Escherichia coli engineering bacteria with high n-hexanoic acid yield and construction method and application thereof

Abstract

This invention belongs to the fields of fermentation and genetic engineering technology, and provides a high-yield hexanoic acid-producing engineered *Escherichia coli* strain, its construction method, and its applications. This engineered strain is *Escherichia coli*... E. coli w3110 was the starting strain, overexpressing the acetyl-CoA transferase gene. act and β-ketothiolase gene bktB Overexpression of trans-enoyl-CoA reductase gene ter 3-Hydroxybutyryl-CoA dehydrogenase gene hbd, and 3-hydroxybutyryl coenzyme A dehydratase gene crt Or, simultaneously not expressing the pyruvate formate lyase gene. pflB or lactate dehydrogenase gene ldhA This engineered bacteria can convert glucose into hexanoic acid, a high-value product, with a yield of 4.968 g / L after 18 hours of fermentation. It adopts aerobic fermentation, resulting in rapid cell growth, a short fermentation cycle, and a high acid production rate. The fermentation process is simple, easy to control, and has low production costs, which is conducive to its promotion and application in industrial production.

Description

Technical Field

[0001] This invention belongs to the fields of fermentation and genetic engineering technology, specifically relating to an engineered Escherichia coli strain that produces high levels of hexanoic acid, its construction method, and its application. Background Technology

[0002] Hexanoic acid is a medium-chain fatty acid and an important raw material for the synthesis of flavorings, fine chemicals, and biofuels. Hexanoic acid and its derivatives have wide applications in the chemical, food, pharmaceutical, feed, and cosmetic industries. Hexanoic acid esters are commonly used as flavor additives in food and beverages, and also in the preparation of flavorings and fragrances. Polymers synthesized from hexanoic acid can be used in the manufacture of biodegradable materials, such as environmentally friendly plastics and fibers. Furthermore, hexanoic acid esters and their derivatives exhibit antibacterial and anti-inflammatory activities in the pharmaceutical field, showing potential medicinal value. Meanwhile, hexanoic acid is also receiving increasing attention as a precursor to biofuels such as hexanol.

[0003] Currently, the main methods for producing hexanoic acid include chemical synthesis and bio-fermentation. Chemical synthesis typically uses 2-octanol as a raw material and nitric acid as an oxidant to produce hexanoic acid, resulting in numerous byproducts and significant pollution. In contrast, bio-fermentation utilizes microorganisms to produce hexanoic acid, offering advantages in environmental friendliness and sustainability. The fermentation method primarily employs Clostridium (such as...) Clostridium kluyveri Anaerobic bacteria, such as ethanol and acetic acid, use ethanol and acetic acid as co-substrate to synthesize hexanoic acid.

[0004] However, Clostridium is a strict anaerobe, and its fermentation process is extremely sensitive to oxygen, requiring demanding operating conditions and complex metabolic regulation, making it difficult to improve the yield and efficiency of hexanoic acid. Furthermore, although the genome sequence of Clostridium is known, traditional genetic modification tools are relatively outdated and require further optimization to drive in-depth metabolic engineering.

[0005] In contrast, Escherichia coli ( Escherichia coli With its rapid growth, simple culture conditions, and mature genetic manipulation system, *E. coli* has become a more promising alternative host. However, research on the production of hexanoic acid using *E. coli* is still in the exploratory stage, and the yield reported so far is low. Using glucose as a substrate, the yield after 36 hours of fermentation is about 0.5 g / L (Bioresource Technology, 2018, 247:1253-1257).

[0006] In conclusion, developing a microbial fermentation method for the efficient synthesis of hexanoic acid using glucose as a single carbon source is of great significance for achieving the green and low-cost industrial production of hexanoic acid. However, no successful reports of this method have been published to date. Summary of the Invention

[0007] This invention provides an engineered Escherichia coli strain that produces high levels of hexanoic acid, its construction method, and its applications.

[0008] This invention is achieved by the following technical solution: a high-yield hexanoic acid-producing engineered Escherichia coli strain, wherein the engineered strain is Escherichia coli (… Escherichia coli w3110 was the starting strain, which overexpressed the acetyl-CoA transferase gene. act and β-ketothiolase gene bktB Overexpression of trans-enoyl-CoA reductase gene ter 3-Hydroxybutyryl-CoA dehydrogenase gene hbd, and 3-hydroxybutyryl coenzyme A dehydratase gene crt Or, simultaneously not expressing the pyruvate formate lyase gene. pflB or lactate dehydrogenase gene ldhA .

