Engineering bacterium for high yield of D-pantothenic acid based on sugar uptake regulation and preparation method of engineering bacterium

Abstract

The invention relates to the technical field of genetic engineering, and discloses an engineering bacterium for high-yield production of D-pantothenic acid based on sugar uptake regulation and a preparation method of the engineering bacterium. The method comprises the following steps: firstly, stabilizing an RNAP promoter open complex formed on an rRNA (Ribonucleic Acid) gene by intensively expressing a necessary global regulation transcription factor CarD of mycobacterium neoaurum; secondly, performing enhanced expression on mycobacterium neoaurum Rpf / Sps family protein RpfB which serves as a resuscitation promoting factor to play a role and catalyze peptidoglycan synthesis to accelerate cell wall growth; meanwhile, the inactivated cell wall lipoprotein Lpp accumulates the outer membrane vesicles; in addition, lipoprotein NlpI responsible for adjusting the activity of endopeptidase MopS and PBP4 is deleted, and the super vesicles are generated; in addition, the mutant membrane lipid asymmetric pathway protein MlaE disturbs the homeostasis of the outer membrane and increases the formation of vesicles; in addition, the membrane anchoring protein TolA is destroyed to improve the membrane permeability; and finally, the escherichia coli gene engineering strain with greatly improved D-pantothenic acid production performance is obtained.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an engineering bacterium capable of highly producing D-pantothenic acid based on regulating a sugar uptake pathway and a preparation method thereof. Background Art

[0002] The bio-fermentation production method has attracted much attention due to its advantages such as cheap substrate, easy separation and low toxicity. The performance of the strain plays a crucial role in the fermentation process, directly affecting the fermentation cycle and time-space conversion rate in the production process. Therefore, improving the growth rate of the strain and stabilizing the yield and conversion rate of the product are still major challenges at present.

[0003] Pantothenic acid (D-PA), also known as vitamin B5, is a component of coenzyme A and plays an important role in important biochemical reactions such as energy metabolism and the citric acid cycle. At present, D-PA for industrial applications is mainly produced by chemical methods using the condensation of D-pantoic acid lactone and β-alanine in methanol or ethanol. However, the use of chemical separation or enzyme-catalyzed esters involves toxic chemical reagents and expensive separation methods. With the continuous development of genetic engineering and synthetic biology, the production of D-PA using biosynthetic methods that are environmentally friendly, cheap and simple has attracted widespread attention from scholars. However, there are still defects in the current production of D-PA using biological methods, such as unstable fermentation process and low yield. Summary of the invention

[0004] In order to solve the technical problem of low D-pantothenic acid production, the present invention provides an engineering bacterium for high-yield D-pantothenic acid production based on regulating the sugar uptake pathway and a preparation method thereof.

[0005] The specific technical scheme of the present invention is: In a first aspect, the present invention provides an engineered bacterium that produces high D-pantothenic acid by regulating the sugar uptake pathway. The genome of the engineered bacterium includes: Enhanced expression of carD gene and rpfB gene; Knock out the mgsA gene, adhE gene, lpp gene, nlpI gene, mlaE gene, and tolA gene.

[0006] Preferably, the carD gene is derived from Mycobacterium novoa new golden branch.

[0007] Preferably, the rpfB gene is derived from Mycorrhiza uralensis.

[0008] The invention firstly stabilizes the RNAP promoter open complex formed on the rRNA gene by enhancing the expression of the necessary global regulatory transcription factor CarD of the new gold mycobacterium; secondly, enhances the expression of the Rpf / Sps family protein RpfB of the new gold mycobacterium to function as a recovery promoting factor, catalyzes the synthesis of peptidoglycan and accelerates the growth of the cell wall; at the same time, inactivates the cell wall lipoprotein Lpp to accumulate outer membrane vesicles; in addition, deletes the lipoprotein NlpI responsible for regulating the activity of the endopeptidase MepS and PBP4 to produce super vesicles; furthermore, mutates the membrane lipid asymmetry pathway protein MlaE to disrupt the outer membrane homeostasis and increase the vesicle formation; furthermore, destroys the membrane anchor protein TolA to improve the membrane permeability; finally, a genetically engineered Escherichia coli strain with greatly improved D-pantothenic acid production performance is obtained.

