CN112442452A - Strain for producing 2-phenethyl alcohol and application thereof - Google Patents
Strain for producing 2-phenethyl alcohol and application thereof Download PDFInfo
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- WRMNZCZEMHIOCP-UHFFFAOYSA-N 2-phenylethanol Chemical compound OCCC1=CC=CC=C1 WRMNZCZEMHIOCP-UHFFFAOYSA-N 0.000 title claims abstract description 77
- 238000000855 fermentation Methods 0.000 claims abstract description 54
- 230000004151 fermentation Effects 0.000 claims abstract description 54
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims abstract description 28
- 241000235048 Meyerozyma guilliermondii Species 0.000 claims abstract description 26
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 16
- 239000008103 glucose Substances 0.000 claims abstract description 16
- 229960005190 phenylalanine Drugs 0.000 claims abstract description 14
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 239000002609 medium Substances 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000011218 seed culture Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 238000010564 aerobic fermentation Methods 0.000 claims description 6
- WSSMOXHYUFMBLS-UHFFFAOYSA-L iron dichloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Fe+2] WSSMOXHYUFMBLS-UHFFFAOYSA-L 0.000 claims description 6
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 229930003270 Vitamin B Natural products 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 235000019156 vitamin B Nutrition 0.000 claims description 4
- 239000011720 vitamin B Substances 0.000 claims description 4
- 238000001994 activation Methods 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 13
- 238000012216 screening Methods 0.000 abstract description 13
- 239000003112 inhibitor Substances 0.000 abstract description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052799 carbon Inorganic materials 0.000 abstract description 4
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 238000005457 optimization Methods 0.000 abstract 1
- 238000000034 method Methods 0.000 description 14
- 230000008569 process Effects 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000011068 loading method Methods 0.000 description 6
- 239000002689 soil Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 241000220317 Rosa Species 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000235058 Komagataella pastoris Species 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 241000109329 Rosa xanthina Species 0.000 description 3
- 235000004789 Rosa xanthina Nutrition 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000234435 Lilium Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical compound C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 2
- -1 ester derivative of phenethyl alcohol Chemical class 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000002304 perfume Substances 0.000 description 2
- MDHYEMXUFSJLGV-UHFFFAOYSA-N phenethyl acetate Chemical compound CC(=O)OCCC1=CC=CC=C1 MDHYEMXUFSJLGV-UHFFFAOYSA-N 0.000 description 2
- 230000001603 reducing effect Effects 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 239000000341 volatile oil Substances 0.000 description 2
- SZIFAVKTNFCBPC-UHFFFAOYSA-N 2-chloroethanol Chemical compound OCCCl SZIFAVKTNFCBPC-UHFFFAOYSA-N 0.000 description 1
- 125000000094 2-phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 description 1
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 1
- 241000978183 Elixia Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 235000010254 Jasminum officinale Nutrition 0.000 description 1
- 240000005385 Jasminum sambac Species 0.000 description 1
- 241000234479 Narcissus Species 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- AWMVMTVKBNGEAK-UHFFFAOYSA-N Styrene oxide Chemical compound C1OC1C1=CC=CC=C1 AWMVMTVKBNGEAK-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- PEZDGNIESNXEDE-UHFFFAOYSA-N benzene;oxirane Chemical compound C1CO1.C1=CC=CC=C1 PEZDGNIESNXEDE-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
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- 235000009508 confectionery Nutrition 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
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- 239000000796 flavoring agent Substances 0.000 description 1
- 231100000171 higher toxicity Toxicity 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
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- 235000019462 natural additive Nutrition 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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Abstract
The invention relates to a strain for producing 2-phenethyl alcohol and application thereof, wherein the strain for producing 2-phenethyl alcohol is classified and named as Pichia guilliermondii (Meyerozyma guilliermondii) YLG18 with the preservation number of CCTCC NO: m2020638. According to the invention, the flat plate containing high-concentration alcohol inhibitors and the shake flask are utilized for fermentation screening, the obtained screened strain takes glucose as a carbon source, and 3.21 g/L of 2-phenethyl alcohol can be finally produced through fermentation condition optimization and fermentation tank amplification fermentation experiments. The strain has strong stress resistance, high product yield and good repeatability, and is an excellent strain suitable for producing 2-phenethyl alcohol by using L-phenylalanine fermentation.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a strain for producing 2-phenethyl alcohol and application thereof.
