Method for producing D-panthenol through fermentation

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a method for producing D-panthenol through fermentation. The D-panthenol is produced by fermenting the modified DFC01 strain, a multi-stage control fermentation process suitable for large-scale fermentation production of the D-panthenol is designed on the basis of the DFC01 strain, the concentration of glucose in fermentation liquor is controlled within the range of 0.5-2 g / L in the fermentation medium period of 12-24 hours, and the concentration of glucose in the fermentation liquor is controlled within the range of 0.5-2 g / L in the fermentation later period of 24 hours to the fermentation end period. The concentration of dissolved oxygen in the fermentation liquor is controlled to be in a range of 28-40%, so that the yield of D-panthenol and the conversion rate of sugar acid are improved while the accumulation of byproducts in the fermentation process is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a method for producing D-panthenol by fermentation. Background Art

[0002] Panthenol is also known as provitamin B5, with the chemical formula C9H 19 NO4, relative molecular weight 205.25, the viscosity of its solution is relatively high, the viscosity at 40 ℃ is above 10000 centipoise. Panthenol can be divided into three forms: racemic isomer (DL-type), right-hand isomer (D-type) and left-hand isomer (L-type), among which only D-panthenol has biological activity. D-panthenol is the precursor of vitamin B5 (pantothenic acid) and is also an equivalent of D-pantothenic acid. It is widely used in medicine, food, liquid preparations, and cosmetics industries. At present, it is of great practical significance to find a method for efficiently synthesizing D-panthenol.

[0003] D-panthenol is widely present in microorganisms, plants, and animals. It is produced by D-pantolactone and 3-aminopropanol under the action of pantolactone hydrolase, and the gene encoding pantolactone hydrolase in organisms is mostly panC gene. D-panthenol can generate D-pantothenic acid (vitamin B5) through a series of biochemical reactions, and then enters the TCA cycle as an important precursor for the synthesis of acyl carrier protein (ACP) and coenzyme A (CoA).

[0004] The existing methods for preparing D-panthenol are mainly chemical synthesis and microbial enzyme methods. The chemical synthesis method mostly uses D-pantolactone and 3-aminopropanol as raw materials to synthesize D-panthenol through acylation reaction in the presence or absence of solvent. The microbial enzyme method mostly uses microorganisms to overexpress the corresponding enzymes, which are separated and purified for the production of D-panthenol.

[0005] In the prior art, a Chinese patent application with publication number CN101851171A discloses a method for preparing D-panthenol without solvent. In this patent, D-pantolactone and 3-aminopropanol are reacted without solvent to obtain high-purity D-panthenol. Although this method omits the step of separating the solvent and the optical purity of the obtained D-panthenol is high, this method has high requirements for raw materials, and the viscosity of the D-panthenol obtained without solvent is high, which causes the direct reaction of the raw materials to gradually slow down, resulting in a low yield of the product. In addition, its high viscosity is not conducive to subsequent operation and utilization, and the actual utilization rate of the obtained product is low.

[0006] Preparation of D-panthenol by microbial enzyme method is mentioned in patent CN1173042C, which uses a strain of Fusarium moniliforme that produces high D-pantolactone hydrolase for fermentation to obtain D-pantolactone hydrolase, and uses the enzyme produced by the strain to hydrolyze substrates D-pantolactone and 3-aminopropanol to obtain the target product D-panthenol. This method overcomes the shortcomings of high cost, pollution, toxicity and low optical purity of D-panthenol produced by chemical methods. However, this method also has many disadvantages. First, the enzyme produced by the microorganism needs to be separated and purified to a certain extent to catalyze the reaction of D-pantolactone and 3-aminopropanol to produce panthenol, which greatly increases the operating cost and production efficiency; second, the stability of the enzyme is poor, and the catalytic activity of the enzyme can only be best exerted under the most suitable conditions, and the enzyme is easily inactivated, which is not conducive to large-scale industrial production and application. Summary of the invention

[0007] In order to solve the above technical problem of efficiently synthesizing D-panthenol, the present invention provides a method for producing D-panthenol by fermentation.