[0009] In a second aspect, the present invention provides a method for constructing an engineered bacterium that regulates the sugar uptake pathway to produce high D-pantothenic acid, comprising the following steps: (1) Replace the mgsA gene in the genome of the fungus with the carD gene and enhance the expression of the carD gene; (2) Replace the adhE gene in the genome of the fungus with the rpfB gene and enhance the expression of the rpfB gene; (3) knocking out the lpp gene in the genome of Chaetococcus; (4) knocking out the nlpI gene in the genome of Chaetococcus spp.; (5) Knockout of the mlaE gene in the genome of the fungus; (6) Knock out the tolA gene in the genome of the fungus.

[0010] Preferably, the bottom plate bacteria is Escherichia coli.

[0011] Preferably, the genotype of the bottom plate bacteria is: E.coli W3110 derivative, Trc-panCpanEpanB / ilvC / ilvG* / ΔavtA / ilvE* / coaA* / ΔpoxB / Δpta / ΔplfB / ΔldhA / Trc-pykA / ilvN* / ilvH* / spoT* / Trc-spoT* / Trc-lpd / Trc-ilvD / ΔlacI / Trc-alaS / Trc-ilvD / ptsG::Trc-glk,galP.

[0012] Preferably, in step (1), the carD gene is derived from Mycobacterium neoaureum.

[0013] Preferably, in step (2), the rpfB gene.

[0014] Preferably, in step (1) and step (2), the enhanced expression is: replacing the original promoter of the gene to be enhanced with the Trc promoter.

[0015] The principles of each step (1) to (6) of the above method of the present invention are as follows: (1) The carD gene regulated by the Trc promoter of pTrc99A from the new golden branched stem replaced the original gene mgsA on the genome of the starting strain DPA-SA, blocking the production of methylglyoxal, D-lactic acid and ethanol mediated by the mgsA gene, while strengthening the global regulation of the carD gene on the cell; (2) The rpfB gene derived from the new golden branched stem and regulated by the Trc promoter of pTrc99A replaced the original adhE gene on the genome of the engineered bacteria SA-W1, blocking the production of acetaldehyde and ethanol mediated by the adhE gene while strengthening the function of the rpfB gene in restoring cell dormancy; (3) knocking out the lpp gene in the genome of Chaetobacterium chaete, inactivating the murein lipoprotein and accumulating outer membrane vesicles; (4) knocking out the nlpI gene in the genome of Chasma subtilisinus to delete the lipoprotein responsible for regulating the activity of endopeptidases MepS and PBP4 to produce super vesicles; (5) Knockout of the mlaE gene in the genome of Chasmodium fumigatus disrupts outer membrane homeostasis and increases vesicle formation; (6) Knock out the tolA gene in the genome of the fungus to disrupt the membrane anchor protein and improve membrane permeability.

[0016] The present invention regulates the sugar uptake pathway of Escherichia coli through the combined action of the above steps, thereby obtaining a D-pantothenic acid producing strain with greatly improved production performance, and the D-pantothenic acid of the strain is also greatly improved.

[0017] In a third aspect, the present invention provides the use of the genetically engineered bacteria constructed by the above construction method in the preparation of D-pantothenic acid.

[0018] Compared with the prior art, the present invention has the following technical effects: (1) The present invention first stabilizes the RNAP promoter open complex formed on the rRNA gene by enhancing the expression of the necessary global regulatory transcription factor CarD of the new gold Mycobacterium; secondly, the expression of the Rpf / Sps family protein RpfB of the new gold Mycobacterium is enhanced to act as a recovery-promoting factor, catalyzing peptidoglycan synthesis and accelerating cell wall growth; at the same time, the cell wall lipoprotein Lpp is inactivated to accumulate outer membrane vesicles; in addition, the lipoprotein NlpI responsible for regulating the activity of endopeptidases MepS and PBP4 is deleted to produce super vesicles; furthermore, the membrane lipid asymmetry pathway protein MlaE is mutated to disrupt the outer membrane homeostasis and increase vesicle formation; furthermore, the membrane anchor protein TolA is destroyed to improve membrane permeability; through the combined action of the above operations, a genetically engineered Escherichia coli strain with greatly improved D-pantothenic acid production performance is finally obtained.

