CN108611383B - Method for improving fermentation yield of artemisinic acid - Google Patents

Method for improving fermentation yield of artemisinic acid Download PDF

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CN108611383B
CN108611383B CN201810449836.8A CN201810449836A CN108611383B CN 108611383 B CN108611383 B CN 108611383B CN 201810449836 A CN201810449836 A CN 201810449836A CN 108611383 B CN108611383 B CN 108611383B
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陈伟
滕云
徐彬
徐美冬
陈亚军
郑玲辉
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Zhejiang Hisun Pharmaceutical Co Ltd
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Abstract

The invention provides a method for improving fermentation yield of artemisinic acid, which comprises the steps of taking saccharomyces cerevisiae engineering bacteria capable of producing the artemisinic acid as production strains, and fermenting in a fermentation culture medium containing lactose hydrolysate capable of being hydrolyzed into glucose and galactose, mevalonic acid and/or citral and other exogenous factors.

Description

Method for improving fermentation yield of artemisinic acid
Technical Field
The invention relates to the technical field of industrial microbial fermentation, in particular to a method for improving the fermentation yield of artemisinic acid.
Background
Artemisinin is sesquiterpene lactone medicine with peroxy group extracted from stem and leaf of composite inflorescence plant Artemisia annua, and is discovered in 1971 by U-yo of Chinese pharmacy15H22O5. Artemisinin is the most effective antimalarial specific drug after acetamiprid, chloroquine and primaquine, particularly has the characteristics of quick response and low toxicity for cerebral malaria and chloroquine malaria, and has been called as the 'only effective malaria treatment drug in the world' by the world health organization. Artemisinin was widely used over the next 40 years, and statistics show that artemisinin and its derivatives are sold worldwide in the number of $ 15 billion each year. In recent years, artemisinin has shown attractive prospects in the treatment of other diseases such as schistosomiasis resistance, regulation or inhibition of the immune function of body fluid, improvement of the conversion rate of lymphocytes, cholagogue, phlegm elimination, cough relief, asthma relief and the like. Therefore, the artemisinin has very wide market prospect.
The artemisinin is mainly prepared by three production methods, one method is the traditional method for extracting the artemisinin from the sweet wormwood herb, and the method is greatly influenced by climate and regions and has unstable yield; secondly, the compound is obtained by chemical total synthesis, so that the method has great difficulty and high cost; the third method is to adopt genetic engineering technology to obtain arteannuic acid through microbial fermentation, and then chemically synthesize artemisinin, and the method has high yield, stable production, low production cost and green and environment-friendly process; the third method is to adopt genetic engineering technology to obtain arteannuic acid by microbial fermentation and then synthesize artemisinin, namely the arteannuic acid is obtained by a biosynthesis method, and then artemisinin is produced by chemical conversion. In 2013, the WHO also approved the artemisinin produced by the process to be applied to clinic.
The third method has great influence on the yield and cost of final products due to arteannuic acid, a key intermediate of artemisinin. The development of the microbial fermentation method of artemisinic acid starts from a supporting project of the 2004 Ganz Foundation, and the technologies adopted at home and abroad are yeast fermentation processes developed on the basis of the supporting project, but the actual industrial production has more problems, and the difficulty in realizing the industrial production is still high.
A model for the use of synthetic biology in pharmaceutical development (Chris J.Paddon and Jay D.Keasling, 2014, volume 12, p355) discloses that the requirement for fermentation to produce artemisinic acid is the presence of galactose, since the engineered Saccharomyces cerevisiae requires the initiation of a galactose-induced promoter to achieve the biosynthesis of artemisinic acid when constructing synthetic genes of artemisinic acid. In actual production, the addition amount of galactose is large, and the cost is very high (about 180-250 yuan/kg), so that the use of a large amount of galactose in the fermentation process directly causes the high fermentation cost of the arteannuic acid, and finally causes the cost of the biosynthetic artemisinin to still be unable to counterbalance the cost of the naturally extracted product, so that the large-scale industrial production of the semi-synthetic artemisinin is finally failed to be formed.
High-level semi-synthetic production of the potential anti-microbial artemisinine (CJ Paddon, PJ Westfall, DJ Pitera, et al. Nature,2013,496(7446): 528-. The Production of amorpha-4, 11-diene from amorpha-4, 11-diene to artemisinic acid, and its conversion to dihydroartemisinic acid (PJ Westfall, DJ Pitera, JR Lenihan, et al. Procedents of the National Academy of sciences.2012,109(3): E111-E118) describes a method of genetically modifying Saccharomyces cerevisiae engineering bacteria to achieve high yield of amorpha-4, 11-diene, but the conversion of amorpha-4, 11-diene to artemisinic acid is difficult to achieve in vivo, the conversion of amorpha-4, 11-diene to artemisinic acid needs to be achieved chemically in vitro, the conversion process is complex, the yield is low, new impurities can be introduced, the cost is high, and therefore, the industrial application is poor. Patent CN101338309A discloses a method for producing artemisinic acid by introducing CYP71AV1P450 gene and P450 reductase CPR gene into saccharomyces cerevisiae cells to obtain artemisinic acid genetic engineering bacteria, the method has less process optimization and yield description on artemisinic acid fermentation yield, and the patent does not describe the application aspect of whether the strains can be industrially produced.
