Application of transporter yaaJ in increasing yield of strain D-pantothenic acid

Abstract

The invention discloses an application of a transporter yaaJ in increasing the yield of strain D-pantothenic acid. The amino acid sequence of the transporter yaaJ is as shown in SEQ ID NO: 2. The transporter yaaJ can significantly improve the D-pantothenic acid fermentation yield of a recombinant strain, and has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to the application of a transport protein yaaJ in improving the D-pantothenic acid yield of a strain. Background Art

[0002] D-Pantothenic acid (DPA), often called D-calcium pantothenate, is a water-soluble B vitamin, also known as vitamin B5, 3-pyridinecarboxylic acid amide. It is widely found in animals and plants. It participates in the metabolism of fat degradation, citric acid cycle, antibody synthesis and fatty acid degradation in the human body, playing an important physiological function. Therefore, it is widely used in medicine, health products, cosmetics, food, feed and other fields.

[0003] At present, there are two industrial production processes for calcium D-pantothenate, namely, full chemical synthesis and a combination of chemical synthesis and enzyme catalysis.

[0004] Both processes use the intermediate DL-pantolactone, which is mostly split by chemical methods. Chiral splitting is expensive and uses toxic cyanide, which causes environmental pollution and toxicity. China's chemical industry has increasingly stringent environmental protection requirements, and has more environmental protection requirements for cyanide, which is not conducive to the expansion of the industry.

[0005] In recent years, many researchers at home and abroad have been studying the production of calcium pantothenate by fermentation using genetically engineered bacteria. In our patent document with publication number CN118165902A, we disclosed an E. coli fermentation strain CGMCC No.29714 (number Ecoli W21), whose D-calcium pantothenate fermentation yield can reach 49.95g / L. However, compared with the chemical method, the cost of producing D-pantothenic acid by fermentation using glucose and β-alanine is still very high, and the D-calcium pantothenate fermentation level needs to be further improved. Summary of the invention

[0006] In our ongoing research on the production of D-pantothenate calcium by fermentation of Escherichia coli, we systematically investigated the regulatory factors / transporters that may positively regulate D-pantothenate synthesis genes based on the biosynthetic pathway of D-pantothenate calcium, and found that the transporter yaaJ (GeneBank No. 944745) can promote the D-pantothenate calcium fermentation level of Escherichia coli engineered bacteria including Ecoli W21 (CGMCC No. 29714) disclosed in the document CN118165902A, suggesting that the transporter yaaJ may be widely used in the improvement of D-pantothenate production bacteria. Specifically, the present invention includes the following technical solutions.

[0007] The first aspect of the present invention provides the use of the transport protein yaaJ in improving the D-pantothenic acid production of a strain.

[0008] The above application can be a method for increasing the D-pantothenic acid production of a strain, comprising the following steps: causing the D-pantothenic acid producing strain to overexpress the transport protein yaaJ (GeneBank No. 944745), wherein the amino acid sequence of the transport protein yaaJ is SEQ ID NO: 2.

[0009] MPDFFSFINSVLWGSVMIYLLFGAGCWFTFRTGFVQFRYIRQFGKSLKNSIHPQPGGLTSFQSLCTSLAARVGSGNLAGVALAITAGGPGAVFWMWVAAFIGMATSFAECSLAQLYKERD VNGQFRGGPAWYMARGLGMRWMGVLFAVFLLIAYGIIFSGVQANAVARALSFSFDFPPLVTGIILAVFTLLAITRGLHGVARLMQGFVPLMAIIWVLTSLVICVMNIGQLPHVIWSIFES AFGWQEAAGGAAGYTLSQAITNGFQRSMFSNEAGMGSTPNAAAAAASWPPHPAAQGIVQMIGIFIDTLVICTASAMLILLAGNGTTYMPLEGIQLIQKAMRVLMGSWGAEFVTLVVILFA FSSIVANYIYAENNLFFLRLNNPKAIWCLRICTFATVIGGTLLSLPLMWQLADIIMACMAITNLTAILLLSPVVHTIASDYLRQRKLGVRPVFDPLRYPDIGRQLSPDAWDDVSQE(SEQ ID NO:2).

[0010] Preferably, the D-pantothenic acid producing bacteria are engineered Escherichia coli bacteria.

