Genetically-engineered bacterium highly-producing d-pantothenic acid independent of the addition of β-alanine, construction method therefor and use thereof

Abstract

The present invention relates to the technical field of bioengineering, and particularly relates to a genetically-engineered bacterium highly-producing D-pantothenic acid independent of the addition of β-alanine, a construction method therefor and the use thereof. By means of gene editing technology, the expression level of a key enzyme in a biological generation pathway of D-pantothenic acid and the expression level of a key enzyme in a biological generation pathway of β-alanine are further enhanced on the basis of existing engineering bacteria, so as to obtain a high-yield bacterial strain which does not need adding β-alanine during a fermentation process. Among all bacterial strains, the maximum shake flask titer reaches 2.97 g / L. Compared with a parent strain, the present invention omits the addition of exogenous β-alanine during the fermentation process, thus greatly reducing the fermentation cost, simplifying the fermentation process, and facilitating large-scale industrial production and application. In addition, the pantothenic acid yield in shake flask fermentation of the genetically-engineered bacterium ZPA18 producing D-pantothenic acid increases to 3.34 g / L, and the yield after replenishment fermentation for 83 h in a 5 L fermentation tank reaches 46.15 g / L.

Description

A high-yield D-pantothenic acid genetically engineered bacterium that does not rely on β-alanine addition, its construction method and application Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a high-yield D-pantothenic acid genetic engineering bacterium that does not rely on the addition of beta-alanine, a construction method and an application thereof. Background Art

[0002] Pantothenic acid exists in nature in two main configurations: D-pantothenic acid and L-pantothenic acid. Only D-pantothenic acid is biologically active in organisms. D-Pantothenic acid, also known as vitamin B5, is a water-soluble vitamin in the B vitamin family. It is widely found in many foods and is one of the 13 essential vitamins for humans. D-Pantothenic acid serves as a precursor for coenzyme A (CoA) in the body and plays a key role in nearly all biological processes, driving energy metabolism and energy exchange. The phosphorylation product of pantothenic acid, mercaptoethylamine, can be synthesized into 4-phosphopantheine, a component of coenzyme A (CoA) and acyl carrier protein (ACP). CoA and ACP, as coenzymes of acyltransferases, are widely involved in sugar, lipid, and protein metabolism and in liver biotransformations, significantly promoting the growth of organisms. It is widely used in food, medicine, feed, cosmetics, and other fields.

[0003] Among the known methods for synthesizing D-pantothenic acid, biofermentation has attracted attention due to its advantages such as low substrate costs and low toxicity. However, the current bio-based production of D-pantothenic acid still has drawbacks, such as the need to add a large amount of β-alanine during fermentation, which limits the development of pantothenic acid cell factories. In the early stages of strain construction, in order to achieve the concentration of carbon metabolism on the D-pantothenic acid metabolic pathway, the D-pantothenic acid metabolic pathway was modified, while the β-alanine metabolic pathway was ignored. Therefore, the construction of a strain that can produce D-pantothenic acid at a higher yield without the need for exogenous β-alanine addition remains a major challenge.

[0004] Summary of the Invention

[0005] The present invention aims to overcome the deficiencies in the prior art by providing a high-yield D-pantothenic acid-independent genetically engineered bacterium and a construction method thereof, and applying the same to the fermentation production of D-pantothenic acid, thereby overcoming the problem in the prior art that D-pantothenic acid-producing strains require a large amount of exogenous β-alanine to be added during the fermentation production of D-pantothenic acid.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides a genetically engineered bacterium that produces high levels of D-pantothenic acid and is independent of the addition of β-alanine, which is constructed by the following method:

[0008] The strain E. coli W3110 Trc-panC / Trc-panE / Trc-panB / Trc-ilvC / ilvG* / △avtA / ilvE* / coaA* / ilvA* / Trc-lpd / △glk is used as a chassis strain, the panB gene derived from Corynebacterium glutamicum is integrated into the pseudogene ycjV site in its genome, and the panD gene derived from Bacillus subtilis and the aspB gene and pyc gene derived from Corynebacterium glutamicum are overexpressed in the chassis strain, thereby obtaining the high-yield D-pantothenic acid genetically engineered bacteria.

[0009] The present invention is based on the E. coli W3110 Trc-panC / Trc-panE / Trc-panB / Trc-ilvC / ilvG* / ΔavtA / ilvE* / coaA* / ilvA* / Trc-lpd / Δglk, comprehensively utilizes a systems metabolic engineering strategy, and utilizes regulatory elements to optimize the expression levels of key genes in the pantoate synthesis pathway, the β-alanine synthesis pathway, and the pantoate and β-alanine condensation pathway in E. coli, further balancing the carbon flux to pantoate and β-alanine, and increasing the synthesis of D-pantothenate. Ultimately, an engineered strain that produces a high amount of D-pantothenate without adding β-alanine is obtained, specifically:

[0010] (1) Using the laboratory-preserved strain DPA11A (E. coli W3110 Trc-panC / Trc-panE / Trc-panB / Trc-ilvC / ilvG* / △avtA / ilvE* / coaA* / ilvA* / Trc-lpd / △glk) as the chassis strain (this strain has been disclosed in patent CN113637618A), the alsS gene from Bacillus subtilis, the panB gene from Corynebacterium glutamicum, and the panB gene from Escherichia coli were mutated in the pantoate biosynthesis branch pathway. coli) panE, ilvC, and ilvD genes were constructed into the pTrc99a plasmid (Kan resistance) to obtain pTrc99a / alsS, pTrc99a / panB, pTrc99a / panE, pTrc99a / ilvC, and pTrc99a / ilvD, which were respectively introduced into the DPA11A chassis strain to obtain genetically engineered D-pantothenic acid-producing bacteria, designated ZPA1-5.