Aiming at the defects of low yield, high cost, incapability of realizing industrial production, unstable strain performance and the like in the fermentation production of the artemisinic acid in the prior art, the artemisinic acid fermentation process which has high yield, low cost, no harm to human bodies, environmental friendliness, high strain genetic stability and capability of realizing industrial production is necessary to be found.
Disclosure of Invention
The invention provides a method for improving fermentation yield of artemisinic acid, which comprises the following steps: the method comprises the steps of taking saccharomyces cerevisiae engineering bacteria capable of producing artemisinic acid as production strains, and fermenting in a fermentation culture medium containing lactose hydrolysate and exogenous factors, wherein the lactose hydrolysate is a solution obtained by hydrolyzing lactose into glucose and galactose through enzyme, and the exogenous factors are one or two of mevalonic acid and citral.
In a preferred embodiment, the enzyme is a β -galactosidase.
In a preferred embodiment, the mass ratio of β -galactosidase to lactose is 0.1-2: 100.
in a preferred embodiment, the mass-to-volume ratio of the amounts of glucose and galactose in the lactose hydrolysate to the fermentation medium is 15-30: 1 (unit is g/L).
In a preferred embodiment, the lactose is hydrolyzed by enzyme at a hydrolysis temperature of 50-55 deg.C for 3-5h with a hydrolysis rate of 90% -95% and a hydrolysis pH of 4-7, preferably 5.5.
In a preferred embodiment, the concentration of the exogenous factor in the fermentation medium is 0.1-0.5 g/L.
In a preferred embodiment, the fermentation medium further comprises: 8-15g/L of ammonium sulfate; 5-8g/L potassium dihydrogen phosphate; 3-6.2g/L magnesium sulfate heptahydrate; 8-12ml/L of vitamin solution; 6-10ml/L of metal ion solution; CuSO4·5H2O20-40 ug/L, water in balance, and pH of 5.0-5.5. Regulating the pH value by using an aqueous solution containing 13-14.5mol/L of ammonia;
the metal ion solution comprises the following components: ZnSO4·7H2O 5.75g/L；MnCl2·4H2O 0.32g/L；CoCl2·6H2O 0.47g/L；NaMoO4·2H2O 0.48g/L；CaCl2·2H2O 2.9g/L；FeSO4·7H2O2.8 g/L; 80ml/L of 0.5M EDTA, and the balance of water;
the vitamin solution comprises the following components: biotin 0.05 g/L; 1g/L of calcium pantothenate; 1g/L of nicotinic acid; inositol 25 g/L; vitamin B11 g/L; 1g/L pyridoxal; 0.2g/L of p-aminobenzoic acid, and the balance of water.
In a preferred embodiment, the fermentation time is between 84 and 120h and the fermentation temperature is between 22 and 35 ℃.
In a preferred embodiment, the fermentation further comprises feeding a carbon source, a nitrogen source and a water-insoluble oily liquid, wherein the water-insoluble oily liquid is one or two of isopropyl palmitate and oleic acid.
In a preferred embodiment, the carbon source is fed-batch for 10-18h from fermentation to fermentation end, and the carbon source is one or more of lactose hydrolysate, glycerol aqueous solution, sucrose aqueous solution and ethanol aqueous solution.
In a preferred embodiment, the concentration of the lactose hydrolysate is 400-700g/L, the concentration of the glycerol aqueous solution is 400-700g/L, the concentration of the sucrose aqueous solution is 400-700g/L, and the concentration of the ethanol aqueous solution is 80-99% (V/V).
In a preferred embodiment, the rate of feeding the carbon source is controlled such that the volume of carbon source fed per hour is 0.2% to 2% of the initial volume of the fermentation medium.
In a preferred embodiment, the time of feeding the nitrogen source is from 36 to 45 hours from the beginning of the fermentation to the end of the fermentation, and the fed nitrogen source is one or more of yeast powder soaking water solution, peptone water solution and sodium glutamate water solution.
In a preferred embodiment, the concentration of the yeast extract powder water solution is 200-500g/L, the concentration of the peptone water solution is 200-500g/L, and the concentration of the sodium glutamate water solution is 200-500 g/L.
In a preferred embodiment, the rate of feeding the nitrogen source is controlled such that the volume of the nitrogen source fed per hour is 0.05% to 0.5% of the initial volume of the fermentation medium.
In a preferred embodiment, after the time for feeding the water-insoluble oily liquid is 48-55h, the feeding mode is a one-time feeding mode.
In a preferred embodiment, the volume of the fed-in water-insoluble oily liquid is 5% to 40% of the initial volume of the fermentation medium.
