Preparation and application of ketone pantoic acid hydroxymethyltransferase mutant for D-pantothenic acid production

Abstract

The invention discloses preparation and application of a ketone pantoic acid hydroxymethyltransferase mutant for D-pantothenic acid production, and belongs to the technical field of gene engineering and enzyme engineering. The escherichia coli ketopantoic acid hydroxymethyltransferase is mutated by means of genetic engineering and enzyme engineering, and the obtained single mutation and double mutation are expressed in improved chassis cells, so that the yield of the escherichia coli D-pantothenic acid can be remarkably increased, and conditions are created for expanding industrial application of the escherichia coli D-pantothenic acid.

Description

Technical Field

[0001] The invention relates to the preparation and application of a ketopantoate hydroxymethyltransferase mutant for producing D-pantothenic acid, and belongs to the technical field of genetic engineering and enzyme engineering. Background Art

[0002] D-pantothenic acid (D-PA), also known as pantothenic acid and vitamin B5, is an important water-soluble B vitamin. D-PA participates in the metabolism of carbohydrates, proteins, fatty acids and other substances in organisms, and is widely used in food, animal feed, cosmetics and pharmaceutical industries. Pantothenic acid is most widely used in the field of animal feed and the pharmaceutical industry. Plants and microorganisms can produce pantothenic acid themselves, but humans and animals cannot produce pantothenic acid. Humans can usually ingest D-pantothenic acid through food, so pantothenic acid deficiency does not occur in humans under normal circumstances. However, if anti-D-pantothenic acid drugs such as salicylic acid are taken and foods with low D-pantothenic acid content are consumed for a long time, D-pantothenic acid deficiency may occur. D-pantothenic acid deficiency can lead to symptoms such as memory loss, adrenal failure, and arthritis. Pantothenic acid is often used clinically to treat vitamin B deficiency, chronic inflammation and loss of appetite. When pantothenic acid deficiency is severe in livestock and poultry, pigs will have symptoms such as diarrhea, hair loss, and anemia. Pantothenic acid deficiency in chickens will lead to abnormal growth, poor feather growth, and even death in severe cases. Therefore, when raising livestock such as poultry, it is generally necessary to add a certain amount of D-pantothenic acid to the feed.

[0003] D-pantothenic acid is widely used in food, feed and pharmaceutical industries, and has a large market demand. With the growing demand for D-pantothenic acid in domestic and foreign markets, it is undoubtedly very important to study methods for efficiently producing D-pantothenic acid. At present, there are three main methods for producing D-PA: chemical synthesis, enzyme-catalyzed synthesis and microbial fermentation. The chemical synthesis method first prepares two key intermediates, β-alanine and D, L-pantothenic acid lactone, and then uses them to synthesize D-pantothenic acid. Among them, β-alanine is mainly synthesized by acrylonitrile method, acrylic acid method and succinimide method, while D, L-pantothenic acid lactone is mainly obtained by isobutyraldehyde-formaldehyde method, isobutyraldehyde-acetic acid method and isobutyraldehyde-trichloroform method. After obtaining the two intermediates of β-alanine and D, L-pantothenic acid lactone, there are two main methods for producing D-pantothenic acid. The first method is to directly treat β-alanine with calcium hydroxide to form calcium β-aminopropionate, and then calcium β-aminopropionate reacts with D, L-pantothenate lactone in a methanol solution and at high temperature to obtain D, L-pantothenate calcium. High-purity D-pantothenate calcium is obtained by adding D-pantothenate calcium seeds to the D, L-pantothenate calcium methanol solution to induce crystallization, and the excess L-pantothenate calcium is racemized before being split. The second method is to split D, L-pantothenate lactone first. There are two splitting methods: chemical splitting and enzymatic splitting. Chemical splitting mainly uses splitting reagents such as quinine, ephedrine, L-leucine, strychnine, L-1-p-nitrotoluene-2-amino-1,3-propanediol to split D, L-pantothenate lactone. The enzymatic splitting uses L-pantoic acid lactone hydrolase, D-pantoic acid lactone hydrolase, L-pantoic acid lactone dehydrogenase, keto-pantoic acid reductase and alcohol dehydrogenase to split D, L-pantoic acid lactone. After obtaining D-pantoic acid lactone, it is amidated with calcium β-aminopropionic acid in methanol solution and high temperature to obtain calcium D-pantothenate. Enzymatic synthesis mainly uses D-pantothenate synthase to catalyze pantothenic acid and β-alanine to synthesize D-pantothenic acid, which is also the last step of microbial fermentation production of D-pantothenic acid. Enzymatic synthesis of D-PA is greener than chemical synthesis, but it still has problems such as high production cost and strict reaction conditions. The rapid development of synthetic biology has promoted the development of microbial fermentation technology, so the use of microbial fermentation to produce D-PA has attracted more and more attention.

[0004] The conversion of α-ketoisovalerate to ketopantothenate is a key step in the D-pantothenate synthesis pathway, but the natural ketopantoate hydroxymethyltransferase that catalyzes this step has poor activity and stability. Obtaining ketopantoate hydroxymethyltransferase mutants with improved activity and stability through enzyme engineering is of great significance for the production of D-pantothenate by microbial fermentation. Summary of the invention

[0005] The present invention provides a ketopantoate hydroxymethyltransferase mutant, which uses the ketopantoate hydroxymethyltransferase from Escherichia coli as a starting sequence and mutates multiple sites such as position 44, position 123, position 124 of the ketopantoate hydroxymethyltransferase.

