Genetically engineered bacterium with high yield of corac acid as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a gene engineering bacterium for high yield of corac acid and a construction method and application thereof, and belongs to the technical field of gene engineering, the construction method comprises the following steps: sequentially knocking out a waaL gene cluster, a lon gene, an hns gene, a clsA gene, a clsB gene, a clsC gene, a mcbR gene and an opgDGH gene on an escherichia coli E.coli MG1655 genome, and then sequentially overexpressing an rcsA gene, a galU gene, a manA gene and a cpsG gene. According to the invention, through a multiple gene knockout technology and in combination with genome site-specific integration of an exogenous gene and an enhanced expression element, efficient and stable regulation and control of a corac acid synthesis pathway are realized, and the obtained strain has the capacity of high yield of corac acid and has high industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a genetically engineered bacterium that produces high levels of colacid, its construction method, and its applications. Background Technology

[0002] Colanic acid (CA) is an anionic heteropolysaccharide, typically secreted by Enterobacteriaceae. It is a white, fibrous substance soluble in water and dilute salt solutions, which are acidic. Colanic acid polysaccharide is composed of a hexasaccharide repeating unit consisting of D-galactose, D-glucose, D-glucuronic acid, and L-fucose, in a molar ratio of 2:1:1:2. Fucose and galactose are modified with non-stoichiometric oxyacetyl and pyruvate groups.

[0003] Kolac not only improves the growth of host cells under harsh environments and maintains their normal physiological functions, but also, as a polysaccharide with unique physiological activities, holds significant potential applications across various industries. For example, kolac polysaccharide possesses a porous cellulose structure and a large number of hydrophilic groups on its surface, making it an excellent natural hydrogel with superior water retention and a soft texture, a promising candidate for the future cosmetics market. Kolac is rich in fucose, which has various known biological activities, including anti-inflammatory, anti-tumor, anti-cancer, immune-enhancing, whitening, and anti-aging activities. Kolac can also regulate mitochondrial dynamics and unfolded protein responses within the host, thereby delaying mitochondrial aging and cell rupture. The mitochondrial responses and longevity effects induced by kolac are widespread across different species; therefore, kolac also has broad application prospects in functional nutrition and health fields.

[0004] Currently, the main method to influence the synthesis of colacid in E. coli is through genetic engineering. However, existing methods mainly focus on the metabolic pathway of colacid, knocking out some genes to achieve colacid synthesis, which has limited effect on increasing colacid production. Although there are also methods to modify the bypass metabolic pathway, thereby redistributing metabolic flux by perturbing the global metabolic network of the cell, thus further increasing colacid production, there are currently few studies on constructing genetically engineered bacteria with high colacid production through modification of bypass metabolic pathways. Moreover, most existing genetically engineered bacteria with high colacid production rely on plasmids, and plasmid loss occurs in the solution during passage, leading to passage instability and further reducing the potential for industrial application. Summary of the Invention

[0005] This invention provides a genetically engineered bacterium that produces high levels of colacid, its construction method, and its application. By using multiple gene knockout technology and combining exogenous genes and site-specific integration of expression-enhancing elements into the genome, efficient and stable regulation of the colacid synthesis pathway is achieved. The resulting strain has the ability to produce high levels of colacid and has high potential for industrial application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for constructing a genetically engineered bacterium that produces high levels of colacid, comprising the following steps: Sequentially knock out E. coli E. coli The waaL gene cluster and lon gene on the MG1655 genome hns Gene, clsA Gene, clsB Gene, clsC Gene, mcbR Gene, opgDGH Genes, and then overexpressed sequentially. rcsA Gene, galU Gene, manA Gene, cpsG Gene; The nucleotide sequence of the waaL gene cluster is shown in SEQ ID No. 7 in the sequence listing; The nucleotide sequence of the lon gene is shown in SEQ ID No. 16 of the sequence listing; The hns The nucleotide sequence of the gene is shown in SEQ ID No. 25 of the sequence listing; The clsA The nucleotide sequence of the gene is shown in SEQ ID No. 34 of the sequence listing; The clsB The nucleotide sequence of the gene is shown in SEQ ID No. 43 of the sequence listing; The clsC The nucleotide sequence of the gene is shown in SEQ ID No. 52 of the sequence listing; The mcbR The nucleotide sequence of the gene is shown in SEQ ID No. 61 of the sequence listing; The opgDGH The nucleotide sequence of the gene is shown in SEQ ID No. 70 of the sequence listing; The rcsA The nucleotide sequence of the gene is shown in SEQ ID No. 81 of the sequence listing; The galU The nucleotide sequence of the gene is shown in SEQ ID No. 92 in the sequence listing; The manAThe nucleotide sequence of the gene is shown in SEQ ID No. 103 in the sequence listing; The cpsG The nucleotide sequence of the gene is shown in SEQ ID No. 114 of the sequence listing; In overexpression rcsA When using genes, the following methods are employed: endA Gene site integration and utilization of promoter P J23119 Start Expression rcsA Genetic methods; In overexpression galU When using genes, the following methods are employed: yjiP Gene site integration and utilization of promoter P J23119 Start Expression galU Genetic methods; In overexpression manA When using genes, the following methods are employed: yhdW Gene site integration and utilization of promoter P J23119 Start Expression manA Genetic methods; In overexpression cpsG When using genes, the following methods are employed: cheW Gene site integration and utilization of promoter P J23119 Start Expression cpsG Genetic methods; Furthermore, in the case of knockout or overexpression, the construction method consists of the following steps: preparing recombinant DNA fragments, constructing plasmid pGRB, transforming the plasmid and recombinant DNA fragments, and eliminating the plasmid; More preferably, the construction of the pGRB plasmid comprises the following steps: preparing transformed competent cells, constructing a pGRB plasmid containing the target sequence, and transforming the recombinant plasmid into... E. coli DH5α competent cells were used to screen for positive transformants. More preferably, the transformation plasmid and the preparation of the recombinant DNA fragment consist of the following steps: transforming pREDCas9, preparing electrocompetent cells of the target strain containing pREDCas9, transforming pGRB and the recombinant DNA fragment; More preferably, the plasmid removal comprises the following steps: removing plasmid pGRB and removing plasmid pREDCas9.