[0009] The overexpression bktB and act The two genes are ligated into the pEM vector for free expression; the overexpression ter,hbd and crt The three genes were ligated into the pET28a-T5 vector for free expression.

[0010] The gene that does not express pyruvate formate lyase pflB or lactate dehydrogenase gene ldhA To knock out or silence pflB or ldhA .

[0011] Furthermore, the knocking pflB or ldhA To use the CRISPR / Cas9 system to knock out pflB or ldhA .

[0012] The engineered bacteria is Escherichia coli. E. coli w3110 is the originating bacterium. E.coli w3110 carries overexpression act and bktB plasmid pEM-act- bktB and overexpression ter,hbd and crt plasmid pET28a-T5- ter - hbd - crt It was named Hex-1; With Escherichia coli E. coli w3110 is the originating bacterium. E. coli w3110 knockout pflB Genes, for E.coli w3110ΔpflB Carry overexpression act and bktB plasmid pEM-act- bktB and overexpression ter,hbd and crt plasmid pET28a-T5- ter - hbd - crt It was named Hex-2; With Escherichia coli E. coli w3110 is the originating bacterium. E. coli w3110 knockout ldhA Genes, for E.coli w3110Δ ldhA Carry overexpression act and bktB plasmid pEM-act- bktB and overexpression ter,hbd and crt plasmid pET28a-T5- ter - hbd - crt It was named Hex-3.

[0013] Furthermore, the engineered bacteria are Hex-2 or Hex-3.

[0014] The present invention also provides a method for constructing the engineered Escherichia coli strain that produces high levels of hexanoic acid, comprising the following steps: (1) Knockout of pyruvate formate lyase gene pflB Using pTarget-F as a template, design the website http: / / crispr-era.stanford.edu / pflB The N20 sequence was obtained, and primers were designed based on the N20 sequence to amplify pTarget-F- by reverse PCR. pflB The linear vector of plasmid was then phosphorylated and self-ligated to obtain the plasmid pTarget-F- pflB ; by E. coli Using the w3110 genome as a template, according to E. coli w3110 pflB Primers were designed based on the gene sequence to amplify genes containing... pflB The upstream and downstream segments of the homologous arm, after homologous recombination, will yield Δ pflB The fragment is concatenated to pMD19-Tsimple to obtain pMD19-T-Δ pflB Then, using this as a template, Δ is amplified. pflB -1; The above plasmid pTarget-F- pflB and fragment ΔpflB- 1 Electroconversion to pCas9-containing plasmid E. coli In w3110 competent cells, positive transformants were obtained, and pTarget-F- in the positive transformants was eliminated. pflB plasmid obtained pflB Gene knockout bacteria E. coli w3110 Δ pflB ; (2) Knockout of lactate dehydrogenase gene ldhA Using pTarget-F as a template, design the website http: / / crispr-era.stanford.edu / ldhA The N20 sequence was obtained, and primers were designed based on the N20 sequence to amplify pTarget-F- by reverse PCR. ldhA The linear vector of plasmid was then phosphorylated and self-ligated to obtain the plasmid pTarget-F- ldhA ; by E. coli Using the w3110 genome as a template, according to E. coli w3110 ldhA Primers were designed based on the gene sequence to amplify genes containing... ldhA The upstream and downstream segments of the homologous arm, after homologous recombination, will yield Δ ldhA The fragment is concatenated to pMD19-Tsimple to obtain pMD19-T-Δ ldhA Then, using this as a template, Δ is amplified. ldhA -1; The above plasmid pTarget-F- ldhA and fragment Δ ldhA -1 Electroconversion to a pCas9-containing plasmid E. coli In w3110 competent cells, positive transformants were obtained, and pTarget-F- in the positive transformants was eliminated. ldhA plasmid obtained ldhA Gene knockout bacteria E. coli w3110 Δ ldhA ; (3) Preparation of recombinant plasmid pEM- act - bktB : artificially synthesized act The gene was ligated into plasmid pEM using a one-step homologous recombination method to obtain the recombinant plasmid pEM- act Using PCR technology with the genome of Rollstonella H16 as a template, the genome was amplified. bktB Gene fragments were ligated to plasmid pEM- using a one-step homologous recombination method. act The recombinant plasmid pEM- was obtained. act -bktB ; (4) Preparation of recombinant plasmid pET28a-T5- ter - hbd - crt Using PCR technology with the genome of Clostridium acetone-butanol ATCC824 as a template, the following were amplified: ter、hbd、crt The gene fragment was ligated into plasmid pET28a-T5 using a one-step homologous recombination method to obtain the recombinant plasmid pET28a-T5- ter - hbd - crt ; (5) Constructing an engineered Escherichia coli strain that produces high levels of hexanoic acid: The recombinant plasmid pEM- obtained in step (3) is used to construct the strain. act - bktB The recombinant plasmid pET28a-T5- obtained in step (4) ter - hbd - crt Transform to E. coli w3110, obtained from step (1) pflB Gene knockout bacteria [[ID= w3110 Δ ​ or the result of step (2) ​ Gene knockout bacteria ​ w3110 Δ ​ Screening was performed by plating kanamycin and ampicillin-resistant plates, and the positive transformants obtained by colony PCR identification were the Escherichia coli genetically engineered bacteria that produce high levels of hexanoic acid.