[0011] (2) Similarly, using DPA11A as the chassis strain, on the β-alanine branch pathway, the aspC and aspA genes from E. coli, the panD gene from B. subtilis, and the aspB and pyc genes from C. glutamicum were constructed onto the pTrc99a plasmid to obtain pTrc99a / aspC, pTrc99a / aspA, pTrc99a / panD, pTrc99a / aspB, and pTrc99a / pyc, which were respectively introduced into the DPA11A chassis strain to obtain a genetically engineered strain producing D-pantothenic acid, designated ZPA6-10;

[0012] (3) Similarly, using DPA11A as the chassis strain, the panC gene from C. glutamicum was constructed into the pTrc99a plasmid in the pantothenate and β-alanine condensation pathway to obtain pTrc99a / panC, which was then introduced into the DPA11A chassis strain to obtain a D-pantothenate-producing genetically engineered bacterium, designated ZPA11.

[0013] (4) The strains constructed in steps (1) and (2) were fermented in shake flasks, and 2-3 key enzyme genes were selected (preferably, alsS gene and panB gene were selected for the pantothenic acid synthesis branch pathway, and panD gene, aspB gene, and pyc gene were selected for the β-alanine branch pathway). These genes were then serially constructed onto the pTrc99a plasmid to obtain pTrc99a / alsS-panB and pTrc99a / panD-aspB-pyc, which were respectively introduced into the DPA11A chassis strain to obtain genetically engineered bacteria that produce D-pantothenic acid, which were recorded as ZPA12-13;

[0014] (5) The plasmids constructed in steps (3) and (4) were further concatenated and constructed onto the pTrc99a plasmid to obtain pTrc99a / alpB-DaBp-C, which was introduced into the DPA11A chassis bacteria to obtain a genetically engineered bacterium that produces D-pantothenic acid, which was designated ZPA14;

[0015] (6) Using the engineered strain DPA11A as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene yjiP on the genome of the starting strain with the alsS gene regulated by the Ptrc promoter from pTrc99A, thereby enhancing the expression intensity of alsS and obtaining the engineered strain DPA11A derivative, yjip::Ptrc-alsS, which was recorded as engineered strain ZPA15;

[0016] (7) Using the engineered strain ZPA15 as the starting strain, the CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene ycjV on the genome of the starting strain with the panB gene regulated by the Ptrc promoter derived from pTrc99A, further enhancing the expression intensity of panB. The engineered strain ZPA15 derivative, ycjV::Ptrc-panB, was obtained and recorded as engineered strain ZPA16.

[0017] (8) The plasmid pTrc99a / panD-aspB-pyc was introduced into the ZPA15 and ZPA16 chassis strains, respectively, to obtain genetically engineered bacteria that produce D-pantothenic acid, which were designated as ZPA17 and ZPA18.

[0018] The nucleotide sequence of the alsS gene regulated by the Ptrc promoter is shown in SEQ ID NO.1; the nucleotide sequence of the panB gene is shown in SEQ ID NO.2; the nucleotide sequence of the panE gene is shown in SEQ ID NO.3; the nucleotide sequence of the ilvC gene is shown in SEQ ID NO.4; the nucleotide sequence of the ilvD gene is shown in SEQ ID NO.5; the nucleotide sequence of the aspC gene is shown in SEQ ID NO.6; the nucleotide sequence of the aspA gene is shown in SEQ ID NO.7; the nucleotide sequence of the panD gene is shown in SEQ ID NO.8; the nucleotide sequence of the aspB gene is shown in SEQ ID NO.9; the nucleotide sequence of the pyc gene is shown in SEQ ID NO.10; and the nucleotide sequence of the panC gene is shown in SEQ ID NO.11.

[0019] Among them, the panB gene regulated by the Ptrc promoter was integrated into the pseudogene ycjV site in the genome of the DPA11A chassis strain, and the plasmid pTrc99a / panD-aspB-pyc was introduced into the chassis strain to successfully obtain a genetically engineered bacterium that produces high D-pantothenic acid without adding β-alanine, becoming the dominant strain in the present invention. The pantothenic acid production of this genetically engineered bacterium was increased to 3.34 g / L in shake flask fermentation, and the pantothenic acid production reached 36.15 g / L after 72 hours of fed-batch fermentation in a 5L fermentor, an increase of 15.9% compared to ZPA13, and the final yield reached 46.15 g / L after 83 hours of fermentation.

[0020] Preferably, the panB gene is regulated by the Ptrc promoter, and the nucleotide sequence of the panB gene is shown in SEQ ID NO.2.

[0021] Preferably, the nucleotide sequence of the panD gene is shown as SEQ ID NO.8.

[0022] Preferably, the nucleotide sequence of the aspB gene is shown in SEQ ID NO.9, and the nucleotide sequence of the pyc gene is shown in SEQ ID NO.10.

[0023] In a second aspect, the present invention provides a method for constructing the genetically engineered bacteria with high D-pantothenic acid production, comprising the following steps:

[0024] The strain E. coli W3110 Trc-panC / Trc-panE / Trc-panB / Trc-ilvC / ilvG* / △avtA / ilvE* / coaA* / ilvA* / Trc-lpd / △glk was used as the chassis strain, and the CRISPR-Cas9-mediated gene editing technology was used to integrate the panB gene regulated by the Ptrc promoter into the pseudogene ycjV site in its genome. The plasmid expression system was used to clone the panD gene, aspB gene, and pyc gene into the pTrc99a plasmid, and the obtained plasmid pTrc99a / panD-aspB-pyc was transferred into the chassis bacteria, thereby obtaining the high-yield D-pantothenic acid genetically engineered bacteria.

[0025] In a third aspect, the present invention provides the use of the high-yield D-pantothenic acid genetically engineered bacteria or the high-yield D-pantothenic acid genetically engineered bacteria constructed by the method in the production of D-pantothenic acid by microbial fermentation.

[0026] Preferably, the application comprises: inoculating the genetically engineered bacteria into a fermentation medium, fermenting and culturing at 28-30° C. and pH 6.6-6.8, and after the fermentation is completed, separating and purifying the supernatant of the fermentation liquid to obtain D-pantothenic acid.

[0027] The genetically engineered bacteria are inoculated into a fermentation medium, and the initial stirring speed during fermentation is 300-500 r / min; the dissolved oxygen is controlled at 15%-25% by stirring and ventilation; the pH is controlled at 6.6-6.8 by feeding 50% ammonia water; the culture temperature is 30°C; feeding the feed medium is started when the pH is greater than 6.81, and the feed medium is added until the fermentation is completed. After the fermentation is completed, the supernatant of the fermentation liquid is separated and purified to obtain the D-pantothenic acid.

