 constructed in the laboratory earlier, and the plasmid backbone was obtained by double digestion with restriction endonucleases NotI and XhoI. 1339 The promoter expresses the transposase.

[0097] First, plasmid pCYG4 was used as a template and primers p1339-f / himar1C9-r were used to amplify the promoter sequence and transposase P 1339 -himar1c9-itr sequence, and the catP gene sequence was amplified using primers catp-f / catp-r. The bdhA gene sequence was amplified using the genome of Clostridium difficile R20291 as a template, and the bdhA gene sequence was amplified using primers bdhA-f / bdhA-r. The promoter sequence P was amplified using the genome of C.ljungdahlii as a template, and the promoter sequence P was amplified using primers 01440-f / 01440-r 01440 . With gene catP fragment and bdhA fragment and promoter P 01440 The fragment was used as a template and the fragment P was obtained by overlapping PCR. 01440 -bdhA-catP. Plasmid pMTL83151-Ppta-thl-ctfAB was used as a template and primers pta-f / ctfB-r were used to amplify sequence P pta -thl-ctfAB, using plasmid pCYG4 as template, and using primers itr-f / itr-r to amplify the sequence transposase recognition site sequence itr-fdxterminator. pta -thl-ctfAB fragment and itr-fdx terminator fragment were used as templates, and overlapping PCR was used to obtain gene fragment P pta -thl-ctfAB-fdx terminator-itr. Finally, the Yisheng multi-fragment one-step rapid cloning kit ( Plus Multi One Step Cloning Kit) for plasmid backbone, fragment P 1339 -himar1c9-itr, fragment P 01440 -bdhA-catP, fragment P pta-thl-ctfAB-fdx terminator-itr for multi-fragment assembly to obtain plasmid pCYG4-P 1339 -3HB, the nucleotide sequence of which is SEQ ID NO: 1.

[0098] 3.2 Transposition: Plasmid pCYG4-P 1339 -3HB was electrotransformed into Clostridium jundal, and spread on a YTF plate with 5 μg / mL Tm added, and cultured in an anaerobic box at 37°C for 3-5 days. The preparation of Clostridium jundal competent cells and electrotransformation conditions are shown in Example 2. After the transformants grow on the plate, 64 single bacteria were picked and inoculated in 2mL YTF (with 5 μg / mL Tm) and cultured in an anaerobic box at 37°C for 24 hours. After the culture was completed, the inoculation amount of 5% v / v was transferred to 2mL YTF medium (with 5 μg / mL Tm, 1M IPTG) for 24 hours to induce plasmid loss, and the passage was repeated twice. 10 μL of the culture system after passage was drawn and plated in YTF solid medium (with 5 μg / mL Tm), and cultured in an anaerobic box at 37°C for 3 days until a single colony grew. Use a sterile toothpick to pick up 10 single bacteria and streak them on YTF (5μg / mL thiamphenicol Tm) and YTF (10μg / mL erythromycin Em) plates at the same time. If the strain can grow on the YTF (5μg / mL Tm) plate but not on the YTF (10μg / mL Em) plate, it proves that the plasmid has been successfully lost and a transposition insertion event has occurred.

[0099] 3.3 Fructose fermentation of integrated bacteria: The fructose fermentation experiment of Clostridium jundal was carried out in a test tube or a 2mL 96-deep-well plate. A small amount of colonies was dipped with a sterilized toothpick and inoculated into a 2mL YTF medium. The culture was anaerobically cultured for 96 hours until the end of growth. The criterion for judging the end of fermentation was that the precipitated bacteria at the bottom of the test tube were completely white (dead bacteria). The fermentation broth at the end of fermentation was collected and centrifuged, and the supernatant was used to determine the production of 3-hydroxybutyric acid by high-performance liquid chromatography. The genome of the precipitated bacteria was extracted and the transposition insertion site was determined by reverse PCR sequencing.

[0100] 3.4 Extraction of integrated bacterial genome: Collect 3mL of culture system and centrifuge at 12,000rpm for 10min to obtain bacterial cells. Add 200μL TEN buffer to the bacterial cells to resuspend, then add 40μL 50mg / mL lysozyme and mix well, and place at 37℃ for 20min. Add 30μL 10% SDS to the system, mix evenly by turning the EP tube upside down to make the solution transparent. Add 7.5μL 10mg / mL proteinase K to the EP tube, place at 50℃ for 1h, and mix evenly by turning it upside down during the period. Add 75μL 5M potassium acetate to the EP tube and mix evenly by turning it upside down slightly. Add 200μL phenol chloroform and mix evenly by turning it upside down slightly. Centrifuge the mixed system at 12,000rpm for 5min. Take the supernatant and add 0.6 times the volume of isopropanol at the same time, centrifuge at 4℃ and 12,000rpm for 10min. Remove the supernatant, wash the precipitate with 50 μL 70% ethanol, and dry at room temperature after removing the ethanol. This step can also be performed on a 50°C metal bath. When the precipitate is completely transparent, add 50 μL ddH2O to the EP tube, place it at 42°C for 20 minutes to dissolve the precipitate, and finally measure the genome concentration using a Nano Drop spectrophotometer.