[0006] In one embodiment, the starting sequence contains S7A / L8S / Y12C / E15D / A63E / Q124K mutations compared to pure wild-type ketopantoate hydroxymethyltransferase. Unless otherwise specified, the "starting sequence" or "parent" referred to below refers to the ketopantoate hydroxymethyltransferase containing the above mutations.

[0007] In one embodiment, the amino acid sequence of the starting sequence is shown as SEQ ID NO.1.

[0008] In one embodiment, the mutant has at least one of the following mutations based on the sequence shown in SEQ ID NO.1:

[0009] (1) mutating the valine at position 123 to glycine, aspartic acid, tyrosine, threonine, methionine, histidine, cysteine, proline, leucine, isoleucine, glutamine, or alanine;

[0010] (2) Mutate the lysine at position 124 to glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine, or histidine.

[0011] In one embodiment, the mutant is based on the sequence shown in SEQ ID NO.1, with the valine at position 123 mutated to isoleucine, and the lysine at position 124 changed to valine.

[0012] In one embodiment, the mutant is based on the sequence shown in SEQ ID NO.1, with the valine at position 123 being changed to isoleucine, and the lysine at position 124 being changed to tryptophan.

[0013] In one embodiment, the mutant is a mutant in which the valine (Val) at position 123 is changed to threonine (Thr), and the obtained mutant is named V123T.

[0014] In one embodiment, the mutant is a mutant in which valine (Val) at position 123 is changed to glutamine (Gln), and the obtained mutant is named V123Q.

[0015] In one embodiment, the mutant is a mutant in which valine (Val) at position 123 is changed to alanine (Ala), and the obtained mutant is named V123A.

[0016] In one embodiment, the mutant is a mutant in which lysine (Lys) at position 124 is changed to valine (Val), and the obtained mutant is named K124V.

[0017] In one embodiment, the mutant is a mutant in which lysine (Lys) at position 124 is changed to tryptophan (Trp), and the obtained mutant is named K124W.

[0018] In one embodiment, the mutant is a mutant in which lysine (Lys) at position 124 is changed to aspartic acid (Asp), and the obtained mutant is named K124D.

[0019] In one embodiment, the mutant is a mutant in which the valine (Val) at position 123 is changed to isoleucine (Ile), and the lysine (Lys) at position 124 is changed to valine (Val), and the obtained mutant is named V123I / K124V.

[0020] In one embodiment, the mutant is a mutant in which the valine (Val) at position 123 is changed to isoleucine (Ile), and the lysine (Lys) at position 124 is changed to tryptophan (Trp), and the obtained mutant is named V123I / K124W.

[0021] The invention also provides a gene encoding the ketopantoate hydroxymethyltransferase high-producing D-pantothenic acid mutant.

[0022] In one embodiment, the nucleotide sequence encoding mutant V123T is shown as SEQ ID NO.3; the nucleotide sequence encoding mutant V123L is shown as SEQ ID NO.4; the nucleotide sequence encoding mutant V123I is shown as SEQ ID NO.5; the nucleotide sequence encoding mutant K124D is shown as SEQ ID NO.6; the nucleotide sequence encoding mutant K124V is shown as SEQ ID NO.7; the nucleotide sequence encoding mutant K124W is shown as SEQ ID NO.8; the nucleotide sequence encoding mutant V123I / K124V is shown as SEQ ID NO.9; the nucleotide sequence encoding mutant V123I / K124W is shown as SEQ ID NO.10;.

[0023] The invention also provides a recombinant plasmid carrying the gene.

[0024] In one embodiment, the recombinant plasmid uses pACYC as a starting plasmid.

[0025] The present invention also provides a microbial cell expressing the ketopantoate hydroxymethyltransferase mutant or carrying the gene.

[0026] In one embodiment, the recombinant microbial cell is Escherichia coli.

[0027] In one embodiment, the Escherichia coli is based on Escherichia coli PA104 as a starting strain, and the Escherichia coli PA104 and its construction method have been disclosed in the paper "Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering".

[0028] In one embodiment, the Escherichia coli is an Escherichia coli capable of synthesizing D-pantothenic acid.

[0029] In one embodiment, the Escherichia coli having the ability to synthesize D-pantothenic acid is based on Escherichia coli MG1655, in which ketopantothenate hydroxymethyltransferase and pantothenate synthase are expressed, and the Escherichia coli has the following improvements (a) or (b):

[0030] (a) The alanine aminotransferase gene avtA and the glucose kinase gene glk were knocked out;

[0031] (b) The alanine aminotransferase gene avtA, the glucose kinase gene glk, and the acetate dehydrogenase gene poxB were knocked out.

[0032] In one embodiment, the nucleotide sequence of the gene panB encoding ketopantoate hydroxymethyltransferase is shown as SEQ ID NO.2; the nucleotide sequence of the gene panC encoding pantothenate synthase is shown as SEQ ID NO.11.

