Construction and application of recombinant escherichia coli engineering strain with high yield of D-pantothenic acid

Abstract

The invention discloses construction and application of a recombinant escherichia coli engineering strain with high yield of D-pantothenic acid, and belongs to the technical field of genetic engineering and biological engineering. According to the invention, acetic acid branch pathway genes are knocked out, so that accumulation of precursor pyruvic acid is improved; carrying out free expression and integrated expression on panBC respectively; by weakening a cofactor 5, 10-CH2-THF degradation pathway and strengthening an de novo synthesis pathway of the cofactor 5, 10-CH2-THF, supply of 5, 10-CH2-THF is promoted, and the yield of the constructed engineering strain D-pantothenic acid can reach 4.83 g / L. The Escherichia coli recombinant strain with high yield of D-pantothenic acid, constructed by the invention, lays a foundation for subsequent biosynthesis of D-pantothenic acid and derivatives, and has potential value and significance for development of synthetic biology.

Description

Technical Field

[0001] The invention relates to the construction and application of a recombinant Escherichia coli engineering strain with high production of D-pantothenic acid, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] D-pantothenic acid (D-PA), also known as pantothenic acid and vitamin B5, is an important water-soluble B vitamin. It 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. In recent years, with the in-depth study of D-PA, it has also received close attention in the treatment of neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. Due to the wide range of applications of D-PA, research on D-PA synthesis has received widespread attention.

[0003] At present, there are three main methods for producing D-PA: chemical synthesis, enzyme catalysis synthesis and microbial fermentation. The mainstream method for synthesizing D-PA is still chemical synthesis, but there are problems such as high production cost and large environmental pollution using chemical synthesis of D-PA. Enzyme catalysis synthesis of D-PA is greener than chemical synthesis, but there are still the above problems. The rapid development of synthetic biology has promoted the development of microbial fermentation technology, so it is necessary to develop a method for producing D-PA and improving D-PA output by microbial fermentation. Summary of the invention

[0004] The present invention provides an engineered Escherichia coli strain for efficiently synthesizing D-pantothenic acid, which is based on the original strain and has at least one of the following improvements:

[0005] (1) Reduce the expression of poxB, a pyruvate dehydrogenase gene, and reduce the accumulation of acetic acid as a byproduct;

[0006] (2) Expression of panB and panC, the key enzymes in the D-pantothenate biosynthesis process in Escherichia coli;

[0007] (3) reducing the expression of the formyltetrahydrofolate deformylase gene purU or the 5,10-methylenefolate reductase gene metF;

[0008] (4) Express the 5,10-methylenetetrahydrofolate dehydrogenase gene folD to increase the supply of 5,10-CH2-THF.

[0009] In one embodiment, the starting strain is a chassis cell constructed by knocking out the gene avtA (shown in Gene ID: 948087) and the gene glk (shown in Gene ID: 946858) on the basis of Escherichia coli MG1655.

[0010] In one embodiment, the starting strain is Escherichia coli PA104, which has been disclosed in the paper "Development of avitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering".

[0011] In one embodiment, the engineered Escherichia coli is based on E. coli PA104, with the pyruvate dehydrogenase gene poxB knocked out.

[0012] In one embodiment, the nucleotide sequence of the gene poxB is shown as Gene ID: 946132.

[0013] In one embodiment, the engineered Escherichia coli uses pACYCDuet as a vector to express ketopantoate hydroxymethyltransferase panB and pantothenate synthetase panC.

[0014] In one embodiment, the engineered Escherichia coli bacteria integrate and express ketopantoate hydroxymethyltransferase panB and pantothenate synthase panC at the yjiR site of the genome.

[0015] In one embodiment, the nucleotide sequence of the gene panBC encoding ketopantoate hydroxymethyltransferase panB and pantothenate synthase panC is shown in SEQ ID NO.1.

[0016] In one embodiment, the sequence of the 5,10-CH2-THF biosynthetic pathway gene folD is shown as SEQ ID NO.2.

[0017] In one embodiment, the genetically engineered bacteria have knocked out the formyltetrahydrofolate deformylase gene purU or the 5,10-methylenefolate reductase gene metF.

[0018] In one embodiment, the nucleotide sequence of the gene purU is shown as Gene ID: 945827.

[0019] In one embodiment, the nucleotide sequence of the gene metF is shown as Gene ID: 948432.

[0020] In one embodiment, the gene folD is integrated at the genomic metF site.