Background
2-phenylethyl alcohol is an aromatic alcohol with a rose smell, and is naturally present in essential oils of various plants such as roses and lilies. At present, 2-phenethyl alcohol is widely applied to industries such as medicine, food, cosmetics, tobacco, daily chemical products and the like at home and abroad. The raw materials can be used for synthesizing a plurality of derivatives with high application value, for example, phenethyl acetate which is an ester derivative of phenethyl alcohol is an important aromatic substance, has sweet fragrance like rose, and can be used as daily chemical essence and edible essence. The annual output of 2-phenylethyl alcohol is nearly ten thousand tons, and most of the 2-phenylethyl alcohol is synthesized by adopting cheap chemical raw materials, namely a benzene-ethylene oxide synthesis method and a styrene oxide hydrogenation method, except that a small part of the 2-phenylethyl alcohol is extracted from natural roses. However, many raw materials used in chemical synthesis belong to carcinogenic substances and have great harm to human health and environment. In addition, chemically synthesized phenethyl alcohol often contains some byproducts which are difficult to remove, for example, biphenyl, beta-chloroethylene, chloroethanol and the like have bad odor, the product quality of the 2-phenethyl alcohol is seriously affected, and the quality standard of edible and daily perfumes is difficult to achieve.
From the end of the last century, with the increasing demands for living quality and health concerns, consumers increasingly pay more attention to the safety of foods, daily necessities and the like, and prefer to "green" and "natural", and the production of foods, cosmetics and the like also increasingly tends to use natural additives. By "natural", it is meant that the substance must be of natural origin, and in the united states and europe, the flavoring and perfuming agents which can be labeled "natural" must be physically extracted from natural materials and enzymatically or microbially fermented, which are also more expensive to sell.
Although natural 2-phenylethyl alcohol exists in refined oil of many flowers and plants such as jasmine, narcissus, lily and the like, the concentration is too low to extract in most cases. The only exception is rose essential oil, however, the rose is opened once a year, the production period of extracting natural 2-phenethyl alcohol from the rose is long, the production cost is expensive, large-scale industrial production cannot be carried out, and the market demand is difficult to meet. The microorganism has the characteristics of small volume, quick propagation, more absorption, quick conversion, strong adaptability and the like, the pure and natural characteristics of the product are reserved by adopting the microorganism conversion method to produce the natural 2-phenethyl alcohol, and the microorganism has the advantages of low cost, short period, high efficiency and the like, and the quality of the product meets the international standard. Research shows that yeast cells have a metabolic pathway for de novo synthesis of 2-phenylethyl alcohol and can also directly convert L-phenylalanine into 2-phenylethyl alcohol through an amino acid catabolic pathway. In 1907, L-phenylalanine was added to the yeast culture in Elixia in order to greatly increase the yield of 2-phenylethyl alcohol, and the method has made a hope for industrialization of producing natural 2-phenylethyl alcohol by yeast biotransformation. In developed countries in Europe and America, healthy and natural living concepts are deeply focused, people pay attention to biosynthesis of natural substances very early, and the biosynthesis of 2-phenethyl alcohol is earlier researched and reaches a higher level at present. Although China has a plurality of research reports about L-phenylalanine biosynthesis, the research on 2-phenethyl alcohol biosynthesis is still in the beginning stage, and related literature reports are few, which is far away from foreign countries.