[0019] (2) The present invention destroys almost all parts between the inner and outer membranes of the cell wall, tampers with the integrity of the cell wall membrane, and excessively damages the cell wall. However, cell growth is not inhibited but promoted, thereby stabilizing the production performance of the slower growing chassis bacteria, which provides a new strategy for the optimization of industrial strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a metabolic pathway diagram related to mgsA and adhE genes; Figure 2 Schematic diagram of rpfB gene related functions of carD; Figure 3 This is a schematic diagram of the metabolic pathway of D-pantothenic acid; Figure 4 The distribution of lpp, nlpI, mlaE and tolA in the cell wall of E. coli; Figure 5 OD for SA-W1 600 and changes in D-pantothenic acid titer; Figure 6 OD for SA-W2 600 and changes in D-pantothenic acid titer; Figure 7 OD for SA-W3 600 and changes in D-pantothenic acid titer; Figure 8 OD for SA-W4 600 and changes in D-pantothenic acid titer; Fig. 9 OD for SA-W5 600 and changes in D-pantothenic acid titer; Fig.10 OD for SA-W6 600 and changes in D-pantothenic acid titer. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0022] The present invention genetically modifies the bottom plate bacteria Escherichia coli, inserts RNA polymerase binding transcription factor carD and pro-resuscitation factor rpfB controlled by trc promoter from Mycobacterium neoglyoxal at the methylglyoxal synthase mgsA gene site and the alcohol dehydrogenase adhE gene site, respectively, so that the overexpression of the double genes greatly improves the growth rate of the strain; then, inactivates four membrane genes lpp, nlpI, mlaE and tolA, improves membrane permeability, and induces and triggers the production of vesicles. The produced outer membrane vesicles can be used as carriers for secreting D-pantothenic acid, promoting the release of D-pantothenic acid from the intracellular to the extracellular; finally, through the above-mentioned transformation, high yield of D-pantothenic acid is achieved. At the same time, the engineered bacteria of the present invention have a high growth rate and are more adaptable to industrial production. Among them, Figure 1 This is the metabolic pathway diagram of mgsA and adhE genes; Figure 2 Schematic diagram of rpfB gene related functions of carD; Figure 3 This is a schematic diagram of the metabolic pathway of D-pantothenic acid; Figure 4 The distribution of lpp, nlpI, mlaE and tolA in the cell wall of E. coli. The E. coli chassis parent strain used in the embodiment of the present invention is E. coli W3110, from the Yale University CGSC Collection Center (Coli Genetic Stock Center), with a deposit date of August 5, 1975, a deposit number of CGSC#4474, and has been disclosed in patents US2009 / 0298135 A1 and US2010 / 0248311 A1.

[0023] In the embodiment of the present invention, the final concentration of kanamycin in the culture medium is 0.05 mg / L, and the final concentration of spectinomycin in the culture medium is 0.05 mg / L.

[0024] In the embodiment of the present invention, the genes and corresponding pathways involved in gene editing are shown in Table 1. Wherein, the accession number is an identifier used to uniquely identify a gene in the Entrez Gene database of the National Center for Biotechnology Information (NCBI) of the United States.

[0025] Table 1 Genes involved in gene editing and corresponding pathways Gene name Accession Number Involved pathways oeLh 7439001 Synthesis of D-lactic acid and ethanol adhE 7437200 Acetaldehyde and ethanol synthesis carD 71063603 rRNA global regulation rPb 17916746 Promote cell recovery lpp 946175 murein nlP 947673 Regulates the activity of endopeptidases MepS and PBP4 mxD 947732 Membrane lipid asymmetry pathway tolA 7438542 Membrane anchored protein The primer sequence information involved in the embodiments of the present invention is shown in Table 2.

[0026] Table 2 Primer sequences Example 1D - HPLC determination of pantothenic acid content The detection method is as follows: Chromatographic conditions: C 18 Column (250 × 4.6 mm, particle size 5 μm, Agilent Technologies Co., Santa Clara, CA, USA), detection wavelength: 200 nm, column temperature: 30 °C; Sample treatment: Dilute the sample with ultrapure water to keep the D-pantothenic acid content between 0.05 g / L and 0.40 g / L; Mobile phase: acetonitrile / water / phosphoric acid (volume ratio: 50 / 949 / 1); Data collection time: 18 minutes.