[0011] Optionally, the host bacteria of the Escherichia coli engineered bacteria is the strain Ecoli W21 reported in the patent document with publication number CN118165902A, namely CGMCC No.29714.

[0012] In one embodiment, the nucleotide sequence of the gene encoding the above-mentioned transport protein yaaJ (GeneBank No. 944745) is SEQ ID NO:1.

[0013] In the above method, the Escherichia coli engineered bacteria overexpressing the transport protein yaaJ (GeneBank No. 944745) can be implemented in the following manner:

[0014] A. inserting a strong promoter upstream of the transporter protein yaaJ in the genome of the D-pantothenic acid producing host bacteria;

[0015] B. integrating one or more, preferably two or more, three or more, four or more, six or more, or eight or more copies of the gene encoding the transporter yaaJ into the genome of the D-pantothenic acid producing host bacteria; and / or

[0016] C. The gene encoding the transporter protein yaaJ is expressed in the form of a plasmid in the genome of the D-pantothenic acid producing host bacteria.

[0017] Optionally, the above step A or B is achieved by transforming a recombinant plasmid containing the transport protein yaaJ encoding gene into a host bacterium, and preferably the plasmid vector is pTrc99a. The recombinant plasmid is, for example, pTrc99a-yaaJ herein.

[0018] In one embodiment, the method of inserting the strong promoter in the above step A and the method of integrating the transport protein yaaJ encoding gene in the genome in step B are selected from plasmid transformation and gene editing technology.

[0019] After the recombinant plasmid is transformed into a host bacterial cell, the transport protein yaaJ encoding gene can be integrated into a multi-copy sequence site or multiple sites on the chromosome.

[0020] The above-mentioned gene editing technology can be selected from the following groups: homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.

[0021] The second aspect of the present invention provides a recombinant plasmid (or recombinant vector) for constructing the above-mentioned Escherichia coli engineered bacteria that overexpress the transport protein yaaJ. The plasmid vector is a plasmid suitable for expressing foreign genes in bacteria such as Escherichia coli, and contains the transport protein yaaJ encoding gene.

[0022] The plasmid vector / backbone of the recombinant plasmid is pTrc99a or selected from the PET series, such as the vector is pET22b, pET24a, pET28a, etc., but is not limited thereto.

[0023] Preferably, the recombinant plasmid is pTrc99a-yaaJ herein, and its nucleotide sequence is shown in SEQ ID NO:3.

[0024] The third aspect of the present invention provides an engineered Escherichia coli that overexpresses the above-mentioned transport protein yaaJ, wherein the transport protein yaaJ encoding gene as described above is integrated into its genome; or a transformant in which the recombinant plasmid as described above is transformed in a host bacterium, and the host bacterium itself is a D-pantothenic acid producing bacterium, such as Escherichia coli with a preservation number of CGMCC No. 29714.

[0025] The fourth aspect of the present invention provides the use of the above-mentioned engineered Escherichia coli in the production of D-pantothenic acid by fermentation.

[0026] The present invention discovered that the transporter protein yaaJ (GeneBank No. 944745) derived from Escherichia coli, such as Escherichia coli MG1655, has a positive regulatory effect on the synthesis of D-pantothenic acid by the strain. Experiments have shown that overexpression of the transporter protein in D-pantothenic acid-producing bacteria can effectively increase the yield of D-pantothenic acid, and the fermentation level can be increased by 20% compared with the original, which has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a structural map of the recombinant plasmid pTrc99a-yaaJ used to express the transporter protein yaaJ (GeneBank No. 944745).

[0028] Figure 2 It is a comparative bar chart examining how some regulatory factors / transporters promote the improvement of fermentation levels of D-pantothenic acid producing bacteria.

[0029] Figure 3 It is the growth curve of fermentation of two D-pantothenic acid producing strains Ecoli W21 and Ecoli W21-yaaJ.