Like many alcohols, the high-concentration 2-phenylethyl alcohol has certain toxicity to microorganisms, and the 2-phenylethyl alcohol with the concentration of 2-3 g/L can completely inhibit the growth of various bacteria and fungi, so that the 2-phenylethyl alcohol can be used as a bactericide in the pharmaceutical industry. In addition, yeast can also produce a large amount of ethanol in addition to 2-phenylethyl alcohol in the fermentation process, and the combined action of ethanol and 2-phenylethyl alcohol has higher toxicity than the sum of the toxicity produced by the ethanol and the 2-phenylethyl alcohol, which is the main reason of low product concentration in the process for producing 2-phenylethyl alcohol by a microbial conversion method and is also the bottleneck of producing natural 2-phenylethyl alcohol by a biological method.
Therefore, in the process of producing 2-phenylethyl alcohol with high yield, the toxicity of 2-phenylethyl alcohol and ethanol to yeast must be considered, and the screening of excellent underpan cells with high stress resistance is one of the key means for improving the yield of 2-phenylethyl alcohol.
Disclosure of Invention
The invention aims to provide a Pichia guilliermondii strain with high stress resistance, which has high tolerance to 2-phenethyl alcohol and obviously improves the yield of a final product of fermentation.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a strain for producing 2-phenethyl alcohol, which is classified and named as Pichia guilliermondii (Meyerozyma guilliermondii) YLG18 with the preservation number of CCTCC NO: m2020638.
The novel Pichia pastoris (Pichia pastoris) for synthesizing 2-phenylethyl alcoholMeyerozyma guilliermondii) YLG18 is prepared by taking several wild strains screened from soil in basalt lake park of Nanjing City of Jiangsu province in 2019 for 6 months as original strains, screening by using a flat plate containing high-concentration 2-phenethyl alcohol to obtain strains with high tolerance to inhibitors and strong reducing power, and finally taking L-phenylalanine as a substrate, and screening by aerobic fermentation to obtain a Pichia guilliermondii target strain with high product yield.
Another object of the present invention is to provide the use of the 2-phenylethyl alcohol-producing strain in the production of 2-phenylethyl alcohol.
The application comprises the following steps: and sequentially carrying out plate activation, seed culture and fermentation culture on the 2-phenethyl alcohol producing strain to obtain the 2-phenethyl alcohol.
Further, the temperature of the plate activation and the seed culture is 28-30 ℃, and preferably 30 ℃; the culture time is 12-24 h.
Further, the fermentation culture is aerobic fermentation, wherein the fermentation temperature is 28-30 ℃, and preferably 30 ℃; the fermentation culture time is 72-120 h.
Further, the inoculation amount of the fermentation culture is 1-10% (v/v).
Further, the culture medium for seed culture comprises the following components in percentage by mass: glucose 4%, YNB 0.05%, FeCl2.4H2O 0.00015%、NaCl 0.1%、MgCl2.6H2O 0.05%、KH2PO4 0.02%、KCl 0.03%、CaCl2.2H20.0015 percent of O, 10.0002 percent of vitamin B, 0.5 percent of L-phenylalanine and the balance of water.
Further, the culture medium for fermentation culture comprises the following components in percentage by mass: glucose 4-10%, YNB 0.05%, FeCl2.4H2O 0.00015%、NaCl 0.1%、MgCl2.6H2O 0.05%、KH2PO4 0.02%、KCl 0.03%、CaCl2.2H20.0015% of O, 10.0002% of vitamin B, 0.5-1% of L-phenylalanine and the balance of water.
The invention has the beneficial effects that:
the invention obtains the pichia guilliermondii through screening, utilizes a flat plate containing high-concentration alcohol inhibitors to screen strains which have stronger tolerance to the alcohol inhibitors, can directly utilize L-phenylalanine to ferment and produce 2-phenethyl alcohol and have high product yield. The strain and the process are utilized for fermentation, glucose is used as a carbon source, 3.21 g/L2-phenethyl alcohol can be produced by fermenting for 84 hours in a fermentation tank, and the highest yield of 2-phenethyl alcohol synthesized by using pichia guilliermondii is reported at present; has great social significance and economic value.
Drawings
FIG. 1 shows the biosynthesis of 2-phenylethyl alcohol by strain YLG18 using different substrates.
FIG. 2 shows the results of 2-phenylethyl alcohol tolerance test of strain YLG 18.