[0028] Preferably, the fermentation medium comprises: glucose 10-20 g / L, (NH4)2SO4 12-16 g / L, KH2PO4 1-2 g / L, MgSO4 0.3-0.5 g / L, yeast extract 1-2 g / L, β-alanine 0.1-0.5 g / L, trace metal ion solution 0.5-1 ml / L, Kan antibiotic 50-75 mg / L, IPTG 0.1-0.2 mM, the solvent is deionized water, and the pH value is natural.

[0029] Preferably, the trace metal ion solution comprises: 5-10 g / L CoCl2, 5-10 g / L FeSO4·7H2O, 0.5-1 g / L ZnSO4·7H2O, 0.10-0.20 g / L CuSO4, 0.01-0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

[0030] Preferably, fermentation medium is used at the start of fermentation, and the feed medium is added to the fermentation system according to a 20% dissolved oxygen feedback feeding strategy. The feed medium composition includes: 400-500 g / L of glucose, 8-10 g / L of ammonium sulfate, 1-3 g / L of yeast extract, 10-14 g / L of potassium dihydrogen phosphate, 6-8 g / L of anhydrous magnesium sulfate, 2-4 g / L of betaine, 0.01 g / L of VB1, 0.004 g / L of VB12, 100-160 mg / L of isoleucine, 1-2 mL / L of salt solution, 94-98 mg / L of isopropyl-β-D-thiogalactoside, and 80-100 mg / L of kanamycin sulfate.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention uses gene editing technology to further enhance the expression levels of key enzymes in the D-pantothenic acid biosynthesis pathway and the β-alanine biosynthesis pathway on the basis of existing engineered bacteria, thereby obtaining a high-yield strain that does not add β-alanine during the fermentation process. The shake flask titer of all strains of the present invention reached a maximum of 2.97g / L. Compared with the starting strain, the addition of exogenous β-alanine is eliminated during the fermentation process, which greatly saves fermentation costs and simplifies the fermentation process. It is suitable for large-scale industrial production and has great value for promotion and application. In addition, the D-pantothenic acid-producing genetically engineered bacteria ZPA18 of the present invention increased the pantothenic acid production to 3.34g / L in shake flask fermentation. After 72h of fed-batch fermentation in a 5L fermentor, the pantothenic acid production reached 36.15g / L, which is 15.9% higher than that of ZPA13. The final yield reached 46.15g / L after 83h of fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1 is a schematic diagram of the D-pantothenic acid biosynthesis pathway in recombinant Escherichia coli.

[0034] Figure 2 shows the OD values ​​of strains DPA11A, ZPA1, ZPA2, ZPA3, ZPA4, ZPA5, and ZPA11 with and without β-alanine. 600 and changes in D-pantothenic acid titer.

[0035] Figure 3 shows the OD values ​​of strains DPA11A, ZPA6, ZPA7, ZPA8, ZPA9, and ZPA10 with and without β-alanine. 600 and changes in D-pantothenic acid titer.

[0036] Figure 4 shows the OD values ​​of strains DPA11A, ZPA1, ZPA2, and ZPA12 when exogenously added with β-alanine. 600 and D-pantothenic acid titer changes, as well as the OD values ​​of strains ZPA8, ZPA9, ZPA10, ZPA13, and ZPA14 without the addition of β-alanine 600 and changes in D-pantothenic acid titer.

[0037] Figure 5 shows the OD values ​​of strains DPA11A, ZPA11, ZPA12, ZPA13, and ZPA14 without adding β-alanine. 600 and changes in D-pantothenic acid titer.

[0038] Figure 6 shows the biomass OD of strain ZPA13 in a 5-L fermenter fed-batch fermentation. 600 , concentration curves of residual sugar and DPA.

[0039] Figure 7 shows the OD of strains 11A, ZPA15, and ZPA16 when β-alanine was added. 600 and D-pantothenic acid titer changes, as well as the OD values ​​of strains ZPA13, ZPA17, and ZPA18 without the addition of β-alanine 600 and changes in D-pantothenic acid titer.

[0040] Figure 8 shows the biomass OD of strain ZPA18 in a 5-L fermenter fed-batch fermentation for 84 h. 600 , concentration curves of residual sugar and DPA. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only a portion of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0042] The following examples use strain DPA11A (E. coli W3110 Trc-panC / Trc-panE / Trc-panB / Trc-ilvC / ilvG* / ΔavtA / ilvE* / coaA* / ilvA* / Trc-lpd / Δglk) as the chassis strain (this strain was constructed by Zhejiang University of Technology and disclosed in patent CN113637618A), and subsequent steps are carried out on this basis. The nucleotide sequence of the alsS gene, regulated by the Ptrc promoter, is shown in SEQ ID NO. 1; the nucleotide sequence of the panB gene is shown in SEQ ID NO. 2; the nucleotide sequence of the panE gene is shown in SEQ ID NO. 3; the nucleotide sequence of the ilvC gene is shown in SEQ ID NO. 4; the nucleotide sequence of the ilvD gene is shown in SEQ ID NO. 5; the nucleotide sequence of the aspC gene is shown in SEQ ID NO. 6; the nucleotide sequence of the aspA gene is shown in SEQ ID NO. 7; the nucleotide sequence of the panD gene is shown in SEQ ID NO. 8; the nucleotide sequence of the aspB gene is shown in SEQ ID NO. 9; the nucleotide sequence of the pyc gene is shown in SEQ ID NO. 10; and the nucleotide sequence of the panC gene is shown in SEQ ID NO. 11. The genes involved in gene editing and the corresponding pathways are shown in Table 1, the primer sequences are shown in Table 2, and a schematic diagram of the D-pantothenic acid biosynthesis pathway in recombinant Escherichia coli is shown in Figure 1.