[0033] The present invention also provides a method for increasing the D-pantothenic acid production of Escherichia coli, wherein the method comprises mutating one or more sites at positions 123 and 124 of the amino acid sequence of the ketopantoate hydroxymethyltransferase of Escherichia coli.

[0034] In one embodiment, the method is to increase the content of D-pantothenic acid in Escherichia coli by performing at least one of the following mutations on ketopantothenate hydroxymethyltransferase:

[0035] (1) mutating the valine at position 123 to glycine, aspartic acid, tyrosine, threonine, methionine, histidine, cysteine, proline, leucine, isoleucine, glutamine or alanine;

[0036] (2) Mutating the lysine at position 124 to glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine or histidine.

[0037] In one embodiment, the method is to mutate the 123rd valine of ketopantoate hydroxymethyltransferase to isoleucine, and to mutate the 124th lysine to valine.

[0038] In one embodiment, the method is to mutate valine at position 123 to isoleucine and lysine at position 124 to tryptophan in ketopantoate hydroxymethyltransferase.

[0039] In one embodiment, the ketopantoate hydroxymethyltransferase has an amino acid sequence as shown in SEQ ID NO.1.

[0040] The invention also provides application of the Escherichia coli engineering bacteria in producing D-pantothenic acid.

[0041] In one embodiment, the application is to ferment the engineered Escherichia coli strain in a culture medium for a period of time and collect D-pantothenic acid in the fermentation broth.

[0042] In one embodiment, the fermentation is carried out at 35-40°C.

[0043] In one embodiment, the culture medium contains carbon sources, nitrogen sources, and inorganic salts required for the growth of Escherichia coli.

[0044] In one embodiment, the culture medium is LB medium.

[0045] In one embodiment, the culture medium contains glucose, tryptone, yeast extract, and inorganic salts; the inorganic salts include sodium salts and ammonium salts; optionally, the sodium salt is sodium chloride, and the ammonium salt is ammonium sulfate.

[0046] In one embodiment, the culture medium contains glucose, corn steep liquor, yeast extract powder, peptone, ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and β-alanine.

[0047] In one embodiment, the fermentation process is also fed with glucose and β-alanine.

[0048] The invention also provides application of the Escherichia coli engineering bacteria in producing products containing D-pantothenic acid.

[0049] In one embodiment, the product includes but is not limited to medicine, health care product or feed.

[0050] Beneficial effects:

[0051] (1) The present invention targets ketopantoate hydroxymethyltransferase (KPHMT) in Escherichia coli MG1655, transforms the amino acid sequence of KPHMET by site-directed mutagenesis, and obtains mutants V123T, V123L, V123I, V123Q, K124D, K124V, and K124W by well plate primary screening and shake flask rescreening. The D-pantothenic acid production of the recombinant bacteria expressing the above mutants is 2.7 g / L, 2.62 g / L, 3.19 g / L, 1.7 g / L, 2.71 g / L, 3.25 g / L, and 3.38 g / L, respectively, which is 11.5%, 8.2%, 31.8%, 12%, 34.3%, and 39.7% higher than that of the parent strain expressing ketopantoate hydroxymethyltransferase, respectively.

[0052] (2) The present invention also constructed a recombinant Escherichia coli expressing the double mutant V123I / K124W, and the D-pantothenic acid production can reach 48.99 g / L; the fermentation intensity of the strain was significantly improved, and the production intensity of the mutant V123I / K124W in the first 24 hours of fermentation was increased by 87% compared with the starting sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of the D-pantothenic acid metabolism of Escherichia coli.

[0054] Figure 2 Figure 2 is a plasmid map carrying the parent gene fragment of ketopantoate hydroxymethyltransferase.

[0055] Figure 3 OD of the parent strain and the recombinant strain of the 44-amino acid ketopantoate hydroxymethyltransferase mutant in 24-well plate culture 600 and a graph of D-pantothenic acid production.

[0056] Figure 4 OD of the parent strain and the recombinant strain of the 123-amino acid ketopantoate hydroxymethyltransferase mutant in 24-well plate culture 600 and a graph of D-pantothenic acid production.

[0057] Figure 5 OD of the parent strain and the recombinant strain of the 124-amino acid ketopantoate hydroxymethyltransferase mutant in 24-well plate culture 600 and a graph of D-pantothenic acid production.

[0058] Figure 6 The strain OD of the parent strain and the recombinant strain with high-yielding D-pantothenate ketopantoate hydroxymethyltransferase mutant was screened by shake flask 600 and a graph of D-pantothenic acid production.

[0059] Figure 7The relative yields of the parent strain and the recombinant strain with high-yielding D-pantothenate ketopantoate hydroxymethyltransferase mutant were screened in shake flasks.

[0060] Figure 8 OD of the parent and double mutant V123I / K124W in a 5L fermenter 600 , residual sugar and D-pantothenic acid production graph. DETAILED DESCRIPTION

[0061] 1. Culture medium:

[0062] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride. Add 15 g / L agar powder to prepare a solid medium.

[0063] Shake flask medium: glucose 25g / L, yeast powder 5g / L, peptone 10g / L, ammonium sulfate 16g / L, β-alanine 4g / L. Depending on the situation, it is sometimes necessary to add antibiotics with a final concentration of 0.1%. β-alanine and glucose need to be sterilized separately and then added to the culture medium.