[0027] Example 2 Construction and shake flask fermentation of SA-W1 (DPA-SA derivative, mgsA::carD) DPA-SA (E. coli W3110 derivative, Trc-panCpanEpanB / ilvC / ilvG * / ΔavtA / ilvE * / coaA * / △poxB / Δpta / ΔplfB / ΔldhA / Trc-pykA / ilvN* / ilv H* / spoT* / Trc-spoT* / Trc-lpd / Trc-ilvD / ΔlacI / Trc-alaS / Trc-ilvD / ptsG::Trc-glk,galP) was used as the starting strain, and CRISPR-Cas9-mediated gene editing technology was used to knock out the Escherichia coli methylglyoxal synthase gene mgsA by gene knockout, and the carD gene from Mycobacterium neoaurea regulated by the trc promoter was inserted at the gene site.

[0028] (1) Construction of pT-mgsA plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and mgsA-sgRNA-F / mgsA-sgRNA-R were used as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. Spectinomycin plates were used for screening and sequencing to verify the correct pT-mgsA plasmid, which was used for subsequent ligation of mgsA-up, Trc-carD, and mgsA-down.

[0029] (2) Construction of pTarget-mgsA::Trc-carD plasmid: Using the E. coli W3110 genome as a template, mgsA-up-F and mgsA-up-R were used as primers to amplify mgsA-up, and mgsA-down-F and mgsA-down-R were used as primers to amplify mgsA-down. Using the M. neoaurum B3805 genome as a template, carD-F and carD-R were used as primers to amplify Trc-carD. The pTarget-mgsA plasmid was amplified using PT-line-F and PT-line-R as primers to obtain pTarget-line-mgsA. The DNA fragment was recovered using the Cleanup kit; pTarget-line-mgsA, mgsA-up, Trc-carD and mgsA-down were connected together according to the instruction manual of (One step cloning kit, Vazyme Biotech, Nanjing, China) and the pTarget-mgsA::Trc-carD plasmid was obtained by sequencing verification.

[0030] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into DPA-SA, and the single clone was transferred to an LB test tube containing 0.05 mg / L kanamycin and cultured at 30°C overnight; then, the inoculum was inoculated into a 250 mL shake flask containing 50 mL LB medium at a volume concentration of 1%, and 500 μl 1 mol / L L-arabinose was added, and the culture was carried out at 150 rpm and 30°C until the OD 600 0.4~0.6; 4000rpm, 4℃ centrifugation for 10min to collect cells and prepare electroporation competent cells. For detailed process, see (Molecular Cloning: A Laboratory Manual, 3ed Edition, 99-102).

[0031] (4) Take 200 ng of pTarget-mgsA::Trc-carD plasmid and mix it with 100 μl of electroporated competent cells, transfer it into a pre-cooled 2 mm electroporation cup, place it on ice for about 1 min, and use an electroporator (MicroPluser TM , BIO-RAD) for electroporation transformation. After the electroporation, 1 mL of LB medium was immediately added and gently aspirated, and the plate was transferred to a 1.5 mL centrifuge tube. After resuscitation at 30°C for 2-3 hours, it was coated with LB plates containing 0.05 mg / L kanamycin and 0.05 mg / L spectinomycin, and inverted culture was carried out at 37°C for 12-16 hours. Colony PCR was performed using mgsA-VF and mgsA-VR as primers to verify the colony. If a fragment of about 1500 bp could be successfully cloned, it was proved to be a DPA-SA derivative, mgsA::carD positive colony.

[0032] (5) Plasmid elimination: Pick a positive single colony and inoculate it into 1 mM IPTG and 0.05 mg / L kanamycin were added to LB test tubes and cultured at 30°C overnight. The next day, the bacterial solution was streaked onto LB plates containing 0.05 mg / L kanamycin and cultured at 30°C for 24 h. Single colonies were picked and streaked onto LB plates containing 0.05 mg / L spectinomycin. The pTarget-mgsA::Trc-carD plasmid of the single colonies that could not be successfully eliminated on the LB plates containing 0.05 mg / L spectinomycin was successfully eliminated. Single colonies with successfully eliminated pTarget-mgsA::Trc-carD plasmid were picked and placed in LB test tubes and cultured at 37°C overnight. The next day, the bacterial solution was streaked onto LB plates and cultured at 37°C for 12 h. Single colonies were picked and streaked onto LB plates containing 0.05 mg / L kanamycin. The pCas plasmid of the single colonies that could not be successfully eliminated on the LB plates containing 0.05 mg / L kanamycin was successfully eliminated, and finally a plasmid-free DPA-SA derivative, mgsA::carD, was obtained.