[0015] The present invention also provides the application of the engineered Escherichia coli strain that produces high levels of hexanoic acid in the production of hexanoic acid.

[0016] Furthermore, the engineered Escherichia coli is used in the fermentation production of hexanoic acid using glucose as a single carbon source.

[0017] The specific method is as follows: After activation, the engineered E. coli bacteria are inoculated into TB medium and cultured in a shaker at 37°C and 200 rpm; culture until OD... 600 When the bacterial culture volume reaches 0.6-0.8, add 0.1% isopropyl-β-D-thiogalactoside IPTG and induce culture at 25℃ and 200rpm for 18h. The TB medium formula is: glucose 20g / L, yeast extract 24g / L, peptone 12g / L, glycerol 4mL / L, KH2PO4 2.31g / L, K2HPO4 12.54g / L.

[0018] In this invention: the β-ketothiolase gene ​The amino acid sequence encoded by the gene shown in NCBI accession number 34309143; the acetyl-CoA transferase gene. ​ The amino acid sequence encoded by SEQ ID NO.1; the trans-enoyl-CoA reductase ​ The amino acid sequence encoded by the gene shown in NCBI accession number 4EUF_A; the 3-hydroxybutyryl-CoA dehydrogenase. ​ The amino acid sequence encoded by the gene shown in NCBI accession number KHD37023.1; the 3-hydroxybutyryl-CoA dehydratase ​ The amino acid sequence encoded by the gene shown in NCBI accession number NP_349318.1. This is the pyruvate-formate lyase gene. ​ The nucleotide sequence of the gene shown in NCBI accession number 945514; the lactate dehydrogenase gene. ​ This is the nucleotide sequence of the gene shown in NCBI accession number 946315.

[0019] Compared with existing technologies, this invention can convert glucose into high-value product hexanoic acid, with a maximum yield of 4.968 g / L after 18 hours of fermentation. This invention uses aerobic fermentation, resulting in rapid cell growth, a short fermentation cycle, and a high acid production rate. The fermentation process is simple, easy to control, and has low production costs, which is conducive to the promotion and application of industrial production. This invention also provides a new approach for constructing high-yield hexanoic acid strains. Attached Figure Description

[0020] ​ The hexanoic acid synthesis pathway constructed in the Escherichia coli cell factory includes: atoB: acetyl-CoA thiolytic enzyme; hbd: 3-hydroxybutyryl-CoA dehydrogenase; crt: 3-hydroxybutyryl-CoA dehydratase; ter: trans-enoyl-CoA reductase; bktB: β-ketothiolytic enzyme gene; and act: acetyl-CoA transferase. ​ Construction process of recombinant plasmid pEM-act-bktB; ​ The image shows the recombinant plasmid pEM-act-bktB. ​ Construction process of recombinant plasmid pET28a-T5-ter-hbd-crt; ​ The spectrum of the recombinant plasmid pET28a-T5-ter-hbd-crt; ​ This is an SDS identification diagram of recombinant proteins from genetically engineered bacteria. In the diagram: lane 1 is pET28a-T5-ter-hbd-crt, lane 2 is pET28a-T5, lane 3 is pEM, lane 4 is pEM-ACT, lane 5 is BktB, and M is the protein marker. ​ This is the standard curve for hexanoic acid. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0023] Equivalent technologies of the specific embodiments described herein that are apparent to those skilled in the art through routine experimentation are included in this application.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0025] In the following examples, "codon optimization" refers to gene redesign that utilizes preferred codons and avoids codons with low utilization or rarity. Every organism exhibits some degree of codon utilization difference or preference, with the most frequently used codons being preferred codons.

