CN114736815A - High-yield sphingolipid microbial strain, screening method and application thereof - Google Patents
High-yield sphingolipid microbial strain, screening method and application thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The application relates to a Wickerhamomyces ciferrii (CGMCC No: 19562), its screening method, and its application in producing Tetraacetylphytosphingosine (TAPS).
Description
Technical Field
The application relates to the technical field of microbial engineering, in particular to a yeast of Wickerhamomyces ciferrii with the preservation number of CGMCC No:19562, a screening method thereof and application thereof in producing tetraacetyl phytosphingosine (TAPS).
Background
Sphingolipids (Sphingolipids) refer to Lipids (Lipids) having a long-chain base (long-chain base) as a backbone, and include four types, i.e., free thiols, ceramides, sphingophospholipids, and glycosphingolipids. Sphingolipids are mainly present in the membranes of animal, plant and microbial cells, and they play many important roles not only in the important components of biological membranes but also in living cells. In addition, sphingolipids are the main component of the stratum corneum on human skin, and constitute a specific lamellar quasicrystal structure with cholesterol, cholesterol sulfate and free fatty acids, which is the skin's permeability barrier (Menon et al (2012)). Therefore, the compounds have high application potential in various application fields such as antibacterial drugs, antifungal drugs, active pharmaceutical ingredients in therapeutics, active ingredients in cosmeceutical or nutritional preparations and the like.
At present, sphingolipid preparations used in cosmetics are mainly extracted from animals and plants. Obviously, this is a very costly process on an industrial level. The chemical synthesis method has the defects of complicated steps, more byproducts, lower synthesis efficiency and the like, and is difficult to realize industrial production.
Strain h. Ciferri NRRL Y-1031 was isolated by Ciferri, Lodder (1932) and filamentous forms of this strain were observed by Bedford (1942) and Wickerham (1951). Ascospore isolates can be obtained from ascospores of this strain according to the method of Wickerham (1958) and Burton (1954) and the sex of the ascospore isolates determined. In 1956, the H.ciferri NRRL Y-1031 mating type 11 strain was cultivated and a large amount of crystals, tetraacetylphytosphingosine, was first found, a compound that has never been previously reported from natural sources (Wickerham and stodora, 1960).
Furthermore, the production of phytosphingosine and/or acetylated derivatives thereof has been shown in the yeasts Candida utilis (Candida utilis) and Saccharomyces cerevisiae (Saccharomyces cerevisiae) (Wangner and Zofcsik, 1966; Oda and Kamiya,1958), Hansenlaspora valbyensis (Braun and snell, 1967), Aspergillus saxatilis (Aspergillus sydow) and Penicillium notatum (Stodola and Wickerham, 1960).
Unlike all known organisms, Wickerhamomyces ciferrii (i.e.H.ciferri NRRL Y-1031, also known as Pichia ciferrii) has the ability to produce fully acetylated sphingoid bases, in particular plant sphingoid bases, and to secrete them in large quantities (Wickerham and Stodola 1960). Making this non-traditional yeast a suitable host for biotechnological production of sphingolipids. Fermentation with the w.ciferrii mating type strain F-60-10 started as early as 1962, with yields of up to 233mg (TAPS)/L and 5mg TAPS per g glucose (Maister et al, 1962).
In recent years, considerable progress has been made in attempts to optimize the yield of TAPS using fermentation technology and mutagenesis technology, and strains and processes that can be produced by fermentation at an industrial level have been obtained. WO94/10131A1(PCT/GB93/02230, 1994) discloses that the highest yield of 2083mg of TAPS/L fermentation broth can be obtained by fed-batch fermentation at 30 ℃ by taking Pichia ciferri NRRL Y-1031 mating strain F-60-10 as a preferred strain, adding ethanol and methanol as inducers, serine and palmitoyl as precursors, glycerol as a carbon source and Tween and Triton as surfactants. Patent EP0688871A2 discloses a method for preparing tetraacetylphytosphingosine by using Wickerhamomyces ciferrii mating strain F-60-10, the strain is subjected to mutagenesis, a high-yield strain is obtained through a specific screening program, a culture medium formula and a fermentation process are optimized, and the yield of TAPS is more than 1.0 g/L. The patent W09512683A1(PCT/EP94/03652) provides a method for producing microbial strains capable of producing higher levels of sphingosine, sphinganine, phytosphingosine or derivatives thereof by mutagenesis and selection techniques (starting with Wickerhamomyces ciferrii NRRL Y-1031F-60-10). Compared with the parent strain cultured under the same condition, the yield level of the mutant strain obtained by screening is 40-60% higher than that of the parent strain.
In 2001, patent US6194196B1 concluded that a completely new Pichia mating strain could be produced with a higher TAPS yield than the F-60-10 mating strain by analyzing early studies of Wickerham and co-workers (Wickerham and Stodola, 1960) that genetic recombination during meiosis of diploid Pichia mating forms mating spores. Based on the method, a screening method of a high-yield strain is developed, a strain (KCCM-10131) with high yield is finally screened, and the maximum TAPS yield can reach 14g/L through fermentation optimization. However, both the mating type screening method reported in this patent and the mating type strain screening method employed by Wickerham and colleagues thereof have problems that the cap spore-obtaining efficiency is low (the cap spore-forming efficiency of this patent is 6% to 8%), the difficulty of screening mating type strains is too high, it is difficult to obtain culturable mating type strains in the actual operation, or the number of strains obtained is small, and it is difficult to screen high-yielding strains therefrom.
Based on the above results, we hypothesized that during meiosis of diploid Wickerhamomyces ciferrii to form ascospores, recombination and mismatching of genetic material leads to the formation of thousands of different spores (theoretically infinite number of spores), wherein strains with TAPS yields much higher than those of mating types F-60-10 and KCCM-10131 may be present. Therefore, a brand-new spore-producing culture medium (WCCB culture medium) is adopted to improve the spore yield of the diploid strain Wickerhamomyces ciferrii to 50.0% -75.0%, and simultaneously, the enrichment of spores and a verification method of mating type strains are optimized.