[0033] (6) DPA-SA derivative, mgsA::carD, with DPA-SA as the control group, were inoculated into 10 mL of LB medium and cultured at 37°C and 200 rpm as pre-cultures; after 8 to 12 hours, 1 mL of the pre-culture was inoculated into a 500 mL shake flask containing 50 mL of MS medium, and then fermented in a constant temperature shaker at 30°C and 200 rpm for 48 hours; after the fermentation was completed, 1 mL of the fermentation broth was taken to measure the OD 600 1 mL of fermentation broth was taken and centrifuged at 12000 rpm for 3 min at room temperature. The fermentation supernatant was diluted 10 times and HPLC was performed according to Example 1. OD 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 5 shown.

[0034] Depend on Figure 5 It can be seen that knocking out the mgsA gene in the DPA-SA genome and overexpressing the RNA polymerase-binding transcription factor carD controlled by the trc promoter significantly promoted cell growth. 600 It increased from 4.23 (indicating that the growth ability of DPA-SA was weak) to 5.61, while the D-pantothenic acid titer increased from 3.83 g / L to 4.40 g / L, which indicated that the overexpression of carD accelerated cell growth and accumulated more rRNA, which was beneficial to the growth of E. coli and the synthesis of D-pantothenic acid.

[0035] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water, pH value is natural.

[0036] MS medium: glucose 20g / L, ammonium sulfate 16g / L, KH2PO4 0.8g / L, MgSO4 0.5g / L, yeast extract 2g / L, β-alanine 2.5g / L, CaCO3 10g / L (sterilized separately), 1mL / L trace element solution, the solvent is deionized water, and the pH value is natural; the trace element solution composition: 10g / L CuCl2, 10g / L FeSO4·7H2O, 1g / L ZnSO4·7H2O, 0.20g / LCuSO4, 0.02g / L NiCl2·7H2O, the solvent is deionized water.

[0037] Example 3 Construction and shake flask fermentation of SA-W2 (SA-W1 derivative, adhE::rpfB) Using SA-W1 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to knock out the Escherichia coli alcohol dehydrogenase gene adhE gene through a gene knockout method, and the rpfB gene from Mycobacterium neogoldensis regulated by the trc promoter was inserted into the gene site.

[0038] (1) Construction of pT-adhE plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and adhE-sgRNA-F / adhE-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The product was screened on a spectinomycin plate and sequenced to obtain the correct pT-adhE plasmid for subsequent ligation of adhE-up, Trc-rpfB, and adhE-down.

[0039] (2) Construction of pTarget-adhE::Trc-rpfB plasmid: Using the E. coli W3110 genome as a template, adhE-up-F and adhE-up-R were used as primers to amplify adhE-up, and adhE-down-F and adhE-down-R were used as primers to amplify adhE-down. Using the M. neoaurum B3805 genome as a template, rpfB-F and rpfB-R were used as primers to amplify Trc-rpfB. The pT-adhE plasmid was amplified using PT-line-F and PT-line-R as primers to obtain pTarget-line-adhE. The DNA fragment was recovered using a Clean up kit; pTarget-line-adhE, adhE-up, Trc-rpfB and adhE-down were connected together according to the instruction manual of the kit (One step cloning kit, Vazyme Biotech, Nanjing, China), and the pTarget-adhE::pTrc-rpfB plasmid was obtained by sequencing verification.

[0040] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-W1 competent cell obtained in Example 2 (5). The preparation method of SA-W1 competent cell was the same as that in Example 2 (3).

[0041] (4) SA-W2 positive colonies were obtained by constructing them using the same method as in Example 2 (4).

[0042] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-W2.

[0043] (6) The constructed SA-W2 engineered strain was used as a control group with the SA-W1 constructed in Example 2, and the shake flask test and detection were carried out according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 6 shown.

[0044] Depend on Figure 6 It can be seen that knocking out adhE in the SA-W1 genome and overexpressing the rpfB gene regulated by the trc promoter increased the cell OD 600 From 5.61 to 6.08, and the D-pantothenic acid titer increased from 4.40g / L to 4.72g / L. It can be seen that the D-pantothenic acid titer of the initial strain is closely related to cell growth, and the growth rate directly affects the production of D-pantothenic acid, which becomes the limiting factor of the bacteria, thereby improving the growth rate of the strain and the ability of D-pantothenic acid to release.