[0021] In one embodiment, the genetically engineered bacteria further express the serine glycine cleavage pathway gene serA or gene glyA.

[0022] In one embodiment, plasmid pACYCDuet is used as an expression vector to express gene serA or glyA.

[0023] In one embodiment, the nucleotide sequence of the serine glycine cleavage pathway gene serA is shown as SEQ ID NO.3; the nucleotide sequence of the gene glyA is shown as SEQ ID NO.4.

[0024] The present invention also provides a method for improving the D-pantothenic acid production capacity of Escherichia coli, the method comprising one or more of (1) to (5):

[0025] (1) Knockout the pyruvate dehydrogenase gene poxB to reduce the accumulation of byproduct acetic acid;

[0026] (2) Expression of ketopantoate hydroxymethyltransferase panB and pantothenate synthase panC;

[0027] (3) knocking out the purU gene, a formyltetrahydrofolate deformylase gene, or the metF gene, a 5,10-methylenefolate reductase gene;

[0028] (4) expressing the 5,10-methylenetetrahydrofolate dehydrogenase gene folD to increase the supply of 5,10-CH2-THF;

[0029] (5) Fermentation in a culture medium containing betaine and / or serine.

[0030] In one embodiment, the method further comprises expressing the serine glycine cleavage pathway gene serA or the gene glyA.

[0031] In one embodiment, plasmid pACYCDuet is used as an expression vector to express gene serA or glyA.

[0032] The invention also provides application of the Escherichia coli engineering bacteria in producing D-pantothenic acid.

[0033] In one embodiment, the engineered Escherichia coli is fermented in a culture medium at 30° C. to 37° C. for at least 24 hours.

[0034] In one embodiment, the engineered Escherichia coli is fermented in a culture medium at 30° C. to 37° C. for 24 to 48 hours.

[0035] In one embodiment, the culture medium contains glucose, yeast powder, peptone, ammonium sulfate, and β-alanine.

[0036] In one embodiment, the culture medium further contains betaine.

[0037] In one embodiment, the concentration of betaine in the culture medium is ≥4 g / L.

[0038] In one embodiment, the culture medium further contains serine.

[0039] In one embodiment, the concentration of serine in the culture medium is ≥ 0.06 g / L.

[0040] In one embodiment, the concentration of serine in the culture medium is 0.06-0.1 g / L.

[0041] The present invention also claims to protect the application of the engineered Escherichia coli in the fields of food, medicine and chemical industry in producing products containing D-pantothenic acid derivatives.

[0042] In one embodiment, the application is for preparing health products or medicines containing D-pantothenic acid, or preparing cosmetics containing D-pantothenic acid.

[0043] Beneficial effects:

[0044] (1) The present invention uses Escherichia coli PA104 as a host, and improves the synthesis capacity of D-pantothenic acid by knocking out the poxB gene, optimizing the expression levels of the ketopantoate hydroxymethyltransferase gene panB and the pantothenate synthase gene panC, and significantly improves the yield, which is 63% to 216% higher than that of the starting strain.

[0045] (2) The present invention weakens the endogenous 5,10-CH2-THF degradation pathway and effectively increases the production of D-pantothenic acid by strengthening the supply of 5,10-CH2-THF, a cofactor of ketopantothenic acid strong methyltransferase, so that the production of D-pantothenic acid by the constructed strain in shake flask fermentation reaches 4.83 g / L.

[0046] (3) The present invention optimizes the culture medium components and adds betaine to the culture medium to promote the synthesis of D-pantothenic acid. Compared with the fermentation conditions without adding betaine, the yield obtained is increased by 12.9%. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the metabolism of D-pantothenic acid in Escherichia coli.

[0048] Figure 2 The chromatographic detection results of D-pantothenic acid produced by the engineered strain in shake flask culture medium.

[0049] Figure 3 This is the mass spectrum of D-pantothenic acid detected in the fermentation broth.

[0050] Figure 4 The OD of the engineered strains DPA03 and DPA04 in shake flask culture medium600 and D-pantothenic acid production.

[0051] Figure 5 OD of engineered strains DPA04, DPA05, and DPA06 after fermentation in shake flask medium 600 and a graph of D-pantothenic acid production.

[0052] Figure 6 OD of engineered strain DPA06 after fermentation in medium containing different concentrations of magnesium ions, vitamin C, betaine, isoleucine or corn steep liquor 600 and a graph of D-pantothenic acid production.

