Yeast sphinganine c4-hydroxylase sur2 mutant and use thereof

Abstract

A yeast sphinganine C4-hydroxylase SUR2 mutant and a use thereof. The mutant is obtained by modifying a gene of an enzyme by means of a mutagenesis technique such as error-prone PCR, followed by screening according to a specific modification objective. By using this mutant, ceramides can be efficiently produced. In addition, the fermentation condition is relatively simple during production. The present invention is easy to implement and control in industry, has less byproducts, facilitates subsequent ceramide extraction, and reduces the production cost.

Description

A mutant of yeast sphingosine C4-hydroxylase SUR2 and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese invention patent application with application number 202311597439.2, filing date November 28, 2023, and invention name “A mutant of yeast dihydrosphingosine C4-hydroxylase SUR2 and its application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of genetic engineering technology, and in particular to a mutant of yeast sphingosine C4-hydroxylase SUR2 and its application. Background Art

[0004] Ceramide, a sphingolipid metabolite, is a key component of cell and organelle membranes. Present in all eukaryotic cells, ceramide plays a crucial role in regulating vital processes such as cell differentiation, proliferation, apoptosis, and aging. Ceramide is an intrinsic component of the human body and serves as a strong skin barrier, preventing the invasion of harmful substances and isolating the skin from water loss.

[0005] With the increasing use of skincare ingredients, ceramides for moisturizing and anti-aging are becoming increasingly popular, and the global ceramide market is growing steadily. Currently, ceramide products used in cosmetics are primarily extracted from plants. However, this method is limited by plant growth cycles and environmental factors, resulting in low yields and, at an industrial level, high costs. Chemical synthesis, on the other hand, is complex, produces numerous byproducts, and is inefficient, environmentally unfriendly, making industrial production difficult. Research has shown that Saccharomyces cerevisiae can directly produce ceramides, but the yields are relatively low.

[0006] In view of this, this application is hereby filed.

[0007] Summary of the Invention

[0008] The purpose of the present application is to provide a mutant of yeast dihydrosphingosine C4-hydroxylase SUR2 and its application. The mutant is obtained by modifying the enzyme gene through mutagenesis techniques such as error-prone PCR, and then screening according to specific modification purposes. The mutant can be used to efficiently produce ceramide.

[0009] This application is implemented as follows:

[0010] In a first aspect, the present application provides a mutant of yeast dihydrosphingosine C4-hydroxylase SUR2, which is obtained by mutating the wild-type yeast dihydrosphingosine C4-hydroxylase with the amino acid sequence shown in SEQ ID NO.1, wherein the mutation is any one or a combination of the following (1)-(7):

[0011] (1) mutating the M at position 153 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to I, L or Y;

[0012] (2) mutating the F at position 127 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to Y;

[0013] (3) mutating the 124th amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to an L.

[0014] (4) mutating Y at position 152 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to F;

[0015] (5) mutating the P at position 121 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to I;

[0016] (6) mutating Y at position 123 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to M;

[0017] (7) The M at position 224 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase is mutated to K.

[0018] Yeast sphinganine C4-hydroxylase gene (SUR2) exists in yeast. The present application obtains the above-mentioned SUR2 mutant by mutating wild-type SUR2, and the mutant can be used to synthesize ceramide.

[0019] The amino acid sequence shown in SEQ ID NO.1 is the SUR2 gene from Saccharomyces cerevisiae. Wild-type SUR2 from other sources can be used to synthesize ceramide by undergoing any one or a combination of the above mutation methods (1)-(7).