[0007] The method for constructing a genetically engineered bacterium that produces high levels of colacid of the present invention first eliminates precursor competition and metabolic flux redirection by knocking out [a specific organism] in *E. coli*. waaThe gene cluster completely blocks the synthesis of the core polysaccharide of lipopolysaccharide, eliminating its major competitive consumption of key precursors UDP-glucose and UDP-galactose, thus efficiently directing metabolic flux to the kola catechin synthesis pathway. Then, it relieves transcriptional and post-translational repression in the kola catechin synthesis pathway, and knocks out the lon gene to eliminate the ATP-dependent protease that degrades the kola catechin positive regulator protein RcsA, significantly improving the intracellular stability of RcsA protein and thus continuously activating the synthesis pathway; and knocks out... hns The gene was targeted to break the silencing effect of the global transcriptional repressor protein on the colacid synthesis gene cluster (cps), enabling it to achieve a basic high level of expression; then, cell membrane structure and physiological function were optimized, and the gene was knocked out. clsA , clsB , clsC Cardiolipin synthase genes, by altering cell membrane phospholipid composition, optimize membrane fluidity and function, thereby enhancing the activity of membrane-bound proteins (such as MdoB) related to the synthesis and transport of colacid precursors, indirectly promoting precursor supply and colacid synthesis; then knocking out mcbR The gene was used to unblock the inhibition of the cysteine ​​synthesis pathway, enhance the intracellular library capacity of the thioacryl donor, and provide an optimized intracellular environment for maintaining the function of proteins related to colacid synthesis and export. Furthermore, the periplasmic dextran synthesis operon opgGHD was knocked out, blocking other competitive polysaccharide synthesis pathways and disrupting the competitive pathway of periplasmic β-1,2-glucan, which consumes large amounts of the precursor UDP-glucan, thus achieving further enrichment of the precursor. Additionally, the following gene expression cassette, driven by the constitutively strong promoter PJ23119, was integrated: integration of the positive regulator PJ23119-... rcsA Constitutive overexpression of positive transcription regulators RcsA , and endogenous RCSB Formation of activating complex, powerfully driving cps Transcription of gene clusters; integration of key precursor supply genes PJ23119- for enhanced expression galU Overexpression of UDP-glucose pyrophosphorylase directly enhances the conversion from glucose-1-phosphate to UDP-glucose, solidifying the precursor foundation; overexpression of phosphogannase isomerase strengthens the metabolic flux from fructose-6-phosphate to GDP-mannose; and integrated enhanced expression of the key enzyme PJ23119- for coratase synthesis... cpsG Overexpression of the initial glycosyltransferase responsible for transferring glycosyl groups from UDP-glucose to the lipid carrier enhances the initiation step of the synthetic pathway.

[0008] Secondly, the present invention provides a genetically engineered bacterium that produces high levels of colacid by the aforementioned construction method; Thirdly, the present invention provides the application of a genetically engineered bacterium that produces high levels of colacid, constructed by the aforementioned construction method, in the fermentation production of colacid.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: The method for constructing a genetically engineered bacterium that produces high levels of colacid of the present invention uses genetic modifications (knockout and integration) that are stable on the genome and are completely independent of plasmids. This fundamentally solves the problems of plasmid loss, the need to add antibiotics, and instability in passage, making it highly suitable for large-scale industrial fermentation applications. In shake flask fermentation, the colacid content in the fermentation broth can reach 1.75 g / L after 48 hours of fermentation, and in fermenter fermentation, the colacid content in the fermentation broth can reach 25 g / L after 48 hours of fermentation. Detailed Implementation

[0010] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0011] The method for constructing the engineered bacteria in Example 1 is based on the method for constructing the engineered bacteria in Examples 2-13. That is, the construction process of the engineered bacteria in Examples 2-13 is carried out in accordance with the method for constructing the engineered bacteria in Example 1.

[0012] Used in Examples 1-13 E. coli MG1655 was purchased from Sangon Biotech.

[0013] Example 1: Method for constructing engineered bacteria The gene editing method was performed in accordance with the literature Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic engineering. 2015, 31: 13-21.

[0014] The plasmid pREDCas9 carries the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of λ phage, the Cas9 protein expression system, and zimexazole resistance (working concentration: 100 mg / L), and needs to be cultured at 32℃; pGRB uses pUC18 as its backbone and includes the J23100 promoter, gRNA-Cas9 binding region sequence, and terminator sequence, as well as ampicillin resistance (working concentration: 100 mg / L), and needs to be cultured at 37℃.

[0015] The specific gene editing steps are as follows: 1. Preparation of recombinant DNA fragments The recombinant fragment used for knockout consists of upstream and downstream homologous arms of the gene to be knocked out (upstream homologous arm - downstream homologous arm); the recombinant fragment used for integration consists of upstream and downstream homologous arms of the integration site and the gene fragment to be integrated (upstream homologous arm - target gene - downstream homologous arm). Primer design software Primer5 was used to design primers for upstream and downstream homologous arms (amplification length approximately 400-600 bp) using the upstream and downstream sequences of the gene to be knocked out or the integration site as templates; primers for amplifying the integration gene were designed using the gene to be integrated as a template. After amplifying the upstream and downstream homologous arms and the target gene fragment separately by PCR, the recombinant fragment was prepared by overlap PCR and used as the donor DNA fragment. The PCR system and method are shown in Table 1. Table 1

[0016] The system for overlap PCR is shown in Table 2: Table 2

[0017] Note: The template consists of equimolar amounts of amplified fragments from upstream and downstream homologous arms and the target gene, and the total amount does not exceed 10 ng.

[0018] PCR reaction conditions (PrimeSTAR HS enzyme): pre-denaturation (95℃) for 5 min; then 30 cycles: denaturation (98℃) for 10 s, annealing ((Tm-3 / 5)℃) for 15 s, extension at 72℃ (this enzyme activity extends by about 1 kb per min); continue extension at 72℃ for 10 min; maintenance (4℃).

[0019] 2. Constructing plasmid pGRB (1) Preparation of transformed competent cells ① Using an inoculation loop in a clean bench to preserve... E. coli One loopful of bacterial culture was inoculated into the preservation tube of TOP 10 competent cells and streaked in three zones on an LB agar plate. The cells were then incubated in a 37°C oven for 12 hours. Single colonies were selected using a sterile pipette tip and inoculated into LB shaker tubes. The cells were then incubated overnight at 37°C and 220 rpm.

[0020] ② Take 1 mL of bacterial culture and inoculate it into a round-bottom flask containing 100 mL of LB liquid medium. Incubate at 37°C and shake at 220 rpm. When the OD reaches 0.3, incubate on ice for 15 min while pre-cooling the centrifuge.

