CN112126662B - Method for producing D-pantothenic acid by fermentation - Google Patents
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Abstract
The invention discloses a method for producing D-pantothenic acid by fermentation, which comprises the following steps of: inoculating Escherichia coli of M2018914 into a fermentation medium, fermenting and culturing to produce D-pantothenic acid, feeding a feed supplement medium into a fermentation tank when the fermentation time is up to 13h, controlling the concentration of glucose in the fermentation liquid to be 0.5-2g/L, and controlling the concentration of glucose in the fermentation liquid to be 4-6g/L when the fermentation time is up to 29h until the fermentation is finished; after the feeding of the feed medium is started, simultaneously feeding an isoleucine aqueous solution into the fermentation tank, wherein the concentration of the isoleucine aqueous solution is 30-60g/L, and the total amount of isoleucine added until the end of fermentation is 30-50 g. The method effectively improves the concentration of the produced D-pantothenic acid, improves the conversion rate of saccharic acid and reduces the generation of inhibitory byproduct acetic acid.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for producing D-pantothenic acid by fermentation.
Background
D-pantothenic acid, also known as vitamin B5, has chemical formula C9H17NO5The relative molecular weight of 219.24 is an important water-soluble vitamin, and can be widely used in the fields of medicine, food, feed, cosmetics, etc. D-pantothenic acid is ubiquitous in the organism, is one of the important synthetic precursors of Acyl Carrier Protein (ACP) and coenzyme a (coa), provides a major substrate for the TCA cycle in all cells, and controls cell growth, proliferation and overall histone acetylation, as well as playing a key role in anabolic and catabolic pathways of a variety of important substances, such as carbohydrates, fatty acids, lipids and phospholipids. The compound preparation containing D-pantothenic acid and other components and capable of improving immunity is used for treating diseasesTreating skin diseases such as neuritis, neurasthenia, postoperative intestinal colic, gastrointestinal diseases, respiratory diseases, lupus erythematosus, Huntington chorea, etc.; meanwhile, the D-pantothenic acid is also applied to various health-care foods, such as growth happiness, Scherkang, 21 Kingvitata and the like, and in the field of foods, the D-pantothenic acid is used for enhancing the flavor of distilled liquor and whisky; it also has effects of preventing Mel crystallization in winter, and relieving bitterness of caffeine and saccharin. Is important for livestock and poultry breeding, and if the D-pantothenic acid is lacked, the phenomena of growth retardation, adaptability and disease resistance reduction, reproductive system disorder, hair loss, dark color and the like of the poultry and the livestock can occur.
The existing industrial production method of D-pantothenic acid is mainly a chemical-enzymatic method, wherein isobutyraldehyde and formaldehyde are subjected to aldol condensation under the alkaline and high-temperature conditions to form hydroxytetravaleraldehyde, and then hydrocyanic acid is added to perform an alcohol cyanidation reaction under the acidic condition to form cyanohydrin; hydrolyzing cyanohydrin under acidic condition to obtain DL-pantoic acid lactone, hydrolyzing DL-pantoic acid lactone with L-pantoic acid lactone hydrolase to obtain D-pantoic acid lactone, and converting the produced L-pantoic acid into DL-pantoic acid lactone via chemical lactone and racemization. The obtained D-pantoic acid lactone is condensed with beta-amino calcium propionate to directly prepare the D-calcium pantothenate. The existing production process has high requirements on equipment and the problem of environmental pollution in the synthesis process cannot be avoided. With the increasing environmental protection problem, the microbial fermentation method using green renewable resources as raw materials to produce D-pantothenic acid has received more and more attention. The microbial fermentation method has the advantages of mild conditions, environmental friendliness, stable product quality and development prospect. The existing D-pantothenic acid producing bacteria produced by a microorganism direct fermentation method mainly comprise Escherichia coli (Escherichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum) and the like.