[0008] The specific technical scheme of the present invention is: The present invention provides a method for producing D-panthenol by fermentation, comprising the following steps: Step S1: overexpressing the panC gene in the bottom plate bacteria to obtain the DFC01 strain; Step S2: inoculating and culturing the DFC01 strain to obtain a seed solution; Step S3: inoculating the seed liquid into a fermentation medium for fermentation culture; The fermentation culture is controlled under the following conditions: During 12 to 24 hours, a feed medium is fed, and the feed rate is controlled so that the concentration of glucose in the fermentation broth is in the range of 0.5 to 2 g / L; During the period from 24 hours to the end of the fermentation, the second type of feed medium is fed, and the dissolved oxygen concentration in the fermentation liquid is kept in the range of 28% to 40% by controlling the stirring speed.

[0009] In view of the problem that the optical purity of D-panthenol in the existing process is not high and it is difficult to achieve large-scale industrial application, the present application produces D-panthenol by fermenting the modified strain DFC01, and based on the DFC01 strain, designs a multi-stage controlled fermentation process suitable for large-scale fermentation production of D-panthenol. During the middle fermentation period of 12 to 24 hours, the glucose concentration in the fermentation broth is controlled to be in the range of 0.5 to 2 g / L, and during the late fermentation period of 24 hours to the end of fermentation, the dissolved oxygen concentration in the fermentation broth is controlled to be in the range of 28% to 40%, so as to reduce the accumulation of by-products during the fermentation process while increasing the D-panthenol yield and sugar-acid conversion rate.

[0010] As a preferred embodiment of the above method, a feed medium is fed during 12 to 24 hours, and the feed rate is controlled so that the concentration of glucose in the fermentation broth is in the range of 0.8 to 1.5 g / L.

[0011] As a preferred embodiment of the above method, during the period from 24 hours to the end of fermentation, the second type of feed medium is fed, and the dissolved oxygen concentration in the fermentation broth is kept in the range of 32% to 35% by controlling the stirring speed.

[0012] As a preferred embodiment of the above method, the stirring speed is controlled to be 400 to 700 r / min.

[0013] As a preferred embodiment of the above method, a type of feed culture medium is composed of: glucose 300 g / L, magnesium sulfate 8 g / L, potassium dihydrogen phosphate 14 g / L, ammonium sulfate 10 g / L, vitamin B12 0.002 g / L, vitamin B1 0.005 g / L, isoleucine 0.08 g / L, saline solution 2 mL / L, and IPTG 1 mol / L.

[0014] As a preferred embodiment of the above method, the composition of the second type of feed culture medium is: 200 g / L glucose, 6 g / L magnesium sulfate, 7 g / L potassium dihydrogen phosphate, 15 g / L 3-aminopropanol, 10 g / L ammonium sulfate, 0.002 g / L vitamin B12, 0.005 g / L vitamin B1, 0.08 g / L isoleucine, 2 mL / L salt solution, and 10 mL / L surfactant.

[0015] As a preferred embodiment of the above method, the temperature of the fermentation culture is 28.0-32.0°C, and the pH of the fermentation culture is 6.5-7.0.

[0016] As a preferred embodiment of the above method, in step S1, the panC gene is derived from Escherichia coli W3110, Bacillus subtilis, Bacillus licheniformis, Corynebacterium glutamicum, or Fusarium moniliforme.

[0017] As a preferred embodiment of the above method, in step S2, the culturing time of the seed solution is 10 to 15 hours.

[0018] As a preferred embodiment of the above method, in step S3, the seed liquid inoculation amount is 10% to 15% of the volume of the fermentation medium.

[0019] Compared with the prior art, the present invention has the following technical effects: (1) The present invention produces D-panthenol by fermenting the modified strain DFC01, and based on the DFC01 strain, designs a multi-stage controlled fermentation process suitable for large-scale fermentation production of D-panthenol, by controlling the glucose concentration in the fermentation broth to be in the range of 0.5 to 2 g / L during the middle fermentation period of 12 to 24 hours, and controlling the dissolved oxygen concentration in the fermentation broth to be in the range of 28% to 40% during the late fermentation period of 24 hours to the end of fermentation, thereby reducing the accumulation of by-products during the fermentation process and increasing the D-panthenol yield and sugar-acid conversion rate.

[0020] (2) The method provided by the present invention provides the optimal fermentation conditions for the production of D-panthenol for the DFC01 strain, and adopts a double feeding method, wherein a first feeding is performed during the period of 12h to 24h of inoculation, and a second feeding is performed during the period of 24h of inoculation to the end of fermentation, and 3-aminopropanol and a surfactant are added to allow the strain to grow fully while overexpressing pantothenate synthase, effectively avoiding the situation where only bacteria grow without synthesizing metabolites, improving the overall D-panthenol production capacity of the fermentation tank, and ultimately achieving efficient synthesis of D-panthenol. The method of the present invention can provide guidance for other engineered bacteria to ferment and efficiently produce D-panthenol.