In a preferred embodiment, the production strain may further be subjected to resistance selection on a plate solid medium containing an antibiotic, which is one or more of noursemycin, geneticin and hygromycin B.
In a preferred embodiment, the antibiotic is present in an amount of 50-300. mu.g/L on the plate solid medium.
In a preferred embodiment, the time for the plate culture is 30-50 h.
The fermentation process of the present invention has artemisinic acid yield as high as 42g/L, high artemisinic acid fermentation yield, and lactose hydrolysate capable of being hydrolyzed into glucose and galactose to replace galactose in available technology, so that one molecule of lactose may be hydrolyzed into one molecule of glucose and one molecule of galactose under the catalysis of enzyme, and 342.3g of lactose may be hydrolyzed into 180.16g of glucose and 180.16g of galactose at hydrolysis rate of 100%. Galactose is a promoter inducer of a synthetic gene necessary for fermentation of artemisinic acid, and the using amount is large, but the market price is about 6 times of lactose, so the fermentation cost is greatly reduced.
Compared with the production process of artemisinic acid in the prior art, the invention process of artemisinic acid mainly has the following beneficial effects:
(1) the arteannuic acid prepared by fermentation of the invention has the titer as high as 42g/L and high fermentation titer.
(2) According to the method, the lactose is hydrolyzed to obtain the hydrolysate containing high-concentration glucose and galactose, the glucose can be directly utilized, the galactose can be used as a promoter inducer of the transgene, the hydrolysate is used as a fermentation carbon source, a process of directly adding the galactose is abandoned, and the fermentation cost of the artemisinic acid is greatly reduced.
(3) According to the invention, exogenous factors such as mevalonic acid and/or citral are added into the fermentation medium, so that the fermentation titer of the artemisinic acid is further improved.
(4) According to the invention, non-water-soluble oily liquid such as isopropyl palmitate and/or oleic acid is added once in the fermentation process, the artemisinic acid is promoted to transfer from the fermentation culture medium to the oily liquid, the toxicity of artemisinic acid accumulation on fermentation thalli is reduced, the fermentation yield of the artemisinic acid is further improved, the isopropyl palmitate and the oleic acid are oily liquids without pungent odor and toxicity, the environment is friendly, the price is relatively low, and the industrial large-scale production is facilitated.
(5) The invention carries out resistance screening on the yeast genetic engineering bacteria capable of producing the artemisinic acid, and the genetic stability of the strains is higher.
Detailed Description
The following examples are intended to further illustrate the present invention, but it should be understood that the following examples are only illustrative of the present invention and are not to be construed as limiting the scope of the present invention, which is defined by the appended claims.
The strain used in the invention is any saccharomyces cerevisiae engineering bacteria capable of producing artemisinic acid, can be purchased commercially or constructed in laboratories, for example, saccharomyces cerevisiae purchased from China center for type culture Collection (Wuhan university) with the preservation number of CCTCC AY92011 is taken as a starting strain, gene engineering means is utilized to construct genes of a methylglutaric acid pathway (MVA), ADH1 (artemisinic alcohol dehydrogenase) and ALDH1 (artemisinic aldehyde dehydrogenase), overexpression of tHMGR and ERG20 genes increases the accumulation amount of farnesyl pyrophosphate FPP which is a common precursor of hemiterpenoid substances in yeast, the expression of squalene synthase is inhibited through overexpression of MVA pathway genes and ADS genes, so that the massive synthesis of artemisinic diene is realized, further, an exogenous gene CYP71AV1 (cytochrome P450 enzyme gene) is introduced so as to realize the synthesis of artemisinic acid, and the specific strain construction method can refer to Ro D K, Paradise E M, ouellet M, et al.production of the antigenic drug ingredient in engineered yeast [ J ] Nature,2006,440 (7086). 940-: the detailed Methods described in Methods section 528-532; or Saccharomyces cerevisiae (Saccharomyces cerevisiae) disclosed in Chinese patent with publication No. CN101338309B, preferably Saccharomyces cerevisiae with biological collection number of CGMCC NO. 1861.
The strains used in the embodiment of the invention are saccharomyces cerevisiae engineering bacteria which can be fermented to produce artemisinic acid and are constructed by using genetic engineering means and by taking saccharomyces cerevisiae purchased from China center for type culture Collection (Wuhan university) with the preservation number of CCTCC AY92011 as an original strain, and the specific strain construction methods refer to the following documents Ro D K, Paraise E M, Ouellet M, et al.Production of the anti-mental drug ingredient in encoded yeast [ J ]. Nature,2006, 7086, 940- "943 and Paddon CJ, Westlfa PJ, Pitera DJ, et al.high-level semi-synthetic product of the functional anti-mental ingredient [ J ]. Nature,2013, 7446: methods detailed in 528-532.
The various materials and reagents used in the examples of the present invention are those commonly used in the art and are commercially available in a conventional manner unless otherwise specified.
Noursemycin, available from Solarbio life science; geneticin, purchased from Inalco; hygromycin B, available from Solarbio life science.