[0045] This example also uses DPA-SA as the starting point, uses CRISPR-Cas9-mediated gene editing technology, knocks out the Escherichia coli alcohol dehydrogenase gene adhE gene through gene editing technology, and inserts the rpfB gene from Mycobacterium neogoldensis regulated by the trc promoter at the gene site to obtain the SA-VSW2 strain. Specific reference is made to the construction of SA-W2 in steps (1) to (5) of this example. The results show that the D-pantothenic acid titer of SA-VSW2 is 4.11 g / L, which is an increase compared to 3.83 g / L of DPA-SA, but SA-W2 is increased from 3.83 g / L to 4.72 g / L compared to DPA-SA, which shows that the knockout of the mgsA gene and the overexpression of the carD gene from Mycobacterium neogoldensis in SA-W2 of this example are of great significance for improving the titer of D-pantothenic acid.

[0046] Example 4 Construction and shake flask fermentation of SA-W3 (SA-W2 derivative, Δlpp) Using SA-W2 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to knock out the peptidoglycan lipoprotein synthesis gene lpp gene through the gene knockout method, inactivate the cell wall lipoprotein and accumulate outer membrane vesicles.

[0047] (1) Construction of pT-lpp plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and lpp-sgRNA-F / lpp-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The product was screened on a spectinomycin plate and sequenced to verify the correct pT-lpp plasmid for subsequent lpp-up / lpp-down ligation.

[0048] (2) Construction of pTarget-lpp plasmid: Using the E. coli W3110 genome as a template, lpp-up was amplified using primers lpp-up-F and lpp-up-R to obtain lpp-up, lpp-down was amplified using primers lpp-down-F and lpp-down-R to obtain lpp-down, and pT-lpp plasmid was amplified using primers PT-line-F and PT-line-R to obtain pTarget-line-lpp. The DNA fragments were recovered using a Clean up kit; pTarget-line-lpp, lpp-up and lpp-down were connected together according to the instruction manual of (One step cloning kit, Vazyme Biotech, Nanjing, China) and the pTarget-lpp plasmid was obtained by sequencing verification.

[0049] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-W2 competent cell obtained in Example 3 (5). The preparation method of SA-W2 competent cell was the same as that of Example 2 (3).

[0050] (4) SA-W3 positive colonies were constructed using the same method as in Example 2 (4).

[0051] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-W3.

[0052] (6) The constructed SA-W3 engineered strain was used as a control group with the SA-W2 constructed in Example 3, and the shake flask test and detection were carried out according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 7 shown.

[0053] As shown in the figure, knocking out lpp in the SA-W2 genome increased the cell OD 600 From 6.08 to 6.16, and the D-pantothenic acid titer increased from 4.72 g / L to 4.88 g / L. This shows that this embodiment weakens the synthesis of outer membrane lipoproteins, reduces lipoproteins in the peritoneal space, and generates some vesicles, so that the D-pantothenic acid titer can be increased.

[0054] Example 5 Construction and shake flask fermentation of SA-W4 (SA-W3 derivative, ΔnlpI) In order to prove that the accumulation of vesicles can promote the increase of D-pantothenic acid production, this example further promotes the generation of vesicles. Using SA-W3 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to knock out the peptidoglycan lipoprotein synthesis gene nlpI through gene knockout method, and the lipoprotein responsible for regulating the activity of endopeptidases MepS and PBP4 was deleted to produce super vesicles.

[0055] (1) Construction of pT-nlpI plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and nlpI-sgRNA-F / nlpI-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The product was screened on a spectinomycin plate and sequenced to obtain the correct pT-lpp plasmid for subsequent connection to nlpI-up / nlpI-down.

[0056] (2) Construction of pTarget-nlpI plasmid: Using the E. coli W3110 genome as a template, nlpI-up was amplified using primers nlpI-up-F and nlpI-up-R, nlpI-down was amplified using primers nlpI-down-F and nlpI-down-R, and pT-lpp plasmid was amplified using primers PT-line-F and PT-line-R to obtain pTarget-line-nlpI. The DNA fragments were recovered using a Clean up kit; pTarget-line-nlpI, nlpI-up and nlpI-down were connected together according to the instruction manual of (One step cloning kit, Vazyme Biotech, Nanjing, China) and the pTarget-nlpI plasmid was obtained by sequencing verification.