[0021] (3) The method provided by the present invention is based on microbial fermentation and has the advantages of being green and pollution-free. At the same time, the D-panthenol yield and sugar-acid conversion rate provided by the method provided by the present invention are high, and the optical purity is higher than that of the existing D-panthenol production process, which can reach 99.8%. In addition, the method of the present invention is easy to implement and is suitable for large-scale industrial production of D-panthenol. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0023] Overall embodiment This embodiment provides a method for producing D-panthenol by fermentation, comprising the following steps: Step S1: overexpressing the panC gene in the bottom plate bacteria to obtain the DFC01 strain; Step S2: inoculating and culturing the DFC01 strain to obtain a seed solution; Step S3: inoculating the seed liquid into a fermentation medium for fermentation culture; The fermentation culture is controlled under the following conditions: During 12 to 24 hours, a feed medium is fed, and the feed rate is controlled so that the concentration of glucose in the fermentation broth is in the range of 0.5 to 2 g / L; During the period from 24 hours to the end of the fermentation, the second type of feed medium is fed, and the dissolved oxygen concentration in the fermentation liquid is kept in the range of 28% to 40% by controlling the stirring speed.

[0024] In view of the problem that the optical purity of D-panthenol in the existing process is not high and it is difficult to achieve large-scale industrial application, this embodiment produces D-panthenol by fermenting the modified strain DFC01, and based on the DFC01 strain, designs a multi-stage controlled fermentation process suitable for large-scale fermentation production of D-panthenol. During the middle fermentation period of 12 to 24 hours, the glucose concentration in the fermentation broth is controlled to be in the range of 0.5 to 2 g / L, and during the late fermentation period of 24 hours to the end of fermentation, the dissolved oxygen concentration in the fermentation broth is controlled to be in the range of 28% to 40%, so that the accumulation of by-products during the fermentation process is reduced while the D-panthenol yield and sugar-acid conversion rate are increased.

[0025] As a preferred embodiment of this embodiment, in step S1, the panC gene is derived from Escherichia coli W3110, Bacillus subtilis, Bacillus licheniformis, Corynebacterium glutamicum, Fusarium moniliforme. The nucleotide sequences of the panC genes derived from the above strains belong to the prior art.

[0026] The panC gene is a gene encoding pantothenate synthase, which is a key enzyme in the biosynthesis pathway of D-pantothenic acid, and D-pantothenic acid is a precursor of D-panthenol. Overexpressed panC genes from different strains will produce different fermentation results for this method. Overexpression of panC genes from Bacillus subtilis and Bacillus licheniformis can make the resulting engineered bacteria ferment to produce D-panthenol with a yield of up to 17.4 g / L.

[0027] As a preferred embodiment of this embodiment, a feed medium is fed during 12 to 24 hours, and the feed rate is controlled to keep the glucose concentration in the fermentation broth in the range of 0.8 to 1.5 g / L.

[0028] As a preferred embodiment of this embodiment, during the period from 24 hours to the end of fermentation, the second type of feed medium is fed, and the dissolved oxygen concentration in the fermentation liquid is kept in the range of 32% to 35% by controlling the stirring speed.

[0029] As a preference of this embodiment, the stirring speed is controlled so that the rotation speed is 400-700 r / min.

[0030] As a preferred embodiment of this embodiment, a type of feed medium is: glucose 300g / L, magnesium sulfate 8g / L, potassium dihydrogen phosphate 14g / L, ammonium sulfate 10g / L, vitamin B12 0.002g / L, vitamin B1 0.005g / L, isoleucine 0.08g / L, salt solution 2mL / L, IPTG 1mol / L.

[0031] As a preferred embodiment of the present invention, the second type of feed medium is: glucose 200g / L, magnesium sulfate 6g / L, potassium dihydrogen phosphate 7g / L, 3-aminopropanol 15g / L, ammonium sulfate 10g / L, vitamin B12 0.002g / L, vitamin B1 0.005g / L, isoleucine 0.08g / L, salt solution 2mL / L, surfactant 10mL / L. The surfactant is selected from Tween 80, Triton, and Span, preferably Tween 80.