The liquid phase detection method for detecting the artemisinin titer has the following conditions: a chromatographic column: diamonsil C18(250 mm. times.460 mm,5 μm), mobile phase: acetonitrile: 0.2% (mass to volume, g/L) phosphoric acid aqueous solution 65:35(v/v), gradient elution, retention time: 25min, flow rate: 1mL/min, sample size: 10 μ L, detection wavelength: 212nm, column temperature: (30. + -. 1) DEG C.
The method for detecting and calculating the hydrolysis rate of the lactose hydrolysate comprises the following steps: taking Rg lactose dissolved in QL water solution as an example, after hydrolyzing with a certain amount of beta-galactosidase, taking 5ml of lactose hydrolysate to fix the volume to a 500ml volumetric flask, namely diluting 100 times, taking 1ml of lactose hydrolysate diluted 100 times, detecting the glucose concentration by an enzyme method to obtain the glucose concentration, and calculating the hydrolysis rate Y after T (g/L), wherein the specific calculation formula is as follows:
Figure BDA0001658162720000061
wherein the content of the first and second substances,
t is glucose concentration detected by an enzyme method, g/L;
r is the amount of lactose, g;
q is the volume of water in which lactose is dissolved, L;
342.3 is the molar mass of lactose, g/mol;
180.16 is the molar mass of glucose, g/mol.
The seed culture medium used in the examples of the present invention was: glucose 19.5g/L, (NH)4)2SO4 15g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, 100ml/L succinate buffer (0.5M, pH 5.0), and the balance water, pH 5.0.
The specific formulas of the vitamin solution and the metal ion solution used in the seed culture medium, the flat solid culture medium and the fermentation culture medium in the embodiment of the invention are as follows:
the vitamin solution is as follows: biotin 0.05 g/L; 1g/L of calcium pantothenate; 1g/L of nicotinic acid; inositol 25 g/L; vitamin B11 g/L; 1g/L pyridoxal; 0.2g/L of p-aminobenzoic acid, and the balance of water.
The metal ion solution is: ZnSO4·7H2O 5.75g/L；MnCl2·4H2O 0.32g/L；CoCl2·6H2O 0.47g/L；NaMoO4·2H2O 0.48g/L；CaCl2·2H2O 2.9g/L；FeSO4·7H2O2.8 g/L; 80ml/L of 0.5M EDTA, and the balance of water.
Comparative example 1:
(1) activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.5% (V/V), and culturing for 16h to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-7After doubling, sucking 100 mu L of the strain and coating the strain on a flat solid culture medium containing 200 mu g/L of hygromycin B, and culturing for 40h to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO4 15g/L，KH2PO4 8g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 24 hours to obtain a seed solution;
(4) fermentation culture: inoculating the seed solution obtained in the step (3) into 20L of fermentation medium without adding exogenous factors according to the inoculation amount of 10% (V/V);
the formula of the fermentation medium is as follows: 25g/L of glucose; 25g/L of galactose; ammonium sulfate is 10 g/L; 6g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 5 g/L; 10ml/L of vitamin solution; 8ml/L of metal ion solution; CuSO4·5H2O30 ug/L, and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 14mol/L ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
Continuously adding 700g/L of sucrose aqueous solution from 15h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 2% of the initial volume of 20L of the fermentation culture medium, continuously adding 300g/L of sodium glutamate aqueous solution from 40h of fermentation culture to the end of fermentation, the fed-batch volume per hour is 0.2% of the initial volume of 20L of the fermentation culture medium, and after 52h of fermentation culture, once supplementing isopropyl palmitate and oleic acid, wherein the supplemented volumes are respectively 15% of the initial volume of 20L of the fermentation culture medium; and after fermentation culture for 120h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 24.6 g/L.
Example 1
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.5 percent, and culturing for 16 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-7After doubling, sucking 100 mu L of the strain and coating the strain on a flat solid culture medium containing 200 mu g/L of hygromycin B, and culturing for 40h to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO4 15g/L，KH2PO4 8g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 24 hours to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing mevalonic acid and citral according to the inoculation amount of 10% for culture, wherein the concentrations of the mevalonic acid and the citral in the culture medium are 0.1g/L respectively;
the formula of the fermentation medium is as follows: 0.1L/L lactose hydrolysate; ammonium sulfate is 10 g/L; 6g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 5 g/L; 10ml/L of vitamin solution; 8ml/L of metal ion solution; CuSO4·5H2O30 ug/L, mevalonic acid 0.1 g/L; 0.1g/L of citral; the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 14mol/L ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the following steps: dissolving 1144g lactose in 2L water, adding 12g beta-galactosidase, hydrolyzing at 53 deg.C and pH 4.0 for 4h to obtain hydrolysis rate of 93%, mixing all lactose hydrolysate into fermentation medium with initial volume of 20L to obtain a mass volume ratio of glucose and galactose in lactose hydrolysate to fermentation medium of 28:1(g/L)
Continuously adding 700g/L of sucrose aqueous solution from 15h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 2% of the initial volume of 20L of the fermentation culture medium, continuously adding 300g/L of sodium glutamate aqueous solution from 40h of fermentation culture to the end of fermentation, the fed-batch volume per hour is 0.2% of the initial volume of 20L of the fermentation culture medium, after 52h of fermentation culture, supplementing isopropyl palmitate and oleic acid once, and the supplemented volumes are 15% of the initial volume of 20L of the fermentation culture medium respectively; and after fermentation culture for 120h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 41.8 g/L.