[0043] Table 1 Genes involved in gene editing and corresponding pathways

[0044] Table 2 Primer sequences

[0045] Example 1: HPLC determination of D-pantothenic acid content, the detection method is as follows:

[0046] 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;

[0047] Sample preparation: The fermentation broth of Example 11 was diluted with ultrapure water and centrifuged at 12,000 rpm for 1 min. The supernatant was retained and filtered through a 0.22 μm organic filter membrane to maintain a D-pantothenic acid content between 0.05 g / L and 0.40 g / L.

[0048] Mobile phase: acetonitrile / water / phosphoric acid: (50 / 949 / 1);

[0049] Data collection time: 20 minutes.

[0050] Example 2: Construction of expression plasmid pTrc99a / alsS

[0051] The alsS gene was amplified by PCR using the B. subtilis genome as a template and primers Trc-alsS-F and Trc-alsS-R. The PCR program was as follows: 95°C pre-denaturation for 10 minutes, 95°C denaturation for 30 seconds, 57°C annealing for 30 seconds, and 72°C extension for 1 minute, for a total of 32 cycles, followed by a final extension at 72°C for 10 minutes. The target fragment, alsS, was amplified by PCR.

[0052] The pTrc99a vector was cloned by PCR using the pTrc99a-panBC plasmid as a template and primers pTrc99a-ty-F and pTrc99a-ty-F. The PCR protocol was as follows: 95°C pre-denaturation for 10 minutes, followed by 32 cycles of denaturation at 95°C for 30 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 2 minutes and 30 seconds, with a final extension at 72°C for 10 minutes. The PCR product was verified by gel electrophoresis and incubated with Dpn I at 37°C and 180 rpm for 1 hour. Residual template was removed using a PCR clean-up kit to obtain a relatively pure linearized pTrc99a vector.

[0053] According to the instructions of the one-step cloning kit (ClonExpress Ultra One Step Cloning Kit C115, Vazyme Biotech, Nanjing, China), the linearized pTrc99a vector was ligated with the target fragment alsS and then introduced into DH5α competent cells. Colony PCR verification was performed, and single colonies corresponding to positive clones were picked and sequenced to obtain the pTrc99a / alsS plasmid.

[0054] Example 3: Construction of expression plasmid pTrc99a / panB

[0055] panB was amplified by PCR using the pTrc99a-panBC plasmid as a template and Trc-panB-F and Trc-panB-R as primers. The PCR program was annealed at 61°C and extended at 72°C for 30 seconds. Other operations were the same as those described in Example 1. The product obtained by one-step cloning of the target fragment and the vector was subjected to colony PCR and sequenced to obtain the pTrc99a / panB plasmid.

[0056] Example 4: Construction of expression plasmid pTrc99a / panD

[0057] panD was amplified by PCR using the B. subtilis genome as a template and Trc-panD-F and Trc-panD-R as primers. The PCR program was annealed at 58°C and extended at 72°C for 30 seconds. Other procedures were the same as those described in Example 1. The product obtained by one-step cloning of the target fragment and the vector was subjected to colony PCR and sequenced to obtain the pTrc99a / panD plasmid.

[0058] Example 5: Construction of expression plasmid pTrc99a / aspB

[0059] panD was amplified by PCR using the C. glutamicum genome as a template and Trc-aspB-F and Trc-aspB-R as primers. The PCR program was annealed at 59°C and extended at 72°C for 1 min. Other operations were the same as those described in Example 1. The product obtained by one-step cloning of the target fragment and the vector was subjected to colony PCR and sequenced to obtain the pTrc99a / panB plasmid.

[0060] Example 6: Construction of expression plasmid pTrc99a / pyc

[0061] pyc was amplified by PCR using the genome of C. glutamicum as a template, Trc-pyc-F and Trc-pyc-R as primers, and the PCR program was annealed at 59°C and extended at 72°C for 2 minutes. Other operations were the same as those described in Example 1. The product obtained by one-step cloning of the target fragment and the vector was subjected to colony PCR and sequenced to obtain the pTrc99a / pyc plasmid.

[0062] Example 7: Construction of expression plasmid pTrc99a / panC

[0063] panC was amplified by PCR using the C. glutamicum genome as a template and Trc-panC-F and Trc-panC-R as primers. The PCR program was annealed at 60°C and extended at 72°C for 30 seconds. Other procedures were the same as those described in Example 1. The product obtained by one-step cloning of the target fragment and the vector was subjected to colony PCR and verified by sequencing to obtain the pTrc99a / panC plasmid.

[0064] Example 8: Construction of expression plasmid pTrc99a / alpB. The pTrc99a / alsS plasmid constructed in Example 1 was used as a template and primers pTrc99a-ty-F and trc-alsS-panB-R were used to amplify the linear pTrc99a / alsS vector. The pTrc99a / panB plasmid constructed in Example 2 was used as a template and primers trc-alsS-panB-F and Trc-panB-R were used to amplify the target fragment panB. After the PCR product was verified by nucleic acid gel electrophoresis, it was incubated with Dpn I at 37°C and 180 rpm for 1 hour, and the residual template was removed using a PCR clean up kit to obtain a pure linearized pTrc99a / panB framework and the target fragment panB.

[0065] According to the instructions of the one-step cloning kit (ClonExpress Ultra One Step Cloning Kit C115, Vazyme Biotech, Nanjing, China), the linearized pTrc99a / panB vector was ligated with the target fragment panB and introduced into DH5α competent cells. Colony PCR verification was performed, and single colonies corresponding to positive clones were picked and sequenced to obtain the pTrc99a / alpB plasmid.

[0066] Example 9: Construction of expression plasmid pTrc99a / DaBp The pTrc99a / panD plasmid constructed in Example 3 was used as a template and pTrc99a-ty-F and DaspB-R were used as primers to amplify the linear pTrc99a / panD vector; the pTrc99a / aspB plasmid constructed in Example 4 was used as a template and DaspB-F and aspBpyc-R were used as primers to amplify the target fragment aspB; the pTrc99a / pyc plasmid constructed in Example 5 was used as a template and aspBpyc-F and Trc-pyc-R were used as primers to amplify the target fragment pyc. Other operations were the same as those described in Example 7. The pTrc99a / DaBp plasmid was obtained by sequencing verification.