Disclosure of Invention
1. A yeast strain belonging to the genus Vickers of the genus Wilkhammyces (Wickerhamamymyces ciferrii) has a preservation number of CGMCC No. 19562.
2. The yeast of item 1, wherein the yeast produces at least 700mg of Tetra Acetyl Phytosphingosine (TAPS)/L fermentation broth under batch shake flask culture conditions.
3. The yeast of item 2, wherein the batch shake flask culture conditions are at least 72h in a flask containing YM medium at appropriate rotation speed and temperature; wherein the YM culture medium comprises 8.0-16.0 g/L of glucose, 3.0-7.0 g/L of peptone, 2.0-4.0 g/L of yeast extract and 1.5-4.5 g/L of malt extract.
4. The yeast of item 1, wherein the yeast produces at least 21.0g TAPS per liter of fermentation broth under fed batch culture conditions.
5. The yeast of item 4, wherein the conditions of the fed-batch culture are: the culture medium comprises 3.5-8.5 g/L of yeast extract powder, 3.5-8.5 g/L of L-serine, 15.0-70.0 g/L of glycerol, 1.5-5.5 g/L of peptone and KH2PO42.0-7.5 g/L, 1.5-5.5 g/L malt extract, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO4 1.5~8.5g/L,KCl 0.2~2.4g/L,CaCl20.3-2.6 g/L; controlling the dissolved oxygen to be more than 20% at a proper fermentation temperature and stirring speed; fed-batch mode: feeding operation is carried out when the carbon source is exhausted, and 30-70% (W/W) of carbon source is fed in at a feeding speed of 6-30 ml/L fermentation liquor/h until fermentation is finished after 5 days.
6. A composition comprising saccharomyces weckerhamilthiavalieri (Wickerhamomyces ciferrii) and a fermentation broth comprising Tetraacetylphytosphingosine (TAPS).
7. The composition of item 6, wherein the concentration of TAPS is 700mg/L or greater in the fermentation broth obtained from batch shake flask culture.
8. The composition according to item 7, wherein the culture conditions of the batch shake flask culture are culture in a flask containing YM medium at a suitable rotation speed and temperature for at least 72 hours; wherein the YM culture medium comprises 8.0-16.0 g/L of glucose, 3.0-7.0 g/L of peptone, 2.0-4.0 g/L of yeast extract and 1.5-4.5 g/L of malt extract.
9. The composition of item 6, wherein the concentration of TAPS in the fermentation broth obtained from fed-batch culture is 21.0g/L or more.
10. The composition of item 9, wherein the fed-batch culture is cultured under the following conditions: the culture medium comprises 3.5-8.5 g/L of yeast extract powder, 3.5-8.5 g/L of L-serine, 15.0-70.0 g/L of glycerol, 1.5-5.5 g/L of peptone and KH2PO42.0-7.5 g/L, 1.5-5.5 g/L malt extract, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO4 1.5~8.5g/L,KCl 0.2~2.4g/L,CaCl20.3-2.6 g/L; controlling the dissolved oxygen to be more than 20% at a proper fermentation temperature and stirring speed; fed-batch mode: and (3) feeding the carbon source at the carbon source exhaustion rate, wherein 30-70% (W/W) of the carbon source is fed at the feeding speed of 6-30 ml/L fermentation liquor/h until the fermentation is finished after 5 days.
11. A culture medium, comprising: 3.5-8.5 g/L yeast extract powder, 3.5-8.5 g/L-serine, 15.0-70.0 g/L glycerol, 1.5-5.5 g/L peptone and KH2PO42.0-7.5 g/L, 1.5-5.5 g/L malt extract, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO4 1.5~8.5g/L,KCl 0.2~2.4g/L,CaCl2 0.3~2.6g/L。
12. A screening method of yeast belonging to the genus Sacchrom, belonging to the genus Wickerhamomyces, comprising the following steps:
-inducing ascospores using an induction medium on a parent strain of yeast vickers-simva, which is capable of producing tetraacetylphytosphingosine, with a spore yield of 50.0% to 75.0%;
-isolating mating type strains;
-selecting a tetra-acetylated phytosphingosine-producing strain from said mating type strain by a batch shake flask cultivation experiment.
13. The screening method according to item 12, wherein the induction medium is a WCCB medium comprising: 6.3-10.5 g/L of sodium acetate, 11.7-23.6 g/L of potassium chloride, 0.09-0.34 g/L of magnesium sulfate, 0.43-1.76 g/L of glucose and 12.0-20.0 g/L of agar powder.
14. The screening method of item 13, wherein the WCCB medium comprises: 7.3-9.5 g/L of sodium acetate, 13.7-21.6 g/L of potassium chloride, 0.14-0.30 g/L of magnesium sulfate, 0.63-1.46 g/L of glucose and 14.0-18.0 g/L of agar powder.
15. The screening method according to item 13 or 14, wherein, in the step of inducing ascospores, the following induction culture conditions are used: aerobic culture is carried out for 3-7 days at 23-29 ℃.
16. The screening method according to item 12, wherein the following steps are carried out in the step of isolating a mating type strain:
-preparing a bacterial suspension of ascospores;
adding helicase/zymolyse/cell lysate with a final concentration of 0.5% -5.0% and a certain amount of quartz sand/glass beads with a diameter of 0.1-0.8 mm, shaking the mixture in a shaker at 100-320 rpm at 25-35 ℃ for 0.5-6.0 hours to break the cyst spore wall and release spores in the cyst wall;
adding the spore liquid after the wall breaking treatment into a sterile centrifuge tube, and carrying out water bath at 48-60 ℃ for 10-50 min to inactivate diplocardia in the spore liquid and enrich spores.
17. The screening method according to item 16, wherein the following is performed in the step of isolating a mating type strain:
-preparing a bacterial suspension of ascospores;
adding 1.0-4.0% snailase/zymolyse/cell lysate and a certain amount of quartz sand/glass beads with the diameter of 0.2-0.6 mm, shaking the mixture in a shaking table at 150-260 rpm at the temperature of 28-33 ℃ for 1.0-4.0 hours, breaking the walls of the ascospores, and releasing the spores;
adding the spore liquid after the wall breaking treatment into a sterile centrifuge tube, and carrying out water bath at 50-58 ℃ for 15-40 min to inactivate diplocardia in the spore liquid and enrich spores.