[0032] In this example, a double feeding method was used for the DFC01 strain, in which a first feeding method was performed during the period of 12 h to 24 h of inoculation, and a second feeding method was performed during the period of 24 h of inoculation to the end of fermentation, and 3-aminopropanol and a surfactant were added to allow the strain to grow fully while overexpressing pantothenate synthase, effectively avoiding the situation in which only bacteria grow but no metabolites are synthesized, thereby improving the overall D-panthenol production capacity of the fermentation tank, and ultimately achieving efficient synthesis of D-panthenol.

[0033] As a preference of this embodiment, the temperature of the fermentation culture is 28.0-32.0° C., and the pH of the fermentation culture is 6.5-7.0.

[0034] As a preferred embodiment of this embodiment, in step S2, the culturing time of the seed solution is 10 to 15 hours.

[0035] As a preferred embodiment of the above method, in step S3, the seed liquid inoculation amount is 10% to 15% of the volume of the fermentation medium.

[0036] Specifically, this embodiment and other embodiments use strain Escherichia coli ZJB18003 as the base bacteria to implement and illustrate the method of the present invention. The base bacteria Escherichia coli ZJB18003 is preserved in China Center for Type Culture Collection (CCTCC), address: Luojia Mountain, Wuchang, Wuhan, zip code: 430072, preservation date: December 21, 2018, preservation number: CCTCCNO: M2018914.

[0037] Example 1 This embodiment provides a method for producing D-panthenol by fermentation, comprising the following steps: (1) Construction of the plasmid pTrc99a-BSpanc: The genome of Bacillus subtilis 168 was used as a template and pTrc-rbs-panC(bs)-F 57 and panC(bs)-R 52 were used as primers for amplification; the empty plasmid of pTrc99a was used as a template and pTrc-rbs-R59 / pTrc-F 60 were used as primers for amplification. The product obtained after PCR was purified, and the linearized pTrc99a and panC were transformed into E. coli DH5α competent cells through a one-step cloning ligation. After overnight culture, the plasmid pTrc99a-BSpanc was extracted.

[0038] (2) Obtaining DFC01 strain: The pTrc99a-BSpanc plasmid was introduced into the competent cells of the base bacterium Escherichia coli ZJB18003, spread on an LB plate containing 100 mg / L kanamycin resistance, and cultured at 30°C for 12 h. A single colony was picked and transferred to an LB test tube containing 100 mg / L kanamycin resistance. After culturing at 30°C overnight, DFC01 was obtained. 800 μL of the bacterial solution was taken out with a pipette and added to a bacterial preservation tube, mixed with 50% glycerol in a 1:1 ratio, and stored in a -80°C refrigerator for later use.

[0039] (3) Activation culture: inoculate DFC01 stored in a -80°C refrigerator onto a plate culture medium and activate and culture it in an incubator at 30°C for 12 h to obtain activated bacteria.

[0040] The plate culture medium consists of: 10 g / L peptone, 5 g / L yeast powder, 5 g / L NaCl, 20 g / L agar powder, 100 mg / L kanamycin, pH = 6.8-7.0.

[0041] (4) Seed culture: Take a loop of activated bacteria with good growth and inoculate it into a shake flask containing seed culture medium for culture. The liquid volume is 150 mL / 500 mL. The seed culture conditions are 30°C and 150 r / min shaking for 12 h to obtain seed solution.

[0042] The composition of the seed culture medium is: peptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, kanamycin 100 mg / L, pH = 6.8-7.0.

[0043] (5) Fermentation culture: According to the inoculation amount (volume ratio) of 15%, the seed liquid obtained in step (2) was inoculated into a 5L fermentation tank containing fermentation medium, the liquid volume was 2L, the culture temperature was 30°C, and the initial stirring speed was 300rpm / min. At 12h after the start of fermentation, the first type of feed medium was added, and the stirring speed was increased to 400rpm / min. The residual sugar content of the fermentation liquid was detected by a sensor analyzer. The feeding rate was 2mL / min and the step rate was 2mL / min. When the residual sugar content of the fermentation liquid was higher than 1.5g / L, the feeding was automatically stopped. When the residual sugar content of the fermentation liquid was lower than 0.8g / L, the feeding rate was increased to keep the residual sugar content of the fermentation liquid at 0.8g / L or above. 24 hours after the start of fermentation, the second type of feed medium was added at a feed rate of 5 mL / min and a step rate of 3 mL / min, and the stirring speed was increased to 500 rpm / min to make the DO value in the fermentation liquid about 34%, and the stirring speed was controlled to make the DO value in the fermentation liquid not exceed 32% to 35% until the end of fermentation, during which the stirring speed was in the range of 400 to 700 rpm / min. The fermentation lasted for 96 hours.