[0053] Figure 7 Schematic diagram of the synthesis of Escherichia coli cofactor 5,10-CH2-THF.

[0054] Figure 8 OD of engineered strains DPA06, DPA07, DPA08 and DPA09 after fermentation in shake flasks 600 and a graph of D-pantothenic acid production.

[0055] Fig. 9 OD of engineered strain DPA09 cultured in shake flask medium supplemented with different concentrations of serine 600 and a graph of D-pantothenic acid production.

[0056] Fig.10 The OD of the engineered strains DPA09, DPA10, and DPA11 cultured in shake flask medium 600 and a graph of D-pantothenic acid production. DETAILED DESCRIPTION

[0057] The present invention is further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto:

[0058] 1. Technical terms

[0059] The terms "comprising", "including", "having", and "containing" used in the present invention mean "including but not limited to", "including but not limited to", "having but not limited to", and "containing but not limited to", and can be used interchangeably with corresponding phrases. Unless the context clearly indicates otherwise, the term "or" used in this article is used in this article to mean the term "and / or" and can be used interchangeably with it. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, the examples of suitable methods and materials described below are illustrative only and do not limit the present invention in any way.

[0060] The term "expression" as used herein refers to any step involved in the production of an enzyme, protein or polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0061] The term "specific yield" as used herein refers to the yield of the target product per OD of fermentation broth. In some embodiments, "specific yield" refers to the yield of D-pantothenic acid per OD of fermentation broth, which is calculated by dividing the D-pantothenic acid yield (g / L) in the fermentation broth by the OD value of the fermentation broth.

[0062] (II) Culture medium

[0063] LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L. Add 20g / L agar powder to prepare LB solid medium.

[0064] Shake flask medium: glucose 25g / L, yeast powder 5g / L, peptone 10g / L, ammonium sulfate 16g / L, β-alanine 4g / L.

[0065] (III) The strains involved in the present invention are shown in Table 1.

[0066] Among them, strain PA104 is a chassis cell obtained by genetic modification of Escherichia coli MG1655 such as Δglk and ΔavtA. Strain PA104 and its construction method are disclosed in the paper "Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering".

[0067] Table 1 Strains and genotypes

[0068]

[0069] (IV) D-pantothenic acid HPLC determination:

[0070] Shimadzu high performance liquid chromatography was used for determination. HPLC conditions: the chromatographic column was Aminex HPX-87H Column (Bio-Rad, Richmond, CA, USA), the column oven temperature was 30°C, the mobile phase was 95% 0.02 mol / L potassium dihydrogen phosphate, 5% acetonitrile, and the pH of the mobile phase was adjusted to 4 with phosphoric acid, the mobile phase flow rate was 1 mL / min, the injection volume was 10 μL, the detection wavelength was 200 nm, and the retention time of pantothenic acid was slightly different when detected by different instruments.

[0071] (V) Primer sequences are shown in Table 2.

[0072] Table 2 Primer sequences

[0073]

[0074]

[0075] Example 1 Knockout of genes in the acetic acid synthesis pathway

[0076] 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:

[0077] (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.

[0078] (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.

[0079] (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.

[0080] (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.

[0081] (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.

[0082] (6) Take E.coli PA104 as the control group, use an inoculation loop to take the bacterial solution from the glycerol tube and streak it on the plate, culture it at 37℃ overnight, then pick a single colony and inoculate it in a shake tube containing 2-3mL LB medium, and culture it at 37℃ 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 culture it at 30℃ and 220rpm for 48h. After the fermentation is completed, take 1mL of the fermentation liquid 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 embodiment. 600 and D-pantothenic acid production Figure 4As can be seen from the figure, knocking out poxB has a certain promoting effect on bacterial growth, and the yield has also increased to a certain extent. The D-pantothenic acid yield increased from 0.63g / L to 1.11g / L, and the specific yield increased from 0.041g / L to 0.067g / L. Since poxB encodes acetate dehydrogenase, which catalyzes pyruvate to produce acetate, knocking out this gene is beneficial to reducing the accumulation of acetate and promoting the accumulation of pyruvate, the key precursor of D-pantothenic acid.