18. A culture medium, comprising: 6.3-10.5 g/L of sodium acetate, 11.7-23.6 g/L of potassium chloride, 0.09-0.34 g/L of magnesium sulfate, 0.43-1.76 g/L of glucose and 12.0-20.0 g/L of agar powder.
19. A method for producing Tetraacetylphytosphingosine (TAPS) by fermentation culture using the yeast Veckerhamella (Wickerhamomyces ciferrii) described in item 1.
20. The method of producing Tetraacetylphytosphingosine (TAPS) by a fermentation culture using a yeast, wackerhamomyces ciferrii, as claimed in item 19, wherein the fermentation culture is a batch shake flask culture; at least 700mg/L of TAPS fermentation broth can be produced.
21. The method of producing Tetraacetylphytosphingosine (TAPS) by fermentation culture using a yeast of the genus wilkhammyces ciferrii as described in item 20, wherein the production conditions of the batch shake flask culture are: culturing in a triangular flask containing YM culture medium at proper rotation speed and temperature for at least 72 h; wherein the YM culture medium comprises 8.0-16.0 g/L of glucose, 3.0-7.0 g/L of peptone, 2.0-4.0 g/L of yeast extract and 1.5-4.5 g/L of malt extract.
22. The method of producing Tetraacetylphytosphingosine (TAPS) by a fermentation culture using a yeast strain of the genus wilhelminthomyces ciferrii as described in item 19, wherein the fermentation culture is a fed-batch culture; at least 21.0g/L of TAPS broth can be produced.
23. The method of producing Tetraacetylphytosphingosine (TAPS) by fermentation culture using saccharomyces weckerhamensis (Wickerhamomyces ciferrii) according to item 22, wherein the fed-batch culture is produced under the conditions: the culture medium comprises 3.5-8.5 g/L of yeast extract powder, 3.5-8.5 g/L of L-serine, 15.0-70.0 g/L of glycerol, 1.5-5.5 g/L of peptone and KH2PO42.0-7.5 g/L, 1.5-5.5 g/L malt extract, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO4 1.5~8.5g/L,KCl 0.2~2.4g/L,CaCl20.3-2.6 g/L; controlling the dissolved oxygen to be more than 20% at a proper fermentation temperature and stirring speed; fed-batch mode: feeding operation is carried out when the carbon source is exhausted, and 30-70% (W/W) of carbon source is fed in at a feeding speed of 6-30 ml/L fermentation liquor/h until fermentation is finished after 5 days.
The technical scheme of this application obtains beneficial effect:
the applicant adopts a brand-new spore-producing culture medium (WCCB culture medium) to improve the spore yield of the diploid strain Wickerhamomyces ciferrii to 50.0% -75.0%, and simultaneously optimizes the enrichment of spores and a verification method of mating type strains. And screening the obtained mating type strains at different levels (firstly, primarily screening the obtained mating type strains on a plate, primarily screening the crystal-producing strains in a shake flask culture mode, and finally, carrying out fermentation verification on the strains with higher yield) to obtain high-yield strains.
Drawings
FIG. 1 is a micrograph of ascospores from which a parent yeast Veckham Schiff, which produces TAPS, was induced;
FIG. 2 is a graph showing spore yields of Saccharomyces weckerhamensis (Wickerhamomyces ciferrii) on different spore culture media; wherein, the formula of the McClary culture medium is as follows: 1.00g/L of glucose, 1.80g/L of potassium chloride, 8.20g/L of sodium acetate, 2.50g/L of yeast extract and 1.50g/L of agar powder; the MA5 culture medium formula is as follows: malt extract: 50.00g/L of agar powder and 1.50g/L of agar powder; the Kleyn culture medium formula comprises: 2.50g/L of peptone, 0.62g/L of glucose, 0.62g/L of sodium chloride, 5.00g/L of sodium acetate and 1.50g/L of agar powder; the formula of the Fowell culture medium is as follows: 8.20g/L of sodium acetate, 0.40g/L of raffinose and 1.50g/L of agar powder;
FIG. 3 is a microscopic photograph of aged fermentation liquid of high-yield strain CGMCC No. 19562;
FIG. 4 shows the HPLC detection results of the fermentation product, wherein A is the fermentation extract product and B is the TAPS standard (sigma);
FIG. 5 is the growth curve of high yield strain CGMCC No.19562 in the fermentation process.
Detailed Description
It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
The present application relates in a first aspect to a yeast.
In one embodiment, there is provided a yeast, saccharomyces weckerhamensis (wickerhamamyyces ciferrii); the strain is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 of Xilu No.1 of Beijing university of Chaoyang district, institute of microbiology of China academy of sciences) at 13.4 months in 2020; the preservation number is CGMCC No. 19562.
In the context of this specification, yeast is defined generally in the field of biology as a unicellular fungus, a tiny unicellular microorganism invisible to the naked eye, capable of fermenting sugars to alcohol and carbon dioxide, distributed throughout the natural world, is a typical heterotrophic facultative anaerobic microorganism, capable of surviving both aerobic and anaerobic conditions, and is a natural starter culture.
In one embodiment, a yeast is provided, wherein the yeast is cultured in a 10% liquid content Erlenmeyer flask of YM medium at 220rpm at 30 ℃ for 72h to produce at least 700mg of Tetraacetylphytosphingosine (TAPS)/L fermentation broth; and at least 21.0g TAPS/L of fermentation broth can be produced after 5 days of fed-batch fermentation culture.
In the context of the present specification, "Sphingolipids" conform to the general definition in the field of biochemistry and Sphingolipids (Sphingolipids) refer to Lipids (Lipids) based on long-chain bases (long-chain) and include four types of free-thiols, ceramides, sphingophospholipids and glycosphingolipids. Sphingolipids are mainly present in the membranes of animal, plant and microbial cells, and they play many important roles not only as an important component of biological membranes, but also in living cells. In addition, sphingolipids are the main component of the stratum corneum on human skin, constitute a specific lamellar quasicrystalline structure with cholesterol, cholesterol sulfate and free fatty acids, and are the skin's permeation barrier (Menon et al (2012)).