[0044] Before and after fermentation, the D-panthenol content was determined by high performance liquid chromatography; the residual sugar content in the fermentation broth was detected by a sensor analyzer to calculate the sugar alcohol conversion rate. The sugar alcohol conversion rate is the efficiency of converting the sugar in the raw material (such as glucose, etc.) into the target alcohol product D-panthenol during the fermentation process. The sugar alcohol conversion rate index is an important parameter for measuring the efficiency and production performance of the fermentation process. It reflects the utilization of the raw materials and the output rate of the product.

[0045] The fermentation medium composition is: glucose 15 g / L, ammonium sulfate 4 g / L, yeast extract 7 g / L, peptone 10 g / L, magnesium sulfate heptahydrate 3 g / L, potassium phosphate trihydrate 5 g / L, sodium chloride 5 g / L, calcium carbonate 10 g / L, citric acid monohydrate 3 g / L, ammonium ferric citrate 0.7 g / L, anhydrous betaine 3 g / L, and defoamer 0.8 mL / L.

[0046] The composition of type I feed medium is: glucose 300 g / L, magnesium sulfate 8 g / L, potassium dihydrogen phosphate 14 g / L, 3-aminopropanol 2 g / L, ammonium sulfate 10 g / L, vitamin B12 0.002 g / L, vitamin B1 0.005 g / L, isoleucine 0.08 g / L, salt solution 2 mL / L, IPTG 1 mol / L, and surfactant 1 mL / L.

[0047] The composition of the second type of feed medium is: glucose 200g / L, magnesium sulfate 6g / L, potassium dihydrogen phosphate 7g / L, 3-aminopropanol 15g / L, ammonium sulfate 10g / L, vitamin B12 0.002g / L, vitamin B1 0.005g / L, isoleucine 0.08g / L, saline solution 2mL / L, Tween 80 10mL / L.

[0048] In this example, the yield of D-panthenol at the end of fermentation was 17.4 g / L, the conversion rate of sugar alcohol was 10.2%, and the optical purity of D-panthenol was 99.8%.

[0049] Example 2 This example provides a method for producing D-panthenol by fermentation, which is different from Example 1 mainly in that: in step (5), when the residual sugar content of the fermentation liquid is higher than 2 g / L, the feeding is automatically stopped, and when the residual sugar content of the fermentation liquid is lower than 0.5 g / L, the feeding rate is increased to keep the residual sugar content of the fermentation liquid at 0.5 g / L or above. The rest is the same as Example 1.

[0050] In this example, the yield of D-panthenol at the end of fermentation was 17.2 g / L, the conversion rate of sugar alcohol was 10.1%, and the optical purity of D-panthenol was 99.8%.

[0051] Example 3 This embodiment provides a method for producing D-panthenol by fermentation, which is different from the embodiment 1 mainly in that: in step (5), in the fermentation culture, 24 hours after the start of the fermentation, the second type of feed medium is fed, and the stirring speed is controlled so that the DO value in the fermentation liquid does not exceed the range of 28% to 40% until the end of the fermentation, during which the stirring speed is in the range of 400 to 700 rpm / min. The rest is the same as the embodiment 1.

[0052] In this example, the yield of D-panthenol at the end of fermentation was 17.3 g / L, the sugar alcohol conversion rate was 10.0%, and the optical purity of D-panthenol was 99.7%.

[0053] Example 4 This embodiment provides a method for producing D-panthenol by fermentation, which is different from the embodiment 1 mainly in that: in step (1) constructing the plasmid pTrc99a-ECpanc, the genome of Escherichia coli W3110 is used as a template, and pTrc-rbs-panC(EC)-F 54 and panC(EC)-R 53 are used as primers for amplification; the empty plasmid of pTrc99a is used as a template, and pTrc-rbs-R 59 / pTrc-F 60 are used as primers for amplification. The rest is the same as the embodiment 1.

[0054] In this example, the yield of D-panthenol at the end of fermentation was 13.4 g / L, the conversion rate of sugar alcohol was 9.2%, and the optical purity of D-panthenol was 99.2%.