[0064] Fermentation medium in a 5L fermenter: 25g / L glucose, 5g / L corn steep liquor, 5g / L yeast extract powder, 5g / L peptone, 15g / L ammonium sulfate, 0.5g / L magnesium sulfate, 0.5g / L potassium dihydrogen phosphate, 1g / L potassium hydrogen phosphate, 10g / L β-alanine, 0.0075g / L VB1, 0.02g / L manganese sulfate, 0.01g / L zinc sulfate, 0.02g / L ferrous sulfate; wherein, VB1, manganese sulfate, zinc sulfate and ferrous sulfate are prepared into 1000 times mother liquor, filtered and sterilized, and then added to the fermentation medium at corresponding concentrations. Depending on the situation, corresponding antibiotics may need to be added. β-alanine and glucose are sterilized separately. Sterilization procedure: 115°C, 20min.

[0065] Feed medium 1 (1 L): 250 g / L β-alanine.

[0066] Feed medium 2 (1 L): 500 g / L glucose.

[0067] 2. Production of D-pantothenic acid by well plate fermentation:

[0068] A single full colony was picked and inoculated into a 24-well round-bottom plate. 4.8 mL of shake flask medium was added to each well of the plate and cultured at 30°C and 220 rpm for 48 h.

[0069] 3. Shake flask fermentation to produce D-pantothenic acid:

[0070] Use an inoculation loop to take the bacterial solution from the glycerol tube and streak it on the plate. Incubate at 37°C overnight. Then pick a single colony and inoculate it in a shake tube containing 2-3mL LB medium. Incubate at 37°C and 220rpm for 12h. Take 500μL of the seed solution and inoculate it in a 250mL conical flask containing 24.5mL shake flask medium. Set up three parallel fermentations for each strain and incubate at 30°C and 220rpm for 48h. After the fermentation is completed, take 1mL of the fermentation solution to measure the OD 600 , take another 1 mL of fermentation broth, centrifuge at 12000 rpm for 2 min, dilute the supernatant 5 times with water, and perform detection according to the HPLC detection method in the specific implementation manner.

[0071] 4. Fermentation production of D-pantothenic acid in 5L fermenter:

[0072] Use an inoculation loop to take the bacterial liquid from the glycerol tube and streak it on the plate. Incubate it in a 37℃ incubator overnight. Then pick the colony and inoculate it in the slant culture medium. If necessary, add the corresponding antibiotics and incubate it in a 37℃ incubator for 12h. Then, streak the colony from the slant culture medium and inoculate it into a 2L conical flask. Add 300mL of LB liquid culture medium to the conical flask and incubate it at 37℃ and 220rpm for 10h. During the culture process, keep the air flow rate at 2L / min and the initial speed at 200rpm / min. Adjust the speed according to the needs of dissolved oxygen and stirring. The maximum speed shall not exceed 900rpm / min. When the initial sugar is basically exhausted, choose the continuous feeding method to keep the sugar concentration below 5g / L. Use pure ammonia water to maintain the pH at about 6.80. The fermentation temperature is maintained at 37℃. Slowly add 100mL, 100mL, and 50mL of β-alanine at 15h, 25h, and 35h of fermentation.

[0073] 5. D-pantothenic acid analysis method:

[0074] The biomass of Escherichia coli was determined by ultraviolet spectrophotometry. An appropriate amount of fermentation broth was taken, diluted and mixed with ultrapure water, and the absorbance OD at a wavelength of 600 nm was measured using water as a control. 600 . Take 1mL of fermentation liquid and suck it into an EP tube, place it in a high-speed centrifuge, centrifuge it at room temperature at 12000 for 2 minutes, and transfer the supernatant to a new EP tube. Dilute the supernatant fermentation liquid with ddH2O to a certain multiple, filter it with a 0.22μm inorganic filter membrane to obtain the sample to be tested, and use a residual sugar meter to determine the residual sugar content in the sample to be tested.

[0075] Take 1mL of fermentation liquid and inhale it into an EP tube, place it in a high-speed centrifuge, centrifuge it at 12000rpm for 2 minutes at room temperature, transfer the supernatant to a new EP tube, and use high-performance liquid chromatography to determine the D-pantothenic acid content in the sample. After the supernatant fermentation liquid is diluted to a certain multiple with ddH2O, it is filtered with a 0.22μm inorganic filter membrane to obtain the sample to be tested, and the D-pantothenic acid content in the sample to be tested is determined by high-performance liquid chromatography. Chromatographic conditions: the chromatographic column is Aminex HPX-87H Column (Bio-Rad, Richmond, CA, USA), the column oven temperature is 30°C, the mobile phase is 95% 0.02mol / L potassium dihydrogen phosphate, 5% acetonitrile, and the pH of the mobile phase is adjusted to 4 with phosphoric acid, the mobile phase flow rate is 1mL / min, the injection volume is 10μL, the detection wavelength is 200nm, and the retention time of pantothenic acid is slightly different when detected by different instruments.

[0076] 6. Strain Information

[0077] The strain information is shown in Table 1. Escherichia coli PA104 is a chassis cell obtained by genetic modification of Escherichia coli MG1655 such as Δglk and ΔavtA. The strain and its construction method are disclosed in the paper "Development of avitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering".