[0057] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-W3 competent cell obtained in Example 4 (5). The preparation method of SA-W3 competent cell was the same as that of Example 2 (3).

[0058] (4) SA-W4 positive colonies were constructed using the same method as in Example 2 (4).

[0059] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-W4.

[0060] (6) The constructed SA-W4 engineered strain was used as a control group with the SA-W3 constructed in Example 4, and shake flask tests and detection were performed according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 8 shown.

[0061] As shown in the figure, knocking out nlpI in the SA-W3 genome resulted in a decrease in cell OD 600 From 6.16 to 6.33, and the D-pantothenic acid titer increased from 4.88g / L to 4.94g / L. Similar to the effect of knocking out lpp, nlpI is also in the periplasm of the cell. After knocking out, it can further increase the formation of vesicles and weaken the synthesis of outer membrane lipoproteins. In this regard, more D-pantothenic acid can be transported out of the cell, thereby increasing the D-pantothenic acid titer.

[0062] Example 6 Construction and shake flask fermentation of SA-W5 (SA-W4 derivative, ΔnlpI) Using SA-W4 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to knock out the peptidoglycan lipoprotein synthesis gene mlaE gene through a gene knockout method, disrupting the outer membrane homeostasis and increasing vesicle formation.

[0063] (1) Construction of pT-mlaE plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and mlaE-sgRNA-F / mlaE-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The plasmid was screened on a spectinomycin plate and sequenced to obtain the correct pT-mlaE plasmid for subsequent ligation of mlaE-up / mlaE-down.

[0064] (2) Construction of pTarget-mlaE plasmid: Using the E. coli W3110 genome as a template, mlaE-up was amplified using primers mlaE-up-F and mlaE-up-R, mlaE-down was amplified using primers mlaE-down-F and mlaE-down-R, and pT-mlaE plasmid was amplified using primers PT-line-F and PT-line-R to obtain pTarget-line-mlaE. The DNA fragment was recovered using a Clean up kit; pTarget-line-mlaE, mlaE-up and mlaE-down were connected together according to the instruction manual of (One step cloning kit, Vazyme Biotech, Nanjing, China) and the pTarget-mlaE plasmid was obtained by sequencing verification.

[0065] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-W4 competent cell obtained in Example 5 (5). The preparation method of SA-W4 competent cell was the same as that of Example 2 (3).

[0066] (4) SA-W5 positive colonies were constructed using the same method as in Example 2 (4).

[0067] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-W5.

[0068] (6) The constructed SA-W5 engineered strain was used as a control group with the SA-W4 constructed in Example 5, and shake flask tests and detection were performed according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Fig. 9 shown.

[0069] As shown in the figure, when mlaE was knocked out in the SA-W4 genome, the cell OD 600The D-pantothenic acid titer increased from 6.33 to 6.59, and the D-pantothenic acid titer increased from 4.94 g / L to 5.15 g / L. Compared with the increase in vesicles in the previous two times, the production of D-pantothenic acid has increased significantly this time. Similar to the effects of knocking out lpp and nlpI, the knockout of the mlaE gene disrupts the outer membrane homeostasis and increases vesicle formation. The difference is that although it is located in the periplasmic space of the cell, it is attached to the inner membrane protein. After knocking out the lpp, nlpI and mlaE genes at the same time, the periplasmic space is destroyed, the cell wall becomes thinner, and the vesicle synthesis rate is greatly increased. The D-pantothenic acid production and OD 600 Compared with the control strain, there was also a significant improvement.

[0070] Example 7 Construction and shake flask fermentation of SA-W6 (SA-W5 derivative, ΔnlpI) Using SA-W5 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to knock out the peptidoglycan lipoprotein synthesis gene tolA gene through the gene knockout method, thereby destroying the membrane anchor protein to improve membrane permeability.

[0071] (7) Construction of pT-tolA plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and tolA-sgRNA-F / tolA-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The product was screened on a spectinomycin plate and sequenced to obtain the correct pT-tolA plasmid for subsequent ligation of tolA-up / tolA-down.