[0026] Molecular biology experiments not specifically described in the following examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, which were mainly performed in accordance with *Molecular Cloning: A Laboratory Manual* (3rd edition). PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier.

[0027] The whole-genome synthesis, primer synthesis, and sequencing in the following examples were performed by Tianlin Biotechnology Wuxi Co., Ltd. Host strain ​ w3110 was purchased from ATCC, and the plasmids used in this experiment were synthesized by Tianlin Biotechnology Wuxi Co., Ltd.

[0028] Sources of experimental materials: Bacterial plasmid extraction kit and bacterial genome extraction kit were purchased from Tiangen Biotech (Beijing) Co., Ltd.; SDS-PAGE protein gel kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.; antibiotics, PCR gel extraction kit, agarose, and glycerol were purchased from Sangon Biotech (Shanghai) Co., Ltd.; restriction endonucleases, PCR polymerases, and homologous recombinases were purchased from Dalian Takara Co., Ltd.; tryptone, yeast extract, sodium chloride, agar powder, and hexanoic acid were purchased from Shanghai Aladdin Biotech Co., Ltd.

[0029] Hexanoic acid was detected by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: column: Aminex HPX-87H (9μm 7.8×300mm); mobile phase: 3mM H2SO4; column temperature: 40℃; detection wavelength: 210nm; injection volume: 10μL; flow rate: 0.6mL / min.

[0030] Example 1: Design of the synthetic route for hexanoic acid: as follows ​ As shown, the designed *E. coli* cell factory hexanoic acid synthesis pathway is completed by six enzymes with acetyl-CoA as the metabolic node. First, acetyl-CoA thiolysis enzyme (AtoB) catalyzes the synthesis of acetyl-CoA from acetyl-CoA. Then, 3-hydroxybutyryl-CoA dehydrogenase (Hbd) catalyzes the synthesis of 3-hydroxybutyryl-CoA from acetyl-CoA. Finally, 3-hydroxybutyryl-CoA dehydratase (Crt) catalyzes the synthesis of crotonyl-CoA from 3-hydroxybutyryl-CoA. Finally, trans-enoyl-CoA reductase (Ter) catalyzes the synthesis of crotonyl-CoA from crotonyl-CoA. Acyl-CoA, β-ketothiolysis (BktB) catalyzes the synthesis of 3-ketohexanoyl-CoA from butyryl-CoA, 3-hydroxybutyryl-CoA dehydrogenase (Hbd) catalyzes the synthesis of 3-hydroxyhexanoyl-CoA from 3-ketohexanoyl-CoA, 3-hydroxybutyryl-CoA dehydratase (Crt) catalyzes the synthesis of trans-2-hexenoyl-CoA from 3-hydroxyhexanoyl-CoA, trans-hexenoyl-CoA reductase (Ter) catalyzes the synthesis of hexanoyl-CoA from trans-2-hexenoyl-CoA, and acetyl-CoA transferase (Act) catalyzes the synthesis of hexanoyl-CoA from hexanoic acid.

[0031] Example 2: ​ w3110 pyruvate formate lyase gene ​ Knockout: Based on E. coli from the KEGG database ​ w3110 ​ The pyruvate-formate lyase gene sequence was designed using data from the CRISPR-ERA website (http: / / crispr-era.stanford.edu / ). ​ The N20 sequence was obtained, and primers N20- were designed based on the N20 sequence. ​And N20-A. Then with N20- ​ Using N20-A as primers and pTarget-F as a template, reverse PCR amplification was performed (reaction program: pre-denaturation 95℃ 3 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 2.5 min, 28 cycles, extension 72℃ 5 min) to obtain pTarget-F- ​ The linear vector was phosphorylated and then self-ligated. The ligation product was transformed into JM109 competent cells, plated on LB agar plates containing zithromycin resistance, transformants were picked, plasmids were extracted, and sequenced for verification, yielding the plasmid pTarget-F-. ​ .

[0032] According to E. coli in the KEGG database ​ w3110 ​ (Pyruvate formate lyase gene) gene sequence design, primer U- ​ -S,U- ​ -A, D- ​ -S and D- ​ -A. Then with U- ​ S, U ​ -A is used as a primer, utilizing ​ Using the w3110 genome as a template, the upstream fragment U- containing the homologous arm was amplified. ​ ; with D- ​ -S,D- ​ -A is used as a primer, utilizing ​ Using the w3110 genome as a template, the downstream fragment D- containing homologous arms was amplified. ​ Then, a one-step homologous recombination method is used to connect to obtain Δ. ​ The fragment was further ligated into pMD19-T simple, and the ligation product was transformed into JM109 competent cells. Transformants were plated on LB plates containing ampicillin resistance, picked, and plasmids were extracted and sequenced to obtain the plasmid pMD19-T-Δ. ​ Using U ​ S and D- ​ -A was used as a primer, utilizing pMD19-T-Δ ​ Using a template, PCR amplification was performed (reaction program: pre-denaturation 95℃ 3 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 1 min, 28 cycles, extension 72℃ 5 min) to obtain Δ ​ -1.