[0078] Table 1 Strains involved in the present invention

[0079]

[0080] Table 2 Primer sequences

[0081]

[0082]

[0083]

[0084] Example 1: Construction of chassis cell DPA04

[0085] Using Escherichia coli PA104 (published in the paper "Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering") as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to knock out the pyruvate dehydrogenase gene poxB, reduce the accumulation of byproduct acetic acid, and increase the accumulation of pyruvate, a precursor for pantothenic acid synthesis. The specific steps are:

[0086] (1) Construction of pTarget-poxB plasmid: Using pTarget plasmid as template and pT-poxB-F / pT-poxB-R as primers, the first 20 bp of the PAM site were introduced into the plasmid pTarget as the guide signal of sgRNA. The constructed pTarget-poxB was sent for sequencing verification, and the plasmid with correct sequencing was kept for subsequent operations.

[0087] (2) Construction of poxB knockout fusion homology arms: Using E. coli MG1655 as a template, two pairs of primers (poxB-up-F / poxB-up-R, poxB-down-F / poxB-down-R) were used to PCR amplify the upstream and downstream homology arms. Using the upstream and downstream homology arms as templates and poxB-up-F / poxB-down-R as primers, the upstream and downstream homology arms were connected by overlapping extension PCR. Agarose gel electrophoresis was then performed to verify, and the PCR products with the correct band size were purified and recovered.

[0088] (3) The pCas plasmid was introduced into E. coli PA104, and the single clone was transferred to an LB test tube containing 0.05 mg / L kanamycin and cultured at 37°C overnight. Then, the inoculum was inoculated into a 250 mL conical flask containing 50 mL LB medium at a volume concentration of 1%, and 500 μL of 1 mol / L arabinose was added. The culture was cultured at 220 rpm and 37°C until the OD 600 0.4-0.6, and then add 500 μL of 1 mol / L arabinose. Then, centrifuge at 5000 rpm, 4°C to collect the strain, wash it three times with 10% glycerol, and then pack it into aliquots to prepare electroporation competent cells.

[0089] (4) Take 500 ng of the pTarget-poxB plasmid constructed in step (1) and the poxB knockout fusion homology arm constructed in step (2) and add them to 100 μL of electroporation competent medium, transfer them into a pre-cooled 1 mm electroporation cup, and perform electroporation transformation using an electroporator. Immediately after the electroporation, add 1 mL of LB culture medium and gently pipette to mix, then aspirate and transfer to a 1.5 mL centrifuge tube, culture on a shaker at 220 rpm and 37°C for 1.5-2 hours, and culture on an LB plate containing 0.05 mg / L kanamycin and streptomycin at 37°C for 16 hours. Perform colony PCR using poxB-up-F and pxoB-down-R as primers. If the band size is 1000 bp, it means that the poxB gene has been successfully knocked out.

[0090] (5) Plasmid elimination: The verified colonies were transferred to LB medium containing 1mM rhamnose and 0.05mg / L kanamycin and cultured overnight to remove the plasmid pTarget-poxB. After overnight culture, streak the colonies on a kanamycin-resistant plate and culture them overnight. After the positive colonies grow up, streak them on a kanamycin-resistant plate and a streptomycin-resistant plate respectively and mark them. The bacteria that can grow on the kanamycin-resistant plate but not on the streptomycin-resistant plate indicate that the plasmid pTarget-poxB has been removed. Then transfer the colonies with the plasmid pTarget-poxB removed to LB medium containing 1% (1M) sucrose to eliminate the pCas9 plasmid. After overnight culture, streak the plate on a non-antibiotic plate and culture it overnight. After the positive colonies grow up, streak the plate on a non-antibiotic plate, a kanamycin-resistant plate, and a streptomycin-resistant plate, and mark them. Bacteria that can grow on the non-antibiotic plate but not on the kanamycin-resistant plate or the streptomycin-resistant plate indicate that the plasmids pCas9 and pTarget have been removed. The constructed strain is named DPA04.

[0091] Example 2: Preparation of a single mutant containing the ketopantoate hydroxymethyltransferase gene

[0092] panB shown in SEQ ID NO.2 and panC shown in SEQ ID NO.11 were connected to plasmid pACYC (SEQ ID NO.12) to construct plasmid pACYC-panBC. Plasmid pACYC was used as a template and reverse PCR amplification was performed with the primers in Table 2 to obtain a linearized pACYC-panBC fragment. After the PCR product was verified to be correct, it was digested with Dpn I at 37°C for 1h and transformed into Escherichia coli JM109 competent cells. The clones were selected for sequencing to confirm whether the encoding gene of the ketopantoate hydroxymethyltransferase single mutant was correct. The mutant plasmids with correct sequencing were transformed into Escherichia coli DPA04 competent cells, and the single mutants G44L, G44V, G44T, G44C, G44A, G44K, G44D, G44S, V123K, V123G, V123D, V123Y, V123T, V123M, V123H, V123C, V123P, V123L, V123A, and V123S were constructed and expressed. strains of 23R, V123E, V123N, V123F, V123I, V123Q, K124D, K124G, K124M, K124T, K124A, K124N, K124E, K124L, K124S, K124V, V124R, K124Q, K124I, K124P, K124Y, K124C, K124H, K124F, and K124W.