[0101] 3.5 Reverse PCR: First, it is necessary to select the high-frequency restriction sites of the Clostridium jundal genome. The restriction sites selected in this study are the recognition sites of the restriction endonucleases HindIII and VspI. The genome is digested with restriction endonucleases HindIII or VspI. The restriction system is 50μL, including 5μLFD buffer, 2μL restriction endonuclease, and 10μg genomic DNA. After overnight digestion at 37℃, the restriction endonuclease is inactivated in a 65℃ water bath, and then the restriction system is diluted to a final concentration of 5ng / μL of DNA in the system. The diluted enzyme system is ligated with 1μLT4 DNA ligase (Takara, Japan). The total amount of enzyme-ligated DNA is 220ng, and the enzyme-ligated system is placed at 16℃ overnight. Inactivate T4 DNA ligase in a 65℃ water bath for 30min, and a circular DNA containing a known fragment will be obtained in the system. The enzyme-linked system was amplified using reverse PCR primers reverse PCR-f / reverse PCR-r and KOD Fx DNA polymerase. The amplified product was recovered by agarose gel electrophoresis and sent to Platinum sequencing.

[0102] 3.6 Integrated bacteria syngas fermentation and growth curve drawing: The syngas fermentation conditions were the same as those in Example 2, the main difference being that PETC 1754 medium was used and no antibiotics were added.

[0103] 3.7 Determination of the yield of 3-hydroxybutyric acid as a syngas fermentation product of integrated bacteria: The method is the same as in Example 2.

[0104] 3.8 Testing pathway gene expression by RT-qPCR: It should be noted that all RNA-related equipment used in this experiment, including gun tips and EP tubes, must use RNase free equipment and reagents.

[0105] Prepare samples: First, prepare the culture medium of the strain to be tested. The preparation process is similar to the gas fermentation experiment of Clostridium jundal. The difference is that the serum bottle used has a volume of 500mL, the fermentation system is 150mL, the atmospheric pressure is 0.1MPa, the ventilation time is 2min, the filling time is 2min, and the gas in the serum bottle is replaced every 12h. After 36h of fermentation, use a 50mL centrifuge tube to collect the bacteria at 4℃ and 5,000rpm for 10min. The collected bacteria are wrapped in tin foil and stored in liquid nitrogen.

[0106] Grinding samples: Place the mortar and grinding rod on a plate, introduce about 2 / 3 of the mortar volume of anhydrous ethanol into each mortar, ignite to burn the alcohol dry, and wait for the mortar to cool. Then prepare several 1mL Trizon and place them on ice. Pour liquid nitrogen into the mortar twice to cool the mortar, and then fill the mortar with liquid nitrogen again after the liquid nitrogen evaporates completely. Quickly take out the sample from the liquid nitrogen tank and transfer it to the mortar. When the liquid nitrogen is almost evaporated, accelerate the grinding. When the liquid nitrogen evaporates completely, add liquid nitrogen to grind again. Repeat this 3 times. After the grinding, the sample is transferred to the pre-cooled 1mL Trizon, and vortex oscillation for 1 minute to mix the sample and Trizon evenly.

[0107] RNA extraction: Add 200 μL chloroform to the Trizon solution, vortex and let stand on ice for 5 minutes; centrifuge at 4°C, 12,000 rpm for 10 minutes, discard the supernatant and add 1 mL 75% ethanol to the centrifuge tube. Take the supernatant after centrifugation and add an equal volume of isopropanol and mix well, place in a -20°C refrigerator for precipitation for 30 minutes, centrifuge at 4°C, 12,000 rpm for 10 minutes, and discard the supernatant; dry at room temperature, add 50 μL RNase free water, let stand at 60°C for 10 minutes until the RNA is completely dissolved, and measure the RNA concentration by Nanodrop.

[0108] Reverse transcription PCR: After dilution, aspirate 1 μg / μL mRNA and perform reverse transcription using the Transcript kit, and then perform PCR using primers RT bdhA-f / RT bdhA-r, RT rho-f / RT rho-r, RT thl-f / RT thl-r, RT ctfA-f / RT ctfA-r, and RT ctfB-f / RT ctfB-r.