[0033] Δ ​ -1 and pTarget-F- ​ Electroconversion to pCas9-containing plasmids​ w3110 competent cells. After rapid resuscitation in 1 mL LB medium at 37°C and 150 rpm for 1 h, the cells were plated onto LB agar plates containing kanamycin and zizomycin antibiotics. After inverted culture for 24 h, identification primer U- was used. ​ -2S and D- ​ Positive transformants were identified by colony PCR (reaction program: pre-denaturation 95℃ 10 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 1.5 min, 30 cycles, extension 72℃ 5 min). Single colonies were picked and cultured with IPTG for 12 h, which induced the localization of the sgRNA-pMB1 sequence to pTarget-F-. ​ Above, eliminate pTarget-F- ​ Plasmids were spread on LB agar plates containing kanamycin, and single colonies were selected to obtain pTarget-F- culprits. ​ The bacterial strain containing the plasmid. Single colonies were selected and inoculated into LB liquid medium. After overnight incubation at 42°C, single colonies that could grow on antibiotic-free plates but not on kanamycin-containing plates were screened and identified using the U- primer. ​ -2S and D- ​ -A was used for colony PCR verification. Correctly verified strains were named... E. ​ w3110 Δ ​ Primer sequences are shown in Table 1.

[0034] Table 1 Primer sequences used for PCR amplification of knockout fragments and overexpressed genes. Example 3: ​ w3110 lactate dehydrogenase gene ​ Knockout: Based on E. coli from the KEGG database ​ w3110 ​ (Lactate dehydrogenase gene) sequence design, using data from the CRISPR-ERA website (http: / / crispr-era.stanford.edu / ). ​ The N20 sequence was obtained, and primers N20- were designed based on the N20 sequence. ​ And N20-A. Then with N20- ​ Using N20-A as primers and pTarget-F as a template, reverse PCR amplification was performed (reaction program: pre-denaturation 95℃ 3 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 2.5 min, 28 cycles, extension 72℃ 5 min) to obtain pTarget-F- ​The linear vector was phosphorylated and then self-ligated. The ligation product was transformed into JM109 competent cells, plated on LB agar plates containing zithromycin resistance, transformants were picked, plasmids were extracted, and sequenced for verification, yielding the plasmid pTarget-F-. ​ .

[0035] According to E. coli in the KEGG database ​ w3110 ​ (Lactate dehydrogenase gene) Gene sequence design, primer U- ​ S, U- ​ A, D ​ S and D- ​ -A. Then with U- ​ S, U- ​ A is used as a primer, utilizing ​ Using the w3110 genome as a template, the upstream fragment U containing the homologous arm was amplified. ​ ; with D- ​ -S,D ​ A is used as a primer, utilizing ​ Using the w3110 genome as a template, the downstream fragment D- containing homologous arms was amplified. ​ Then, a one-step homologous recombination method is used to connect to obtain Δ. ​ The fragment was further ligated into pMD19-T simple, and the ligation product was transformed into JM109 competent cells. Transformants were plated on LB plates containing ampicillin resistance, picked, and plasmids were extracted and sequenced to obtain the plasmid pMD19-T-Δ. ​ Using U ​ -S and D- ​ A was used as a primer, and pMD19-T- ​ Using a template, PCR amplification was performed (reaction program: pre-denaturation 95℃ 3 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 1.5 min, 28 cycles, extension 72℃ 5 min) to obtain Δl ​ 1.