[0067] Example 10: Construction of expression plasmid pTrc99a / alpB-DaBp-C Using the example pTrc99a / alsS-panB plasmid as a template, pTrc99a-ty-F and alB-DaBp-R were used as primers to amplify the linear vector pTrc99a / alpB; using the pTrc99a / panD-aspB-pyc plasmid as a template, alB-DaBp-F and DaBp-DR were used as primers to amplify the target fragment DaBp; using the pTrc99a / panC plasmid as a template, DaBp-DF and Trc-panC-R were used as primers to amplify the target fragment panC. Other operations were the same as those described in Example 7. The pTrc99a / alpB-DaBp-C plasmid was obtained by sequencing verification.

[0068] Example 11: Construction of strain ZPA2

[0069] Use a pipette tip to dip the bacterial solution from the glycerol cryovial of the plasmid-free DPA11A strain and streak it on an antibiotic-free LB plate, and culture it in a constant temperature incubator at 37°C overnight; select the correct single colony from the plate and inoculate it into 5 mL of LB medium, and culture it in a constant temperature shaker incubator at 37°C and 200 rpm overnight; then aspirate 400 mL of the bacterial solution and transfer it to 40 mL of LB medium, and culture it in a constant temperature shaker at 37°C and 200 rpm until the OD is between 0.4 and 0.6; place the shake flask containing the bacterial solution in ice and cool it for about 10 minutes, and centrifuge it at 4°C and 4500 rpm for 5 minutes to collect the bacteria; add 30 mL of pre-cooled 0.1 mol / L CaCl2 sterile solution, resuspend it in an ice water bath, and then ice bath it for 30 minutes; centrifuge it at 4000 rpm for 5 minutes, and then add 1 mL of pre-cooled 0.1 mol / L A sterile solution of CaCl2 and 15% glycerol was resuspended in an ice-water bath, then aliquoted into sterile 1.5mL centrifuge tubes (100μL per tube) and stored at -80°C to obtain competent cells of the plasmid-free DPA11A strain. 3μL of the successfully constructed plasmid pTrc99a / panB was introduced into the competent cells of the plasmid-free DPA11A strain and allowed to stand on ice for 30 minutes. The transformation product was heat-shocked in a 42°C water bath for 90 seconds, quickly cooled on ice for 5 minutes, and then added to 600μL of LB liquid medium. The cells were incubated at 37°C and 180 rpm for 1 hour, centrifuged at 12,000 rpm for 1 minute, and 600μL of the supernatant was discarded and resuspended. 100μL of this bacterial solution was evenly spread onto a solid LB plate containing kanamycin at a final concentration of 50μg / mL. After the bacterial solution was completely absorbed by the medium, the cells were incubated inverted at 37°C for 12 hours. A single colony was picked to obtain strain ZPA2.

[0070] Example 12: Shake flask fermentation and cell mass determination of strains DPA11A, ZPA1, ZPA2, ZPA3, ZPA4, ZPA5, and ZPA11. Strain ZPA1, strain ZPA2, strain ZPA3, strain ZPA4, ZPA5, and strain ZPA11 were obtained as described in Example 11. Strain DPA11A was used as the control group, and they were inoculated into 10 mL of LB medium and cultured at 37°C and 200 rpm for use as seed liquid. After 8-12 h, 200 μL of preculture and 0.2 mM IPTG were inoculated into a 250 mL shake flask containing 20 mL of fermentation medium, and then cultured at 30°C and 150 rpm for 48 h. After the fermentation was completed, samples were taken to detect the cell mass.

[0071] After fermentation, take 200 μL of fermentation broth to a 2 mL centrifuge tube and dilute 10-fold with 1.8 mL of sterile water. Centrifuge at 12,000 rpm for 1 minute at room temperature. Remove the supernatant and add 200 μL of 0.3 M hydrochloric acid to dissolve the calcium carbonate. Similarly, add 1.8 mL of sterile water to return the volume to 2 mL. Measure the OD using a spectrophotometer at a wavelength of 600 nm. 600 The measured value × dilution factor is the actual OD 600 The shake flask fermentation results of each strain with and without β-alanine are shown in Figure 2, where the error bars represent the deviation between the standard and triplicate experiments. The figure shows that strains ZPA1 and ZPA2 have better pantothenic acid production in the pantothenic acid biosynthesis branch pathway.

[0072] Example 13: Shake flask fermentation and cell mass determination of strains DPA11A, ZPA6, ZPA7, ZPA8, ZPA9, and ZPA10. Strains ZPA6, ZPA7, ZPA8, ZPA9, and ZPA10 were obtained using the method described in Example 11. The shake flask fermentation results of each strain with and without the addition of β-alanine are shown in Figure 3, where the error bars represent the deviation between the standard and triplicate experiments. It can be seen from the figure that strains ZPA8, ZPA9, and ZPA10 in the β-alanine synthesis branch pathway have better pantothenic acid production.

[0073] Example 14: Shake-flask Fermentation and Cell Mass Determination of Strains DPA11A, ZPA1, ZPA2, ZPA12, ZPA8, ZPA9, ZPA10, and ZPA13. Strains ZPA1, ZPA2, ZPA12, ZPA8, ZPA9, ZPA10, and ZPA13 were obtained using the methods described in Example 11. Shake-flask fermentation procedures were performed as described in Example 12. The shake-flask fermentation results for each strain are shown in Figure 4 , where error bars represent the deviation from the standard and triplicate experiments. The results showed that strain ZPA12 exhibited increased pantothenate production compared to ZPA1 and ZPA2 in the pantothenate biosynthesis pathway, as shown in Figure 4A . Furthermore, strain ZPA13 exhibited increased pantothenate production compared to ZPA8, ZPA9, and ZPA10 in the β-alanine biosynthesis pathway, as shown in Figure 4B .

[0074] Example 15: Shake-flask Fermentation and Cell Mass Determination of Strains DPA11A, ZPA11, ZPA12, ZPA13, and ZPA14 Shake-flask fermentations of strains ZPA11, ZPA12, ZPA13, and ZPA14 were performed as described in Example 11. The shake-flask fermentation results for each strain are shown in Figure 5 , where error bars represent the deviation from the standard and triplicate experiments. The final data showed that ZPA13 and ZPA14 both produced high levels of D-pantothenic acid, with yields of 2.97 g / L and 1.68 g / L, respectively, without the addition of β-alanine.