[0021] ③ In a clean bench, slowly pour the bacterial solution after the ice bath into a pre-cooled 50mL sterile centrifuge cup, centrifuge at 4℃ and 6500rpm for 6min, and collect the bacterial cells.

[0022] ④ Resuspend the bacterial cells at the bottom of the centrifuge cup in a pre-cooled 0.1 mol / L CaCl2 solution in a clean bench, incubate on ice for 20 min, and then centrifuge at 4℃ and 6500 rpm for 6 min to collect the bacterial cells.

[0023] ⑤ In a clean bench, discard the supernatant, add 4 mL of CaCl2 solution and 1 mL of 80% glycerol, and resuspend the bacterial cells thoroughly using a pipette. Aliquot 100 μL into 1.5 mL EP tubes to obtain... E. coli The top 10 competent cells were stored in a -80°C freezer.

[0024] (2) Constructing pGRB plasmids containing target sequences The plasmid pGRB was constructed by recombination and ligation of a DNA fragment containing the target sequence with a linearized vector fragment.

[0025] ① Design target sequence Using CRISPR RGEN Tools, a specific 20bp target sequence was selected. The target sequence primer design format is as follows: 5' linearized vector terminal sequence (15bp) + restriction site + target sequence (20bp) + linearized vector terminal sequence (15bp) + 3' and its reverse complementary primer.

[0026] ②Preparing DNA fragments containing the target sequence The designed target sequence primers, namely 5'-linearized vector terminal sequence (15 bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector terminal sequence (15 bp)-3' and its reverse complementary primer, were used to prepare a DNA fragment containing the target sequence by annealing single-stranded DNA. Reaction conditions: pre-denaturation 95℃, 5 min; annealing 50℃, 1 min. The annealing system is shown in Table 3. Table 3

[0027] ③ Preparation of linear carriers Primers were designed using the empty plasmid pGRB as a template, and the linearized pGRB vector was obtained by inverse PCR amplification.

[0028] ④ Connect the linearized pGRB vector to the target sequence The linearized pGRB vector was ligated with a DNA fragment containing the target sequence using the ClonExpress® II One Step Cloning Kit. The recombination conditions were 37°C for 30 min to obtain the recombinant plasmid. The recombination system is shown in Table 4.

[0029] Table 4

[0030] (3) Transfer the recombinant plasmid to E. coli DH5α competent cells ① Place the recombinant plasmid in an ice bath for 5 minutes, then slowly add the recombinant plasmid from the ice bath into the clean bench. E. coli In the TOP 10 competent cells, the mixture was pipetted and aspirated evenly, and then placed in an ice bath for 20 minutes.

[0031] ② After the EP tubes were in an ice bath, they were placed in a 42°C water bath for 45 seconds and then immediately placed in an ice box for 3 minutes to obtain the transformed competent cells.

[0032] ③ Using a pipette in a clean bench, add the transformed competent cells into an EP tube containing 900 mL of SOC resuscitation solution. Place the EP tube in a 37°C shaker at 220 rpm for 1.5 h to obtain the resuscitated cells.

[0033] ④ Centrifuge the revived bacterial cells at 8000 rpm for 1 min, spread all the bacterial cells on LB plates containing ampicillin resistance in a clean bench, spread evenly, and then place in a 37℃ constant temperature incubator for 15 h to obtain transformed competent cells.

[0034] (4) Screening for positive transformants ① Configure the colony PCR system according to Table 5, with a primer concentration of 10 µmol / L.

[0035] Table 5

[0036] ② In a clean bench, use a sterile toothpick to select single colonies of transformed competent cells. Draw oblique lines and spot them onto LB agar plates containing ampicillin resistance, while simultaneously spotting them into the colony PCR system. Perform the PCR reaction under the following conditions: pre-denaturation at 95℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 35 s, and further extension at 72℃ for 10 min. Maintain this denaturation-annealing-extension cycle for 30 cycles at 22℃ to obtain the bacterial culture.

[0037] ③ Use a pipette to aspirate 5 μL of bacterial culture into the wells of a 1% agarose gel for electrophoresis. Inoculate positive strains into small shaker tubes containing 5 mL of LB liquid medium using a sterile toothpick, and add 100 μg / L ampicillin resistance. Incubate at 37°C and 220 rpm for 15 h to preserve the bacteria. Meanwhile, extract plasmid pGRB from the remaining bacterial culture using a kit (TaKaRaMiniBEST Plasmid Purification Kit Ver.4.0) for subsequent electroporation experiments.

[0038] 3. Transformation plasmids and recombinant DNA fragments (1) Transformation of pREDCas9 The plasmid pREDCas9 (purchased from Jingfeng Biotechnology) was electroporated into competent cells. After cell resuscitation and culture, the cells were plated on LB agar plates containing 100 μg / L zizomycin and incubated overnight at 32°C. Single colonies growing on the antibiotic-resistant plates were subjected to colony PCR using identification primers to screen for positive recombinants containing pREDCas9.

[0039] (2) Preparation of electrotransformation competent cells of the target strain containing pREDCas9 The positive recombinants containing pREDCas9 were cultured at 32°C until OD. 600 When the concentration reaches 0.2, add 0.1M IPTG solution (to bring the final concentration to 0.1mM) and continue culturing until OD reaches 0.2. 600 Competent cells were prepared at a concentration of 0.6 to obtain electrotransformed competent cells containing pREDCas9. The purpose of adding IPTG was to induce the expression of the recombinase on plasmid pREDCas9. The culture medium and preparation process for competent cells followed standard operating procedures.

[0040] (3) Transformation of pGRB and recombinant DNA fragments Donor DNA fragments and plasmid pGRB were simultaneously electroporated into electroporated competent cells containing pREDCas9. The revived cells after electroporation were plated on LB agar plates containing 100 μg / L ampicillin and 100 μg / L zizomycin, respectively, and incubated overnight at 32°C. Colony PCR was performed using upstream primers for the upstream homologous arm and downstream primers for the downstream homologous arm to verify the colonies. Positive recombinants containing both the donor DNA fragment and pGRB were screened and maintained.

[0041] 4. Eliminate plasmids (1) Elimination of plasmid pGRB The positive recombinants containing donor DNA fragments and pGRB obtained from screening were inoculated into LB medium containing 0.2% arabinose and 100 μg / L zidimecrolimus resistance in shaker tubes and incubated at 32°C and 220 rpm for 15 h. Using an inoculation loop, three zones were streaked on LB plates containing 100 μg / L zidimecrolimus resistance and incubated overnight at 32°C. The colonies were then spotted onto LB plates containing 100 μg / L ampicillin resistance and 100 μg / L zidimecrolimus resistance, respectively. Single colonies that did not grow on ampicillin-resistant plates but grew on zidimecrolimus-resistant plates were selected as positive transformants that eliminated pGRB and were then preserved.