[0030] Figure 4 This is the HPLC spectrum of the product D-calcium pantothenate in the fermentation broth of the engineered bacteria Ecoli W21-yaaJ. DETAILED DESCRIPTION

[0031] In view of the various studies on the use of transporters to improve the efficiency of intracellular transport, in the screening of regulatory factors / transporters with the potential to enhance the biosynthesis of D-pantothenate calcium, we investigated 75 transporters from Escherichia coli based on bioinformatics data, first screened by well plate screening, then by shake flask screening, and finally by fermentation tank comparison verification, and finally screened out the transporter yaaJ (GeneBank No. 944745), and applied it to the host strain Ecoli W21 (deposit No. CGMCC No. 29714) reported in CN118165902A for overexpression. The engineered strain Ecoli W21-yaaJ overexpressing the YaaJ gene was fermented in a fermentation tank, and the yield of D-pantothenate calcium in the fermentation broth reached 60.62 g / L, which was 20.1% higher than that of the control group (CK), confirming the promoting effect of the transporter yaaJ on increasing the yield of D-pantothenate calcium.

[0032] In this article, for the sake of simplicity of description, a certain protein, such as transport protein yaaJ (GeneBank No. 944745), is sometimes mixed with its coding gene (DNA) yaaJ. Those skilled in the art should understand that they represent different substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context. For example, for transport protein yaaJ, when used to describe the function or category of the protein, it refers to the protein; when described as a gene, it refers to the gene encoding the protein.

[0033] The terms "Escherichia coli (genetic) engineered bacteria", "engineered bacteria (strain)" and "recombinant bacteria (strain)" herein have the same meaning and can be used interchangeably. Similarly, the terms "recombinant vector" and "recombinant plasmid" have the same meaning and can be used interchangeably.

[0034] Correspondingly, for the convenience of description, the starting strain that has not overexpressed yaaJ, such as Ecoli W21, CGMCC No. 29714, can be referred to as the "host strain" or "control strain".

[0035] Through genetic engineering modification, such as integrating a single copy or multiple copies of the transporter gene yaaJ into the genome of the D-pantothenic acid producing bacteria and / or placing it under the regulation of a strong promoter to promote the overexpression of the transporter protein yaaJ, the D-pantothenic acid production can be effectively increased.

[0036] The terms "(D-pantothenic acid production) increase", "enhance" or "enhance" can mean an increase of at least 10% compared to a reference level (such as a starting strain / host bacterium), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or any increase between 10%-100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level.

[0037] In the description of the technical solutions of the present invention, the terms used, such as "A and / or B", "A and / or B", "and / or", "and / or", are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0038] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0039] Example

[0040] The examples herein involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0041] For experimental methods without specific conditions specified in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0042] Materials and methods

[0043] The gene and primer synthesis and sequencing in the examples were all completed by Suzhou Genewise Biotechnology Co., Ltd.

[0044] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0045] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0046] LB medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, pH 7.2. (LB solid medium plus 20g / L agar powder.)

[0047] TB medium: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K2HPO4.3H2O, 2.31g / L KH2PO4, 5g / L glycerol, pH 7.0-7.5. (TB solid medium plus 20g / L agar powder.)

[0048] Well plate fermentation medium: corn steep liquor 20 g / L, yeast extract 10 g / L, ammonium sulfate 10 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 4 g / L, glucose 30 g / L, Beta-alanine 5 g / L, PH 7.0.

[0049] Shake flask fermentation medium: calcium carbonate 5g / L, corn steep liquor 20g / L, yeast extract 10g / L, ammonium sulfate 10g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 2g / L, dipotassium hydrogen phosphate 4g / L, glucose 30g / L, Beta-alanine 5g / L.

[0050] Fermentation medium in fermenter: calcium carbonate 1g / L, corn steep liquor 20g / L, yeast extract 10g / L, ammonium sulfate 10g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 2g / L, dipotassium hydrogen phosphate 5g / L, glucose 30g / L, Beta-alanine 5g / L, salt solution 1ml / L. The composition of the salt solution is as follows: FeSO4·7H2O 10g / L, CuCl2 0.2g / L, MnSO4·5H2O 1g / L, CaCl2·2H2O 0.2g / L, (NH4)6Mo7O24 2g / L. Feed medium 1: glucose 600g / L, feed medium 2: Beta-alanine 100g / L.

[0051] HPLC detection conditions of product D-calcium pantothenate:

[0052] Agilent 1260 liquid chromatograph; chromatographic column AMINEX HPX-87H 300mm×7.8mm; detection wavelength 260nm; mobile phase: 5mM sulfuric acid; temperature 40℃; flow rate 0.5ml / min.