[0083] Example 2D - Optimization of expression intensity of key enzymes in pantothenate synthesis

[0084] panB and panC are key enzymes in the D-PA synthesis pathway. Both are transcribed and expressed in the form of gene clusters in Escherichia coli. Their expression levels and activities significantly affect the synthesis of D-pantothenic acid. Previous reports in the prior art usually adopted a strong expression method of plasmid free expression, but plasmid free expression has disadvantages such as instability and the need for exogenous addition of antibiotics. In comparison, integrated expression is more stable and does not require exogenous addition of antibiotics. Therefore, this embodiment expresses the panBC gene freely on the plasmid and integrates the panBC gene cluster on the Escherichia coli pseudogene site yjiR (the nucleotide sequence is shown in SEQ ID NO.1). The specific steps are:

[0085] (1) Construction of pACYCDuet-panBC plasmid:

[0086] Using panBC-pACYC-R / panBC-pACYC-F as primers and pACYCDuet plasmid as template, linearized pACYCDuet plasmid was amplified. Using panBC-F / panBC-R as primers and E. coli MG1655 genome as template, panBC gene fragment with homologous sequence was amplified. The fragment was purified using DNA purification kit and then used The kit completed the ligation and obtained the plasmid pACYCDuet-panBC.

[0087] The constructed pACYCDuet-panBC plasmid was transformed into the strain DPA04 constructed in Example 1 to obtain the strain DPA05.

[0088] (2) panBC genome integration:

[0089] Construction of pTarget-yjiR plasmid: The construction steps refer to step (1) of Example 1.

[0090] Construction of yjiR integrated panBC homology arms: Using E. coli MG1655 as a template, two pairs of primers (yjiR-up-F / yjiR-up-R, yjiR-down-F / yjiR-down-R) were used to PCR amplify the upstream and downstream homology arms, and the panBC gene was amplified using yjiR-panBC-F / yjiR-panBC-R. Using the upstream and downstream homology arms and the synthetic panBC gene cluster as templates, poxB-up-F / poxB-down-R were used as primers to connect the upstream and downstream homology arms and the target gene through overlap extension PCR. Afterwards, agarose gel electrophoresis was performed for verification, and the PCR product with the correct band size was purified and recovered.

[0091] The pCas plasmid was introduced into the strain DPA04 constructed in Example 1. The specific steps were as described in step (3) of Example 1. E. coli PA104 (ΔpoxB, yjiR:: panBC) positive colonies were obtained by construction. The construction method was the same as step (4) of Example 1. Plasmid elimination was performed by referring to the method of step (5) of Example 1. The constructed E. coli PA104 (ΔpoxB, yjiR:: panBC) was named strain DPA06.

[0092] Using DPA04 constructed in Example 1 as a control, strains DPA05 and DPA06 were subjected to shake flask fermentation verification according to the method described in step (6) of Example 1. 600 And D-pantothenic acid content see Figure 5 . The D-pantothenic acid production of strains DPA05 and DPA06 was 3.27 g / L and 3.77 g / L, respectively, which was 172% and 216% higher than that of the control DPA04. It can be seen that both strong and weak expression of the panBC gene significantly promoted the production of D-pantothenic acid, but the single-copy expression yield of gene integration was higher than the high-copy expression of the plasmid. Therefore, the weak expression level of panBC may be more conducive to the production of D-pantothenic acid in the strain than the strong expression level.

[0093] Example 3D - Optimization of pantothenic acid fermentation medium

[0094] The process of microbial synthesis of many natural and non-natural products is affected by many factors, and the culture medium is an important environmental factor in the growth of microorganisms, which has an important impact on various metabolic processes of cells. By changing certain nutrients in the culture medium, various cell reactions can be affected without changing other environmental and genetic factors, thereby controlling the growth of microorganisms and the production of target products.

[0095] The shake flask fermentation medium was used as the basal medium, and the strain DPA06 constructed in Example 2 was used as the control. Magnesium ions (final concentrations were 0 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, and 2 g / L, respectively), or vitamin C (final concentrations were 0 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, and 2 g / L, respectively), or betaine (final concentrations were 0 g / L, 0.5 g / L, 1 g / L, 2 g / L, and 4 g / L, respectively), or isoleucine (final concentrations were 0 g / L, 0.02 g / L, 0.04 g / L, 0.08 g / L, and 0.16 g / L, respectively), or corn steep liquor (final concentrations were 0 g / L, 1 g / L, 2 g / L, 4 g / L, and 8 g / L, respectively) were added to the basal medium to verify the effects of these substances on strain growth and production. Shake flask fermentation was performed according to the method of step (6) of Example 1. Strain OD 600 And D-pantothenic acid content see Figure 6 The results showed that the addition of betaine at a concentration of 4g / L significantly increased the yield of D-pantothenic acid, which was 12.9% higher than the control. Since the addition of betaine can enhance the strain's resistance to adverse external factors, the reason for the increase in yield may be that the strain's resistance to certain toxic substances has been enhanced.