[0036] Δ ​ -1 and pTarget-F- ​ Electroconversion to pCas9-containing plasmids ​ w3110 competent cells. After rapid resuscitation in 1 mL LB medium at 37°C and 150 rpm for 1 h, the cells were plated onto LB agar plates containing kanamycin and zizomycin antibiotics. After inverted culture for 24 h, identification primer U- was used. ​ -2S and D- ​-A Positive transformants were identified using colony PCR. Single colonies were picked and cultured with IPTG for 12 hours, which induced the localization of the sgRNA-pMB1 sequence to pTarget-F-. ​ Above, eliminate pTarget-F- ​ Plasmids were spread on LB agar plates containing kanamycin, and single colonies were selected to obtain pTarget-F- culprits. ​ The bacterial strain containing the plasmid. Single colonies were selected and inoculated into LB liquid medium. After overnight incubation at 42°C, single colonies that could grow on antibiotic-free plates but not on kanamycin-containing plates were screened and identified using the U- primer. ​ -2S and D- ​ -A was used for colony PCR verification (reaction program: pre-denaturation 95℃ 10min; cycling phase 98℃ 10s, 55℃ 30s, 72℃ 1.5min, 30 cycles, extension 72℃ 5min). The correctly verified strain was named... ​ w3110 Δ ​ The primer sequences used are shown in Table 1.

[0037] Example 4: Construction of plasmid pEM- ​ - ​ The build process is as follows ​ As shown, based on the amino acid sequence of acetyl-CoA transferase ACT (amino acid sequence as shown in SEQ ID NO.1), the codons of the acetyl-CoA transferase gene were optimized (gene sequence as shown in SEQ ID NO.2), and the gene was artificially synthesized and ligated into the pEM plasmid to obtain the plasmid pEM-act.

[0038] According to the NCBI database, Rawlstonella Ralstonia eutropha Using the H16 genome as a template, primers were used... bktB -A and bktB -S will come from Ralstonia eutropha The β-ketothiolase gene of H16 (NCBI accession number: AM260479.1) was amplified by PCR (reaction program: pre-denaturation 95℃ 3 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 1.2 min, 28 cycles, extension 72℃ 5 min). Primer sequences are shown in Table 1.

[0039] The amplified fragment was ligated into the pEM-act plasmid digested with XhoI via one-step homologous recombination. The ligation product was transformed into JM109 competent cells, plated on LB agar plates containing ampicillin resistance, transformants were picked, plasmids were extracted, and sequenced for verification, yielding the plasmid pEM-act- bktB The diagram is as followsFigure 3 As shown.

[0040] Example 5: Construction of plasmid pET28a-T5- ter - hbd - crt The construction process is shown in Figure 4, based on the NCBI database. Clostridium acetobutylicum Using the ATCC824 genome as a template, primer 1- ter (CA)-S and 2- ter( Ca)-S will come from Clostridium acetobutylicum The trans-enoyl-CoA reductase gene of ATCC824 (NCBI accession number: 4EUF_A) was amplified by PCR (reaction program: pre-denaturation 95℃ 3 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 1.2 min, 28 cycles, extension 72℃ 5 min), using primers 1- hbd (Ca)-s and 2- hbd (CA)-s amplification of the 3-hydroxybutyryl-CoA dehydrogenase gene (NCBI accession number: KHD37023.1) was performed by PCR (reaction program: pre-denaturation 95℃ 3 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 0.8 min, 28 cycles, extension 72℃ 5 min), using primer 1- Crt (Ca)-S and 2- Crt (Ca)-S amplification of the 3-hydroxybutyryl-CoA dehydratase gene (NCBI accession number: NP_349318.1) was performed by PCR (reaction program: pre-denaturation 95℃ 3 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 0.8 min, 28 cycles, extension 72℃ 5 min). Primer sequences are shown in Table 1.

[0041] The amplified fragment was ligated into the pET28a-T5 plasmid, which was double-digested with PstI and XhoI, using a one-step homologous recombination method. The ligation product was transformed into JM109 competent cells, plated on LB plates containing kanamycin resistance, and transformants were picked. The plasmid was extracted and sequenced for verification, yielding the pET28a-T5 plasmid. ter - hbd - crt The diagram is as follows Figure 5 As shown.

[0042] Example 6: Construction of dual-plasmid engineered bacteria: The plasmid pET28a-T5- obtained above was used to construct the dual-plasmid engineered bacteria. ter - hbd - crtand pEM-act- bktB Converted to E.coli w3110, E.coli w3110 Δ pflB and E.coli w3110 Δ ldhA Competent cells were plated with kanamycin and ampicillin-resistant plates for screening. Positive transformants obtained by colony PCR (reaction program: pre-denaturation 95℃ 10 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 2 min, 30 cycles, extension 72℃ 5 min) are the *E. coli* genetically engineered bacteria described in this invention, named Hex-1. E.coli w3110 carries pEM-act- bktB and pET28a-T5- ter - hbd - crt Hex-2 E.coli w3110 Δ pflB Carrying pEM-act- bktB and pET28a-T5- ter - hbd - crt Hex-3 E.coli w3110 Δ ldhA Carrying pEM-act- bktB and pET28a-T5- ter - hbd - crt Simultaneously convert pET28a-T5 and pEM to E.coli Competent cells of w3110 were screened by plating on kanamycin and ampicillin-resistant plates. Positive transformants obtained by colony PCR (reaction program: pre-denaturation 95℃ 10 min; cycling phase 98℃ 10 s, 55℃ 30 s, 72℃ 1 min, 30 cycles, extension 72℃ 5 min) were used as the control strain and named Hex-C. E.coli w3110 carries pEM and pET28a). SDS-PAGE identification of the recombinant proteins from the genetically engineered bacteria is shown in the figure below. Figure 6 As shown.