The biological material is classified and named as Pichia pastoris (A)Meyerozyma guilliermondii) YLG18, which is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: m2020638, deposit date of 26/10/2020, address: wuhan, Wuhan university.
Detailed Description
The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the embodiments are described only for illustrating the present invention and should not limit the present invention described in detail in the claims.
Example 1 this example illustrates a method of screening for pichia guilliermondii.
Wherein the formula of the used culture medium (% by mass):
(1) liquid culture medium: 1% of yeast powder, 2% of peptone, 2% of glucose and the balance of water, and the pH value is 6.
(2) Solid plate medium: 1% of yeast powder, 2% of peptone, 2% of glucose, 2% of agar and the balance of water, wherein the pH value is 6.
(3) 2-phenethyl alcohol plate medium: 1% of yeast powder, 2% of peptone, 2% of glucose, 0.2% of 2-phenethyl alcohol and 2% of agar, the balance being water and pH 6.
(4) 2-phenethyl alcohol plate synthesis medium: 0.5 percent of L-phenylalanine, 4 percent of glucose, 0.2 percent of 2-phenethyl alcohol and VB1 0.0002%，FeCl2.4H2O 0.00015%，NaCl 0.1%，MgCl2.6H2O 0.05%，KH2PO4 0.02%，KCl 0.03%，CaCl2.2H20.0015 percent of O and the balance of water.
(5) Shake flask fermentation screening culture medium: 0.5 percent of L-phenylalanine, 4 percent of glucose, 0.05 percent of YNB and VB1 0.0002%，FeCl2.4H2O 0.00015%，NaCl 0.1%，MgCl2.6H2O 0.05%，KH2PO4 0.02%，KCl 0.03%，CaCl2.2H20.0015 percent of O and the balance of water.
A screening step:
1: collecting samples: according to the characteristic that 2-phenethyl alcohol has the fragrance of roses, taking a piece of soil with the area of 4cm x 5cm and the thickness of 6cm from the soil at the root of a rosebush of a basalt lake;
2: inhibitor plate screening
Enriching collected soil samples by using a liquid culture medium, diluting the soil samples by using normal saline to different concentrations, coating the soil samples on a conventional solid culture medium plate containing an alcohol inhibitor (0.2%), carrying out aerobic culture at the temperature of 28-30 ℃ for 30 h, and selecting a plurality of bacterial colonies which can grow on a screening plate and have large bacterial colonies.
3: 2-phenethyl alcohol flat plate double screen
Inoculating the screened strain into 20 mL of a 100 mL triangular flask with liquid loading capacity, carrying out aerobic culture at 28-30 ℃ for 10-14 h, preparing a bacterial suspension with the concentration OD =0.6 by using sterile normal saline, sucking 2 uL of the bacterial suspension, dripping the bacterial suspension onto a synthetic medium plate containing an alcohol inhibitor (0.2%), carrying out aerobic culture at 28-30 ℃ for 12-24 h, and selecting a bacterial colony with the bacterial colony larger than a growing bacterium.
The final strain YLG18 showed strong inhibitor tolerance and reducing activity.
4: shake flask fermentation screening
Inoculating the strain YLG18 into a seed culture medium for amplification culture, wherein the culture temperature is 30 ℃, the liquid loading capacity of a 250 mL triangular flask is 50 mL, and the aerobic culture time is 12 h. Then carrying out aerobic fermentation by using a fermentation medium, wherein the inoculation amount is 1% (v/v), the fermentation temperature is 30 ℃, the liquid loading amount of a 500 mL triangular flask is 100 mL, and the yield of the 2-phenylethyl alcohol of the strain is detected after the fermentation time is 120h and is shown in figure 1:
the strain YLG18 obtained by plate combination screening can synthesize 1.2 g/L2-phenethyl alcohol by using L-phenylalanine as a substrate in the fermentation process.
Example 2 this example illustrates the passaging stability of the selected strain YLG 18.