[0042] (2) Elimination of plasmid pREDCas9 Positive transformants that eliminated pGRB were transferred into LB liquid medium containing zirconia-resistant strains and incubated for 12 hours at 42°C and 220 rpm in a shaker. The resulting inoculum was then streaked into three zones on LB plates and incubated overnight at 37°C. Single colonies were selected from LB plates containing both non-resistant and zirconia-resistant strains. These single colonies, which did not grow on the non-resistant LB plates, were selected as the target strains and preserved.

[0043] Example 2: Construction of engineered bacteria E. coli MG1655 CA-1 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-1. Details are as follows: 1. Prepare the WaaLQ gene knockout fragment according to the method in step 1 of Example 1 for preparing recombinant DNA fragments; specifically: by E. coli Using the MG1655 genome as a template, according to waaLUZYROBSPGQ Design upstream homologous arm primers (UPs) for upstream and downstream sequences of gene clusters. -waaLQ- S、Up- waaLQ -A) and downstream homologous arm primer (DN- waaLQ- S, DN- waaLQ -A), UP -waaLQ- The nucleotide sequence of S is shown in SEQ ID No. 1 of the sequence listing, Up- waaLQ The nucleotide sequence of -A is shown in SEQ ID No. 2 of the sequence listing, DN- waaLQ- The nucleotide sequence of S is shown in SEQ ID No. 3 of the sequence listing, DN- waaLQ The nucleotide sequence of -A is shown in SEQ ID No. 4 of the sequence listing; The upstream and downstream homologous arm fragments of WaaLQ were amplified by PCR. The nucleotide sequence of the upstream homologous arm fragment of WaaLQ is shown in SEQ ID No. 5 of the sequence listing, and the nucleotide sequence of the downstream homologous arm fragment of WaaLQ is shown in SEQ ID No. 6 of the sequence listing. The above fragments were fused by overlap PCR to obtain the WaaLQ gene knockout fragment. The nucleotide sequence of the WaaLQ gene knockout fragment is shown in SEQ ID No. 7 of the sequence listing.

[0044] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- waaLQ -S and gRNA- waaLQ gRNA was obtained by annealing A. waaLQThe nucleotide sequence of -S is shown in SEQ ID No. 8 of the sequence listing, gRNA- waaLQ The nucleotide sequence of -A is shown in SEQ ID No. 9 of the sequence listing.

[0045] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment are wild-type. E. coli MG1655 was obtained and verified by PCR. Successful PCR verification confirms the presence of the strain. E. coli MG1655CA-1.

[0046] Example 3: Construction of engineered bacteria E. coli MG1655 CA-2 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-2. Details are as follows: 1. Prepare the Lon gene knockout fragment according to the method in step 1 of Example 1 for preparing recombinant DNA fragments; specifically: by E. coli Using the MG1655 genome as a template, according to lon Design upstream homologous arm primers (UPs) based on gene upstream and downstream sequences. -lon- S、Up- lon -A) and downstream homologous arm primer (DN- lon -S、DN- lon- A), UP -lon- The nucleotide sequence of S is shown in SEQ ID No. 10 of the sequence listing, Up- lon The nucleotide sequence of -A is shown in SEQ ID No. 11 of the sequence listing. lon The nucleotide sequence of -S is shown in SEQ ID No. 12 of the sequence listing, DN- lon- The nucleotide sequence of A is shown in SEQ ID No. 13 of the sequence listing; PCR amplification of its upstream and downstream homologous arm fragments yielded lon The nucleotide sequence of the upstream homologous arm fragment is shown in SEQ ID No. 14 of the sequence listing. lon The nucleotide sequence of the downstream homologous arm fragment is shown in SEQ ID No. 15 of the sequence listing; the above fragments were fused by overlap PCR to obtain... lon The gene knockout fragment obtained lon The nucleotide sequence of the gene knockout fragment is shown in SEQ ID No. 16 of the sequence listing.

[0047] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- lon -S and gRNA- lon gRNA was obtained by annealing A. lon The nucleotide sequence of -S is shown in SEQ ID No. 17 of the sequence listing, gRNA- lon The nucleotide sequence of -A is shown in SEQ ID No. 18 of the sequence listing.

[0048] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 2. E. coli MG1655CA-1 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was successfully obtained. E. coli MG1655CA-2.

[0049] Example 4: Construction of engineered bacteria E. coli MG1655 CA-3 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-3. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. hns The gene knockout segment; specifically: by E. coli Using the MG1655 genome as a template, according to hns Design upstream homologous arm primers (UPs) based on gene upstream and downstream sequences. -hns- S、Up- hns -A) and downstream homologous arm primer (DN- hns -S、DN- hns- A), UP -hns- The nucleotide sequence of S is shown in SEQ ID No. 19 of the sequence listing, Up- hns The nucleotide sequence of -A is shown in SEQ ID No. 20 of the sequence listing, DN- hns The nucleotide sequence of -S is shown in SEQ ID No. 21 of the sequence listing, DN- hns- The nucleotide sequence of A is shown in SEQ ID No. 22 of the sequence listing; PCR amplification of its upstream and downstream homologous arm fragments yielded hns The nucleotide sequence of the upstream homologous arm fragment is shown in SEQ ID No. 23 of the sequence listing. hns The nucleotide sequence of the downstream homologous arm fragment is shown in SEQ ID No. 24 of the sequence listing; the above fragments were fused by overlap PCR to obtain... hns The gene knockout fragment obtained hns The nucleotide sequence of the gene knockout fragment is shown in SEQ ID No. 25 of the sequence listing.

[0050] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- hns -S and gRNA- hns gRNA was obtained by annealing A. hns The nucleotide sequence of -S is shown in SEQ ID No. 26 of the sequence listing, gRNA- hns The nucleotide sequence of -A is shown in SEQ ID No. 27 of the sequence listing.

[0051] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 3. E. coli MG1655CA-2 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was successfully obtained. E. coli MG1655CA-3.