[0043] Example 7: Production of hexanoic acid using genetically engineered *E. coli* strains: The engineered strains preserved in glycerol tubes were inoculated onto LB solid medium. Single colonies were selected and transferred to LB medium (5 mL / 20 mL test tubes). After incubation at 37°C and 200 rpm for 12 h, TB medium (50 mL / 250 mL Erlenmeyer flasks) was inoculated at a 10% inoculation rate (V / V). The culture was maintained at 37°C and 200 rpm until OD500 was reached. 600When the concentration reached 0.6-0.8, 0.1% isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added by volume, and the mixture was induced and cultured at 25℃ and 200 rpm for 18 h. The yield of hexanoic acid was determined by high-performance liquid chromatography (HPLC), and the hexanoic acid standard curve is shown below. Figure 7 As shown in Table 2, the calculated acid yields of the genetically engineered strains Hex-1, Hex-2, and Hex-3 of this invention were 4.226 g / L, 4.582 g / L, and 4.968 g / L, respectively, while the control strain Hex-C accumulated a small amount of hexanoic acid.

[0044] Table 2: Product yield (unit: g / L) Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-yield hexanoic acid-producing engineered Escherichia coli, characterized in that: The engineered bacteria is Escherichia coli (Escherichia coli) Escherichia coli w3110 was the starting strain, which overexpressed the acetyl-CoA transferase gene. act and β-ketothiolase gene bktB Overexpression of trans-enoyl-CoA reductase gene ter 3-Hydroxybutyryl-CoA dehydrogenase gene hbd, and 3-hydroxybutyryl coenzyme A dehydratase gene crt Or, simultaneously not expressing the pyruvate formate lyase gene. pflB or lactate dehydrogenase gene ldhA .

2. The engineered Escherichia coli strain with high hexanoic acid production according to claim 1, characterized in that: The overexpression bktB and act The two genes are ligated into the pEM vector for free expression; the overexpression ter, hbd and crt The three genes are ligated into the pET28a-T5 vector for free expression.

3. The engineered Escherichia coli strain with high hexanoic acid production according to claim 1, characterized in that: The gene that does not express pyruvate formate lyase pflB or lactate dehydrogenase gene ldhA To knock out or silence pflB or ldhA .

4. The engineered Escherichia coli strain with high hexanoic acid production according to claim 3, characterized in that: The knock pflB or ldhA To use the CRISPR / Cas9 system to knock out pflB or ldhA .

5. The engineered Escherichia coli strain with high hexanoic acid production according to any one of claims 1-4, characterized in that: The engineered bacteria is Escherichia coli. E. coli w3110 is the originating bacterium. E. coli w3110 carries overexpression act and bktB plasmid pEM-act- bktB and overexpression ter, hbd and crt plasmid pET28a-T5- ter - hbd - crt It was named Hex-1; With Escherichia coli E. coli w3110 is the originating bacterium. E. coli w3110 knockout pflB Genes, for E. coli w3110Δ pflB Carry overexpression act and bktB plasmid pEM-act- bktB and overexpression ter, hbd and crt plasmid pET28a-T5- ter - hbd - crt It was named Hex-2; With Escherichia coli E. coli w3110 is the originating bacterium. E. coli w3110 knockout ldhA Genes, for E. coli w3110Δ ldhA Carry overexpression act and bktB plasmid pEM-act- bktB and overexpression ter, hbd and crt plasmid pET28a-T5- ter - hbd - crt It was named Hex-3.

6. The engineered Escherichia coli strain with high hexanoic acid production according to claim 5, characterized in that: The engineered bacteria are Hex-2 or Hex-3.