[0072] (8) Construction of pTarget-tolA plasmid: Using the E. coli W3110 genome as a template, tolA-up-F and tolA-up-R were used as primers to amplify tolA-up, tolA-down-F and tolA-down-R were used as primers to amplify tolA-down, and pT-tolA plasmid was amplified using PT-line-F and PT-line-R as primers to obtain pTarget-line-tolA, and the DNA fragment was recovered using a Clean up kit; pTarget-line-tolA, tolA-up and tolA-down were connected together according to the instruction manual of the kit (One step cloning kit, Vazyme Biotech, Nanjing, China), and the pTarget-tolA plasmid was obtained by sequencing verification.

[0073] (9) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-W5 competent cell obtained in Example 6 (5). The preparation method of SA-W5 competent cell was the same as that of Example 2 (3).

[0074] (10) SA-W6 positive colonies were constructed using the same method as in Example 2 (4).

[0075] (11) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-W6.

[0076] (12) The constructed SA-W6 engineered strain was used as a control group with the SA-W5 constructed in Example 6, and shake flask tests and detection were performed according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Fig.10 shown.

[0077] As shown in the figure, knocking out tolA in the SA-W5 genome resulted in a decrease in cell OD 600 From 6.59 to 6.64, and the D-pantothenic acid titer increased from 5.15g / L to 5.21g / L. From the perspective of the tolA gene alone, it is located in the peptidoglycan layer of the cell wall, destroying membrane anchoring proteins to improve membrane permeability, but knocking out lpp, nlpI, mlaE and tolA almost destroyed all parts between the inner and outer membranes of the cell wall, and by tampering with the membrane integrity, the E. coli cell wall became thinner, and the vesicles produced by excessive cell wall damage were sufficient to transport intracellular D-pantothenic acid out of the cell. In addition, cell growth was not only not inhibited, but promoted, stabilizing the production performance of a strain that grew too slowly, and also providing a new strategy for the optimization of industrial strains.

[0078] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0079] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. An engineered bacterium for high production of D-pantothenic acid by regulating the sugar uptake pathway, characterized in that: The genome of the engineered bacteria includes: Strengthen expression carD Gene, rPb Gene; Knockout oeLh Gene, adhE Gene, lpp Gene, nlP Gene, mxD Gene, tolA Gene.

2. The engineered bacteria according to claim 1, characterized in that: carD The gene is derived from the new golden branching rod.

3. The engineered bacteria according to claim 1, characterized in that: rPb The gene is derived from the new golden branching rod.

4. A method for constructing an engineered bacterium for high production of D-pantothenic acid based on balanced sugar utilization, characterized in that: The following steps are involved: (1) Take carD Gene replacement in the genome of Chassis bacteria oeLh Genes and enhance expression carD Gene; (2) Take rPb Gene replacement in the genome of Chassis bacteria adhE Genes and enhance expression rPb Gene; (3) Knockout in the genome of the fungus lpp Gene; (4) Knockout in the genome of the fungus nlP Gene; (5) Knockout in the genome of the fungus mxD Gene; (6) Knockout in the genome of the fungus tolA Gene.

5. The construction method according to claim 4, characterized in that: The bottom plate bacteria is Escherichia coli.

6. The construction method according to claim 5, characterized in that: The genotype of the chassis bacteria is: E. coli W3110derivative, Trc-panCpanEpanB / ilvC / ilvG* / ΔavtA / ilvE* / coaA* / △poxB / Δpta / ΔplfB / ΔldhA / Trc-pykA / ilvN* / ilvH* / spoT* / Trc-spoT* / Trc-lpd / Trc-ilvD / ΔlacI / Trc-alaS / Trc-ilvD / ptsG::Trc-glk, galP.

7. The construction method according to claim 5 or 6, characterized in that: In step (1), the carD The gene is derived from the new golden branching rod.

8. The construction method according to claim 5 or 6, characterized in that: In step (2), the rPb Gene.

9. The construction method according to claim 5 or 6, characterized in that: In step (1) and step (2), the enhanced expression is: replacing the original promoter of the gene to be enhanced with the Trc promoter.

10. Use of the genetically engineered bacterium according to any one of claims 1 to 3, or the genetically engineered bacterium constructed by the construction method according to any one of claims 4 to 9 in the preparation of D-pantothenic acid.

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