[0052] Example 5: Construction of engineered bacteria E. coli MG1655 CA-4 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-4. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. clsA The gene knockout segment; specifically: by E. coli Using the MG1655 genome as a template, according to clsA Design upstream homologous arm primers (UPs) based on gene upstream and downstream sequences. -clsA- S、Up- clsA -A) and downstream homologous arm primer (DN- clsA -S、DN- clsA- A), UP -clsA- The nucleotide sequence of S is shown in SEQ ID No. 28 of the sequence listing, Up- clsA The nucleotide sequence of -A is shown in SEQ ID No. 29 of the sequence listing. clsAThe nucleotide sequence of -S is shown in SEQ ID No. 30 of the sequence listing, DN- clsA- The nucleotide sequence of A is shown in SEQ ID No. 31 of the sequence listing; PCR amplification of its upstream and downstream homologous arm fragments yielded clsA The nucleotide sequence of the upstream homologous arm fragment is shown in SEQ ID No. 32 of the sequence listing. clsA The nucleotide sequence of the downstream homologous arm fragment is shown in SEQ ID No. 33 in the sequence listing; the above fragments were fused by overlap PCR to obtain... clsA The gene knockout fragment obtained clsA The nucleotide sequence of the gene knockout fragment is shown in SEQ ID No. 34 of the sequence listing.

[0053] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- clsA -S and gRNA- clsA gRNA was obtained by annealing A. clsA The nucleotide sequence of -S is shown in SEQ ID No. 35 of the sequence listing, gRNA- clsA The nucleotide sequence of -A is shown in SEQ ID No. 36 of the sequence listing.

[0054] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 4. E. coli MG1655CA-3 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was successfully obtained. E. coli MG1655CA-4.

[0055] Example 6: Construction of engineered bacteria E. coli MG1655 CA-5 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-5. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. clsB The gene knockout segment; specifically: by E. coli Using the MG1655 genome as a template, according to clsB Design upstream homologous arm primers (UPs) based on gene upstream and downstream sequences. -clsB- S、Up- clsB-A) and downstream homologous arm primer (DN- clsB -S、DN- clsB- A), UP -clsB- The nucleotide sequence of S is shown in SEQ ID No. 37 of the sequence listing, Up- clsB The nucleotide sequence of -A is shown in SEQ ID No. 38 of the sequence listing. clsB The nucleotide sequence of -S is shown in SEQ ID No. 39 of the sequence listing. clsB- The nucleotide sequence of A is shown in SEQ ID No. 40 of the sequence listing; PCR amplification of its upstream and downstream homologous arm fragments yielded clsB The nucleotide sequence of the upstream homologous arm fragment is shown in SEQ ID No. 41 of the sequence listing. clsB The nucleotide sequence of the downstream homologous arm fragment is shown in SEQ ID No. 42 in the sequence listing; the above fragments were fused by overlap PCR to obtain... clsB The gene knockout fragment obtained clsB The nucleotide sequence of the gene knockout fragment is shown in SEQ ID No. 43 in the sequence listing.

[0056] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- clsB -S and gRNA- clsB gRNA was obtained by annealing A. clsB The nucleotide sequence of -S is shown in SEQ ID No. 44 of the sequence listing, gRNA- clsB The nucleotide sequence of -A is shown in SEQ ID No. 45 of the sequence listing.

[0057] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 5. E. coli MG1655CA-4 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was obtained. E. coli MG1655CA-5.

[0058] Example 7: Construction of engineered bacteria E. coli MG1655 CA-6 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-6. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. clsC The gene knockout segment; specifically: by E. coli Using the MG1655 genome as a template, according to clsC Design upstream homologous arm primers (UPs) based on gene upstream and downstream sequences. -clsC- S、Up- clsC -A) and downstream homologous arm primer (DN- clsC -S、DN- clsC- A), UP -clsC- The nucleotide sequence of S is shown in SEQ ID No. 46 of the sequence listing, Up- clsC The nucleotide sequence of -A is shown in SEQ ID No. 47 of the sequence listing. clsC The nucleotide sequence of -S is shown in SEQ ID No. 48 of the sequence listing. clsC- The nucleotide sequence of A is shown in SEQ ID No. 49 of the sequence listing; PCR amplification of its upstream and downstream homologous arm fragments yielded clsC The nucleotide sequence of the upstream homologous arm fragment is shown in SEQ ID No. 50 of the sequence listing. clsC The nucleotide sequence of the downstream homologous arm fragment is shown in SEQ ID No. 51 of the sequence listing; the above fragments were fused by overlap PCR to obtain... clsC The gene knockout fragment obtained clsC The nucleotide sequence of the gene knockout fragment is shown in SEQ ID No. 52 in the sequence listing.

[0059] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- clsC -S and gRNA- clsC gRNA was obtained by annealing A. clsC The nucleotide sequence of -S is shown in SEQ ID No. 53 of the sequence listing, gRNA- clsC The nucleotide sequence of -A is shown in SEQ ID No. 54 in the sequence listing.

[0060] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 6. E. coli MG1655CA-5 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was successfully obtained. E. coliMG1655CA-6.

[0061] Example 8: Construction of engineered bacteria E. coli MG1655 CA-7 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-7. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. mcbR The gene knockout segment; specifically: by E. coli Using the MG1655 genome as a template, according to mcbR Design upstream homologous arm primers (UPs) based on gene upstream and downstream sequences. -mcbR- S、Up- mcbR -A) and downstream homologous arm primer (DN- mcbR -S、DN- mcbR- A), UP -mcbR- The nucleotide sequence of S is shown in SEQ ID No. 55 of the sequence listing, Up- mcbR The nucleotide sequence of -A is shown in SEQ ID No. 56 of the sequence listing. mcbR The nucleotide sequence of -S is shown in SEQ ID No. 57 of the sequence listing. mcbR- The nucleotide sequence of A is shown in SEQ ID No. 58 of the sequence listing; PCR amplification of its upstream and downstream homologous arm fragments yielded mcbR The nucleotide sequence of the upstream homologous arm fragment is shown in SEQ ID No. 59 of the sequence listing. mcbR The nucleotide sequence of the downstream homologous arm fragment is shown in SEQ ID No. 60 of the sequence listing; the above fragments were fused by overlap PCR to obtain... mcbR The gene knockout fragment obtained mcbR The nucleotide sequence of the gene knockout fragment is shown in SEQ ID No. 61 of the sequence listing.

[0062] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- mcbR -S and gRNA- mcbR gRNA was obtained by annealing A. mcbR The nucleotide sequence of -S is shown in SEQ ID No. 62 of the sequence listing, gRNA- mcbR The nucleotide sequence of -A is shown in SEQ ID No. 63 in the sequence listing.