7. A method for constructing the engineered Escherichia coli strain with high hexanoic acid production as described in claim 5, characterized in that: Includes the following steps: (1) Knockout of pyruvate formate lyase gene pflB Designed using pTarget-F as a template on the website http: / / crispr-era.stanford.edu / pflB The N20 sequence was obtained, and primers were designed based on the N20 sequence to amplify pTarget-F- by reverse PCR. pflB The linear vector of plasmid was then phosphorylated and self-ligated to obtain the plasmid pTarget-F- pflB ; by E. coli Using the w3110 genome as a template, according to E. coli w3110 pflB Primers were designed based on the gene sequence to amplify the gene containing... pflB The upstream and downstream segments of the homologous arm, after homologous recombination, will yield Δ pflB The fragment is concatenated to pMD19-Tsimple to obtain pMD19-T-Δ pflB Then, using this as a template, Δ is amplified. pflB -1; The above plasmid pTarget-F- pflB and fragment Δ pflB- 1 Electroconversion to pCas9-containing plasmid E. coli In w3110 competent cells, positive transformants were obtained, and pTarget-F- in the positive transformants was eliminated. pflB plasmid obtained pflB Gene knockout bacteria E. coli w3110 Δ pflB ; (2) Knockout of lactate dehydrogenase gene ldhA Designed using pTarget-F as a template on the website http: / / crispr-era.stanford.edu / ldhA The N20 sequence was obtained, and primers were designed based on the N20 sequence to amplify pTarget-F- by reverse PCR. ldhA The linear vector of plasmid was then phosphorylated and self-ligated to obtain the plasmid pTarget-F- ldhA ; by E. coli Using the w3110 genome as a template, according to E. coli w3110 ldhA Primers were designed based on the gene sequence to amplify the gene containing... ldhA The upstream and downstream segments of the homologous arm, after homologous recombination, will yield Δ ldhA The fragment is concatenated to pMD19-Tsimple to obtain pMD19-T-Δ ldhA Then, using this as a template, Δ is amplified. ldhA -1; The above plasmid pTarget-F- ldhA and fragment Δ ldhA -1 Electroconversion to a pCas9-containing plasmid E. coli In w3110 competent cells, positive transformants were obtained, and pTarget-F- in the positive transformants was eliminated. ldhA plasmid obtained ldhA Gene knockout bacteria E. coli w3110 Δ ldhA ; (3) Preparation of recombinant plasmid pEM- act - bktB : artificially synthesized act The gene was ligated into plasmid pEM using a one-step homologous recombination method to obtain the recombinant plasmid pEM- act Using PCR technology with the genome of Rollstonella H16 as a template, the genome was amplified. bktB Gene fragments were ligated to plasmid pEM- using a one-step homologous recombination method. act The recombinant plasmid pEM- was obtained. act - bktB ; (4) Preparation of recombinant plasmid pET28a-T5- ter - hbd - crt Using PCR technology with the genome of Clostridium acetone-butanol ATCC 824 as a template, the following were amplified: ter, hbd, crt The gene fragment was ligated into plasmid pET28a-T5 using a one-step homologous recombination method to obtain the recombinant plasmid pET28a-T5- ter - hbd - crt ; (5) Constructing an engineered Escherichia coli strain that produces high levels of hexanoic acid: The recombinant plasmid pEM- obtained in step (3) is used to construct the strain. act - bktB The recombinant plasmid pET28a-T5- obtained in step (4) ter - hbd - crt Transform to E. coli w3110, obtained from step (1) pflB Gene knockout bacteria E. coli w3110 Δ pflB or the result of step (2) ldhA Gene knockout bacteria E. coli w3110 Δ ldhA Screening was performed by plating with kanamycin and ampicillin-resistant plates, and the positive transformants obtained by colony PCR identification were the Escherichia coli genetically engineered bacteria that produce high levels of hexanoic acid.

8. The application of the engineered Escherichia coli strain with high hexanoic acid production as described in claim 5 in the production of hexanoic acid.

9. The application according to claim 8, characterized in that: The application of the engineered Escherichia coli in the fermentation production of hexanoic acid using glucose as the single carbon source.

10. The application according to claim 9, characterized in that: The method was as follows: After activation, engineered Escherichia coli was inoculated into TB medium and cultured in a shaker at 37°C and 200 rpm; cultured until OD... 600 When the bacterial culture reaches a pH of 0.6-0.8, add 0.1% isopropyl-β-D-thiogalactoside IPTG by volume and induce culture at 25℃ and 200rpm for 18h. The TB medium formula is: glucose 20g / L, yeast extract 24g / L, peptone 12g / L, glycerol 4mL / L, KH2PO4 2.31g / L, K2HPO4 12.54g / L.

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