The microbial fermentation method synthesizes D-pantothenic acid or its derivatives by the metabolism of microorganisms and secretes them into a fermentation liquid, and the fermentation strain mainly metabolizes saccharides (glucose, maltose, etc.). And (2) sterilizing a fermentation medium containing carbon source substances, inorganic nitrogen sources, inorganic salts and the like at high temperature, inoculating, fermenting, separating and purifying the fermentation liquor to obtain the D-calcium pantothenate. The existing research on microbial fermentation methods mainly focuses on strain construction and metabolic research, but the production of D-pantothenic acid by Escherichia coli still has a significant obstacle. For example, although many viable engineered bacterial construction schemes have been proposed, the conversion of glucose to D-pantothenate is still low. The further improvement of the microbial synthesis yield of the D-pantothenic acid is limited by a fermentation process, few reports are reported for producing the D-pantothenic acid by the fermentation method at present, and no special report is found for optimizing the D-pantothenic acid fermentation process aiming at researching the production technology of a D-pantothenic acid fermentation factory. Control of the D-pantothenic acid fermentation process is critical for the overall production. The microorganisms may have different requirements on environmental conditions at different stages of growth, at different stages of production of the desired metabolite.
The previous patent application of the applicant, CN109868254A, discloses a genetically engineered bacterium for producing pantothenic acid in high yield, a construction method and application. In the former strain construction work, the aim is to realize the concentration of carbon metabolism to a D-pantothenic acid metabolic pathway and relieve the inhibition effect of valine, isoleucine and leucine on a key enzyme AHAS in the D-pantothenic acid metabolic pathway. The key pathway genes of valine and isoleucine, avtA and ilvA, were knocked out, while the expression level of the leucine key pathway gene, ilvE, was reduced. At the same time, the downstream consuming gene avtA of D-pantothenic acid is cut off, preventing the conversion and consumption of D-pantothenic acid to CoA. The conversion of D-pantothenic acid is an important source of intracellular CoA. CoA combines with acetate to form acetyl CoA, entering the oxidation process. Lack of CoA prevents the conversion of intracellular acetate to acetyl-CoA, and the reduced availability of intracellular acetyl-CoA results in reduced activity of TCA cycle enzymes, which in turn inhibits the TCA cycle to provide energy and NADPH for bacterial growth and D-pantothenate formation. In addition, intracellular deficiencies of valine, isoleucine and leucine in previous metabolic engineering also prevented the conversion of these amino acids into the succinyl-coa metabolic pathway. It is well known that succinyl-coa generates oxaloacetate through a series of enzymatic reactions, which is extremely important for bacterial growth and formation of target metabolites, since the concentration of oxaloacetate directly affects the cycling rate (TCA cycle). Insufficient flux of TCA cycle results in insufficient intracellular availability of NADPH involved in the D-pantothenate pathway. As a result, the glycolytic pathway and the central carbon metabolism are not balanced, and carbon metabolism overflows to the acetate pathway, which is manifested by the accumulation of a large amount of acetate during fermentation of the recombinant D-pantothenic acid-producing strain. In the technical scheme, the problems of low D-pantothenic acid yield, low saccharic acid conversion and high acetic acid accumulation in the fermentation process also exist.
Disclosure of Invention
The application aims at the problems of low D-pantothenic acid yield, low saccharic acid conversion and high acetic acid accumulation amount in the fermentation process in the prior art, improves and optimizes the problems in the prior fermentation process of a single strain, and provides a method for producing D-pantothenic acid.
A process for the fermentative production of D-pantothenic acid comprising the steps of:
(1) activation culture: the preservation number is CCTCC NO: inoculating the Escherichia coli of M2018914 to a plate culture medium for activation culture to obtain activated bacteria;
(2) seed culture: inoculating the activated bacteria obtained in the step (1) into a seed culture medium for seed culture to obtain a seed solution;
(3) inoculating the seed liquid into a fermentation culture medium for fermentation culture to produce D-pantothenic acid, wherein the stirring speed is 400-; controlling the dissolved oxygen to be 10-25% by stirring and ventilating; controlling the pH value to be 6.5-7.0 by adding ammonia water in a flowing manner; the culture temperature is 25.0-32.0 ℃; when the fermentation time is 13 hours, feeding a supplemented medium into the fermentation tank, controlling the concentration of glucose in the fermentation liquid to be 0.5-2g/L, and when the fermentation time is 29 hours, controlling the concentration of glucose in the fermentation liquid to be 4-6g/L until the fermentation is finished; after the feeding of the feed medium is started, simultaneously feeding an isoleucine aqueous solution into the fermentation tank, wherein the concentration of the isoleucine aqueous solution is 30-60g/L, and the total amount of isoleucine added until the end of fermentation is 30-50 g.