[0093] The specific steps of competent cell preparation and transformation are:

[0094] (1) Take out the preserved bacteria from the -80℃ refrigerator, dip a small amount of bacterial solution with a sterile inoculation needle on a sterile clean bench, and streak on a plate of LB solid medium without any antibiotics. Place the streaked plate in a constant temperature incubator at 37℃ and culture for about 12 hours.

[0095] (2) After 12 hours, a single colony with good growth was picked and inoculated into a 50 mL shake flask containing 5 mL LB liquid medium, and cultured in a shaking incubator at 37°C and 220 rpm for 8-10 hours.

[0096] (3) Use a pipette to draw 500 μL (1% inoculation volume) of culture solution and inoculate it into a 250 mL shake flask containing 50 mL LB liquid culture medium, and place a 50 mL centrifuge tube in a -20°C refrigerator for precooling.

[0097] (4) Place the 250 mL shake flask in a shaker at 37°C and 220 rpm for about 2 h until the OD 600 In the range of 0.4 to 0.6.

[0098] (5) Transfer the bacterial solution in the shake flask to a 50 mL centrifuge tube, place it in an ice box for 10-15 min, and precool the 50 mL centrifuge to 4°C.

[0099] (6) Place a 50 mL centrifuge tube in a 50 mL centrifuge and centrifuge at 4000 rpm for 5 min. After centrifugation, pour out the supernatant.

[0100] (7) In a sterile clean bench, use a pipette to draw up 5 mL of solution A, add it to a 50 mL centrifuge tube, and resuspend the cells.

[0101] (8) Repeat step (6).

[0102] (9) Use a pipette to draw 5 mL of solution B in a sterile clean bench, add it to a 50 mL centrifuge tube, and resuspend the bacteria. Use a pipette to draw 100 μL of the bacterial solution and add it to a sterile EP tube placed on ice for aliquoting. After aliquoting, place the competent E. coli cells in a -80°C refrigerator for storage.

[0103] (10) Take out the competent cells from the -80°C freezer and place them in an ice box for 5-10 min to thaw. Use a pipette to draw 1 μL of the correctly sequenced mutant plasmid and add it to the EP tube containing the competent cells. Slowly blow 3-4 times to mix it evenly, and then place it in ice for 30 min.

[0104] (11) Remove the EP tube from the ice box, heat shock it in a 42°C constant temperature water bath for 1-2 min, and then quickly place it on ice for 2 min.

[0105] (12) Use a pipette to add 1 mL of antibiotic-free LB liquid culture medium into the EP tube on a sterile clean bench. Mix gently and transfer to a constant temperature shaker at 37 °C and 220 rpm and culture for 40-60 min.

[0106] (13) Remove the EP tube from the shaker and transfer it to a centrifuge. Centrifuge at 5000 rpm for 3 min. Pour off part of the supernatant in a sterile clean bench and keep about 70-100 μL. Use a pipette to blow and mix the cells and add them to the LB solid medium containing the corresponding antibiotics. Use a sterile spreader to spread evenly. After marking, invert and culture in a 37°C constant temperature incubator overnight.

[0107] Example 3: Production of D-pantothenate by fermentation of a single ketopantoate hydroxymethyltransferase mutant in a well plate

[0108] The mutant strains constructed in Example 2 were inoculated into round-bottom 24-well plates containing shake flask medium, 100 μg / L ampicillin was added as needed, and fermented at 30°C and 220 rpm for 48 h. The fermentation broth was collected to detect the OD of the strain. 600 The DPA04 strain expressing the starting sequence (panBC) was used as the control (WT) and fermented in the same manner as above.

[0109] The results showed that the 44-site mutant strain had no improvement over the parent strain, while the strains with single-point mutation at the 123 amino acid site (V123G, V123D, V123Y, V123T, V123H, V123C, V123P, V123L, V123A, V123I, V123Q, V123N, and V123F) had higher D-pantothenic acid production than the strain expressing the starting sequence. Among them, the D-pantothenic acid production of the better mutants V123L, V123I, or V123Q were 1.77 g / L, 1.33 g / L, and 1.48 g / L, respectively, which were 197%, 122%, and 149% higher than the control, respectively. The D-pantothenic acid production of the mutants with single-point mutation at amino acid position 124 (K124D, K124G, K124M, K124T, K124E, K124L, K124V, K124Q, K124I, K124P, K124R, K124Y, K124C, K124H, and K124W) was higher than that of the strain expressing the starting sequence. Among them, the D-pantothenic acid production of the better mutants K124D or K124V was 1.48 g / L and 1.56 g / L, respectively, which were 148% and 164% higher than that of the control.