Comparative example 1 is different from example 1 in that (i) lactose hydrolysate is added to the fermentation medium of example 1 and galactose is added to comparative example 1, (ii) exogenous factors mevalonic acid and citral are added to example 1 and no exogenous factors are added to comparative example 1. Compared with the comparative example 1, the lactose hydrolysate is used in the fermentation culture medium of the example 1 to replace the galactose in the comparative example 1, when the lactose hydrolysate is used in the culture medium, a large amount of galactose does not need to be added separately, so that the fermentation cost is obviously reduced, and in addition, the titer of the comparative example 1 is 24.6g/L, the titer of the example 1 is 41.8g/L, and the yield of the artemisinic acid is obviously improved. Therefore, the use of the lactose hydrolysate greatly reduces the fermentation cost on the premise of ensuring the arteannuic acid yield, and the addition of the exogenous factors significantly improves the arteannuic acid fermentation yield.
Example 2
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.1% (V/V), and culturing for 30h to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-5After doubling, sucking 100 mu L of the culture medium, coating the culture medium on a flat solid culture medium containing 300 mu g/L of nourseothricin, and culturing for 30h to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO4 15g/L，KH2PO4 8g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinic acid buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 16h to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step 3) into a 20L fermentation culture medium containing mevalonic acid according to the inoculation amount of 10% (V/V) for culture, wherein the concentration of mevalonic acid in the culture medium is 0.3 g/L;
the formula of the fermentation medium is as follows: 0.05L/L lactose hydrolysate; 8g/L of ammonium sulfate; 5g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 3 g/L; the vitamin solution is 8 ml/L; 6ml/L of metal ion solution; CuSO4·5H2O20 ug/L; 0.3g/L of mevalonic acid and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the following steps: 633g of lactose is dissolved in 1L of water, 0.633g of beta-galactosidase is added, hydrolysis is carried out for 5h under the conditions that the pH is 7.0 and the temperature is 50 ℃, the hydrolysis rate is 90%, all lactose hydrolysate is mixed into a fermentation medium, the initial volume of the fermentation medium is 20L, and the mass volume ratio of glucose and galactose in the lactose hydrolysate to the fermentation medium is respectively 15:1 (g/L).
Continuously adding 400g/L lactose hydrolysate from 10h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 1% of the initial volume of 20L of the fermentation culture medium, continuously adding 200g/L yeast extract powder aqueous solution from 36h of fermentation culture to the end of fermentation, the fed-batch volume per hour is 0.05% of the initial volume of 20L of the fermentation culture medium, and after 48h of fermentation culture, supplementing isopropyl palmitate once, wherein the supplemented volume is 5% of the initial volume of 20L of the fermentation culture medium; and after fermentation culture for 84h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
The preparation method of the lactose hydrolysate by feeding comprises the following steps: 8000g of lactose is dissolved in 20L of water, 168.5g of beta-galactosidase is added, and the hydrolysis is carried out for 5h under the conditions that the pH is 5.5 and the temperature is 50 ℃, the hydrolysis rate is 95 percent, and then 400g/L of lactose hydrolysate is obtained.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 38.9 g/L.
Example 3
(1) Preparing strain liquid: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.3 percent, and culturing for 20 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: subjecting the product obtained in the step (1)Diluting the obtained strain liquid 10-6After doubling, sucking 100 mu L of the culture medium, coating the culture medium on a flat solid culture medium containing 250 mu g/L of geneticin, and culturing for 35 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO4 15g/L，KH2PO4 8g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 20h to obtain a seed solution;
(4) fermentation culture: inoculating the seed solution obtained in the step 3) into a 20L fermentation culture medium containing citral according to the inoculation amount of 10% for culture, wherein the concentration of the citral in the culture medium is 0.4 g/L;
the formula of the fermentation medium is as follows: 0.075L/L of lactose hydrolysate; 15g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 6.2 g/L; the vitamin solution is 12 ml/L; 10ml/L of metal ion solution; CuSO4·5H2O40 ug/L; 0.4g/L of citral and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 14.5mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the following steps: dissolving 800g of lactose in 1.5L of water, adding 10g of beta-galactosidase, hydrolyzing for 3h under the conditions of pH 5.5 and 55 ℃, wherein the hydrolysis rate is 95%, mixing all lactose hydrolysate into a fermentation medium, and the initial volume of the fermentation medium is 20L, so that the mass-to-volume ratio of glucose and galactose in the lactose hydrolysate to the fermentation medium is respectively 20: 1 (g/L).