[0075] Example 16: Fed-batch fermentation of strain ZPA13 in a 5 L fermentor:

[0076] As described in Example 11, the successfully constructed plasmid pTrc99a / panD-aspB-pyc was transformed on a plate, a single colony was picked, inoculated into LB test tube culture medium, and cultured at 37°C and 10 rpm for 10 h. The seed liquid was inoculated into a triangular flask LB culture medium (liquid volume 100 mL / 500 mL) at a 1% inoculum volume, cultured at 37°C and 180 rpm for 10 h, and then inoculated into a fermenter at 1%.

[0077] Fermentation culture: inoculation volume 1%, fermentation tank liquid volume 2L / 5L, culture temperature 30℃, pH 6.8, initial rotation speed 400rpm, initial ventilation volume 4L / min, stirring and dissolved oxygen control after 12h, appropriately increase the ventilation volume to a maximum of 8L / min, maintain dissolved oxygen at 15%-25%; start feeding and pH control when pH>6.8.

[0078] After 72 h of fermentation, D-pantothenic acid was measured by high performance liquid chromatography.

[0079] Initial fermentation medium: glucose 20g / L, (NH4)2SO4 16g / L, KH2PO4 2g / L, MgSO4 0.5g / L, yeast extract 2g / L, betaine 2g / L, 1ml / L trace metal ion solution, 50-75mg / L Kan antibiotic, 0.1-0.2mM IPTG, solvent is deionized water, pH value is natural; trace element solution composition is: 5-10g / L CoCl2, 5-10g / L FeSO4·7H2O, 0.5-1g / L ZnSO4·7H2O, 0.10-0.20g / L CuSO4, 0.01-0.02g / L NiCl2·7H2O, solvent is deionized water.

[0080] Feed medium: glucose 500 g / L, ammonium sulfate 10 g / L, yeast extract 2 g / L, potassium dihydrogen phosphate 14 g / L, anhydrous magnesium sulfate 8 g / L, betaine 4 g / L, VBl 0.01 g / L, VBl2 0.004 g / L, isoleucine 160 mg / L, saline solution 2 mL / L, isopropyl-β-D-thiogalactoside 96 mg / L, kanamycin sulfate 100 mg / L.

[0081] The activation culture medium consists of 10 g / L peptone, 10 g / L yeast powder, 10 g / L NaCl, 2% agar, and a pH of 6.8-7.0.

[0082] The composition of the seed culture medium is as follows: peptone 10 g / L, yeast powder 10 g / L, NaCl 10 g / L, pH 6.8-7.0.

[0083] The fermentation results are shown in FIG6 . As can be seen from the figure, after 72 h of fermentation, the OD value was 81.3, the pantothenic acid production was 31.19 g / L, and the β-alanine production was 22.34 g / L.

[0084] Excessive β-alanine production indicated a weak pantoate pathway and needed to be strengthened. Overexpressing both the pantoate and β-alanine pathways on a single plasmid resulted in an overly large plasmid, creating a metabolic burden. Furthermore, the expression of each enzyme on the same plasmid interfered with each other, resulting in lower yields in ZPA14 compared to ZPA13 in shake flasks. Therefore, we chose to strengthen the pantoate pathway on the genome.

[0085] Example 17: Construction and shake flask fermentation of strain ZPA15 (DPA11A derivative, yjip::Ptrc-alsS) Using DPA11A as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene yjip in the genome of the starting strain with the alsS gene (nucleotide sequence shown in SEQ ID No. 1) regulated by the Ptrc promoter derived from pTrc99A by gene knock-in to enhance the expression intensity of alsS.

[0086] (1) Construction of pTarget-yjip plasmid: Using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-yjip-F / R as primers, PCR amplification was performed. After the PCR product was verified by nucleic acid gel electrophoresis, it was incubated with Dpn I digestion enzyme at 37°C and 180 rpm for 1 h. The residual template was removed using a PCR clean up kit and transformed into E. coli DH5α competent cells. After SD resistance LB solid plate screening and sequencing verification, the correct pTarget-yjip plasmid was obtained for subsequent ligation of Donor DNA.

[0087] (2) Construction of pTD-alsS plasmid: First, using the E. coli W3110 genome as a template, primers yjip-P1-F / R were used to amplify the upstream part (F1) of the donor DNA, and primers yjip-P2-F / R were used to amplify the downstream part (F2) of the donor DNA. Then, using the B. subtilis genome as a template, the alsS gene fragment (F3) with the promoter pTrc was amplified using primers pTrc-alsS-JYZ-F / R. The PCR fragments F1, F2, and F3 were purified by gel extraction. The plasmid pTarget-yjip was amplified using pTyjip-VF / R to obtain linearized pTarget-yjip. After verification by nucleic acid gel electrophoresis, the PCR product was incubated at 37°C for 1 hour using Dpn I. The residual template was removed using a PCR clean-up kit to obtain a relatively pure linearized pTarget-yjip plasmid framework. The linearized pTarget-yjip vector, fragments F1, F2, and F3 were ligated together according to the instructions of the one-step cloning kit (ClonExpress Ultra One Step Cloning Kit C115, Vazyme Biotech, Nanjing, China), and the pTD-alsS plasmid was verified by sequencing.