Passage stability of the selected strain YLG18 was tested in a fermentation medium using glucose as a carbon source (the fermentation medium was the same as the shake flask fermentation selection medium of example 1). The results of the passage fermentation test of strain YLG18 are shown in Table 1:
TABLE 1 results of passage fermentation test of strain YLG18
| Number of passages | 1 | 2 | 3 | 4 | 5 | 6 |
| 2-PE (g/L) | 1.18 | 1.25 | 1.21 | 1.16 | 1.22 | 1.20 |
From experimental results, the 2-phenethyl alcohol yield of the screened strain YLG18 is stable after 6 continuous passages, has better passage stability, and can be used as a production strain for further research and development.
Example 3 this example illustrates the 2-phenylethyl alcohol tolerance of the screened strain YLG 18.
Respectively adding 2-phenethyl alcohol with different concentrations into a fermentation culture medium taking glucose as a carbon source, and detecting the 2-phenethyl alcohol tolerance of the screened strain YLG 18. The results of the strain YLG18 tolerant fermentation experiments are shown in FIG. 2.
According to the experimental results, the 2-phenethyl alcohol tolerance of the strain YLG18 is very high through a concentration gradient test of an inhibitor 2-phenethyl alcohol, the strain has very good 2-phenethyl alcohol synthesis potential, and the strain can be used as a production strain for further research and development.
Example 4 this example illustrates Pichia guilliermondii (R) ((R))Meyerozyma guilliermondii) A process for producing 2-phenethyl alcohol by fermenting YLG 18.
The formula of the plate culture medium in the embodiment is the same as that of the solid plate culture medium in the embodiment 1; the seed medium and the fermentation medium were the same as those in the shake flask fermentation screening medium described in example 1.
Pichia guilliermondii (R)Meyerozyma guilliermondii) YLG18 was inoculated to a plate medium for aerobic culture at 30 ℃ for 12 h. Inoculating plate-cultured YLG18 into a seed culture medium, wherein the liquid loading amount of a 250 mL triangular flask is 50 mL, the culture temperature is 30 ℃, and the culture time is 12 h; inoculating the seeds into a fermentation medium, wherein the inoculation amount is 1% (v/v), the fermentation temperature is 30 ℃, the liquid loading amount of a 500 mL triangular flask is 100 mL, and after aerobic fermentation culture is carried out for 120h, the detected yield of the 2-phenethyl alcohol reaches 1.55 g/L, which is increased by 29.2% compared with the original fermentation condition.
Example 5 this example illustrates Pichia guilliermondii (R) ((R))Meyerozyma guilliermondii) A process for producing 2-phenethyl alcohol by fermenting YLG 18.
This example differs from example 4 only in that the fermentation medium contains 10% by mass of glucose.
The detection shows that the yield of the 2-phenethyl alcohol reaches 1.95 g/L, and is improved by 62.5 percent compared with the original fermentation condition.
Example 6 this example illustrates Pichia guilliermondii (R) ((R))Meyerozyma guilliermondii) A process for producing 2-phenethyl alcohol by fermenting YLG 18.
This example differs from example 5 only in that the glucose mass percentage in the fermentation medium is 6%.
The detection shows that the yield of the 2-phenethyl alcohol reaches 2.17 g/L, which is improved by 80.8 percent compared with the original fermentation condition.
Example 7 this example illustrates Pichia guilliermondii (R) ((R))Meyerozyma guilliermondii) A process for producing 2-phenethyl alcohol by fermenting YLG 18.
This example differs from example 6 only in that the fermentation medium contains 0.7% by mass of L-phenylalanine.
The detection shows that the yield of the 2-phenethyl alcohol reaches 2.21 g/L, which is improved by 84.17% compared with the original fermentation condition.
Example 8 this example illustrates Pichia guilliermondii (R) ((R))Meyerozyma guilliermondii) A process for producing 2-phenethyl alcohol by fermenting YLG 18.
This example differs from example 6 only in that the fermentation medium contains 1% by mass of L-phenylalanine.
The detection shows that the yield of the 2-phenethyl alcohol reaches 2.32 g/L, which is 93.33 percent higher than the original fermentation condition.