In the context of this specification, "Tetraacetylphytosphingosine (TAPS)" is a subordinate concept of "sphingolipids", which is known in the english name Tetraacetylphytosphingosine, and is a nonionic surfactant (belonging to esters) which is useful as a conditioner and the like, and is used in the field of personal care products. Has good effects of anti-aging, anti-wrinkle, whitening, moisturizing and eliminating black eye circles. Has good compatibility with skin.
In the context of this specification, "fed-batch fermentation" refers to fed-batch fermentation (also known as "semi-continuous fermentation" or "fed-batch fermentation"), and refers to a fermentation technique in which a fermentation broth is discharged outward discontinuously while a certain amount of material is fed into a fermentation system in a certain manner during microbial batch fermentation, and is a fermentation technique between batch fermentation and continuous fermentation.
The present application relates in a second aspect to a composition.
In a specific embodiment, a composition is provided comprising a yeast strain of Wickerhamomyces ciferrii and a fermentation broth comprising Tetraacetylphytosphingosine (TAPS), wherein the concentration of TAPS in the fermentation broth is at least 700mg/L under batch shake flask culture conditions and at least 21.0g/L under fed-batch culture conditions.
Here, the medium used in the fed-batch culture includes: 3.5-8.5 g/L yeast extract powder, 3.5-8.5 g/L-serine, 15.0-70.0 g/L glycerol, 1.5-5.5 g/L peptone and KH2PO42.0-7.5 g/L, malt extract 1.5-5.5 g/L, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO4 1.5~8.5g/L,KCl 0.2~2.4g/L,CaCl20.3~2.6g/L。
In a third aspect, the present application relates to a method for screening yeast.
In one embodiment, there is provided a method for screening saccharomyces weckerhamensis (wickerhamamyyces ciferrii), comprising the steps of:
-inducing ascospores of the parent yeast vickers-schiff, which is able to produce TAPS, with a spore yield of 50.0-75.0%;
isolating mating type strains.
In the context of the present specification, "ascospore (ascospore)" refers to a spore produced within an ascomycete ascospore. Immediately before sporulation, nuclear fusion and meiosis take place within the asco, producing 1-8 spores in the asco, or an n-th power of 2, with 8 spores in a typical asco. The 8 spores are produced by meiosis followed by one mitosis, the meiosis divides diploid nucleus into four haploid nuclei, the four nuclei are mitotically divided into eight nuclei respectively and are coated by secretion in the ascomycetes, so that one ascocyst usually has eight ascospores, but the number of the spores is still exceptional, for example, the ascomycetes of cordyceps can have 20-60 ascospores, the ascomycetes usually has 1-4 ascospores, and the ascomycetes usually has four ascospores. The shape is generally an ellipse, and some are needle-like bodies or have a partition wall. Lines, thorns, meshes, etc. are visible on the surface. Different features, their colors are also varied: some are colorless, and some are light yellow, light red, orange, brown and black. The spores are generally arranged in a row within the asco, with half being of a different genus to the other half.
In the context of the present specification, "induction" means in particular "induction of sporulation by a fungus".
In one embodiment, in the inducing step, WCCB medium is used as the inducing medium, which comprises: 6.3-10.5 g/L of sodium acetate, specifically 6.3g/L, 6.5g/L, 6.7g/L, 6.9g/L, 7.1g/L, 7.3g/L, 7.5g/L, 7.6g/L, 7.7g/L, 7.9g/L, 8.1g/L, 8.3g/L, 8.5g/L, 8.7g/L, 8.9g/L, 9.1g/L, 9.3g/L, 9.5g/L, 9.7g/L, 9.9g/L, 10.1g/L, 10.3g/L, 10.5 g/L; 11.7-23.6 g/L potassium chloride, specifically 11.7g/L, 12.7g/L, 13.7g/L, 14.7g/L, 15.7g/L, 16.7g/L, 17.4g/L, 17.7g/L, 18.7g/L, 19.7g/L, 20.7g/L, 21.7g/L, 22.7g/L and 23.6 g/L; magnesium sulfate is 0.09-0.34 g/L, specifically 0.09g/L, 0.14g/L, 0.18g/L, 0.24g/L, 0.29g/L, 0.34 g/L; glucose 0.43-1.76 g/L, specifically 0.43g/L, 0.76g/L, 0.9g/L, 1.1g/L, 1.43g/L, 1.76 g/L; 12.0-20.0 g/L of agar powder, specifically 12g/L, 13g/L, 14g/L, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L and 20 g/L. Further, the medium consists of: 6.3-10.5 g/L of sodium acetate, 11.7-23.6 g/L of potassium chloride, 0.09-0.34 g/L of magnesium sulfate, 0.43-1.76 g/L of glucose and 12.0-20.0 g/L of agar powder.
In one embodiment, in the inducing step, WCCB medium is used as the inducing medium, which comprises: 7.3-9.5 g/L of sodium acetate, specifically 7.3g/L, 7.5g/L, 7.7g/L, 7.9g/L, 8.1g/L, 8.3g/L, 8.5g/L, 8.7g/L, 8.9g/L, 9.1g/L, 9.3g/L and 9.5 g/L; 13.7-21.6 g/L potassium chloride, specifically 13.7g, 14.7g, 15.7g, 16.7g, 17.7g, 18.7g, 19.7g, 20.7g, 21.6 g; magnesium sulfate is 0.14-0.30 g/L, specifically 0.14g, 0.19g, 0.24g, 0.30 g; glucose is 0.63-1.46 g/L, specifically 0.63g/L, 0.76g/L, 1.1g/L, 1.46 g/L; 14.0-18.0 g/L of agar powder, specifically 14g/L, 15g/L, 16g/L, 17g/L and 18 g/L.
In one embodiment, in the inducing step, the following inducing culture conditions are used: aerobic culture is carried out for 3-7 days at 23-29 ℃. Specifically, the culture can be carried out at 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 29 ℃ for 3 days, 4 days, 5 days, 6 days or 7 days.