[0088] (3) Preparation of DPA11A / pCas electroporation competent medium: pCas plasmid (Addgene Plasmid #62225) was introduced into DPA11A electroporation competent medium to obtain a plate of strain DPA11A / pCas. Monoclonal colonies were transferred to 10 mL LB tubes containing Kan resistance and arabinose, and cultured at 30°C and 180 rpm for 10-12 h. Then, 1% inoculum was inoculated into 50 mL LB medium containing Kan resistance and arabinose, and cultured at 180 rpm and 30°C until OD 600 between 0.6 and 0.8; place the shake flask containing the bacterial solution in an ice water bath for 15-30 minutes, transfer the bacterial solution in the shake flask to a 50ml pre-cooled centrifuge tube in a clean bench, centrifuge at 4000rpm for 5 minutes at 4°C, remove the supernatant in the clean bench and retain the bacteria; add 40mL of pre-sterilized and pre-cooled ultrapure water to the centrifuge tube, resuspend in an ice water bath (gently), and centrifuge at 4500rpm for 8 minutes, discard the supernatant in the clean bench again and add 40mL of pre-sterilized and pre-cooled ultrapure water, resuspend in an ice water bath, and centrifuge at 450rpm for 8 minutes at 4°C; discard the supernatant in the clean bench and add 1mL of pre-cooled 10% sterile glycerol, resuspend in an ice water bath, and then divide into 1.5mL sterile centrifuge tubes (100μL per tube) in the clean bench and store in a -80°C ultra-low temperature refrigerator for later use.

[0089] (4) Use a pipette to draw an appropriate amount of pTD-alsS plasmid (about 200 ng) and mix it with 100 μL of electroporation competent cells prepared in advance, and transfer them together into a pre-cooled 2 mm electroporation cup. After ice bathing for about 1 minute, wipe the surface of the electroporation cup with a tissue paper and use an electroporator (MicroPluser TM , BIO-RAD) for electroporation. Immediately after electroporation, 800 μL of LB medium was added and aspirated, and the tube was transferred to a 1.5 mL sterile EP tube. The tube was revived at 30°C and 180 rpm for 2.5-3.5 h. 200 μL was then spread on a LLB solid plate containing Kan and SD, and the plate was incubated inverted at 30°C for 20-24 h. Colony PCR verification was performed using alsS-YZ-F / R as primers. Single colonies with the correct bands were ZPA15 (DPA11A derivative, yjip::Ptrc-alsS)-positive colonies.

[0090] (5) Plasmid elimination: Use an inoculation loop to pick up positive single colonies and inoculate them into LB liquid test tubes containing 0.5mM IPTG and 0.05mg / L Kan. Culture at 30℃ and 200rpm for 12-16h. The next day, the bacterial liquid is streaked onto Kan-resistant LB solid plates and cultured at 30℃ overnight. When the bacteria grow to a certain size, pick some single colonies and streak them onto Kan and SD double-resistant plates and culture at 30℃ for 12h. The single colonies that cannot grow on the double-resistant plates are the single colonies in which the pTarget-yjip plasmid has been successfully eliminated. If all single colonies can grow on LLB solid medium (Kan and SD double-resistant), select the single colony with the least growth and re-inoculate and induce, and repeat the subsequent process.

[0091] After that, single colonies that had successfully eliminated the pTarget-yjip plasmid were picked and placed in a non-resistance LB tube and cultured overnight at 42°C to eliminate the pCas plasmid. The next day, the bacterial liquid was streaked onto a non-resistance LB plate. After culturing at 37°C for 12 hours, some single colonies were streaked onto an LB plate containing Kan resistance and cultured at 30°C for 12 hours. Those that could not be found on the Kan resistance plate were single colonies that had successfully eliminated the pCas plasmid. Colony PCR verification was then performed again and sent to the company for sequencing. After successful verification, the plasmid-free strain ZPA15 (DPA11A derivative, yjip::Ptrc-alsS) was obtained.

[0092] (6) Shake flask fermentation: ZPA15 (DPA11A derivative, yjip::Ptrc-alsS) was used as a control group, and the starting strain DPA11A was used for shake flask testing and detection according to the method of Example 12. The shake flask fermentation results of each strain are shown in Figure 7, where the error bars represent the deviation between the standard and the triplicate experiment. As can be seen from Figure 7A, when β-alanine was added, after the alsS gene copy number was increased in the genome by gene knockin, the D-pantothenic acid shake flask titer increased to 2.50 g / L, which was an increase of 12.66% compared with 11A. The effect was not obvious, so the D-pantothenic acid pathway synthesis capacity is still insufficient and needs to be further strengthened.

[0093] Example 18: Construction of strain ZPA16 (DPA11A derivative, yjip::Ptrc-alsS)

[0094] (1) Construction of pTarget-ycjV plasmid: Using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-yjiV-F / R as primers, PCR amplification was performed. The PCR product was verified by nucleic acid gel electrophoresis and incubated with Dpn I digestion enzyme at 37°C and 180 rpm for 1 h. It was then transformed into E. coli DH5α and screened on LB solid plates for SD resistance. The correct pTarget-yjiV plasmid was obtained by sequencing and verified for subsequent ligation with Donor DNA.

[0095] (2) Construction of pTD-panB plasmid: First, using the E. coli W3110 genome as a template, ycjV-P1-F / R primers were used to amplify the upstream part (F1) of the donor DNA, and ycjV-P2-F / R primers were used to amplify the downstream part (F2) of the donor DNA. Then, using the C. glutamicum genome as a template, the panB gene fragment (F3) with the promoter pTrc was amplified using primers pTrc-panB-JYZ-F / R, and the PCR fragments F1, F2, and F3 were purified by gel recovery. The plasmid pTarget-ycjV was amplified using pTycjV-VF / R to obtain linearized pTarget-ycjV. After the PCR product was verified by nucleic acid gel electrophoresis, Dpn I was used to incubate the reaction at 37°C for 1 hour, and the residual template was removed using a PCR clean up kit to obtain a relatively pure linearized pTarget-ycjV plasmid framework. According to the instructions of the one-step cloning kit (ClonExpress Ultra One Step Cloning Kit C115, Vazyme Biotech, Nanjing, China), the linearized pTarget-ycjV vector, fragments F1, F2, and F3 were connected together, and the pTD-panB plasmid was obtained by sequencing verification.

[0096] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the ZPA15 competent cells obtained in Example 2. The ZPA15 competent cells were prepared by electroporation as in Example 17 (3).

[0097] (4) ZPA16-positive colonies were constructed using the same method as in Example 17(4).

[0098] (5) Plasmid elimination: The implementation method is the same as Example 17(5) to obtain plasmid-free ZPA16.