The preservation number is CCTCC NO: m2018914, which is described in the applicant's earlier patent application: the publication number is CN109868254A, the invention name is 'a genetic engineering bacterium for producing pantothenic acid with high yield, a construction method and application'.
Preferably, the plate culture medium in step (1) is: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
Preferably, the seed culture medium in step (2) is: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 6.8-7.0 of pH.
Preferably, the inoculation amount of the seed liquid in the step (3) is 6-15% of the volume of the fermentation medium.
Preferably, the inoculation amount of the seed liquid in the step (3) is 8-12% of the volume of the fermentation medium; the dissolved oxygen is controlled at 14-18%; controlling the pH value to be 6.8-7.0 by adding ammonia water in a flowing manner; the culture temperature is controlled to be 29.0-31.0 ℃.
Preferably, the fermentation medium in step (3) is: 15-50g/L of glucose, 10-20g/L of ammonium sulfate, 1-4g/L of yeast extract, 0.5-2g/L of monopotassium phosphate, 0.3-1g/L of magnesium sulfate, 1-3g/L of beta-alanine, 5-15g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 0.5-2mL/L of salt solution, 80-200mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside;
the salt solution is: CuCl2 10g/L、FeSO4·7H2O 10g/L、ZnSO4·7H2O 1g/L、CuSO40.20g/L、NiCl2·7H2O 0.02g/L。
More preferably, the fermentation medium in step (3) is: 18-22g/L of glucose, 12-16g/L of ammonium sulfate, 1.5-2.5g/L of yeast extract, 0.6-0.8g/L of monopotassium phosphate, 0.4-0.6g/L of magnesium sulfate, 1.2-2g/L of beta-alanine, 8-12g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 150mg/L of serine 100-beta-galactoside and 0.1mmol/L of isopropylthiogalactoside.
Preferably, the feed medium in step (3) is: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the salt solution is: CuCl2 10g/L、FeSO4·7H2O 10g/L、ZnSO4·7H2O 1g/L、CuSO40.20g/L、NiCl2·7H2O 0.02g/L。
Preferably, the control strategy of the concentration of glucose in the fermentation liquid during the fermentation culture in the step (3) is as follows:
in the early stage of fermentation, the time is 0-13h, and the concentration of glucose is not controlled;
in the middle stage of fermentation, the time is 13-29h, and the concentration of glucose is controlled to be 0.8-1.2 g/L;
at the later stage of fermentation, after 29h, the glucose concentration is controlled to be 4.5-5.5 g/L.
Preferably, the concentration of the isoleucine aqueous solution in step (3) is 35-55g/L, and the total amount of isoleucine added until the end of fermentation is 34-45 g.
The invention has the beneficial effects that:
the synthesis pathway of the D-pantothenic acid is complex and is influenced by a plurality of factors, and the growth problem often occurs when the Escherichia coli is transformed by means of metabolic engineering to accumulate the D-pantothenic acid; in order to avoid the cell growth lag phase caused by the knockout of a metabolic byproduct pathway of a strain, a proper amount of isoleucine is supplemented in a culture medium, so that TCA cycle can be promoted to provide more energy for the production of D-pantothenic acid and stimulate the growth of thalli while reducing hydrogen, and the proper amount of isoleucine can balance glycolysis pathway and central metabolic capacity, thereby inhibiting the formation of the byproduct acetic acid and promoting the accumulation of amino acid; various factors cooperate with each other to improve the fermentation yield of the D-pantothenic acid; meanwhile, in order to solve the problems of premature senility and low acid production efficiency of the thalli and improve the vitality of thalli in the middle and later periods, a multi-stage glucose fed-batch strategy is explored based on different requirements of thalli growth and product synthesis on the environment in different stages of fermentation, the acid production capacity of cells in the later period is improved, and the yield of D-pantothenic acid is further improved. The finally obtained fermentation strategy of bringing the isoleucine feeding system into the glucose feeding control framework method successfully solves the problems of substrate inhibition, product feedback inhibition, overhigh acetic acid concentration in the fermentation process of metabolic engineering bacteria and the like in the fermentation process, and the yield of D-pantothenic acid is greatly improved, so that the method has important industrial application value and has certain guiding significance for the fermentation production of other amino acids and related compounds thereof.