Continuously feeding 450g/L of glycerol aqueous solution from 12h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 0.5 percent of the initial volume of 20L of the fermentation culture medium, continuously feeding 250g/L of peptone aqueous solution from 38h of fermentation culture to the end of fermentation, the fed-batch volume per hour is 0.1 percent of the initial volume of 20L of the fermentation culture medium, and replenishing oleic acid once after 50h of fermentation culture, wherein the replenished volume is 10 percent of the initial volume of 20L of the fermentation culture medium; and after fermentation culture for 96h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 39.7 g/L.
Example 4
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.8 percent, and culturing for 18 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-7After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media containing 150 mu g/L of nourseothricin and geneticin respectively, and culturing for 45 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO4 15g/L，KH2PO4 8g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 28h to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing mevalonic acid and citral according to the inoculation amount of 10% for culture, wherein the concentrations of the mevalonic acid and the citral in the culture medium are 0.2g/L respectively;
the formula of the fermentation medium is as follows: 0.1L/L lactose hydrolysate; 12g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; 6g/L magnesium sulfate heptahydrate; the vitamin solution is 11 ml/L; 8ml/L of metal ion solution; CuSO4·5H2O35 ug/L; 0.2g/L of mevalonic acid; 0.2g/L of citral and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13.5mol/L of ammonia to ensure that the fermentation is carried outThe pH is maintained between 5.0 and 5.5 during the fermentation.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the following steps: 1212g of lactose was dissolved in 2L of water, 24.2g of beta-galactosidase was added and the mixture was hydrolyzed at 52 ℃ at pH 5.0 for 3h with a hydrolysis rate of 94%, all lactose hydrolysate was mixed into the fermentation medium with an initial volume of 20L, so that the mass to volume ratios of glucose and galactose in the lactose hydrolysate to the fermentation medium were 30: 1 (g/L).
Continuously adding 65% (V/V) ethanol aqueous solution from 16h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 1.2% of the initial volume of 20L of the fermentation culture medium, continuously adding 350g/L yeast extract powder aqueous solution and 350g/L peptone aqueous solution from 42h of fermentation culture to the end of fermentation, wherein the fed-batch volume of the nitrogen source per hour is 0.25% of the initial volume of 20L of the fermentation culture medium, supplementing isopropyl palmitate and oleic acid once after 55h of fermentation culture, and the supplementing volume is 20% of the initial volume of 20L of the fermentation culture medium; and after fermentation culture for 84h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of artemisinic acid of 40.6 g/L.
Example 5
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.4 percent, and culturing for 24 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-6After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media containing 50 mu g/L of nourseothricin and hygromycin B respectively, and culturing for 50h to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO4 15g/L，KH2PO4 8g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0)5M, pH 5.0), agar 20g/L, balance water.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 30h to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing mevalonic acid according to the inoculation amount of 10% for culture, wherein the concentration of mevalonic acid in the culture medium is 0.5 g/L;
the formula of the fermentation medium is as follows: 0.075L/L of lactose hydrolysate; 15g/L of ammonium sulfate; 7g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 5.2 g/L; the vitamin solution is 12 ml/L; 9ml/L of metal ion solution; CuSO4·5H2O40 ug/L; 0.5g/L of mevalonic acid and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13.8mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the following steps: dissolving 800g of lactose in 1.5L of water, adding 10g of beta-galactosidase, hydrolyzing for 3h under the conditions of pH 5.5 and 55 ℃, wherein the hydrolysis rate is 95%, mixing all lactose hydrolysate into a fermentation medium, and the initial volume of the fermentation medium is 20L, so that the mass-to-volume ratio of glucose and galactose in the lactose hydrolysate to the fermentation medium is respectively 20: 1 (g/L).
Continuously adding 500g/L lactose hydrolysate and 500g/L glycerol aqueous solution from 18h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 0.8 percent of the initial volume of the fermentation culture medium of 20L, continuously adding 400g/L peptone aqueous solution and 400g/L sodium glutamate aqueous solution from 45h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 0.2 percent of the initial volume of the fermentation culture medium of 20L, and supplementing isopropyl palmitate once after 50h of fermentation culture, wherein the supplemented volume is 15 percent of the initial volume of the fermentation culture medium of 20L; and after fermentation culture for 96h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
The preparation method of the lactose hydrolysate by feeding comprises the following steps: 10000g of lactose is dissolved in 20L of water, 210g of beta-galactosidase is added, and the hydrolysis is carried out for 5h under the conditions that the pH is 5.5 and the temperature is 50 ℃, the hydrolysis rate is 95 percent, and then the lactose hydrolysate of 500g/L is obtained.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 42.1 g/L.