[0063] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 7. E. coli MG1655CA-6 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was obtained. E. coli MG1655CA-7.

[0064] Example 9: Construction of engineered bacteria E. coli MG1655 CA-8 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-8. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. opgDGH The gene knockout segment; specifically: by E. coli Using the MG1655 genome as a template, according to opgDGH Design upstream homologous arm primers (UPs) based on gene upstream and downstream sequences. -opgDGH- S、Up- opgDGH -A) and downstream homologous arm primer (DN- opgDGH -S、DN- opgDGH- A), UP - opgDGH- The nucleotide sequence of S is shown in SEQ ID No. 64 of the sequence listing, Up- opgDGH The nucleotide sequence of -A is shown in SEQ ID No. 65 of the sequence listing. opgDGH The nucleotide sequence of -S is shown in SEQ ID No. 66 of the sequence listing, DN- opgDGH- The nucleotide sequence of A is shown in SEQ ID No. 67 of the sequence listing; PCR amplification of its upstream and downstream homologous arm fragments yielded opgDGH The nucleotide sequence of the upstream homologous arm fragment is shown in SEQ ID No. 68 of the sequence listing. opgDGH The nucleotide sequence of the downstream homologous arm fragment is shown in SEQ ID No. 69 of the sequence listing; the above fragments were fused by overlap PCR to obtain... opgDGH The gene knockout fragment obtained opgDGH The nucleotide sequence of the gene knockout fragment is shown in SEQ ID No. 70 of the sequence listing.

[0065] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- opgDGH -S and gRNA- opgDGH gRNA was obtained by annealing A. opgDGH The nucleotide sequence of -S is shown in SEQ ID No. 71 of the sequence listing, gRNA- opgDGH The nucleotide sequence of -A is shown in SEQ ID No. 72 in the sequence listing.

[0066] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 8. E. coli MG1655CA-7 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was successfully obtained. E. coli MG1655CA-8.

[0067] Example 10 Construction of engineered bacteria E. coli MG1655 CA-9 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-9. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. rcsA The integrated segment of the gene; specifically: by E. coli Using the MG1655 genome as a template, according to end Design upstream homologous arm primers (UPs) based on gene upstream and downstream sequences. -endA- S、Up- end -A) and downstream homologous arm primer (DN- end -S、DN- endA- A), UP -endA- The nucleotide sequence of S is shown in SEQ ID No. 73 of the sequence listing, Up- end The nucleotide sequence of -A is shown in SEQ ID No. 74 of the sequence listing. end The nucleotide sequence of -S is shown in SEQ ID No. 75 of the sequence listing. endA- The nucleotide sequence of A is shown in SEQ ID No. 76 of the sequence listing; PCR amplification of its upstream and downstream homologous arm fragments yielded... end The nucleotide sequence of the upstream homologous arm fragment is shown in SEQ ID No. 77 of the sequence listing. end The nucleotide sequence of the downstream homologous arm fragment is shown in SEQ ID No. 78 in the sequence listing.

[0068] according to rcsA Gene design primers ( rcsA -S、 rcsA -A), rcsA The nucleotide sequence of -S is shown in SEQ ID No. 79 of the sequence listing. rcsA The nucleotide sequence of -A is shown in SEQ ID No. 80 of the sequence listing. Amplification rcsA Gene fragments obtained rcsA fragment The nucleotide sequence is shown in SEQ ID No. 81 in the sequence listing.

[0069] Among them, promoter P J23119 Design downstream primers in the upstream homologous arm and rcsA In the upstream primer of the gene. The above fragments are fused using overlap PCR to obtain... rcsA Integration fragment of gene (upstream homologous arm - P) J23119 - rcsA -Downstream homologous arm).

[0070] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- end -S and gRNA- end gRNA was obtained by annealing A. end The nucleotide sequence of -S is shown in SEQ ID No. 82 of the sequence listing, gRNA- end The nucleotide sequence of -A is shown in SEQ ID No. 83 in the sequence listing.

[0071] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 9. E. coli MG1655CA-8 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was successfully obtained. E. coli MG1655CA-9.

[0072] Example 11 Construction of engineered bacteria E. coli MG1655 CA-10 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-10. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. stay The integrated segment of the gene; specifically: according to E. coli MG1655 yjiP Gene sequence and PJ23119 Promoter sequence design integration stay The primer for the upstream homologous arm is UP- yjiP- S and UP- yjiP -A, UP- yjiP- The nucleotide sequence of S is shown in SEQ ID No. 84, UP- yjiP The nucleotide sequence of -A is shown in SEQ ID No. 85 in the listing; the downstream homologous arm primer is DN- yjiP -S and DN- yjiP -A, DN- yjiP The nucleotide sequence of -S is shown in SEQ ID No. 86 of the nucleotide sequence listing, DN- yjiP The nucleotide sequence of -A is shown in SEQ ID No. 87 in the listing; its upstream and downstream homologous arm fragments were amplified by PCR to obtain... yjiP The nucleotide sequence of the upstream homologous arm is shown in SEQ ID No. 88 in the listing. yjiP The nucleotide sequence of the downstream homologous arm is shown in SEQ ID No. 89 in the listing.

[0073] Amplification stay The primers required for inserting the fragment are stay -S and stay -A, stay The nucleotide sequence of -S is shown in SEQ ID No. 90 of the listing. stay The nucleotide sequence of -A is shown in SEQ ID No. 91 of the PCR amplification table. stay The nucleotide sequence of the fragment is shown in SEQ ID No. 92.

[0074] UP- yjiP -A、 stay -S and stay -A、DN- yjiP The -S primer pairs each have a complementary base overlap region of approximately 40 bp in length, which allows for overlapping PCR to splice upstream and downstream homologous arms and inserted gene fragments.

[0075] The above yjiP Upstream homologous arm, yjiP Downstream homologous arm, stay Fragments were fused using overlap PCR to obtain stay Integration fragments of genes.

[0076] 2. Construct pGRB- according to the method in step 2 of Example 1 for constructing plasmid pGRB- yjiP The primers (gRNA-) required for designing recombinant plasmids yjiP -S and gRNA- yjiP -A), gRNA- yjiP The nucleotide sequence of -S is shown in SEQ ID No. 93 in the sequence listing, gRNA- yjiP The nucleotide sequence of -A is shown in SEQ ID No. 94 of the sequence listing, and the primer contains... yjiP Target site sequence, then containing yjiP The target sequence DNA fragment and the pGRB linearized vector fragment were ligated via homologous recombination to construct pGRB- yjiP Recombinant plasmid.