[0055] Example 5 This embodiment provides a method for producing D-panthenol by fermentation, which is different from the embodiment 1 mainly in that: in step (1) constructing the plasmid pTrc99a-ECpanc, the genome of Corynebacterium glutamicum is used as a template, and pTrc-rbs-panC(CG)-F 52 and panC(CG)-R 53 are used as primers for amplification; the empty plasmid of pTrc99a is used as a template, and pTrc-rbs-R 59 / pTrc-F 60 are used as primers for amplification. The rest is the same as the embodiment 1.

[0056] In this example, the yield of D-panthenol at the end of fermentation was 12.4 g / L, the conversion rate of sugar alcohol was 10.2%, and the optical purity of D-panthenol was 99.3%.

[0057] Example 6 This embodiment provides a method for producing D-panthenol by fermentation, which is different from that of embodiment 1 mainly in that: in step (1) constructing the plasmid pTrc99a-ECpanc, the genome of Bacillus licheniformis is used as a template, and pTrc-rbs-panC(BL)-F 52 and panC(BL)-R 53 are used as primers for amplification; the empty plasmid of pTrc99a is used as a template, and pTrc-rbs-R 59 / pTrc-F 60 are used as primers for amplification. The rest is the same as that of embodiment 1.

[0058] In this example, the yield of D-panthenol at the end of fermentation was 17.4 g / L, the sugar alcohol conversion rate was 17.2%, and the optical purity of D-panthenol was 99.8%.

[0059] Example 7 This example provides a method for producing D-panthenol by fermentation, which is different from Example 1 mainly in that: in step (1) constructing the plasmid pTrc99a-ECpanc, the genome of Fusarium moniliforme is used as a template, and pTrc-rbs-panC(FM)-F 52 and panC(FM)-R 53 are used as primers for amplification; the empty plasmid of pTrc99a is used as a template, and pTrc-rbs-R 59 / pTrc-F 60 are used as primers for amplification. Other steps are the same as Example 1.

[0060] In this example, the yield of D-panthenol at the end of fermentation was 15.4 g / L, the conversion rate of sugar alcohol was 14.3%, and the optical purity of D-panthenol was 99.6%.

[0061] Example 8 This example provides a method for producing D-panthenol by fermentation, which is different from Example 1 mainly in that in step (5) of fermentation culture, Tween 80 in the second type feed medium is replaced by Triton. Others are the same as Example 1.

[0062] In this example, the yield of D-panthenol at the end of fermentation was 8.4 g / L, the sugar alcohol conversion rate was 7.3%, and the optical purity of D-panthenol was 99.3%.

[0063] Example 9 This example provides a method for producing D-panthenol by fermentation, which is different from Example 1 mainly in that in step (5) of fermentation culture, Tween 80 in the second type of feed medium is replaced by Span. Other contents are the same as Example 1.

[0064] In this example, the yield of D-panthenol at the end of fermentation was 8.3 g / L, the sugar alcohol conversion rate was 7.4%, and the optical purity of D-panthenol was 99.3%.

[0065] Comparative Example 1 This comparative example provides a method for producing D-panthenol by fermentation, which is different from Example 1 mainly in that: in step (5), in the fermentation culture, 12 hours after the start of fermentation, a type of feed medium is added, the feed rate is 1 mL / min, and the step rate is 1.2 mL / min, so that during the period of 12 hours to 24 hours after the start of fermentation, the feed rate is appropriately adjusted, and the residual sugar content of the fermentation liquid is maintained at about 0.4 g / L. Others are the same as Example 1.

[0066] In this comparative example, the yield of D-panthenol at the end of fermentation was 7.2 g / L, the sugar alcohol conversion rate was 6.7%, and the optical purity of D-panthenol was 99.1%.

[0067] Comparative Example 2 This comparative example provides a method for producing D-panthenol by fermentation, which is mainly different from Example 1 in that: in step (5), in the fermentation culture, 12 hours after the start of fermentation, a type of feed medium is added, the feed rate is 3 mL / min, and the step rate is 2.5 mL / min, so that during the period of 12 hours to 24 hours after the start of fermentation, the feed rate is appropriately adjusted, and the residual sugar content of the fermentation liquid is maintained at about 2.3 g / L. Others are the same as Example 1.

[0068] In this comparative example, the yield of D-panthenol at the end of fermentation was 7.1 g / L, the conversion rate of sugar alcohol was 6.9%, and the optical purity of D-panthenol was 99.1%.