[0096] Example 4 Enhanced Supply of Ketopantoate Hydroxymethyltransferase Cofactor 5,10-CH2-THF

[0097] Ketopantoate hydroxymethyltransferase KPHMT (EC 2.1.2.11) encoded by panB can transfer a hydroxymethyl group in 5,10-CH2-THF to α-ketoisovalerate to generate ketopantoate. 5,10-CH2-THF is a methyl donor for the synthesis of D-PA and an essential cofactor for KPHMT. In order to strengthen the supply of KPHMT cofactor 5,10-CH2-THF, the formyltetrahydrofolate deformylase gene purU gene was knocked out on the basis of strain DPA06 to construct strain DPA07. Further, the 5,10-methylenefolate reductase gene metF gene was knocked out on the basis of DPA07 to construct strain DPA08. On the basis of strain DPA07, the 5,10-methylenetetrahydrofolate dehydrogenase gene folD gene (nucleotide sequence as SEQ ID NO.2) was integrated at the metF site to construct strain DPA09. The specific steps are:

[0098] (1) Construct pTarget-purU and pTarget-metF plasmids respectively: the construction steps refer to step (1) of Example 1.

[0099] (2) Construction of purU and metF knockout homology arms and folD integration homology arms: The steps for constructing purU and metF knockout homology arms are the same as step (1) of Example 1, and the steps for constructing folD integration homology arms are the same as step (1) of Example 2.

[0100] (3) Introducing the pCas plasmid into the strain, the specific steps are the same as step (3) in Example 1.

[0101] (4) Constructing positive colonies of E. coli PA104 (ΔpoxB, yjiR::panBC, ΔpurU), E. coli PA104 (ΔpoxB, yjiR::panBC, ΔpurUΔmetF), and E. coli PA104 (ΔpoxB, yjiR::panBC, metF::folD), and the construction method is the same as step (4) of Example 1.

[0102] (5) Plasmid elimination: The elimination method is the same as step (5) of Example 1.

[0103] (6) The constructed E. coli PA104 (ΔpoxB, yjiR::panBC, ΔpurU) was named DPA07, the E. coli PA104 (ΔpoxB, yjiR::panBC, ΔpurUΔmetF) was named DPA08, and the E. coli PA104 (ΔpoxB, yjiR::panBC, metF::folD) was named DPA09. DPA06 was used as a control and shake flask verification was performed according to the method of step (6) of Example 1. Strain OD 600 And D-pantothenic acid content see Figure 8 . The shake flask results showed that the yield of DPA07 with the purU gene knocked out was slightly increased, 0.23 g / L higher than the control DPA06. The yield of DPA08 with the metF gene knocked out was 0.35 g / L higher than that of strain DPA07, and the shake flask yield of strain DPA08 was 4.35 g / L. The strain DPA09 with the folD gene integrated was 0.84 g / L higher than the control strain DPA06, and the final shake flask yield of strain DPA09 could reach 4.83 g / L, an increase of 21.1% over the control. It can be seen that the enhanced supply of 5,10-CH2-THF can effectively increase the activity of ketopantothenate hydroxymethyltransferase, thereby promoting the production of D-pantothenic acid.

[0104] Example 5: Increasing Serine Supply to Increase D-Pantothenic Acid Content

[0105] DPA09 was used as the fermentation strain, and different concentrations of serine were added to the culture medium. Shake flask fermentation was carried out according to the method of step (6) of Example 1. The OD of the strain was 600 And D-pantothenic acid content see Fig. 9 The results showed that adding 0.1 g / L serine to the culture medium was beneficial to the production of D-PA.

[0106] Example 6 Strengthening the Serine Glycine Cleavage System to Produce 5,10-CH2-THF and Increase D-Pantothenic Acid Content

[0107] In addition to strengthening the de novo synthesis pathway of 5,10-CH2-THF and weakening the degradation pathway, the prior art also reports a method for promoting D-pantothenic acid by strengthening the serine glycine cleavage system pathway to produce 5,10-CH2-THF. In order to verify whether this pathway can promote the production of D-pantothenic acid, the key genes in this pathway, serine synthase (PGDH) serA (nucleotide sequence such as SEQ ID NO.3) and serine hydroxymethyltransferase (SHMT) glyA (nucleotide sequence such as SEQ ID NO.4) were overexpressed by plasmid to enhance the serine glycine cleavage system and thus enhance the supply of cofactor 5,10-CH2-THF. The specific steps are:

[0108] Referring to the method of step (1) of Example 2, plasmids pACYCDuet-serA and pACYCDuet-glyA were constructed, and the constructed plasmids were transformed into strain DPA09, and the constructed strains were named DPA10 and DPA11, respectively.