Example 6
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.6 percent, and culturing for 16 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-7After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media containing 100 mu g/L of geneticin and hygromycin B respectively, and culturing for 35 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO4 15g/L，KH2PO4 8g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 24 hours to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing citral according to the inoculation amount of 10% to culture, wherein the concentration of the citral in the culture medium is 0.2 g/L;
the formula of the fermentation medium is as follows: 0.075L/L of lactose hydrolysate; 15g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; 6g/L magnesium sulfate heptahydrate; the vitamin solution is 12 ml/L; 10ml/L of metal ion solution; CuSO4·5H2O40 ug/L; 0.2g/L of citral and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13.5mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the following steps: 633g of lactose is dissolved in 1.5L of water, then 3.2g of beta-galactosidase is added, the hydrolysis is carried out for 5h under the conditions that the pH is 5.5 and the temperature is 55 ℃, the hydrolysis rate is 90 percent, all lactose hydrolysate is mixed into a fermentation medium, the initial volume of the fermentation medium is 20L, and the mass-to-volume ratio of glucose and galactose in the lactose hydrolysate to the fermentation medium is 15:1 (g/L).
Starting from 17h of fermentation culture until the end of fermentation, continuously adding 600g/L of glycerol aqueous solution and 600g/L of sucrose aqueous solution, wherein the fed-batch volume per hour is 0.7 percent of the initial volume of the fermentation culture medium of 20L respectively, starting from 40h of fermentation culture until the end of fermentation, continuously adding 450g/L of yeast extract powder aqueous solution, 450g/L of peptone aqueous solution and 450g/L of sodium glutamate aqueous solution, wherein the fed-batch volume per hour is 0.15 percent of the initial volume of the fermentation culture medium of 20L respectively, supplementing oleic acid once after 48h of fermentation culture, and the supplemented volume is 20 percent of the initial volume of the fermentation culture medium of 20L; and after fermentation culture for 120h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 38.5 g/L.
Example 7
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.7 percent, and culturing for 20 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-7After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media containing 80 mu g/L of geneticin and hygromycin B respectively, and culturing for 45 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO4 15g/L，KH2PO4 8g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 28h to obtain a seed solution;
(4) fermentation culture: inoculating the seed solution obtained in the step 3) into a 20L fermentation culture medium containing citral according to the inoculation amount of 10% for culture, wherein the concentration of the citral in the culture medium is 0.1 g/L;
the formula of the fermentation medium is as follows: 0.1L/L lactose hydrolysate; 13g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 6.2 g/L; 10ml/L of vitamin solution; 10ml/L of metal ion solution; CuSO4·5H2O30 ug/L; 0.1g/L of citral and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 14mol/L ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the following steps: dissolving 1239g of lactose in 2L of water, adding 5.2g of beta-galactosidase, hydrolyzing for 5h under the conditions of pH 5.5 and 52 ℃, wherein the hydrolysis rate is 92%, mixing all lactose hydrolysate into a fermentation medium, and the initial volume of the fermentation medium is 20L, so that the mass-to-volume ratios of glucose and galactose in the lactose hydrolysate to the fermentation medium are respectively 30: 1 (g/L).
Continuously adding 700g/L of sucrose aqueous solution and 70% (V/V) of ethanol aqueous solution from the beginning of fermentation culture for 13h until the end of fermentation, wherein the adding volume per hour is respectively 0.1% of the initial volume of the fermentation culture medium for 20L, continuously adding 500g/L of yeast extract powder aqueous solution and 500g/L of sodium glutamate aqueous solution from the beginning of fermentation culture for 44h until the end of fermentation, wherein the adding volume per hour is respectively 0.2% of the initial volume of the fermentation culture medium for 20L, and after the fermentation culture for 53h, supplementing isopropyl palmitate and oleic acid for one time, and the supplementing volume is respectively 18% of the initial volume of the fermentation culture medium for 20L; and after fermentation culture for 90 hours, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 36.7 g/L.
Example 8
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.2 percent, and culturing for 30 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-5After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media respectively containing 60 mu g/L of nourseothricin, geneticin and hygromycin B, and culturing for 48 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO4 15g/L，KH2PO4 8g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 25h to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing mevalonic acid and citral according to the inoculation amount of 10% for culture, wherein the concentrations of the mevalonic acid and the citral in the culture medium are 0.25g/L respectively;
the formula of the fermentation medium is as follows: 0.1L/L lactose hydrolysate; 15g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; 6g/L magnesium sulfate heptahydrate; the vitamin solution is 11 ml/L; 8ml/L of metal ion solution; CuSO4·5H2O38 ug/L; 0.25g/L of mevalonic acid; 0.25g/L of citral; the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13.5mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the following steps: 1156g of lactose is dissolved in 2L of water, 9.8g of beta-galactosidase is added, hydrolysis is carried out for 5h under the conditions that the pH is 5.5 and the temperature is 50 ℃, the hydrolysis rate is 92%, all lactose hydrolysate is mixed into a fermentation medium, the initial volume of the fermentation medium is 20L, and the mass-to-volume ratios of glucose and galactose in the lactose hydrolysate to the fermentation medium are respectively 28:1 (g/L).