[0020] In this application, amino acid sequence refers to the type and arrangement of amino acid residues that constitute a protein or polypeptide. Amino acid residues are usually represented by the three-letter method commonly used in the art, and the single-letter method commonly used in the art can also be used to represent amino acid residues. Those skilled in the art should be able to convert the three-letter amino acid sequence with the single-letter amino acid sequence. It should be clear that no matter which method is used in this application, those skilled in the art can understand and convert. For example: alanine, single letter A, three letters are Ala; arginine, single letter R, three letters are Arg; aspartic acid, single letter D, three letters are Asp; cysteine, single letter C, three letters are Cys; glutamine, single letter Q, three letters are Gln; glutamic acid, single letter E, three letters are Glu; histidine, single letter H, three letters are His; isoleucine, single letter I, three letters are Ile; glycine, single letter G, three letters are Gly; asparagine, single letter N, three letters are A sn; leucine, single letter L, three letters Leu; lysine, single letter K, three letters Lys; methionine, single letter M, three letters Met; phenylalanine, single letter F, three letters Phe; proline, single letter P, three letters Pro; serine, single letter S, three letters Ser; threonine, single letter T, three letters Thr; tryptophan, single letter W, three letters Trp; tyrosine, single letter Y, three letters Tyr; valine, single letter V, three letters Val.

[0021] In some embodiments, the SUR2 mutant is mutated in the following manner:

[0022] For example, the amino acid sequence of SEQ ID NO.1 in which the M at position 153 is mutated to I, and the amino acid sequence of the mutant is shown in SEQ ID NO.2; or

[0023] For example, in the amino acid sequence shown in SEQ ID NO.1, the M at position 153 is mutated to I, and the M at position 124 is mutated to L, and the amino acid sequence of the mutant is shown in SEQ ID NO.3; or

[0024] For example, in the amino acid sequence shown in SEQ ID NO.1, the M at position 153 is mutated to L, and the Y at position 152 is mutated to F, and the amino acid sequence of the mutant is shown in SEQ ID NO.4; or

[0025] For example, in the amino acid sequence shown in SEQ ID NO.1, the M at position 153 is mutated to Y, the Y at position 123 is mutated to M, and the M at position 224 is mutated to K, and the amino acid sequence of the mutant is shown in SEQ ID NO.5; or

[0026] For example, the F at position 127 in the amino acid sequence shown in SEQ ID NO.1 is mutated to Y, and the amino acid sequence of the mutant is shown in SEQ ID NO.6; or

[0027] In the amino acid sequence shown in SEQ ID NO.1, F at position 127 is mutated to Y, and P at position 121 is mutated to I. The amino acid sequence of the mutant is shown in SEQ ID NO.7.

[0028] That is, the amino acid mutation of the mutant of the present application is M153I or M153I / M124L or M153L / Y152F or F127Y or F127Y / P121I or M153Y / Y123M / M224K.

[0029] In a second aspect, the present application provides a method for screening the above-mentioned mutants, which comprises: introducing nucleotide mutations into the SUR2 gene in vitro using error-prone PCR technology, obtaining target mutants through multiple rounds of recombination and random mutagenesis, and directed evolution.

[0030] Specifically, the screening method is:

[0031] (1) The error-prone PCR amplification product was purified using a DNA purification kit and ligated with the pESC-URA plasmid digested with restriction endonucleases BamHI and HindIII. The product was then transformed into Saccharomyces cerevisiae competent cells using the lithium acetate method. The cells were spread on SD-URA plates, a solid medium lacking uracil nutrition, and inverted and cultured at 25-30°C for 2-5 days.

[0032] (2) Using the method of step (1), multiple rounds of error-prone PCR are performed using the mutant gene as a template to construct a mutant library.

[0033] (3) Screening of SUR2 mutant library:

[0034] The transformants grown on the SD-URA plate were inoculated into a 96-well deep-well plate containing SD-URA liquid culture medium, cultured at 25-30°C and 180-200 rpm for 18-24 hours, and the bacterial liquid was transferred to a 96-well deep-well plate containing induction culture medium. At the same time, wild-type cerevisiae yeast was inoculated as a control and cultured in a shaker at 25-30°C and 600-800 rpm for 2-3 days. The fermentation liquid after induced expression for 2-3 days was taken, and ceramide was extracted and analyzed for its production. The strain with a ceramide production significantly higher than that of wild-type cerevisiae yeast under the same conditions was selected as the target strain.