[0053] Example 1: Construction of a transporter gene expression library

[0054] Based on bioinformatics data, for the regulatory factors and transporters in the biosynthetic pathway of D-calcium pantothenate, 75 transporters were screened out by query and comparison, primers were designed for each, and they were constructed into the low copy vector pTrc99a (the vector pTrc99a is the same as the commercial vector pTrc99a disclosed in patent document CN118165902A) by seamless cloning to obtain 75 transporter expression plasmids pTR01-pTR75. Taking pTR39 (i.e., pTrc99a-yaaJ) as an example, the construction method is briefly described as follows.

[0055] Using the Escherichia coli MG1655 genome as a template and yaaJ-5 and yaaJ-3 as primers, PCR amplified a yaaJ fragment of about 1.5 kb.

[0056] Primer yaaJ-5: tttcacacaggaaacagaccATGCCAGATTTTTTTCTCCTTTATTAAC,

[0057] Primer yaaJ-3: ccgccaaaacagccaagcttTTACTCCTGCGAAACATCATC.

[0058] Using Takara Biotechnology (Beijing) Co., Ltd. (Takara China) PCR amplification was performed using the GXL DNA polymerase kit. Reaction system: 10 μL of 5× Buffer, 10 mM dNTP Mixture, 1 μL each of forward primer and reverse primer, 0.5 μL of template, 2 μL of DNA polymerase, and ddH2O was added to make up to 50 μL.

[0059] PCR amplification conditions: denaturation at 95°C for 5 min; (denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1.5 min) for a total of 10 cycles; (denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1.5 min) for a total of 20 cycles; extension at 72°C for 7 min.

[0060] Using plasmid pTrc99A as template and trc99aA-5 and trc99A-3 as primers, PCR amplified the TRC99A backbone of about 4.1 kb.

[0061] Primer trc99aA-5: aagcttggctgttttggcgg,

[0062] Primer trc99a-3: ggtctgtttcctgtgtgaaattg.

[0063] After the TRC99A backbone and yaaJ fragment were recombined, the recombinant plasmid pTrc99a-yaaJ was constructed, and its structure map is shown in Figure 1 As shown, the nucleotide sequence is SEQ ID NO:3.

[0064] The recombinant plasmid pTrc99a-yaaJ was transformed into DH5a chemically competent, and the bacteria were spread on LB plates containing 100μg / ml Amp resistance, cultured overnight at 37°C, and the clones grown were identified by colony PCR using primers trc99aA-5 / yaaJ-3. The positive clone was 1431bp, and the clone with correct sequencing was recorded as pTrc99a-trc-yaaJ / DH5a strain. After bacterial culture, plasmid extraction was performed to obtain plasmid pTrc99a-yaaJ, i.e. pTR39. The yaaJ gene was sequenced and its nucleotide sequence was SEQ ID NO:1, and the corresponding amino acid sequence was SEQ ID NO:2. The plasmid pTrc99a-yaaJ was transformed into the host bacteria Ecoli W21 (deposit number CGMCC No.29714) by electrotransformation to obtain the recombinant Escherichia coli engineered bacteria Ecoli W21-yaaJ, and well plate and shake flask fermentation were performed.

[0065] According to the above construction method, the relevant expression plasmids of the transporter library were obtained and transformed into the host Ecoli W21 to obtain recombinant strains overexpressing different transporters. These strains were screened together with the control strain Ecoli W21 (CK) in well plates and rescreened in shake flasks to screen out transporters that help increase the production of D-pantothenate calcium.

[0066] Example 2: Initial screening of transporter expression library well plates

[0067] The clones of recombinant bacteria containing different transporters and CK (control bacteria Ecoli W21) were fermented in 48-well plates.