Continuously adding 550g/L lactose hydrolysate, 550g/L glycerol aqueous solution, 550g/L sucrose aqueous solution and 55% (V/V) ethanol aqueous solution from 15h of fermentation culture to the end of fermentation, wherein the fed-in volume per hour is 0.3 percent of the initial volume of the fermentation culture medium 20L, continuously adding 400g/L yeast extract powder aqueous solution, 400g/L peptone aqueous solution and 400g/L sodium glutamate aqueous solution from 39h of fermentation culture to the end of fermentation, wherein the fed-in volume per hour is 0.1 percent of the initial volume of the fermentation culture medium 20L, and supplementing isopropyl palmitate and oleic acid once after 51h of fermentation culture, wherein the supplemented volume is 5 percent of the initial volume of the fermentation culture medium 20L; and after fermentation culture for 120h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
The preparation method of the lactose hydrolysate by feeding comprises the following steps: 11000g of lactose is dissolved in 20L of water, 232g of beta-galactosidase is added, and hydrolysis is carried out for 5h under the conditions that the pH is 5.5 and the temperature is 50 ℃, the enzymolysis rate is 95 percent, and then 550g/L of lactose hydrolysate is obtained.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 39.4 g/L.

Claims (19)

1. The method for improving fermentation yield of artemisinic acid is characterized by comprising the steps of taking saccharomyces cerevisiae engineering bacteria which need galactose to induce synthesis of the artemisinic acid and produce the artemisinic acid as production strains, fermenting the strains in a fermentation culture medium containing lactose hydrolysate and exogenous factors, wherein the lactose hydrolysate is a solution obtained by hydrolyzing lactose into glucose and galactose through enzyme, and the exogenous factors are one or two of mevalonic acid and citral.
2. The method of claim 1, wherein the enzyme is β -galactosidase.
3. The method according to claim 2, wherein the mass ratio of β -galactosidase to lactose is 0.1-2: 100.
4. the method according to claim 1, wherein the mass-to-volume ratio of the amounts of glucose and galactose in the lactose hydrolysate to the fermentation medium is 15-30: 1, unit is g/L.
5. The process according to claim 1, wherein the lactose is hydrolyzed by the enzyme at a hydrolysis temperature of 50-55 ℃, for a hydrolysis time of 3-5h, at a hydrolysis rate of 90-95% and at a hydrolysis pH of 4-7.
6. The process according to claim 5, wherein the hydrolysis pH of the lactose by enzymatic hydrolysis is 5.5.
7. The method of claim 1, wherein the exogenous factor is present in the fermentation medium at a concentration of 0.1 to 0.5 g/L.
8. The process according to any one of claims 1 to 7, wherein the fermentation time is 84 to 120 hours and the fermentation temperature is 22 to 35 ℃.
9. The method according to any one of claims 1 to 7, wherein the fermentation further comprises feeding a carbon source, a nitrogen source and a water-insoluble oily liquid, wherein the water-insoluble oily liquid is one or both of isopropyl palmitate and oleic acid.
10. The method of claim 9, wherein the feeding of the carbon source is performed for a period of time from fermentation time to fermentation time of 10-18h, and the feeding of the carbon source is performed by one or more of lactose hydrolysate, glycerol aqueous solution, sucrose aqueous solution and ethanol aqueous solution.
11. The method as claimed in claim 10, wherein the concentration of the lactose hydrolysate is 400-700g/L, the concentration of the glycerol aqueous solution is 400-700g/L, the concentration of the sucrose aqueous solution is 400-700g/L, and the concentration of the ethanol aqueous solution is 80-99% (V/V).
12. The method of claim 11, wherein the rate of feeding the carbon source is controlled such that the volume of the carbon source fed per hour is 0.2% to 2% of the initial volume of the fermentation medium.
13. The method of claim 9, wherein the time of feeding the nitrogen source is from 36-45h from fermentation to the end of fermentation, and the nitrogen source is one or more of yeast-rich powder aqueous solution, peptone aqueous solution and sodium glutamate aqueous solution.
14. The method as claimed in claim 13, wherein the concentration of the yeast powder soaking water solution is 200-500g/L, the concentration of the peptone water solution is 200-500g/L, and the concentration of the sodium glutamate water solution is 200-500 g/L.
15. The method of claim 14, wherein the feeding of the nitrogen source is performed at a rate such that the volume of the fed nitrogen source per hour is 0.05 to 0.5% of the initial volume of the fermentation medium.
16. The method as claimed in claim 9, wherein the water-insoluble oily liquid is fed in one time after 48-55h of fermentation.
17. The method of claim 16, wherein the volume of the fed water-insoluble oily liquid is 5% to 40% of the initial volume of the fermentation medium.
18. The method according to any one of claims 1 to 7, wherein the production strain is further subjected to resistance selection on a plate solid medium containing an antibiotic, wherein the antibiotic is one or more of noursemycin, geneticin and hygromycin B.
19. The method of claim 18, wherein the antibiotic is present in an amount of 50-300 μ g/L on the plate solid medium.
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