In yet another embodiment, the following is performed in the separation step:
-preparing a bacterial suspension of ascospores;
adding helicase/zymolyse/cell lysate with a final concentration of 0.5% -5.0% and a certain amount of quartz sand/glass beads with a diameter of 0.1-0.8 mm, specifically, the diameter may be 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8 mm; shaking the mixture for 0.5 to 6.0 hours at 25 to 35 ℃ and 100 to 320rpm, specifically, the temperature can be 25 ℃, 27 ℃, 29 ℃, 31 ℃, 33 ℃ and 35 ℃; specifically, the rotation speed may be 100rpm, 120rpm, 140rpm, 160rpm, 180rpm, 200rpm, 220rpm, 240rpm, 260rpm, 280rpm, 300rpm, 320 rpm; specifically, the time may be 0.5 hour, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours; breaking the ascospore wall to release spores therein;
adding the spore liquid after the wall breaking treatment into a sterile centrifuge tube, and carrying out water bath at 48-60 ℃ for 10-50 min to inactivate diplocardia in the spore liquid and enrich spores.
In the context of the present specification, "diploid" and "diploid" are used interchangeably and refer to an individual organism that develops from a fertilized egg and contains two sets of chromosomes in a somatic cell.
In a fourth aspect, the present application relates to a medium for inducing production of ascospores.
In one embodiment, a culture medium for inducing the production of ascospores is provided, which is a WCCB culture medium, and specifically comprises: 6.3-10.5 g/L of sodium acetate, 11.7-23.6 g/L of potassium chloride, 0.09-0.34 g/L of magnesium sulfate, 0.43-1.76 g/L of glucose and 12.0-20.0 g/L of agar powder.
By using a completely new sporulation medium (WCCB medium), the solution of the present application increases the spore yield of the diploid strain Wickerhamomyces ciferrii up to a level of 62.74%. Generally, with the spore production medium, the spore yield can reach a level of 50.0% -75.0% with moderate adjustment of culture conditions.
In a fifth aspect, the present application relates to a method for producing TAPS using yeast fermentation.
In one embodiment, a method is provided for producing Tetraacetylphytosphingosine (TAPS) by shake flask fermentation of the yeast Wickerhamomyces ciferrii, wherein culture is carried out in 10% liquid charge of YM medium in a Erlenmeyer flask at 220rpm and 30 ℃ for 72h to produce at least 700mg TAPS per L of fermentation broth.
In yet another embodiment, there is provided a fed-batch fermentation process of a yeast strain of the genus saccharomyces weckerhamensis (Wickerhamomyces ciferrii), the medium comprising: 3.5-8.5 g/L yeast extract powder, 3.5-8.5 g/L-serine, 15.0-70.0 g/L glycerol, 1.5-5.5 g/L peptone and KH2PO42.0-7.5 g/L, 1.5-5.5 g/L malt extract, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO4 1.5~8.5g/L,KCl 0.2~2.4g/L,CaCl20.3-2.6 g/L; the fermentation temperature is 25-33 ℃; the stirring speed is 300-900 rpm, and the dissolved oxygen is controlled to be more than 20%; fed batch mode: feeding operation is carried out when the carbon source (glycerol) is exhausted (dissolved oxygen rises back), and 30-70% (W/W) of carbon source (glycerol) is fed at a feeding speed of 6-30 ml/L (fermentation liquor)/hGlycerol until fermentation is complete) to produce Tetraacetylphytosphingosine (TAPS), wherein at least 21.0g TAPS/L fermentation broth is produced at 5 days of culture. Further, the fed-batch medium consisted of: 3.5-8.5 g/L yeast extract powder, 3.5-8.5 g/L-serine, 15.0-70.0 g/L glycerol, 1.5-5.5 g/L peptone and KH2PO42.0-7.5 g/L, 1.5-5.5 g/L malt extract, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO4 1.5~8.5g/L,KCl 0.2~2.4g/L,CaCl2 0.3~2.6g/L。
< examples section >
Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Example 1 acquisition of mating type strains
1. Screening of spore-forming medium:
inoculating an initial strain Wickerhamomyces ciferrii into a YM [ glucose 10.0g/L, peptone 5.0g/L, yeast extract 3.0g/L and malt extract 3.0g/L ] liquid culture medium (agar powder 15.0g/L is added into a solid culture medium, the same holds below) shake flask, culturing for about 24 hours to reach a logarithmic phase, centrifuging the culture solution, collecting bacterial sludge, and washing for 3 times by using sterile normal saline; the cleaned bacterial sludge is respectively coated on Mcclary, WCCB [ sodium acetate 7.60g/L, potassium chloride 17.40g/L, magnesium sulfate 0.18g/L, glucose 0.90g/L, agar powder 15.0g/L ], MA5, Kleyn and Fowell medium plates, and cultured for 7 days at 25 ℃ to obtain ascospores (shown in figure 1). Scraping bacterial sludge on each spore-forming plate, diluting to a certain gradient, counting on a blood counting chamber, and counting the spore-forming rate of the Wickerhamomyces ciferrii on each culture medium, wherein the specific result is shown in figure 2, the spore-forming rate of the WCCB culture medium is 62.74% and is far higher than that of other culture media, so that ascospores required in subsequent screening experiments are induced by the culture medium; FIG. 1 shows a microscopic photograph of ascospores produced by Wickerhamomyces ciferrii on WCCB medium; FIG. 2 shows the spore yields of the strains of Wickerhamomyces ciferrii on different spore-forming media. As can be seen from the experimental comparison of fig. 2, the highest spore yields were obtained using the WCCB of the present application. Wherein the Mcclary, the MA5, the Kleyn and the Fowell culture medium respectively comprise the following components: mcclary media composition: 1.00g/L glucose, 1.80g/L potassium chloride, 8.20g/L sodium acetate, 2.50g/L yeast extract and 1.50g/L agar powder; MA5 medium composition: malt extract: 50.00g/L of agar powder and 1.50g/L of agar powder; kleyn medium components: 2.50g/L of peptone, 0.62g/L of glucose, 0.62g/L of sodium chloride, 5.00g/L of sodium acetate and 1.50g/L of agar powder; fowell medium composition: 8.20g/L of sodium acetate, 0.40g/L of raffinose and 1.50g/L of agar powder.