[0035] (4) Shake flask rescreening of dominant mutants:

[0036] The high-ceramide-producing Saccharomyces cerevisiae strain was streaked onto an SD-URA plate by dipping a colony from the mother plate and incubating it upside down in a 25-30°C incubator for 2-5 days to grow a single colony. The activated single colony was picked and transferred to SD-URA liquid medium, cultured overnight at 25-30°C and 220-250 rpm for 16-20 hours. The bacterial solution in the SD-URA liquid was inoculated into the fermentation medium until the initial OD 600 =0.1-0.2, culture at 25-30°C, 220-250 rpm for 2-3 days; after the culture, take 20-50 μL of bacterial liquid, dilute 40-100 times with deionized water, and measure the absorbance at a wavelength of 600 nm in a spectrophotometer; at the same time, take 200-500 μL of fermentation liquid for ceramide extraction and measure the ceramide production.

[0037] Six strains with significantly improved ceramide production efficiency were obtained through the above screening method. The pESC-SUR2 expression plasmid was extracted from them using a yeast plasmid extraction kit, the SUR2 nucleotide sequence was determined, and the amino acid sequence of SUR2 was inferred using triplet codons. The amino acid changes and relative ceramide production activities are shown in Table 1. The relative ceramide production activity of the wild-type Saccharomyces cerevisiae control was set as 100%, and the relative ceramide production activities of the engineered bacteria in the SUR2 mutant library were determined based on the analysis results of liquid chromatography detection.

[0038] The directed evolution used in this application is a non-rational design. It refers to the modification of the gene of a specific protein enzyme through error-prone PCR and other mutagenesis techniques. Then, valuable natural enzymes are screened according to the specific modification purpose. Currently, directed evolution technology has been successfully used to improve the catalytic reaction activity of enzymes, improve substrate specificity, enhance thermal stability, and improve enantioselectivity. The improvement in enzyme activity in this application can be represented by the efficiency of ceramide production by the engineered bacteria expressing the mutant SUR2. The improvement in the efficiency of ceramide production by the engineered bacteria expressing the mutant SUR2 represents an increase in the ceramide production activity of the mutant SUR2.

[0039] In a third aspect, the present application provides a nucleic acid molecule encoding the above mutant.

[0040] In a fourth aspect, the present application provides an expression vector containing the above-mentioned nucleic acid molecule.

[0041] In a fifth aspect, the present application provides a recombinant bacterium, which is constructed by introducing the above-mentioned nucleic acid molecule into a host bacterium via a plasmid or integrating it into the host bacterium chromosome through genetic engineering means.

[0042] In the present application, the host bacteria include Saccharomyces cerevisiae.

[0043] In some embodiments, the host strain is Saccharomyces cerevisiae PK2-1D.

[0044] In a sixth aspect, the present application provides a cell catalyst containing the above-mentioned recombinant bacteria.

[0045] In a seventh aspect, the present application provides the use of the above-mentioned mutants, biomaterials, recombinant bacteria or cell catalysts in the production of ceramide.

[0046] In an eighth aspect, the present application provides a method for efficiently synthesizing ceramide, which uses glucose as a substrate and utilizes the whole cells of the above-mentioned recombinant bacteria to transform and synthesize ceramide.

[0047] In some embodiments, the production system for whole-cell transformation includes: 5-15 g / L glucose, 15-25 g / L galactose, 1-10 g / L yeast extract, 5-15 g / L peptone, 10-20 g / L α-cyclodextrin, 0.5-1.0 g / L CaCl2, 0.5-1.0 g / L NH4SO4, 0.5-1.0 g / L KH2PO4, 0.5-1.0 g / L MgSO4, 0.1-0.5 g / L, 0.01-0.05 g / L FeSO4 7H2O, 10-50 mg / L niacin, and 1-20 g / L recombinant bacterial cell amount; the temperature for whole-cell transformation is 25-30°C, and the time is 2-3 days.

[0048] In the present application, when whole cells of recombinant bacteria containing the above mutants are used to transform and synthesize ceramide, the method uses glucose as a substrate, galactose mainly as an inducer, and nicotinic acid is used to provide a precursor for the synthesis of the coenzyme NADPH.