[0099] (6) The constructed DPAP8 production strain was used as a control group with the DPAP7 constructed in Example 2, and shake flask testing and detection were performed according to the method of Example 12. The shake flask fermentation results of each strain are shown in Figure 7, where the error bars represent the deviation between the standard and the triplicate experiment. As can be seen from Figure 7A, when β-alanine was added, after the panB gene copy number was increased again in the genome by gene knock-in, the D-pantothenic acid production still increased to about 2.73 g / L, which was an increase of 23.03% compared with 11A, indicating that the D-pantothenic acid pathway synthesis capacity was further enhanced.

[0100] Example 19: Construction of strains ZPA17 and ZPA18 and shake flask fermentation

[0101] As described in Example 11, the successfully constructed plasmid pTrc99a / panD-aspB-pyc was introduced into ZPA15 and ZPA16 for plate transformation, respectively, to obtain strains ZPA17 and ZPA18, which were shake flask tested and detected according to the method of Example 12. 600 The D-pantothenic acid content in the fermentation supernatant is shown in Figure 7B. In the absence of β-alanine, increasing the copy number of the alsS and panB genes in the genome by gene knockin further increased D-pantothenic acid production to approximately 3.34 g / L.

[0102] Example 20: Fed-batch fermentation of strain ZPA18 in a 5 L fermentor

[0103] The method steps were the same as those in Example 16, and the results are shown in FIG8 . As can be seen from FIG8 , the pantothenic acid production of ZPA18 in shake flask fermentation was increased to 3.34 g / L. After 72 h of fed-batch fermentation in a 5 L fermentor, the pantothenic acid production reached 36.15 g / L, an increase of 15.9% compared to ZPA13. The final production reached 46.15 g / L after 83 h of fermentation.

Claims

1. A high-yield D-pantothenic acid genetically engineered bacterium that does not rely on the addition of β-alanine, characterized in that, It is constructed by the following method: Using the strain E. coli W3110 Trc-panC / Trc-panE / Trc-panB / Trc-ilvC / ilvG* / △avtA / ilvE* / coaA* / ilvA* / Trc-lpd / △glk as the chassis strain, the panB gene derived from Corynebacterium glutamicum is integrated into the pseudogene ycjV locus in its genome, and the panD gene derived from Bacillus subtilis, the aspB gene and the pyc gene derived from Corynebacterium glutamicum are overexpressed in the chassis bacterium, thereby obtaining the high-yield D-pantothenic acid genetically engineered bacterium.

2. The high-yield D-pantothenic acid genetic engineering bacterium according to claim 1, characterized in that, The panB gene is regulated by the Ptrc promoter, and the nucleotide sequence of the panB gene is as shown in SEQ ID NO.

2.

3. The high-yield D-pantothenic acid genetically engineered bacterium according to claim 1 or 2, characterized in that, The nucleotide sequence of the panD gene is as shown in SEQ ID NO.

8.

4. The high-yield D-pantothenic acid genetically engineered bacterium according to claim 3, characterized in that, The nucleotide sequence of the aspB gene is as shown in SEQ ID NO.9, and the nucleotide sequence of the pyc gene is as shown in SEQ ID NO.

10.

5. A method for constructing a high-yield D-pantothenic acid genetically engineered bacterium as described in any one of claims 1 to 4, characterized in that, It includes the following steps: Using the strain E. coli W3110 Trc-panC / Trc-panE / Trc-panB / Trc-ilvC / ilvG* / △avtA / ilvE* / coaA* / ilvA* / Trc-lpd / △glk as the chassis strain, applying the CRISPR-Cas9 mediated gene editing technology, integrating the panB gene regulated by the Ptrc promoter into the pseudogene ycjV locus in its genome, and using the plasmid expression system, cloning the panD gene, the aspB gene and the pyc gene into the pTrc99a plasmid, and transferring the obtained plasmid pTrc99a / panD-aspB-pyc into the chassis bacterium, thereby obtaining the high-yield D-pantothenic acid genetically engineered bacterium.

6. Use of the high-yield D-pantothenic acid genetically engineered bacterium according to any one of claims 1 to 4 or the high-yield D-pantothenic acid genetically engineered bacterium constructed by the method according to claim 5 in the microbial fermentation preparation of D-pantothenic acid.

7. The application according to claim 6, characterized in that The use includes: inoculating the genetically engineered bacterium into a fermentation medium, fermenting and culturing at 28 - 30 °C and pH 6.6 - 6.8, and separating and purifying the supernatant of the fermentation broth after fermentation to obtain D-pantothenic acid.

8. The application according to claim 7, wherein The composition of the fermentation medium includes: glucose 10 - 20 g / L, (NH4)2SO4 12 - 16 g / L, KH2PO4 1 - 2 g / L, MgSO4 0.3 - 0.5 g / L, yeast extract 1 - 2 g / L, β-alanine 0.1 - 0.5 g / L, trace metal ion solution 0.5 - 1 ml / L, Kan antibiotic 50 - 75 mg / L, IPTG 0.1 - 0.2 mM, the solvent is deionized water, and the pH value is natural.

9. The application according to claim 8, characterized in that, The composition of the trace metal ion solution includes: 5 - 10 g / L CoCl2, 5 - 10 g / L FeSO4·7H2O, 0.5 - 1 g / L ZnSO4·7H2O, 0.10 - 0.20 g / L CuSO4, 0.01 - 0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

10. The application according to claim 7 or 8, characterized in that At the start of fermentation, a fermentation medium is used, and according to the 20% dissolved oxygen feedback feeding strategy, the feeding medium is added to the fermentation system. The composition of the feeding medium includes: 400 - 500 g / L glucose, 8 - 10 g / L ammonium sulfate, 1 - 3 g / L yeast extract, 10 - 14 g / L potassium dihydrogen phosphate, 6 - 8 g / L anhydrous magnesium sulfate, 2 - 4 g / L betaine, 0.01 g / L VB1, 0.004 g / L VB12, 100 - 160 mg / L isoleucine, 1 - 2 mL / L salt solution, 94 - 98 mg / L isopropyl-β-D-thiogalactoside, 80 - 100 mg / L kanamycin sulfate.

Images