2. Wall breaking of ascospores: preparing ascospore into bacterial suspension, adding snailase with final concentration of 3.0% and 20% quartz sand (diameter of 0.20mm), shaking with shaking table at 220rpm at 30 deg.C for 4.0 hr to release spore;
3. and (3) enrichment of spores: as the yeast spores have stronger stress resistance compared with diplocardia, spore liquid (mixed with ascospores and diplocardia) after wall breaking treatment is added into a sterile centrifuge tube and is subjected to water bath at 60 ℃ for 10 minutes to inactivate the diplocardia and enrich spores.
4. Selection and verification of mating type strains: diluting the spore liquid after water bath treatment to a certain concentration (preferably 10-200 single colonies grow on each plate after the plates are coated), absorbing a small volume of diluent, coating the diluent on a YM solid plate, culturing for about 3-7 days at 25-33 ℃, selecting a single colony with a colony morphology different from that of a Wickerhamomyces ciferrii diploid strain, transferring the single colony to a new YM plate, and standing and culturing at 30 ℃; meanwhile, selecting a small amount of bacterial sludge, transferring the bacterial sludge onto a WCCB culture medium plate, performing static culture at the temperature of 22-27 ℃, and performing sporulation verification; the spore production verification step is to screen whether the picked single colony is a mating type strain, if the single colony is the mating type strain, ascospores cannot be produced on a WCCB culture medium plate, and the spore production verification step can be used for further screening of crystal production; if the strain is not a mating strain, a large amount of ascospores are generated on a WCCB culture medium plate, the strain can be basically determined as a diploid strain, and subsequent crystallization screening is not required.
Example 2 screening of high-yielding TAPS Strain CGMCC19562
Separating and purifying the mating type strains obtained by the previous screening by streaking and the like to obtain single colonies, respectively inoculating the separated and purified mating type strains into a shake flask of YM liquid culture medium, culturing for 3 days in a shaking table at 30 ℃ and 220rpm, collecting culture solution, aging for 3 days in a refrigerator at 4 ℃, and observing the crystal generation condition under a 400-fold microscope (the generated crystal is shown in figure 3), wherein the results are shown in Table 1. FIG. 3 is a microscopic photograph of aged fermentation broth of a high-producing strain. The high-yield strain is preserved in China general microbiological culture Collection center (CGMCC) at 13 months and 4 months in 2020, and the preservation number is CGMCC No. 19562.
TABLE 1 statistics of fractional mating type crystallization
Note: the gamma group represents no crystallization; + represents a small amount of crystallization; + represents a typical amount of crystals; + + + indicates a larger amount of crystals; + + + + + indicates a large amount of crystals and a small amount of flocculent precipitate on the surface of the thallus; + + + + + + +, which indicates a large amount of crystals and a large amount of flocculent precipitates on the surface of the thalli; the evaluation method comprises the following steps: sampling and microscopic examination and visual observation;
performing shake flask fermentation on the strains with high yield of partial crystals, inoculating each strain into a sterilized YM liquid culture medium (12 g/L glucose, 5g/L peptone, 3g/L yeast extract and 3g/L malt extract) in a shake flask, performing shake flask culture at 30 ℃ and 220rpm for 3 days, aging the fermentation broth in a refrigerator at 4 ℃ for 3 days, extracting, detecting the content of TAPS in the fermentation product by High Performance Liquid Chromatography (HPLC) (as shown in FIG. 4), and calculating the fermentation yield, wherein the specific results are shown in Table 2. Wherein, the yield of CGMCC19562 is the highest and can reach 713 mg/L. FIG. 4 shows the HPLC analysis results of the fermentation product, wherein A is the fermentation extract product and B is the TAPS standard (sigma).
TABLE 2 Shake flask fermentation TAPS yields of some of the high producing strains
Strain numbering | Fermentation yield mg/L |
20190925-2 | 311 |
CGMCC19562 | 713 |
20190925-26 | 343 |
20190925-31 | 401 |
20190925-42 | 637 |
EXAMPLE 3 optimization of the Medium for producing TAPS by shaking culture of CGMCC19562 (Condition two)
Inoculating CGMCC19562 strain into sterilized YM liquid culture medium (glucose 8g/L, peptone 3g/L, yeast extract 2g/L, and malt extract 1.5g/L), shaking for 3 days at 30 deg.C and 220rpm, aging the fermentation liquid in refrigerator at 4 deg.C for 3 days, extracting, detecting the content of TAPS in the fermentation product by High Performance Liquid Chromatography (HPLC), and calculating the fermentation yield. The final yield was 702 mg/L.
Example 4 optimization of the Medium for producing TAPS by shaking culture of CGMCC19562 (Condition III)
Inoculating CGMCC19562 strain into sterilized YM liquid culture medium (glucose 16g/L, peptone 7g/L, yeast extract 4g/L, and malt extract 4.5g/L), shaking for 3 days at 30 deg.C and 220rpm, aging the fermentation liquid in a refrigerator at 4 deg.C for 3 days, extracting, detecting the content of TAPS in the fermentation product by High Performance Liquid Chromatography (HPLC), and calculating the fermentation yield. The final yield was 705 mg/L.
COMPARATIVE EXAMPLE 1 optimization of the culture Medium for producing TAPS by Shake flask culture of CGMCC19562 (Condition IV)
Inoculating CGMCC19562 strain into sterilized YM liquid culture medium (glucose 17g/L, peptone 8g/L, yeast extract 5g/L, and malt extract 5.5g/L), shaking and culturing at 30 deg.C and 220rpm for 3 days, aging the fermentation broth in a refrigerator at 4 deg.C for 3 days, extracting, detecting the content of TAPS in the fermentation product by High Performance Liquid Chromatography (HPLC), and calculating the fermentation yield. The final yield was 692 mg/L.