[0049] Beneficial effects of this application:

[0050] The present application constructs a SUR2 mutant for efficient ceramide production through directed evolution. The SUR2 mutant is obtained by directed evolution of the ScSUR2 gene from Saccharomyces cerevisiae using an error-prone PCR method through multiple rounds of recombination and random mutagenesis. The mutant is used to synthesize ceramide with significantly improved yield. Moreover, during production, the fermentation conditions are relatively simple, which is easy to implement and control industrially. There are fewer by-products, which facilitates subsequent ceramide extraction, reduces production costs, and has broad development prospects. DETAILED DESCRIPTION

[0051] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0052] The present application describes SUR2 mutants that efficiently produce ceramide, constructed through directed evolution. These SUR2 mutants were derived from the Saccharomyces cerevisiae ScSUR2 gene (GenBank Accession No. U07171), SEQ ID NO. 1, using error-prone PCR, followed by multiple rounds of recombination and random mutagenesis. The amino acid sequences of these mutants include the following: M153I, M153I / M124L, M153L / Y152F, F127Y, F127Y / P121I, and M153Y / Y123M / M224K. These mutants demonstrated improved ceramide production efficiency, as measured by ceramide yields in engineered bacteria expressing the corresponding SUR2 mutants, compared to control engineered bacteria expressing wild-type ScSUR2.

[0053] The host bacteria used in the examples of the present application is the Saccharomyces cerevisiae PK2-1D strain.

[0054] The culture medium formula involved in this application is as follows:

[0055] SD-URA liquid medium (yeast nitrogen base (YNB) 6.7 g / L; glucose 2 g / L, DO supplement-URA 0.76 g / L, pH 6.0);

[0056] SD-URA solid medium (yeast nitrogen base (YNB) 6.7 g / L; glucose 2 g / L, DO supplement-URA 0.76 g / L, agar powder 2 g / L, pH 6.0);

[0057] Fermentation medium (glucose 10 g / L, galactose 20 g / L, yeast powder 5 g / L, peptone 10 g / L, α-cyclodextrin 15 g / L, CaCl2 1 g / L, NH4SO4 1 g / L, KH2PO4 1 g / L, MgSO4 0.43 g / L, FeSO4 7H2O 0.05 g / L, niacin 50 mg / L).

[0058] The induction medium and fermentation medium formulas in the following examples are the same.

[0059] High-Performance Liquid Chromatography (HPLC-ELSD) Conditions: Qualitative and quantitative analysis of the extracted ceramides was performed using a Shimadzu LC-2050 liquid chromatography system. Products were separated by reversed-phase chromatography on a Shimadzu C8 (3 μm; 4.6 × 250 mm) column using 100% acetonitrile (Solution A) and 0.06% (v / v) trifluoroacetic acid in water (Solution B) as eluents. The sample was diluted with 50% anhydrous ethanol, and the gradient elution program was as follows: 60% Solution A for 2 min, linear elution at 60% / 75% Solution A for 11 min, linear elution at 75% / 93% Solution A for 4 min, elution at 93% Solution A for 3 min, and elution at 60% Solution A for 5 min. The flow rate was 1 mL / min, column temperature was 40°C, injection volume was 10 μL, drift tube temperature was 50°C, drying gas (N2) pressure was 350 kPa, and filtration constant was 10.

[0060] Example 1

[0061] This example is about mutant library construction: This example constructs a SUR2 mutant that efficiently produces ceramide through directed evolution. The SUR2 mutant is obtained by directed evolution of the ScSUR2 gene from Saccharomyces cerevisiae using the error-prone PCR method through multiple rounds of recombination and random mutagenesis.