[0110] Example 4: Production of D-pantothenate by fermentation of single ketopantoate hydroxymethyltransferase mutants in shake flasks

[0111] The expression mutants screened in Example 3 are G44L, G44V, G44T, G44C, G44A, G44K, G44D, G44S, V123K, V123G, V123D, V123Y, V123T, V123M, V123H, V123C, V123P, V123L, V123A, V123R, V123E, V123N, V123F, V123I, V123Q, K124D, K124G, and K Among the strains of 124M, K124T, K124A, K124N, K124E, K124L, K124S, K124V, V124R, K124Q, K124I, K124P, K124Y, K124C, K124H, K124F or K124W, the mutants V123T, V123L, V123I, V123Q, K124D, K124V and K124W with higher yields verified by well plate were selected for shake flask rescreening verification. The specific steps are:

[0112] Pick out full recombinant E. coli and inoculate them in a shaking tube containing 2-3mL LB medium, and culture at 37℃ and 220rpm for 12h to obtain seed solution. Take 500μL of the seed solution and inoculate it in a 250mL conical flask containing 24.5mL shaking flask medium. Set up three parallel fermentations for each strain and culture at 30℃ and 220rpm for 48h. Collect the fermentation broth to detect the OD value of the strain. 600 and D-pantothenic acid production.

[0113] The results showed that the D-pantothenic acid production of strain WT reached 2.42 g / L after 48 h of shake flask fermentation, and the D-pantothenic acid production of mutants V123T, V123L, V123I, V123Q, K124D, K124V, and K124W were 2.7 g / L, 2.62 g / L, 3.19 g / L, 1.7 g / L, 2.71 g / L, 3.25 g / L, and 3.38 g / L, respectively. Except for V123Q, the other mutants were better than the starting sequence and basically consistent with the well plate results. V123T, V123L, V123I, K124D, K124V, and K124W increased by 11.5%, 8.2%, 31.8%, 12%, 34.3%, and 39.7% respectively compared with the strain expressing ketopantothenate hydroxymethyltransferase parent.

[0114] Example 5: Preparation of Ketopantoate Hydroxymethyltransferase Double Mutant

[0115] Using the plasmid containing mutant V123I constructed in Example 2 as a template, primers for introducing V123I / K124V and V123I / K124W mutants were designed and synthesized. Site-directed mutagenesis was performed by PCR linearization template. After the PCR product was verified to be correct, it was digested with Dpn I at 37°C for 1h and transformed into Escherichia coli JM109 competent state, and clones were selected for sequencing to confirm whether the coding gene of the double mutant of ketopantoic acid hydroxymethyltransferase was correct. The mutant plasmid with correct sequencing was transformed into Escherichia coli DPA04 competent cells to construct strains expressing double mutants V123I / K124V and V123I / K124W. The specific steps of competent state preparation and transformation are shown in Example 2.

[0116] Example 6: Shake flask fermentation of ketopantoate hydroxymethylase double mutants for production of D-pantothenic acid

[0117] The strains expressing V123I / K124V and V123I / K124W constructed in Example 5 were screened and verified by shaking flasks. Pick full E. coli and inoculate them in a shaking tube containing 2-3mL LB medium, and culture at 37°C and 220rpm for 12h to obtain seed liquid. Take 500μL of the seed liquid and inoculate it in a 250mL conical flask containing 24.5mL shaking flask medium. Set up three parallel fermentations for each strain and culture at 30°C and 220rpm for 48h. Collect the fermentation broth to detect the OD of the strain. 600 The strain DPA04 in which the ketopantoate hydroxymethyltransferase parent (pACYC-panBC) was expressed using the pACYC plasmid was used as a control strain.

[0118] The results showed that the D-pantothenic acid production of the control strain WT reached 2.52g / L, and the D-pantothenic acid production of the strains expressing single mutations V123I, K124V, and K124W were 3.16g / L, 3.31g / L, and 3.52g / L, respectively. The strain expressing the double mutant V123I / K124V did not work well, with a D-pantothenic acid production of only 3.29g / L. However, the D-pantothenic acid production of the double mutant strain V123I / K124W increased significantly, with a D-pantothenic acid production of 4.16g. This was an increase of 65.1% over the starting sequence, and an increase of 31.6% and 18.2% over the single mutants V123I and K124W, respectively.

[0119] Example 7: Ketopantoate hydroxymethyltransferase and its mutants were used to produce D-pantothenic acid in a 5L fermenter

[0120] The mutant strains constructed in Examples 1 to 5 were streaked on a plate by taking bacterial liquid from a glycerol tube with an inoculation loop, and cultured in a 37°C incubator overnight. Then, colonies were picked and inoculated on a slant medium. If necessary, corresponding antibiotics were added, and cultured in a 37°C incubator for 12 hours. Then, colonies were streaked from the slant medium and inoculated into a 2L conical flask. 300 mL of LB liquid medium was added to the conical flask, and the seed solution was obtained by culturing at 37°C and 220 rpm for 10 hours.

[0121] The seed liquid was inoculated into the fermentation medium at a 10% inoculation amount (initial OD = 3). During the culture process, the air flow rate was maintained at 2L / min, the initial rotation speed was 200rpm / min, the rotation speed was adjusted according to the needs of dissolved oxygen and stirring, and the maximum rotation speed should not exceed 900rpm / min. When the initial sugar was basically exhausted, the feed medium 2 was added by continuous feeding to keep the sugar concentration at ≤5g / L. The pH was maintained at 6.80±0.1 with pure ammonia water. The fermentation temperature was maintained at 37°C. 100mL, 100mL, and 50mL of feed medium 1 were slowly added at 15h, 25h, and 35h of fermentation, respectively. The strain expressing the ketopantoic acid hydroxymethyltransferase parent using the pACYC plasmid in strain DPA04 was used as the control strain.