COMPARATIVE EXAMPLE 2 optimization of the culture Medium for producing TAPS by Shake flask culture of CGMCC19562 (Condition five)
Inoculating CGMCC19562 strain into sterilized YM liquid culture medium (glucose 7g/L, peptone 2g/L, yeast extract 1.5g/L, and malt extract 1g/L), shaking for 3 days at 30 deg.C and 220rpm, aging the fermentation liquid in a refrigerator at 4 deg.C for 3 days, extracting, detecting the content of TAPS in the fermentation product by High Performance Liquid Chromatography (HPLC), and calculating the fermentation yield. The final yield was 687 mg/L.
TABLE 3 optimization of the medium for producing TAPS by shake-flask culture of CGMCC19562
And (4) conclusion: in order to achieve the set technical goal (production of at least 700mg Tetra Acetyl Phytosphingosine (TAPS)/L broth), medium optimization experiments in shake flask culture were performed using the optimal strain CGMCC19562, leading to the conclusion that: the YM medium is preferably composed of: 8.0-16.0 g/L glucose, 3.0-7.0 g/L peptone, 2.0-4.0 g/L yeast extract and 1.5-4.5 g/L malt extract.
Example 5 fed-batch fermentation culture of high-producing Strain CGMCC19562
1. Activating strains: taking out a high-yield strain CGMCC19562, transferring to YM solid plate by coating or streaking, and standing at 30 deg.C for 5 days until a large amount of thallus Porphyrae grows on the plate.
2. Preparing shake flask seeds: selecting a small amount of bacterial sludge with inoculating needle, inoculating into shake flask seed culture medium, culturing at 30 deg.C and 220rpm, sampling, and measuring seed liquid OD600When shaking the seed OD600When the culture reaches about 10, the seed liquid culture is finished, and the seed liquid is inoculated into a fermentation tank for culture.
3. Fermentation culture: inoculating the seed liquid into a fermentation tank for fermentation culture, wherein the fermentation culture process comprises the following steps: (1) the fermentation medium comprises yeast extract powder 5.5g/L, L-serine 6.0g/L, glycerol 42.0g/L, peptone 3.5g/L, KH2PO45.5g/L, malt extract 4.0g/L, MgSO4·7H2O 2.4g/L,(NH4)2PO4 3.8g/L,KCl 1.7g/L,CaCl21.9 g/L; (2) the fermentation temperature is 30 ℃, the stirring speed is 600rpm, the dissolved oxygen is controlled to be more than 20 percent, and the method can be realized by adjusting the rotation speed, increasing the ventilation volume, increasing the tank pressure and the like; (3) in a fed-batch fermentation mode, feeding operation is carried out when a carbon source (glycerol) is exhausted (dissolved oxygen is returned to rise), and 50% (W/W) of glycerol is fed at a feeding speed of 10ml/L (fermentation liquor)/h until the fermentation is finished after 5 days. (4) After fermentation, transferring the fermentation liquor to a low-temperature tank for storage for 3 days, and after crystallization is basically secreted out of cells, extracting and purifying the product by using an organic solvent and detecting the fermentation yield. The specific growth curve is shown in FIG. 5, the final yield of the fermentation batch is 22.14g (TAPS)/L (fermentation broth), and the yield increases with the lapse of the fermentation time; however, after 84h of fermentation, the rate of increase in yield began to slow, which is in contrast toThe accumulation of the thalli and the fermentation biomass are in a linear relation.
Example 6 optimization of the Medium for production of TAPS by fed-batch culture of CGMCC19562 (Condition two)
TAPS biosynthesis is carried out by using a high-yield strain CGMCC19562 through a fed-batch culture mode; example 6 differs from example 5 only in that the culture conditions used were a medium using yeast extract powder 3.5g/L, L-serine 3.5g/L, glycerol 15.0g/L, peptone 1.5g/L, KH2PO42.0g/L, malt extract 1.5g/L, MgSO4·7H2O 1.2g/L,(NH4)2PO4 1.5g/L,KCl 0.2g/L,CaCl20.3 g/L; the fermentation temperature is 23 ℃; the stirring speed is 280rpm, and the dissolved oxygen is controlled to be more than 20 percent; fed batch mode: when the carbon source (glycerol) was depleted (dissolved oxygen rise), a feed operation was carried out with 30% (W/W) glycerol at a feed rate of 6ml/L (broth)/h until the end of the fermentation. In this fermentation batch, the final yield was 21.11g (TAPS)/L (fermentation broth).
Example 7 optimization of the Medium for production of TAPS by fed-batch culture of CGMCC19562 (Condition three)
TAPS biosynthesis is carried out by using a high-yield strain CGMCC19562 through a fed-batch culture mode; example 7 differs from example 5 only in that the culture conditions used were 8.5g/L yeast extract, 8.5 g/L-serine, 70.0g/L glycerol, 5.5g/L peptone, KH2PO47.5g/L, malt extract 5.5g/L, MgSO4·7H2O 5.4g/L,(NH4)2PO48.5g/L,KCl 2.4g/L,CaCl22.6 g/L; the fermentation temperature is 23 ℃; the stirring speed is 280rpm, and the dissolved oxygen is controlled to be more than 20 percent; fed batch mode: when the carbon source (glycerol) was depleted (dissolved oxygen rising), the feed was carried out with 70% (W/W) glycerol at a feed rate of 30ml/L (broth)/h until the end of the fermentation. In this batch, the final yield was 21.78g (TAPS)/L (fermentation broth).
COMPARATIVE EXAMPLE 3 optimization of the Medium for the fed-batch culture of CGMCC19562 for TAPS production (Condition IV)
Fed-batch culture using high-producing strain CGMCC19562Performing TAPS biosynthesis; comparative example 3 differs from example 5 only in that the culture conditions used were such that the medium used was 3.4g/L yeast extract, 3.3 g/L-serine, 13.0g/L glycerol, 1.3g/L peptone, KH2PO41.9g/L, malt extract 1.3g/L, MgSO4·7H2O 1.1g/L,(NH4)2PO41.3g/L,KCl 0.15g/L,CaCl20.25 g/L; the fermentation temperature is 23 ℃; the stirring speed is 280rpm, and the dissolved oxygen is controlled to be more than 20 percent; fed batch mode: when the carbon source (glycerol) was depleted (dissolved oxygen rise), a feed operation was carried out with a feed rate of 5ml/L (broth)/h of glycerol of 28% (W/W) until the end of the fermentation. In this fermentation batch, the final yield was 13.76g (TAPS)/L (fermentation broth).