[0062] In this example, the ScSUR2 gene nucleotide mutation method is to introduce nucleotide mutations into the ScSUR2 gene in vitro using error-prone PCR technology:

[0063] The reaction conditions for error-prone PCR are as follows

[0064] Upstream primer F:

[0065] 5'aggagaaaaaaccccggatccATGAACGTAACATCGAATGCAACT 3';

[0066] Downstream primer R:

[0067] 5'gctagccgcggtaccaagcttTTATTTCTCTTTCTTACTTCATTTTCA 3'

[0068] PCR amplification conditions: 94°C for 3 min; 94°C for 1 min, 59°C for 1 min, 72°C for 2 min, 30 cycles; 72°C for 10 min;

[0069] The error-prone PCR amplification product was purified using a DNA purification kit and ligated with the pESC-URA plasmid digested with the restriction enzymes BamHI and HindIII. The product was then transformed into competent Saccharomyces cerevisiae cells using the lithium acetate method. The cells were then plated on SD-URA plates, a solid medium deficient in uracil, and cultured at 30°C for 2 days.

[0070] Using the same method as above, multiple rounds of error-prone PCR were performed with the mutant gene as a template to construct a mutation library.

[0071] Example 2

[0072] This example is a mutant library screening: transformants grown on SD-URA plates were inoculated with sterile toothpicks into 96-well deep-well plates containing 1 mL of SD-URA liquid medium and cultured in a shaking incubator at 30°C, 800 rpm for 24 h. The bacterial culture was then transferred to a 96-well deep-well plate containing induction medium. Saccharomyces cerevisiae PK2-1D / pESC-ScSUR2-WT was also inoculated as a control and cultured in a shaking incubator at 30°C, 800 rpm for 2 days. After fermentation, 500 μL of the 96-well plate fermentation broth from the 2-day induced expression was collected, ultrasonically disrupted, and ceramide was extracted with petroleum ether. Ceramide production was analyzed using high-performance liquid chromatography coupled with evaporative light scattering (ELSD). Under the same conditions, the control engineered strain PK2-1D / pESC-ScSUR2-WT produced only trace amounts of ceramide. The strain with significantly higher ceramide production than the control engineered strain PK2-1D / pESC-ScSUR2-WT under the same conditions was selected as the target strain.

[0073] Example 3

[0074] This example is a shake flask screening of dominant mutants: a high-ceramide-producing Saccharomyces cerevisiae strain was streaked onto an SD-URA plate using a toothpick to pick a colony from the mother plate, which was then incubated upside down at 30°C for 2 days to grow a single colony; the activated single colony was picked and transferred to 3 mL of SD-URA liquid medium, and cultured overnight at 30°C, 250 rpm for 16 hours. The bacterial solution in the SD-URA liquid was inoculated into 25 mL of induction medium until the initial OD 600 =0.2, cultured at 30°C, 250 rpm for 3 days; after the culture, 50 μL of bacterial solution was taken, diluted 40 times with deionized water, and the absorbance at a wavelength of 600 nm was measured in a spectrophotometer; at the same time, 500 μL of fermentation broth was taken, ultrasonically disrupted, and ceramide was extracted with petroleum ether. The sample was filtered through a 0.22 μm filter membrane, and the ceramide yield was determined by HPLC-ELSD.

[0075] Based on the mutant library constructed by the error-prone PCR method, screening was performed to obtain 6 strains with significantly improved ceramide production efficiency. The pESC-SUR2 expression plasmid was extracted using a yeast plasmid extraction kit, and the SUR2 nucleotide sequence was determined. The amino acid sequence of SUR2 was deduced using triplet codons. The amino acid changes and relative ceramide production activities are shown in Table 1. The relative ceramide production activity of the control engineered strain PK2-1D / pESC-ScSUR2-WT was set as 100%, and the relative ceramide production activities of the engineered strains in the SUR2 mutant library were determined based on the analysis results of liquid chromatography-mass spectrometry.

[0076] Table 1 Sequencing results of high-yielding ceramide mutants SUR2 1-6 and their phytoceramide yields

[0077] Note: The yield of phytoceramide in Table 1 is the total amount of ceramide 3 and ceramide 3B, wherein the mass ratio of ceramide 3 to ceramide 3B is 3:1.