[0077] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 10. E. coli MG1655CA-9 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was obtained. E. coli MG1655CA-10.

[0078] Example 12 Construction of engineered bacteria E. coli MG1655 CA-11 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-11. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. meaning The integrated segment of the gene; specifically: by E. coli Using the MG1655 genome as a template, based on its yhdW Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene. yhdW -S、UP- yhdW -A) and downstream homologous arm primer (DN- yhdW -S、DN- yhdW -A), UP- yhdW The nucleotide sequence of -S is shown in SEQ ID No. 95 of the UP- nucleotide sequence listing. yhdW The nucleotide sequence of -A is shown in SEQ ID No. 96 of the nucleotide sequence listing; DN- yhdW The nucleotide sequence of -S is shown in SEQ ID No. 97 of the nucleotide sequence listing, DN- yhdW The nucleotide sequence of -A is shown in SEQ ID No. 98 in the listing; its upstream and downstream homologous arm fragments were amplified by PCR to obtain... yhdW The nucleotide sequence of the upstream homologous arm is shown in SEQ ID No. 99 in the listing. yhdW The nucleotide sequence of the downstream homologous arm is shown in SEQ ID No. 100 in the listing.

[0079] according to meaning Gene design primers ( meaning -S、 meaning -A), meaning The nucleotide sequence of -S is shown in SEQ ID No. 101 of the listing. meaning The nucleotide sequence of -A is shown in SEQ ID No. 102 in the listing. PCR amplification meaning Gene fragments obtained meaning The nucleotide sequence of the gene fragment is shown in SEQ ID No. 103 in the listing; promoter P J23119 Then the design is in yhdW Downstream primer of upstream homologous arm and meaning In the upstream primer of the gene. The above fragments were fused using overlap PCR to obtain... meaning Integration fragment of gene (upstream homologous arm - P) J23119 - meaning -Downstream homologous arm).

[0080] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- yhdW -S and gRNA- yhdW gRNA was obtained by annealing A. yhdW The nucleotide sequence of -S is shown in SEQ ID No. 104 of the sequence listing, gRNA- yhdW The nucleotide sequence of -A is shown in SEQ ID No. 105 of the sequence listing.

[0081] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 11. E. coli MG1655CA-10 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was obtained. E. coli MG1655CA-11.

[0082] Example 13 Construction of engineered bacteria E. coli MG1655 CA-12 Following the method for constructing engineered bacteria in Example 1, strains were constructed. E. coli MG1655 CA-12. Details are as follows: 1. Prepare recombinant DNA fragments according to the method in step 1 of Example 1. cpsG The integrated segment of the gene; specifically: according to E. coli MG 1655 Chew Gene sequence and P J23119 Promoter sequence design integration cpsG The primer for the upstream homologous arm is UP- Chew -S and UP- Chew -A, the downstream homologous arm primer is DN- Chew -S and DN- Chew -A;UP- Chew The nucleotide sequence of -S is shown in SEQ ID No. 106 of the UP- nucleotide sequence listing. Chew -A is shown in SEQ ID No. 107 of the nucleotide sequence listing; DN- Chew The nucleotide sequence of -S is shown in SEQ ID No. 108 of the nucleotide sequence listing, DN- Chew The nucleotide sequence of -A is shown in SEQ ID No. 109 of the listing; its upstream and downstream homologous arm fragments were amplified by PCR, yielding... Chew The nucleotide sequence of the upstream homologous arm is shown in SEQ ID No. 110 of the listing. Chew The nucleotide sequence of the downstream homologous arm is shown in SEQ ID No. 111 in the listing.

[0083] according to cpsG Gene design primers ( cpsG -S、 cpsG -A), cpsG The nucleotide sequence of -S is shown in SEQ ID No. 112 of the listing. cpsG The nucleotide sequence of -A is shown in SEQ ID No. 113 in the listing, PCR amplification. cpsG Gene fragments obtained cpsG The nucleotide sequence of the gene fragment is shown in SEQ ID No. 114 in the listing; UP- Chew -A、 cpsG -S and cpsG -A、DN- Chew The -S primer pairs each have complementary base overlap regions of approximately 40 bp in length, enabling overlapping PCR for the overlapping splicing of upstream and downstream homologous arms and the inserted gene fragment. The fragments are then fused using overlapping PCR to obtain... cpsG Integration fragments of genes.

[0084] 2. Following the method described in step 2 of Example 1 for constructing plasmid pGRB, construct plasmid pGRB containing the target sequence, wherein the DNA fragment containing the target sequence is used via primer gRNA- Chew -S and gRNA- Chew gRNA was obtained by annealing A. ChewThe nucleotide sequence of -S is shown in SEQ ID No. 115 of the sequence listing, gRNA- Chew The nucleotide sequence of -A is shown in SEQ ID No. 116 of the sequence listing.

[0085] 3. Following the method of transforming the plasmid and recombinant DNA fragment in step 3 and eliminating the plasmid in step 4 of Example 1, wherein the competent cells used in step (1) of transforming pREDCas9 in step 3 of transforming the plasmid and recombinant DNA fragment were obtained in Example 11. E. coli MG1655CA-11 was obtained and verified by PCR. The PCR verification was successful, meaning the strain was successfully obtained. E. coli MG1655CA-12.

[0086] Application Example 1 The strains obtained in Examples 2-13 E. coli MG1655CA-1 strain E. coli MG1655 CA-12 was used for shake-flask fermentation to produce colacid, while wild-type colacid was used. E. coli A comparison was made with MG1655, and the specific method is as follows: 1. Slant culture: Take the -80℃ preserved strain and streak it onto the activated slant medium, incubate at 37℃ for 12 hours, and subculture once; The activated slant culture medium consisted of: 17.1 g / L Na2HPO4·12H2O, 3 g / L KH2PO4, 4 g / L glucose, 1 g / L NH4Cl, 0.5 g / L NaCl, 0.24 g / L MgSO4, 0.011 g / L CaCl2, and 25 g / L agar powder. The mixture was heated until the agar powder was completely dissolved, and then dispensed into 9.5 mL tubes. The tubes were then sterilized by moist heat at 121°C for 20 min.

[0087] 2. Scale-up culture: Single colonies from the slant culture were inoculated into 100 mL of LB liquid medium and cultured overnight at 30 °C and 220 r / min with shaking to obtain seed culture; LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, sterilized by moist heat at 121°C for 20 min.