COMPARATIVE EXAMPLE 4 optimization of the Medium for production of TAPS by fed-batch culture of CGMCC19562 (Condition five)
TAPS biosynthesis is carried out by using a high-yield strain CGMCC19562 through a fed-batch culture mode; comparative example 4 differs from example 5 only in that the culture conditions used were 8.6g/L of yeast extract, 8.6g/L of L-serine, 71.0g/L of glycerol, 5.6g/L of peptone, KH2PO47.6g/L, malt extract 5.6g/L, MgSO4·7H2O 5.5g/L,(NH4)2PO4 8.6g/L,KCl 2.5g/L,CaCl22.7 g/L; the fermentation temperature is 35 ℃; the stirring speed is 920rpm, and the dissolved oxygen is controlled to be more than 20 percent; fed batch mode: when the carbon source (glycerol) was depleted (dissolved oxygen rising), 72% (W/W) of glycerol was fed in at a feed rate of 32ml/L (broth)/h until the end of the fermentation. In this fermentation batch, the final yield was 18.53g (TAPS)/L (fermentation broth).
TABLE 3 optimization of the Medium for the production of TAPS by fed-batch culture of CGMCC19562
Media protocol | Fermentation yield g/L |
Example 5 | 22.14 |
Example 6 | 21.11 |
Example 7 | 21.78 |
Comparative example 3 | 13.76 |
Comparative example 4 | 18.53 |
And (4) conclusion: to achieve the set technical goal (production of at least 21g of Tetra Acetyl Phytosphingosine (TAPS)/L broth), a medium optimization experiment of fed-batch culture was performed using the optimal strain CGMCC19562, leading to the conclusion that: the fed-batch medium preferably consists of: 3.5-8.5 g/L yeast extract powder, 3.5-8.5 g/L-serine, 15.0-70.0 g/L glycerol, 1.5-5.5 g/L peptone and KH2PO42.0-7.5 g/L, 1.5-5.5 g/L malt extract, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO4 1.5~8.5g/L,KCl 0.2~2.4g/L,CaCl2 0.3~2.6g/L。
Comparative example 5 fed-batch culture Using less preferred Strain and ideal culture conditions
TAPS biosynthesis was carried out by fed-batch culture according to the fermentation culture conditions of example 5; comparative example 5 differs from example 5 only in that the strain used was the less preferred strain from example 2, numbered 20190925-42. In this fermentation batch, the final yield was 16.21g (TAPS)/L (fermentation broth).
Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-described embodiments and application fields, and the above-described embodiments are illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.
Claims (10)
1. A yeast strain belonging to the genus Vickers of the genus Wilkhammyces (Wickerhamamymyces ciferrii) has a preservation number of CGMCC No. 19562.
2. The yeast of claim 1, wherein the yeast produces at least 700mg Tetraacetylphytosphingosine (TAPS)/L fermentation broth under batch shake flask culture conditions.
3. The yeast of claim 2, wherein the batch shake flask culture conditions are at least 72 hours in a flask containing YM medium at appropriate rotation speed and temperature; wherein the YM culture medium comprises 8.0-16.0 g/L of glucose, 3.0-7.0 g/L of peptone, 2.0-4.0 g/L of yeast extract and 1.5-4.5 g/L of malt extract.
4. The yeast of claim 1, wherein the yeast produces at least 21.0g TAPS/L of fermentation broth under fed-batch culture conditions.
5. The yeast of claim 4, wherein the fed batch culture conditions are: the culture medium comprises 3.5-8.5 g/L of yeast extract powder, 3.5-8.5 g/L of L-serine, 15.0-70.0 g/L of glycerol, 1.5-5.5 g/L of peptone and KH2PO42.0-7.5 g/L, malt extract 1.5-5.5 g/L, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO41.5~8.5g/L,KCl 0.2~2.4g/L,CaCl20.3-2.6 g/L; dissolving at proper fermentation temperature and stirring speedOxygen is controlled to be more than 20 percent; fed-batch mode: feeding operation is carried out when the carbon source is exhausted, and 30-70% (W/W) of carbon source is fed in at a feeding speed of 6-30 ml/L fermentation liquor/h until fermentation is finished after 5 days.
6. A composition comprising saccharomyces weckerhamstring (Wickerhamomyces ciferrii) and a fermentation broth comprising Tetraacetylphytosphingosine (TAPS).
7. The composition of claim 6, wherein the concentration of TAPS is above 700mg/L in the fermentation broth obtained from batch shake flask culture.
8. The composition of claim 7, wherein the culture conditions of the batch shake flask culture are at least 72h in a triangular flask containing YM medium at appropriate rotation speed and temperature; wherein the YM culture medium comprises 8.0-16.0 g/L of glucose, 3.0-7.0 g/L of peptone, 2.0-4.0 g/L of yeast extract and 1.5-4.5 g/L of malt extract.
9. The composition according to claim 6, wherein the concentration of TAPS in the fermentation broth obtained from fed-batch culture is 21.0g/L or more.
10. The composition of claim 9, wherein the fed-batch culture is cultured under the following conditions: the culture medium comprises 3.5-8.5 g/L of yeast extract powder, 3.5-8.5 g/L of L-serine, 15.0-70.0 g/L of glycerol, 1.5-5.5 g/L of peptone and KH2PO42.0-7.5 g/L, 1.5-5.5 g/L malt extract, MgSO4·7H2O 1.2~5.4g/L,(NH4)2PO41.5~8.5g/L,KCl 0.2~2.4g/L,CaCl20.3-2.6 g/L; controlling the dissolved oxygen to be more than 20% at a proper fermentation temperature and stirring speed; fed batch mode: and (3) feeding the carbon source at the carbon source exhaustion rate, wherein 30-70% (W/W) of the carbon source is fed at the feeding speed of 6-30 ml/L fermentation liquor/h until the fermentation is finished after 5 days.
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