[0109] Using DPA09 as the control, DPA10 and DPA11 were fermented in shake flasks according to the method of step (6) of Example 1. The OD of the strain was 600 And D-pantothenic acid content see Fig.10 . The results showed that compared with the control DPA09, the yields of strains DPA10 and DPA11 decreased to a certain extent. Considering that serine accumulation may inhibit the growth of the strain, we measured the serine content of the strain, but no serine accumulation was detected. It is speculated that the reason for the decrease in yield may be that the supply of cofactor 5,10-CH2-THF has been strengthened before, and strengthening its supply again will compete with the D-PA synthesis pathway for carbon sources.

[0110] 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. An engineered Escherichia coli strain for synthesizing D-pantothenic acid, characterized in that: Based on the starting strain, at least one of the following improvements was made: (1) Reduce the expression of poxB, a pyruvate dehydrogenase gene, and reduce the accumulation of acetic acid as a byproduct; (2) Expression of panB and panC, the key enzymes in the D-pantothenate biosynthesis process in Escherichia coli; (3) reducing the expression of the formyltetrahydrofolate deformylase gene purU or the 5,10-methylenefolate reductase gene metF; (4) Express the 5,10-methylenetetrahydrofolate dehydrogenase gene folD to increase the supply of 5,10-CH2-THF.

2. The Escherichia coli engineered bacteria according to claim 1, characterized in that The starting strain is Escherichia coli PA104.

3. The Escherichia coli engineered bacteria according to claim 1, characterized in that: Based on E. coli PA104, the pyruvate dehydrogenase gene poxB was knocked out.

4. The engineered Escherichia coli according to any one of claims 1 to 3, characterized in that: pACYCDuet was used as a vector to express ketopantothenate hydroxymethyltransferase panB and pantothenate synthase panC.

5. The engineered Escherichia coli according to any one of claims 1 to 3, characterized in that: Ketopantoate hydroxymethyltransferase panB and pantothenate synthase panC were integrated and expressed at the yjiR site of the genome.

6. The engineered Escherichia coli according to any one of claims 1 to 5, characterized in that: The nucleotide sequence of the gene panBC encoding ketopantoate hydroxymethyltransferase panB and pantothenate synthase panC is shown in SEQ ID NO.

1.

7. The engineered Escherichia coli according to any one of claims 1 to 6, characterized in that: The serine glycine cleavage pathway gene serA or the gene glyA was expressed.

8. The engineered Escherichia coli according to any one of claims 1 to 7, characterized in that: The gene folD is integrated into the genome at the metF site.

9. A method for improving the D-pantothenic acid production capacity of Escherichia coli, characterized in that: Include one or more of (1) to (5): (1) Knockout the pyruvate dehydrogenase gene poxB to reduce the accumulation of byproduct acetic acid; (2) Expression of ketopantoate hydroxymethyltransferase panB and pantothenate synthase panC; (3) knocking out the purU gene, a formyltetrahydrofolate deformylase gene, or the metF gene, a 5,10-methylenefolate reductase gene; (4) expressing the 5,10-methylenetetrahydrofolate dehydrogenase gene folD to increase the supply of 5,10-CH2-THF; (5) Fermentation in a culture medium containing betaine and / or serine.

10. The method according to claim 9, characterized in that The serine glycine cleavage pathway gene serA or gene glyA was also expressed in the E. coli.

11. A method for preparing D-pantothenic acid, characterized in that: The engineered Escherichia coli according to any one of claims 1 to 8 is fermented in a culture medium at 30° C. to 37° C. for at least 24 hours.

12. The method according to claim 11, characterized in that The culture medium contains glucose, yeast powder, peptone, ammonium sulfate and beta-alanine.

13. The method according to claim 11 or 12, characterized in that: The culture medium also contains betaine and / or serine.

14. Use of the engineered Escherichia coli according to any one of claims 1 to 8 in the preparation of products containing D-pantothenic acid or its derivatives.

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