Example 9 this example illustrates Pichia guilliermondii (R) ((R))Meyerozyma guilliermondii) A process for producing 2-phenethyl alcohol by fermenting YLG 18.
This embodiment differs from embodiment 7 only in that: inoculating plate-cultured YLG18 into a seed culture medium, filling 200 mL of liquid in a 500 mL triangular flask, culturing at 30 ℃ for 24 h; inoculating the seeds into a fermentation medium, wherein the liquid loading capacity of a 5L fermentation tank is 2L, the inoculation amount is 10% (v/v), the fermentation temperature is 30 ℃, and after aerobic fermentation culture is carried out for 84 h, the detected yield of the 2-phenethyl alcohol reaches 3.21 g/L, which is improved by 167.5% compared with the original fermentation condition.
Claims (10)
1. A strain for producing 2-phenethyl alcohol is characterized in that the strain is classified and named as Pichia guilliermondii (A.guilliermondii)Meyerozyma guilliermondii) YLG18 with the preservation number of CCTCC NO: m2020638.
2. Use of the 2-phenylethyl alcohol-producing strain according to claim 1 for the production of 2-phenylethyl alcohol.
3. The use according to claim 2, comprising: and sequentially carrying out plate activation, seed culture and fermentation culture on the 2-phenethyl alcohol producing strain to obtain the 2-phenethyl alcohol.
4. Use according to claim 3, wherein the temperature of the plate activation and seed culture is 28-30 ℃, preferably 30 ℃; the culture time is 12-24 h.
5. The use according to claim 3, wherein the fermentation culture is aerobic fermentation, the fermentation temperature is 28-30 ℃, preferably 30 ℃; the fermentation culture time is 72-120 h.
6. The use according to claim 3, wherein the amount of inoculum for the fermentation culture is 1-10% v/v.
7. The use according to claim 3, wherein the culture medium for seed culture comprises the following components in percentage by mass: glucose 4%, YNB 0.05%, FeCl2.4H2O 0.00015%、NaCl 0.1%、MgCl2.6H2O 0.05%、KH2PO4 0.02%、KCl 0.03%、CaCl2.2H20.0015 percent of O, 10.0002 percent of vitamin B, 0.5 percent of L-phenylalanine and the balance of water.
8. The use according to claim 3, wherein the culture medium of the fermentation culture comprises the following components in percentage by mass: glucose 4-10%, YNB 0.05%, FeCl2.4H2O 0.00015%、NaCl 0.1%、MgCl2.6H2O 0.05%、KH2PO4 0.02%、KCl 0.03%、CaCl2.2H20.0015% of O, 10.0002% of vitamin B, 0.5-1% of L-phenylalanine and the balance of water.
9. Use according to claim 8, characterized in that the percentage by mass of glucose in the fermentation medium is 6%.
10. The use of claim 8, wherein the L-phenylalanine is present in the fermentation medium in an amount of 0.7 to 1% by weight.
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| CN114574374A (en) * | 2022-03-18 | 2022-06-03 | 南京工业大学 | Strain for producing 2-phenethyl alcohol and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106085889A (en) * | 2016-06-24 | 2016-11-09 | 四川大学 | Pichia guilliermondii 3 J15 and application thereof |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106085889A (en) * | 2016-06-24 | 2016-11-09 | 四川大学 | Pichia guilliermondii 3 J15 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| WEI YAN ET AL.,: ""Comprehensive investigations of 2-phenylethanol production by high 2-phenylethanol tolerating Meyerozyma sp. strain YLG18"", 《ENZYME AND MICROBIAL TECHNOLOGY》 * |
| WEI YAN ET AL.,: ""The draft genome sequence of Meyerozyma guilliermondii strain YLG18, a yeast capable of producing and tolerating high concentration of 2‑phenylethanol"", 《3BIOTECH》 * |
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|---|---|---|---|---|
| CN114574374A (en) * | 2022-03-18 | 2022-06-03 | 南京工业大学 | Strain for producing 2-phenethyl alcohol and application thereof |
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