[0109] 3.9 Experimental Results

[0110] After the transposition plasmid is successfully constructed, it is introduced into Clostridium jundal through electroporation for transposition integration. Figure 3 As shown, steps ① and ② are to electro-transform the plasmid carrying the transposase and the synthetic pathway genes into the Clostridium junda cells, and drive the pathway genes to be integrated into the chromosomes of Clostridium junda through the expression of the transposase; steps ③ to ⑤ are to pick the transformants grown on the flat solid culture medium and subculture them in a 96-deep-well plate containing a liquid culture medium (with IPTG added), and induce plasmid elimination while achieving transposition; steps ⑥ to ⑦ are to verify whether the free plasmid in the cell is eliminated; steps ⑧ to ⑩ are to identify the insertion site of the 3-HB synthetic pathway gene cluster on the chromosome of the integrated strain and to detect the 3-HB fermentation yield of the strain.

[0111] A total of 64 strains with integrated 3-HB biosynthesis pathway were selected to identify the production of 3-hydroxybutyrate and the integration sites of pathway genes under fructose fermentation conditions. The results showed that the gene cluster of 3-HB biosynthesis pathway was integrated at different sites on the chromosomes of these strains (see Table 2), with a large degree of randomness (see Figure 4 Subsequently, these 64 strains were used for batch fructose fermentation to test their ability to synthesize 3-HB. Figure 4 As shown in B, all 64 strains can produce 3-HB. Interestingly, their 3-HB production varies greatly, with the highest reaching 3.48 g / L, more than three times the lowest (1.01 g / L), suggesting that there are differences in the expression of the 3-HB synthesis pathway in these strains, which may be caused by the "position effect" of the integration of pathway genes on chromosomes. Next, we selected strains clj-2, clj-13, and clj-48 with high production under fructose as the carbon source and strains clj-45, clj-51, and clj-64 with low production under gas fermentation conditions and tested them. We found that their production was consistent with that under fructose fermentation conditions (see Figure 4 C, D). After testing the expression of pathway genes in the selected strains clj-2, clj-45, and clj-51, we found that there were obvious differences in the expression of pathway genes. The expression of pathway genes in strain clj-2 was significantly higher than that in clj-45 and clj-54, which shows that there is a direct relationship between the integration site and the expression of the gene (see Figure 4 Middle E).

[0112] Table 2. Integration sites of 3-hydroxybutyrate pathway genes in Clostridium jundal

[0113]

[0114]

[0115]

[0116] (Note: The blank in Table 2 means that the insertion site of the pathway gene corresponding to the integrant strain could not be determined by experimental methods. CLJU_cXXXX-YYYY means that the insertion site of the pathway gene is located in the non-coding region between the CLJU_cXXXX and CLJU_cYYYY genes.)

[0117] The experimental results showed that when the 3-HB synthesis pathway gene was integrated into the non-coding region between the CLJU_c05590 and CLJU_c05600 genes in the genome of Clostridium aerovar, the expression level of 3-HB was significantly higher, suggesting that there is a "position effect" in the expression of genes in this pathway.

[0118] Example 4: Functional optimization of high-yield strains

[0119] After comprehensive comparison, strain clj-2 showed good phenotypic advantages under both sugar fermentation conditions and gas fermentation conditions, so this strain was subsequently selected for further optimization of its metabolic network. After testing, it was found that Clostridium ljungdahlii contained 3-hydroxybutyryl-CoA dehydrogenase (3-hydroxybytyryl-CoA dehydrogenase, Hbd2) from Clostridium ljungdahlii DSM 13528, which catalyzes 3-hydroxybutyryl-CoA to produce acetoacetyl-CoA. By overexpressing hbd2 in clj-2, it is expected to increase the production of acetoacetyl-CoA, the precursor compound of 3-HB, thereby increasing the yield of 3-HB. Based on this inference, we overexpressed the hbd2 gene in clj-2 (strain clj-2-hbd2) for verification. The experimental steps are as follows:

[0120] 4.1 Plasmid construction: The transposition plasmid constructed in this study was pMTL83251-P pta -hbd2. The starting plasmid was the plasmid pMTL83251-aor-fadk constructed in the laboratory earlier. First, the plasmid pMTL83251-aor-fadk was digested with restriction endonucleases NotI and XhoI to obtain the plasmid backbone. The C. ljungdahli genome was used as a template, and the promoter sequence of the gene pta was amplified with primers M13-r / pta-hbd2-r to obtain P pta The hbd2 gene fragment was obtained by amplifying the promoter P pta The gene hbd2 was used as template and the fragment P was obtained by overlapping PCR using primers M13-r / hbd2-r. ptaFinally, the fragment P was assembled using the ClonExpress MultiS One Step cloning kit. pta -hbd2 and the plasmid backbone are connected to obtain the plasmid pMTL83251-P pta -hbd2, the nucleotide sequence of which is SEQ ID NO:2.