[0078] As can be seen from Table 1, the mutants obtained in this application contain amino acid mutations M153I, M153I / M124L, M153L / Y152F, F127Y, F127Y / P121I, and M153Y / Y123M / M224K. The mutants produce phytoceramide at a yield 1.64 to 3.45 times that of the wild type.

[0079] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application. Industrial Applicability

[0080] The mutant of yeast dihydrosphingosine C4-hydroxylase SUR2 provided in the present application is obtained by modifying the enzyme gene through mutagenesis techniques such as error-prone PCR, and then screening according to specific modification purposes. The mutant can be used to efficiently produce ceramide.

Claims

1. A mutant of yeast sphingosine C4-hydroxylase SUR2, characterized in that: The mutant is obtained by mutating the wild-type yeast dihydrosphingosine C4-hydroxylase whose amino acid sequence is shown in SEQ ID NO.1, wherein the mutation is any one or a combination of the following (1)-(7): (1) mutating the M at position 153 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to I, L or Y; (2) mutating the F at position 127 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to Y; (3) mutating the 124th M in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to L; (4) mutating the Y at position 152 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to F; (5) mutating the P at position 121 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to I; (6) mutating the Y at position 123 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase to M; (7) The M at position 224 in the amino acid sequence of the wild-type yeast dihydrosphingosine C4-hydroxylase is mutated to K.

2. The mutant according to claim 1, characterized in that The mutation mode of the mutant is: The M at position 153 in the amino acid sequence shown in SEQ ID NO.1 is mutated to I; or For example, in the amino acid sequence shown in SEQ ID NO.1, the M at position 153 is mutated to I, and the M at position 124 is mutated to L; or For example, in the amino acid sequence shown in SEQ ID NO.1, the M at position 153 is mutated to L, and the Y at position 152 is mutated to F; or In the amino acid sequence shown in SEQ ID NO.1, the M at position 153 is mutated to Y, the Y at position 123 is mutated to M, and the M at position 224 is mutated to K; or The F at position 127 in the amino acid sequence shown in SEQ ID NO.1 is mutated to Y; or For example, in the amino acid sequence shown in SEQ ID NO.1, the F at position 127 is mutated to Y, and the P at position 121 is mutated to I.

3. The method for screening mutants according to claim 1 or 2, characterized in that: The error-prone PCR technique was used to introduce nucleotide mutations into the wild-type yeast dihydrosphingosine C4-hydroxylase gene in vitro, and the target mutants were obtained through recombination and random mutagenesis and directed evolution.

4. A nucleic acid molecule encoding the mutant according to claim 1 or 2.

5. An expression vector containing the nucleic acid molecule according to claim 4.

6. A recombinant bacterium, characterized in that: The recombinant bacteria are constructed by introducing the nucleic acid molecule of claim 4 into a host bacteria via a plasmid or integrating it into the chromosome of the host bacteria via genetic engineering; The host bacteria include Saccharomyces cerevisiae.

7. A cell catalyst comprising the recombinant bacteria according to claim 6.

8. Use of the mutant according to claim 1 or 2, the expression vector according to claim 5, the recombinant bacteria according to claim 6, or the cell catalyst according to claim 7 in the production of ceramide.

9. A method for efficiently synthesizing ceramide, characterized in that: Using glucose as a substrate, ceramide is transformed and synthesized using the whole cell of the recombinant bacteria according to claim 6.

10. The method for efficiently synthesizing ceramide according to claim 9, characterized in that: The whole cell conversion production system includes: glucose 5-15 g / L, galactose 15-25 g / L, yeast powder 1-10 g / L, peptone 5-15 g / L, α-cyclodextrin 10-20 g / L, CaCl2 0.5-1.0 g / L, NH4SO4 0.5-1.0 g / L, KH2PO4 0.5-1.0 g / L, MgSO4 0.1-0.5 g / L, FeSO47H2O 0.01-0.05 g / L, nicotinic acid 10-50 mg / L, and recombinant bacterial cell amount 1-20 g / L; The temperature for the whole cell transformation is 25-30°C and the time is 2-3 days.

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