Drawings
FIG. 1 is a graph showing the results of examination of the effect of isoleucine on D-pantothenate fermentation.
Detailed Description
The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914. The strain is described in detail in the applicant's earlier patent applications: the publication number is CN109868254A, the invention name is 'a genetic engineering bacterium for producing pantothenic acid with high yield, a construction method and application'.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaC 15/15 g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 1 g/L.
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 50g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
wherein the salt solution is: CuCl2 10g/L、FeSO4·7H2O 10g/L、ZnSO4·7H2O 1g/L、CuSO40.20g/L、NiCl2·7H2O0.02 g/L (hereinafter, the salt solution is the formulation).
The yield of D-pantothenic acid after the fermentation is finished is 18.4g/L, and the saccharic acid conversion rate is 11.3%.
Example 2: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914. The strain is described in detail in the applicant's earlier patent applications: the publication number is CN109868254A, the invention name is 'a genetic engineering bacterium for producing pantothenic acid with high yield, a construction method and application'.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaC 15/15 g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 1 g/L.
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 15g/L glucose, 14g/L ammonium sulfate, 2g/L yeast extract, 0.8g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate, 1.5g/L beta-alanine, 10g/L calcium carbonate, 120.001g/L VBE, 10.001g/L VBE, 1mL/L saline solution, 100mg/L serine, 0.1mmol/L isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is finished is 17.4g/L, and the saccharic acid conversion rate is 10.2%.
Example 3: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914. The strain is described in detail in the applicant's earlier patent applications: the publication number is CN109868254A, the invention name is 'a genetic engineering bacterium for producing pantothenic acid with high yield, a construction method and application'.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaC 15/15 g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 1 g/L.
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 18g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is finished is 19.8g/L, and the saccharic acid conversion rate is 12.5%.
Example 4: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914. The strain is described in detail in the applicant's earlier patent applications: the publication number is CN109868254A, the invention name is 'a genetic engineering bacterium for producing pantothenic acid with high yield, a construction method and application'.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaC 15/15 g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 1 g/L.
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 22g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is finished is 19.7g/L, and the saccharic acid conversion rate is 11.9%.
Example 5: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 5 g/L.
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 50g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is finished is 24.3g/L, and the saccharic acid conversion rate is 14.5%.
Example 6: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 5 g/L.
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 20g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is finished is 26.1g/L, and the saccharic acid conversion rate is 15.5%.
Example 7: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 1 g/L.
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 20g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is 27g/L, and the sugar-acid conversion rate is 16%.
Example 8: method for producing D-pantothenic acid by fermentation
On the basis of the conventional fermentation of example 1, the effect of isoleucine-addition concentration on the content of D-pantothenic acid and major by-products in the fermentation broth was examined. The concentration gradients of the isoleucine after the addition are set to be 0, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035 and 0.04 respectively, and the unit is g/L. As shown in figure 1, with the increase of isoleucine, the content of D-pantothenic acid in the fermentation broth is steadily increased, while the content of acetic acid as a byproduct is gradually reduced, when the concentration of isoleucine is increased to 0.02g/L, the content of D-pantothenic acid reaches the maximum value, the content of isoleucine is continuously increased, the content of D-pantothenic acid is not obviously increased, at the moment, the content of acetic acid as a byproduct is not continuously reduced, which indicates that the metabolic flows reach equilibrium. During the fermentation process, a proper amount of isoleucine is added in the middle and later stages of the fermentation, so that feedback inhibition can be performed on an acetic acid synthesis pathway, the acetic acid synthesis pathway is blocked, and more metabolic flow flows to a D-pantothenic acid synthesis pathway. Therefore, the preferred isoleucine concentration is between 0.015 and 0.025 g/L.
Example 9: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 5 g/L.
Constant velocity fed isoleucine strategy: after 11h of feed initiation, the feed of concentrated isoleucine solution (40g/L) was started by a computer-coupled feed pump at a feed rate of 10 mL/h.