[0121] 4.2 The preparation of competent cells and electroporation conditions were basically the same as those in Example 2, except that the electroporated strain was the obtained integrated strain clj-2, and the plate coated after electroporation was a YTF plate supplemented with 10 μg / mL erythromycin (Em).

[0122] 4.3 The steps of strain syngas fermentation and growth curve drawing are basically the same as those in Example 2, with the main difference being that PETC 1754 medium supplemented with 10 μg / mL erythromycin is used.

[0123] 4.4 The method for determining 3-HB production in fermentation broth is high performance liquid chromatography, and the determination process is consistent with Example 2.

[0124] 4.5 Experimental results:

[0125] See also Figure 5 The fermentation results showed that the growth phenotype of the overexpressed hbd2 gene in clj-2 (strain clj-2-hbd2) for gas fermentation was not much different ( Figure 5 However, after 96 h of fermentation, the 3-HB production of the clj-2-hbd2 strain was 3.01 g / L, while the 3-HB production of the control strain clj-2 (without overexpression of hbd2) was 1.2 g / L, which was 150% higher than that of the latter ( Figure 5 (middle B).

[0126] The above experiments verified the rationality of the design of the present invention and achieved the expected effect of increasing the production of the target compound.

[0127] It should be noted that the listing and discussion of previously disclosed documents in this specification should not be regarded as an admission that the document is prior art or common knowledge.

Claims

1. A method for constructing an engineered bacterium of Clostridium aerovorum, characterized in that: The steps include: A. Genes required in the biosynthetic pathway for preparing the target product, i.e., pathway genes, form pathway gene clusters; B. embedding the pathway gene cluster obtained in step A into the transposase recognition site of mariner transposon to form a pathway gene expression cassette; C. Select a strong constitutive promoter commonly used in Clostridium jundal to drive the expression of the transposase, load the pathway gene expression cassette obtained in step B into the multiple cloning site of a plasmid suitable for expression in Clostridium, and insert an inducible repressor element upstream of the replicon of the plasmid to obtain a transposase plasmid; D. The transposition plasmid obtained in step C is transformed into Clostridium aerovorum, and positive clones in which the pathway gene cluster is integrated into the chromosome of Clostridium aerovorum are screened to obtain engineered Clostridium aerovorum expressing the target product.

2. The method according to claim 1, characterized in that The aerophagous Clostridium is Clostridium ljungdahlii.

3. The method according to claim 1, characterized in that The pathway gene cluster was integrated into the following site of the C. aerovorans genome: the noncoding region between the CLJU-c05590 and CLJU-c05600 genes.

4. The method according to claim 1, characterized in that Step A also includes inserting a resistance gene in the middle of the pathway gene cluster for use as a resistance selection tag.

5. The method according to claim 1, characterized in that The mariner transposon is the transposase Himar1C9.

6. The method according to claim 1, characterized in that The plasmid is selected from the group consisting of pMTL 82151, pMTL 83151, pMTL 84151, pMTL 85151, pMTL 82251, pMTL 82254, pMTL 83251, pMTL 83254, pMTL84251, pMTL 84254, pMTL 85251, pMTL 85254 and pIMP1.

7. The method according to claim 1, characterized in that The plasmid is pMTL83151; the constitutive strong promoter is P 1339 ; The inducible repressor element is Pfac / Pptb::lacI.

8. The method according to claim 1, characterized in that The target product is 3-hydroxybutyric acid, and accordingly, the biosynthetic pathway is a 3-hydroxybutyric acid biosynthetic pathway; optionally, the transposase plasmid obtained in the above step C is used to express a 3-hydroxybutyric acid biosynthetic pathway gene, and the nucleotide sequence of the transposase plasmid may be SEQ ID NO: 1; preferably, it also includes a plasmid overexpressing 3-hydroxybutyryl-CoA dehydrogenase (GenBank No. CLJU_c37300) from Clostridium ljungdahlii DSM 13528, and its nucleotide sequence may be SEQ ID NO:

2.

9. An engineered bacterium of Clostridium aerovorum, characterized in that: The method is constructed according to any one of claims 1 to 8.

10. Use of the engineered bacterium Clostridium aerovorum as claimed in claim 9 in producing a target product using a carbon gas.

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