[0068] Inoculate 1200 μl of antibiotic-free LB liquid medium into each 48-well plate, then inoculate the clones into the well plate and culture at 37°C, 280 rpm for 30 h as seed solution;

[0069] 200 μl of bacterial solution was transferred from each well to a well plate containing 1200 μl of fermentation medium, and then cultured at 37°C, 280 rpm for 72 hours, and 100 μl of 20% glucose was added every 24 hours; after 72 hours, samples were diluted 10 times for HPLC detection of the product. Four clones were selected for parallel fermentation of each strain, and the results were averaged. The preliminary screening results showed that after 58.7% (44 / 75) of the transporters were overexpressed, the production of D-pantothenic acid was significantly reduced, and only 8% (6 / 75) of the transporters were overexpressed, and the production of D-pantothenic acid was increased, with an increase of 4%-30%, see Figure 2 .

[0070] The initial screening results are shown in the following table:

[0071] Group Transporter D-Calcium pantothenate (g / L) G1-0 CK (control) 3.22 G1-1 d 2.53 G1-2 oeLh 1.53 G1-3 oeLh 3.25 G1-4 Yj Y 0.84 G1-5 YjY 2.00 G1-6 kef 1.50 G1-7 yidE 1.56 G1-8 chac2 1.69 G1-9 mxDV 0.85 G1-10 eO 0.98

[0072] Group Transporter D-Calcium pantothenate (g / L) G2-0 CK (control) 2.95 G2-1 ydP 1.86 G2-2 PcqI 1.35 G2-3 foeA 2.06 G2-4 hydN 0.93 G2-5 ikB 0.16 G2-6 FWf 1.08 G2-7 PqA 1.21 G2-8 ybeX 2.57 G2-9 tolC 0.43 G2-10 wxya 2.44

[0073]

[0074]

[0075] Group Transporter D-Calcium pantothenate (g / L) G4-0 CK (control) 3.16 G4-1 yncD 2.85 G4-2 yd 3.64 G4-3 J 2.22 G4-4 bD 1.09 G4-5 yf 0.55 G4-6 yt 1.36 G4-7 sU 2.94 G4-8 ssB 2.45 G4-9 yaj 4.12 G4-10 Yj 1.56

[0076]

[0077]

[0078] Group Transporter D-Calcium pantothenate (g / L) G6-0 CK (control) 3.65 G6-1 metI 1.33 G6-2 P 1.56 G6-3 oeLh 2.54 G6-4 hisP 3.05 G6-5 pheP 2.99 G6-6 QUR 0.54 G6-7 codB 1.27 G6-8 alsB 0.55 G6-9 Yj Y 0.64 G6-10 ansP 0.81

[0079] Group Transporter D-Calcium pantothenate (g / L) G7-0 CK (control) 2.98 G7-1 Yj Y 2.61 G7-2 Yj 1.32 G7-3 x 0.35 G7-4 y 0.84 G7-5 Yj Y 3.87 G7-6 mnT 1.56 G7-7 Y 2.53 G7-8 Yj Y 0.87 G7-9 nupC 1.11 G7-10 cy 2.81

[0080] Group Transporter D-Calcium pantothenate (g / L) G8-0 CK (control) 2.84 G8-1 Yj 1.66 G8-2 Yj Y 2.15 G8-3 YbT 1.65 G8-4 betT 0.84 G8-5 yd 1.26

[0081] Overexpression of six transporters, including yaaJ, ydcV, thiP, proW, ydcT and cycA, could promote the production of D-pantothenic acid, among which yaaJ had the best promoting effect.

[0082] Example 3: Transporter expression library shake flask rescreening

[0083] Six recombinant strains with different transport proteins and clones of CK (control strain EcoliW21) with improved D-pantothenic acid production were screened in well plates and fermented in shake flasks. Three clones of each strain were selected for parallel fermentation, and the results were averaged.

[0084] The clones that need to be rescreened were inoculated into a test tube containing 5 ml of LB medium and cultured at 37°C, 280 rpm for 20 h as seed solution.

[0085] The inoculum was transferred to a 50 ml shake flask fermentation medium at a 1% v / v inoculum, and then cultured at 37°C, 280 rpm for 72 h, with 4 ml of 20% glucose added every 24 h; after 72 h, a sample was taken and diluted 10 times for HPLC detection of the product.

[0086] The six strains screened initially showed significantly higher yields than the control, including Ecoli W21-ydcV, Ecoli W21-yaaJ, and EcoliW21-ydcT. Among them, Ecoli W21-yaaJ showed a stable high yield of 7.08 g / L, which was 29.2% higher than the control. The results of the rescreening were as follows: Figure 2 shown.