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 20g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is 28.6g/L, and the saccharic acid conversion rate is 14.9%. The accumulated amount of acetic acid was reduced to 16.5g/L compared to 39.48g/L in example 2.
Example 10: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 5 g/L.
Intermittent isoleucine feeding strategy: after 11h of initiation of the feed, 10mL of concentrated isoleucine solution (40g/L) was exogenously added for the first time, and 10mL of concentrated isoleucine solution (40g/L) was added every 11 h.
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 20g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is finished is 32.5g/L, and the sugar-acid conversion rate is 15.8%. The accumulated amount of acetic acid was reduced to 7.44g/L compared to 39.48g/L in example 2.
Example 11: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. The residual glucose concentration was controlled to 5 g/L.
Feedback isoleucine feeding strategy: after 11h, the OD600 value of the cells was measured every 2h, and each time the OD of the cells was measured600When the concentration was not increased or even decreased compared to the previous measurement, 10mL of concentrated isoleucine (40g/L isoleucine) was exogenously added to stimulate growth.
Measurement of microbial body OD 600: the sample was divided into two tubes, one tube was centrifuged at 12000rpm for 5min in a centrifuge tube to collect the supernatant as a standard control for measuring absorbance, and the other tube was used for measuring Optical Density (OD) at 600nm using an Eppendorf BioPhotometer600)。
Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; and detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate.
Fermentation medium: 20g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is finished is 33.8g/L, and the sugar-acid conversion rate is 17.9%. The accumulation of acetic acid (by-product) was reduced to 1.45g/L compared with 39.48g/L in example 2.
Example 12: method for producing D-pantothenic acid by fermentation
Test strain Escherichia coli ZJB 18003, deposited in China Center for Type Culture Collection (CCTCC) and having address: wuhan city Wuchang Lojia mountain, postcode: 430072, preservation date: 21/12/2018, accession number: CCTCC NO: m2018914.
(1) Activation culture: inoculating Escherichia coli to a plate culture medium, and performing activation culture in an incubator at 30 ℃ for 12h to obtain activated bacteria;
plate culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
(2) Seed culture: inoculating a ring of well-grown activated bacteria to a shake flask containing a seed culture medium for culturing, wherein the liquid loading amount is 100mL/500mL, the seed culture condition is 30 ℃, and shake culture is carried out at 200r/min for 12h to obtain a seed liquid;
seed culture medium: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 6.8-7.0 of pH.
(3) Fermentation culture: and (3) inoculating the seed liquid obtained in the step (2) into a 5L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the liquid loading amount is 2L/5L, the culture temperature is 30 ℃, the initial stirring speed is 400rpm/min, and the dissolved oxygen value is maintained to be 15%. Feeding a supplemented medium after fermenting for 13h respectively, detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and adjusting the glucose feeding rate once every 2 hours, wherein the glucose consumption rate is calculated according to the change of the glucose concentration in 2 hours. Middle fermentation stage (13-29 h): controlling the concentration of residual glucose to be 1 g/L; late fermentation (after 29 h): controlling the residual glucose concentration to be 5g/L, and fermenting for 55-70 h. The end time of fermentation is judged by taking the acid production as a criterion. Measuring the content of D-pantothenic acid by high performance liquid chromatography before and after fermentation culture; detecting the content of residual sugar in the fermentation liquor by using a biosensor analyzer, and calculating the sugar-acid conversion rate;
feedback isoleucine feeding strategy: after 11h, the OD600 value of the cells was measured every 2h, and each time the OD of the cells was measured600When the concentration was not increased or even decreased compared to the previous measurement, 10mL of concentrated isoleucine (40g/L isoleucine) was exogenously added to stimulate growth.
Measurement of microbial body OD 600: the sample was divided into two tubes, one tube was centrifuged at 12000rpm for 5min in a centrifuge tube to collect the supernatant as a standard control for measuring absorbance, and the other tube was used for measuring Optical Density (OD) at 600nm using an Eppendorf BioPhotometer600)。
Fermentation medium: 20g/L of glucose, 14g/L of ammonium sulfate, 2g/L of yeast extract, 0.8g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1.5g/L of beta-alanine, 10g/L of calcium carbonate, 120.001g/L of VBE, 10.001g/L of VBE, 1mL/L of salt solution, 100mg/L of serine and 0.1mmol/L of isopropyl thiogalactoside (IPTG);
a supplemented medium: the feed medium comprises: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the yield of D-pantothenic acid after the fermentation is finished is 48.5g/L, and the saccharic acid conversion rate is 23.0%. The accumulated amount of acetic acid was reduced to 0.41g/L compared to 39.48g/L in example 2.