[0069] Comparative Example 3 This comparative example provides a method for producing D-panthenol by fermentation, which is different from Example 1 mainly in that: in step (5), in the fermentation culture, 24 hours after the start of the fermentation, the second type of feed medium is fed, and the stirring speed is controlled so that the DO value in the fermentation liquid does not exceed the range of 20% to 25% until the end of the fermentation, during which the stirring speed is in the range of 200 to 500 rpm / min. The rest is the same as Example 1.

[0070] In this comparative example, the yield of D-panthenol at the end of fermentation was 8.0 g / L, the conversion rate of sugar alcohol was 6.7%, and the optical purity of D-panthenol was 99.0%.

[0071] Comparative Example 4 This comparative example provides a method for producing D-panthenol by fermentation, which is different from Example 1 mainly in that: in step (5), in the fermentation culture, 24 hours after the start of the fermentation, the second type of feed medium is fed, and the stirring speed is controlled to make the DO value in the fermentation liquid above 41%, and the stirring speed is in the range of 500 to 800 rpm / min. The rest is the same as Example 1.

[0072] In this comparative example, the yield of D-panthenol at the end of fermentation was 8.0 g / L, the conversion rate of sugar alcohol was 7.1%, and the optical purity of D-panthenol was 99.4%.

[0073] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0074] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for producing D-panthenol by fermentation, characterized in that: The following steps are involved: Step S1: overexpressing the panC gene in the bottom plate bacteria to obtain the DFC01 strain; Step S2: inoculating and culturing the DFC01 strain to obtain a seed solution; Step S3: inoculating the seed liquid into a fermentation medium for fermentation culture; The fermentation culture is controlled under the following conditions: During 12 to 24 hours, a feed medium is fed, and the feed rate is controlled so that the concentration of glucose in the fermentation broth is in the range of 0.5 to 2 g / L; During the period from 24 hours to the end of the fermentation, the second type of feed medium is fed, and the dissolved oxygen concentration in the fermentation liquid is kept in the range of 28% to 40% by controlling the stirring speed.

2. The method for producing D-panthenol by fermentation according to claim 1, characterized in that: During 12 to 24 hours, a feed medium is fed, and the feed rate is controlled so that the concentration of glucose in the fermentation broth is in the range of 0.8 to 1.5 g / L.

3. The method for producing D-panthenol by fermentation according to claim 1, characterized in that: During the period from 24 hours to the end of the fermentation, the second type of feed medium is fed, and the dissolved oxygen concentration in the fermentation liquid is kept in the range of 32% to 35% by controlling the stirring speed.

4. A method for producing D-panthenol by fermentation according to claim 1 or 3, characterized in that: The stirring speed is controlled to be 400-700 r / min.

5. The method for producing D-panthenol by fermentation according to claim 1, characterized in that: The composition of the first type of feed medium is: glucose 300g / L, magnesium sulfate 8g / L, potassium dihydrogen phosphate 14g / L, ammonium sulfate 10g / L, vitamin B12 0.002g / L, vitamin B1 0.005g / L, isoleucine 0.08g / L, salt solution 2mL / L, IPTG 1mol / L.

6. The method for producing D-panthenol by fermentation according to claim 1, characterized in that: The composition of the second type of feed medium is: glucose 200g / L, magnesium sulfate 6g / L, potassium dihydrogen phosphate 7g / L, 3-aminopropanol 15g / L, ammonium sulfate 10g / L, vitamin B12 0.002g / L, vitamin B1 0.005g / L, isoleucine 0.08g / L, salt solution 2mL / L, and surfactant 10mL / L.

7. The method for producing D-panthenol by fermentation according to claim 1, characterized in that: The temperature of the fermentation culture is 28.0-32.0° C., and the pH of the fermentation culture is 6.5-7.

0.

8. The method for producing D-panthenol by fermentation according to claim 1, characterized in that: In step S1, the panC gene is derived from Escherichia coli W3110, Bacillus subtilis, Bacillus licheniformis, Corynebacterium glutamicum, and Fusarium moniliforme.

9. The method for producing D-panthenol by fermentation according to claim 1, characterized in that: In step S2, the seed solution is cultured for 10 to 15 hours.

10. The method for producing D-panthenol by fermentation according to claim 9, characterized in that: In step S3, the seed liquid inoculation amount is 10% to 15% of the volume of the fermentation medium.