[0087] Example 4: Fermentation investigation in a 5L fermenter

[0088] Ecoli W21 and Ecoli W21-yaaJ strains were selected for fermentation comparison experiments in 5L fermentation tanks.

[0089] The single clone was inoculated into 5 ml LB liquid medium and cultured at 37°C for 20-24 h. Then, it was transferred to LB medium at a 1% v / v inoculum and cultured at 37°C until OD600 was about 1.0. Then, 100 ml of the bacterial solution was transferred to 2 L fermentation medium (glucose 20 g / L, corn steep liquor 10 g / L, ammonium sulfate 20 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate, 10 g / L, β-alanine 2 g / L, yeast extract 20 g / L, thiamine 5 mg / L, vitamin B12 2 mg / L, defoamer 0.7 g / L, trace elements 1 ml / L (trace element formula: CaCl2 0.50 g / L, FeCl3·6H2O 16.70 g / L, ZnSO4·7H2O0.18 g / L, CuSO4·5H2O0.16 g / L, MnSO4·H2O 0.15g / L, CoCl2·6H2O 0.18g / L)) in a 5L fermenter at 30°C, 400-700rpm, aeration 1.8vvm, dissolved oxygen 20-30%, continue culturing, add feed medium 1 and feed medium 2 in the middle flow, culture for 70h, sample the liquid phase to detect the content of D-pantothenate calcium, see Figure 3 and Figure 4 .

[0090] After 70 hours of fermentation, the yield results are shown in the following table:

[0091] Bacteria No. D-calcium pantothenate production (g / L) Sugar conversion rate (%) Ecoli W21 50.47 12.3 Ecoli W21-yaaJ 60.62 15.6

[0092] From the above table and Figure 3 It can be seen that the yield of the control strain Ecoli W21 is 50.47 g / L. After overexpressing the transporter protein yaaJ, the yield of D-pantothenate calcium increased by 20.1%, which has very good prospects for industrial development and application.

[0093] It should be noted that the above is a description of a specific embodiment of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments and features in the embodiments herein may be combined with each other at will.

Claims

1. Application of the transporter protein yaaJ in increasing the D-pantothenic acid production of strains.

2. The use according to claim 1, which is a method for increasing the D-pantothenic acid production of a strain, characterized in that: The method comprises the following steps: causing the D-pantothenic acid producing bacteria to over-express the transport protein yaaJ (GeneBank No. 944745), wherein the amino acid sequence of the transport protein yaaJ is SEQ ID NO:

2.

3. The use according to claim 1, characterized in that The D-pantothenic acid producing bacteria are engineered Escherichia coli bacteria.

4. The use according to claim 1, characterized in that The nucleotide sequence of the gene encoding the transporter protein yaaJ is SEQ ID NO:

1.

5. The use according to claim 1, characterized in that Overexpression of the transporter protein yaaJ was carried out in the following manner: A. inserting a strong promoter upstream of the transporter protein yaaJ in the genome of the D-pantothenic acid producing host bacteria; B. integrating more than one copy of the gene encoding the transporter protein yaaJ into the genome of the D-pantothenic acid producing host bacteria; and / or C. The gene encoding the transporter protein yaaJ is expressed in the form of a plasmid in the genome of the D-pantothenic acid producing host bacteria.

6. The use according to claim 5, characterized in that Step B is achieved by transforming the recombinant plasmid containing the transporter protein yaaJ encoding gene into the host bacteria.

7. A recombinant plasmid as claimed in claim 6, characterized in that The plasmid vector is a plasmid suitable for expressing foreign genes in bacteria such as Escherichia coli, and contains the gene encoding the transporter protein yaaJ.

8. The recombinant plasmid according to claim 7, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

3.

9. An engineered Escherichia coli bacterium overexpressing the transport protein yaaJ as claimed in claim 1, characterized in that: The coding gene of the transport protein yaaJ is integrated into the genome; or the transformant of the recombinant plasmid as claimed in claim 7 or 8 is transformed into the host bacteria, and the host bacteria is a D-pantothenic acid producing bacteria.

10. Use of the engineered Escherichia coli as claimed in claim 9 in the production of D-pantothenic acid by fermentation.

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