[0122] Ferment for 72 hours, and take samples regularly for testing. Figure 8 As shown, the strain expressing the ketopantoate hydroxymethyltransferase parent can produce 42.5g / L of D-pantothenic acid in a 5L fermenter for 72h, while the strain expressing the double mutant V123I / K124W can produce 48.99g / L of D-pantothenic acid, which is 7.49g / L higher than the control strain. In addition, the production intensity of the double mutant strain is much higher than that of the control strain. The control strain only produces 22.08g / L after 24h of fermentation, while the double mutant can reach 41.28g / L after 24h of fermentation. In the first 24h, the production intensity of the control strain was 0.92g / L / h, while the production intensity of the double mutant strain was 1.72g / L / h. The production intensity of the mutant V123I / K124W was 87% higher than that of the control.

[0123] Comparative Example:

[0124] The specific implementation method is the same as that of Examples 2 to 6, except that mutant strains G44L, G44V, G44T, G44C, G44A, G44K, G44D, G44S, V123K, V123M, V123R, V123E, K124A, and K124S were also constructed, and the results showed that the D-pantothenic acid production of these mutant strains was 0.259 g / L, 0.272 g / L, 0.252 g / L, 0.235 g / L, 0.303 g / L, 0.268 g / L, 0.306 g / L, 0.311 g / L, 0.508 g / L, 0.08 g / L, 0.368 g / L, 0.517 g / L, 0.477 g / L, and 0.539 g / L, respectively. The levels were 56.4%, 54.2%, 57.6%, 60.4%, 49%, 54.9%, 48.6%, 47.6%, 14.5%, 86.5%, 38%, 12.9%, 19.7% and 9.3% lower than those of the strain expressing wild ketopantoate hydroxymethyltransferase, respectively.

[0125] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A ketopantoate hydroxymethyltransferase mutant, characterized in that: Based on the amino acid sequence as shown in SEQ ID NO.1, there is at least one of the following mutations: (1) mutating the valine at position 123 to glycine, aspartic acid, tyrosine, threonine, methionine, histidine, cysteine, proline, leucine, isoleucine, glutamine, or alanine; (2) Mutate the lysine at position 124 to glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine, or histidine.

2. The ketopantoate hydroxymethyltransferase mutant according to claim 1, characterized in that Based on the sequence shown in SEQ ID NO.1, the valine at position 123 is mutated to threonine, or the valine at position 123 is mutated to glutamine, or the valine at position 123 is mutated to alanine, or the lysine at position 124 is mutated to valine, or the lysine at position 124 is mutated to tryptophan, or the lysine at position 124 is mutated to aspartic acid.

3. The ketopantoate hydroxymethyltransferase mutant according to claim 1, characterized in that Based on the sequence shown in SEQ ID NO.1, the valine at position 123 is mutated to isoleucine and the lysine at position 124 is changed to valine; or The valine at position 123 was changed to isoleucine and the lysine at position 124 was changed to tryptophan.

4. A gene encoding the ketopantoate hydroxymethyltransferase mutant according to any one of claims 1 to 3.

5. A recombinant plasmid carrying the gene according to claim 4.

6. A microbial cell expressing the ketopantoate hydroxymethyltransferase mutant according to any one of claims 1 to 3 or carrying the gene according to claim 4.

7. A recombinant Escherichia coli, characterized in that Expressing the mutant of any one of claims 1 to 3 in an Escherichia coli capable of synthesizing D-pantothenic acid; the Escherichia coli capable of synthesizing D-pantothenic acid is based on Escherichia coli MG1655, and has the following improvements (a) or (b): (a) The alanine aminotransferase gene avtA and the glucose kinase gene glk were knocked out; (b) The alanine aminotransferase gene avtA, the glucose kinase gene glk, and the acetate dehydrogenase gene poxB were knocked out.

8. A method for increasing the production of D-pantothenic acid in Escherichia coli, characterized in that: Carry out any mutation (a) to (d) on the ketopantoate hydroxymethyltransferase in Escherichia coli: (a) mutating the valine at position 123 to glycine, aspartic acid, tyrosine, threonine, methionine, histidine, cysteine, proline, leucine, isoleucine, glutamine or alanine; (b) mutating the lysine at position 124 to glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine or histidine; (c) mutating the valine at position 123 of ketopantoate hydroxymethyltransferase to isoleucine, and mutating the lysine at position 124 to valine; (d) The ketopantoate hydroxymethyltransferase is mutated by mutating the valine at position 123 to isoleucine and the lysine at position 124 to tryptophan.

9. A method for preparing D-pantothenic acid, characterized in that: The recombinant Escherichia coli according to claim 7 is fermented in a culture medium.

10. The method according to claim 9, wherein the fermentation is carried out at 35-40°C; optionally, glucose and β-alanine are also fed during the fermentation process.

11. Use of the mutant according to any one of claims 1 to 3, or the microbial cell according to claim 6, or the recombinant Escherichia coli according to claim 7, or the method according to any one of claims 8 to 10 in producing D-pantothenic acid or a product containing D-pantothenic acid.

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