In summary, the feedback isoleucine feeding embedded glucose feeding control strategy proposed in this patent can satisfy different periods of producing the target metabolites at different fermentation stages, with different demands for environmental conditions, while balancing the metabolic flows, with the result that the acetic acid synthesis pathway is blocked and more metabolic flows flow to the D-pantothenic acid synthesis pathway. The strategy obviously improves the conversion rate of saccharic acid and the yield of D-pantothenic acid, and obviously reduces the accumulation of acetic acid in the fermentation liquor.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. A process for the fermentative production of D-pantothenic acid comprising the steps of:
(1) activation culture: the preservation number is CCTCC NO: inoculating the Escherichia coli of M2018914 to a plate culture medium for activation culture to obtain activated bacteria;
(2) seed culture: inoculating the activated bacteria obtained in the step (1) into a seed culture medium for seed culture to obtain a seed solution;
(3) inoculating the seed liquid into a fermentation culture medium for fermentation culture to produce D-pantothenic acid, wherein the stirring speed is 400-; controlling the dissolved oxygen to be 10-25% by stirring and ventilating; controlling the pH value to be 6.5-7.0 by adding ammonia water in a flowing manner; the culture temperature is 25.0-32.0 ℃; in the early stage of fermentation, the time is 0-13h, and the concentration of glucose is not controlled; feeding a feed supplement culture medium into the fermentation tank in the middle stage of fermentation for 13-29h, and controlling the concentration of glucose to be 0.8-1.2 g/L; in the later fermentation period, after 29 hours, the concentration of glucose is controlled to be 4.5-5.5 g/L; after feeding of the feed medium is started, simultaneously feeding an isoleucine aqueous solution into the fermentation tank, wherein the concentration of the isoleucine aqueous solution is 35-55g/L, and the total amount of isoleucine added until the fermentation is finished is 34-45 g; the isoleucine concentration in the fermentation liquor after the fed-batch is 0.015-0.025 g/L;
the fermentation medium in the step (3) is as follows: 18-22g/L glucose, 12-16g/L ammonium sulfate, 1.5-2.5g/L yeast extract, 0.6-0.8g/L potassium dihydrogen phosphate, 0.4-0.6g/L magnesium sulfate, 1.2-2g/L beta-alanine, 8-12g/L calcium carbonate, 120.001g/L VBE, 10.001g/L VBE, 1mL/L saline solution, 150mg/L serine 100-;
the feed culture medium in the step (3) is as follows: 500g/L glucose, 8g/L magnesium sulfate, 14g/L potassium dihydrogen phosphate, 40g/L beta-alanine, 10g/L ammonium sulfate, 120.002g/L VB10.005g/L VBE, 0.08g/L Ile, 100mg/L serine, 2mL saline solution and 1mmol/L IPTG;
the salt solution is: CuCl2 10g/L、FeSO4·7H2O 10g/L、ZnSO4·7H2O 1g/L、CuSO4 0.20g/L、NiCl2·7H2O 0.02g/L。
2. The method of claim 1, wherein the plating medium in step (1) is: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 20g/L of agar powder, wherein the pH value is 6.8-7.0.
3. The method of claim 1, wherein the seed medium in step (2) is: 10g/L of peptone, 10g/L, NaCl 5g/L of yeast extract and 6.8-7.0 of pH.
4. The method of claim 1, wherein the seed liquid is inoculated in step (3) in an amount of 6-15% by volume of the fermentation medium.
5. The method of claim 4, wherein the seed liquid is inoculated in step (3) in an amount of 8-12% by volume of the fermentation medium; the dissolved oxygen is controlled at 14-18%; controlling the pH value to be 6.8-7.0 by adding ammonia water in a flowing manner; the culture temperature is controlled to be 29.0-31.0 ℃.
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