[0088] 3. Shake flask fermentation: Inoculate 10% of the seed culture volume into 500mL Erlenmeyer flasks containing fermentation medium (final volume 50mL), seal the flasks, and culture at 30℃ and 220r / min with shaking. At the same time, add 60% (m / V) glucose solution to maintain fermentation and maintain residual sugar at 1g / L. The fermentation cycle is 48h.

[0089] Fermentation medium: 17.1 g / L Na2HPO4·12H2O, 3 g / L KH2PO4, 4 g / L glucose, 1 g / L NH4Cl, 0.5 g / L NaCl, 0.24 g / L MgSO4, 0.011 g / L CaCl2, sterilized by moist heat at 121℃ for 20 min.

[0090] After the shake-flask fermentation was completed, the content of colacid in the fermentation broth was measured, and the results are shown in Table 6. Table 6

[0091] The results in the table above show that the strains E. coli The shake-flask fermentation of MG1655 CA-12 yielded the best results.

[0092] Application Example 2 strains E. coli The MG1655 CA-12 was used for fermentation in a fermenter, and the specific method is as follows: The -80℃ preserved bacterial strain was streaked onto an activated slant culture medium and incubated at 30℃ for 12 hours. The culture was then transferred to a flask and incubated at 30℃ for another 12 hours. The culture was rinsed with 100 mL of sterile water to obtain a seed culture suspension, which was then inoculated into a fermenter containing 2.5 L of fermentation medium at a 10% inoculation rate. Initial stirring speed was controlled at 200 rpm, and airflow at 2 L / min. The fermentation temperature was maintained at 30℃. The pH of the fermentation broth was maintained at 7.0 by automatic ammonia addition. During fermentation, the stirring speed and airflow were adjusted according to cell growth to maintain relative dissolved oxygen at 32%. Furthermore, when the glucose in the initial culture medium was depleted, a glucose solution (800 g / L) was added, with the residual sugar content controlled to be no higher than 10 g / L. The incubation period was 48 hours. After incubation, the content of colacid in the fermentation broth reached 25 g / L.

[0093] The components of the activated slant culture medium are: 1 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2.5 g / L NaCl, and 25 g / L agar powder. After heating until the agar powder is completely dissolved, the medium is dispensed into 9.5 mL tubes and sterilized at 121°C for 20 min.

[0094] The fermentation medium consisted of: 17.1 g / L Na2HPO4·12H2O, 3 g / L KH2PO4, 4 g / L glucose, 1 g / L NH4Cl, 0.5 g / L NaCl, 0.24 g / L MgSO4, and 0.011 g / L CaCl2, sterilized by moist heat at 121℃ for 15 min.

Claims

1. A method for constructing a genetically engineered bacterium that produces high levels of colacid, characterized in that, It consists of the following steps: sequentially knocking out E. coli E. coli The waaL gene cluster and lon gene on the MG1655 genome hns Gene, clsA Gene, clsB Gene, clsC Gene, mcbR Gene, opgDGH Genes, and then overexpressed sequentially. rcsA Gene, galU Gene, manA Gene, cpsG Gene.

2. The method for constructing a genetically engineered bacterium producing high levels of colacid according to claim 1, characterized in that, The nucleotide sequence of the waaL gene cluster is shown in SEQ ID No. 7 in the sequence listing; The nucleotide sequence of the lon gene is shown in SEQ ID No. 16 of the sequence listing; The hns The nucleotide sequence of the gene is shown in SEQ ID No. 25 of the sequence listing; The clsA The nucleotide sequence of the gene is shown in SEQ ID No. 34 of the sequence listing; The clsB The nucleotide sequence of the gene is shown in SEQ ID No. 43 of the sequence listing; The clsC The nucleotide sequence of the gene is shown in SEQ ID No. 52 of the sequence listing; The mcbR The nucleotide sequence of the gene is shown in SEQ ID No. 61 of the sequence listing; The opgDGH The nucleotide sequence of the gene is shown in SEQ ID No. 70 of the sequence listing; The rcsA The nucleotide sequence of the gene is shown in SEQ ID No. 81 of the sequence listing; The galU The nucleotide sequence of the gene is shown in SEQ ID No. 92 in the sequence listing; The manA The nucleotide sequence of the gene is shown in SEQ ID No. 103 in the sequence listing; The cpsG The nucleotide sequence of the gene is shown in SEQ ID No. 114 in the sequence listing.

3. The method for constructing a genetically engineered bacterium producing high levels of colacid according to claim 1, characterized in that, In overexpression rcsA When using genes, the following methods are employed: endA Gene site integration and utilization of promoter P J23119 Start Expression rcsA Genetic methods; In overexpression galU When using genes, the following methods are employed: yjiP Gene site integration and utilization of promoter P J23119 Start Expression galU Genetic methods; In overexpression manA When using genes, the following methods are employed: yhdW Gene site integration and utilization of promoter P J23119 Start Expression manA Genetic methods; In overexpression cpsG When using genes, the following methods are employed: cheW Gene site integration and utilization of promoter P J23119 Start Expression cpsG Genetic pathways.

4. The method for constructing a genetically engineered bacterium producing high levels of colacid according to claim 1, characterized in that, When knocking out or overexpressing, the construction method consists of the following steps: preparing a recombinant DNA fragment, constructing plasmid pGRB, transforming the plasmid and the recombinant DNA fragment, and eliminating the plasmid.

5. The method for constructing a genetically engineered bacterium producing high levels of colacid according to claim 4, characterized in that, The construction of the pGRB plasmid consists of the following steps: preparing transformed competent cells, constructing a pGRB plasmid containing the target sequence, and transforming the recombinant plasmid into... E. coli DH5α competent cells were used to screen for positive transformants.

6. The method for constructing a genetically engineered bacterium producing high levels of colacid according to claim 4, characterized in that, The transformation plasmid and the preparation of the recombinant DNA fragment consist of the following steps: transforming pREDCas9, preparing electrocompetent cells of the target strain containing pREDCas9, transforming pGRB and the recombinant DNA fragment.

7. The method for constructing a genetically engineered bacterium producing high levels of colacid according to claim 4, characterized in that, The plasmid removal process consists of the following steps: removing plasmid pGRB and removing plasmid pREDCas9.

8. A genetically engineered bacterium that produces high levels of colacid, constructed using the construction method described in any one of claims 1-7.

9. The application of a genetically engineered bacterium that produces high levels of colacid, constructed by the construction method according to any one of claims 1-7, in the fermentation production of colacid.

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