阅读说明：本技术 利用混菌发酵生产2-苯乙醇的方法 (Method for producing 2-phenethyl alcohol by mixed fermentation ) 是由 信丰学 姜岷 严伟 章文明 蒋羽佳 周杰 董维亮 于 2020-11-13 设计创作，主要内容包括：本发明公开了利用混菌发酵生产2-苯乙醇的方法,将活化大肠杆菌接种至含葡萄糖发酵培养基中发酵后,将活化的季也蒙毕赤酵母接种至大肠杆菌发酵体系,发酵生产2-苯乙醇；所述大肠杆菌为引入苯丙氨酸合成中关键抗反馈抑制基因的重组菌。该方法是目前首次使用多细胞共培养的方式合成2-苯乙醇,降低了工业生产2-苯乙醇的底物成本,具有重要的应用价值。(The invention discloses a method for producing 2-phenethyl alcohol by mixed fermentation, which comprises the steps of inoculating activated escherichia coli into a fermentation culture medium containing glucose for fermentation, inoculating activated pichia guilliermondii into an escherichia coli fermentation system, and fermenting to produce 2-phenethyl alcohol; the Escherichia coli is a recombinant bacterium introduced with a key anti-feedback inhibition gene in phenylalanine synthesis. The method synthesizes the 2-phenethyl alcohol by using a multi-cell co-culture mode for the first time at present, reduces the substrate cost of industrial production of the 2-phenethyl alcohol and has important application value.)

1. A method for producing 2-phenethyl alcohol by using mixed fermentation is characterized in that activated escherichia coli is inoculated into a fermentation culture medium containing glucose for fermentation, and then activated pichia guilliermondii is inoculated into an escherichia coli fermentation system for fermentation production of 2-phenethyl alcohol;

the Escherichia coli is a tyrosine defect type recombinant strain introduced into a key anti-feedback inhibition gene in phenylalanine synthesis.

2. The method according to claim 1, wherein the recombinant bacterium is obtained by introducing a key anti-feedback inhibition gene in phenylalanine synthesis into escherichia coli in which a key enzyme gene in a tyrosine synthesis pathway is knocked out; the key enzyme gene of the tyrosine synthesis pathway istyrAThe key anti-feedback inhibition gene in the phenylalanine synthesis isaroFAndpheAa gene.

3. The method according to claim 1 or 2, wherein the recombinant strain is constructed by a method comprising:

(1) knock-out of E.coli K12 by Crispr gene editing technologytyrAObtaining tyrosine defect type Escherichia coli K12;

(2) the genearoFAndpheAintroducing pTrc99a plasmid, introducing the recombinant plasmid into tyrosine-deficient Escherichia coli K12, and expressing to obtain recombinant bacteria.

4. The method of claim 1, wherein the pichia guilliermondii is pichia guilliermondii (pichia guilliermondii)) (Meyerozyma guilliermondii) YLG18 with the preservation number of CCTCC NO: m2020638.

5. The method according to claim 1, wherein the activated escherichia coli is inoculated into a fermentation medium containing glucose and fermented for 24-48 hours, and then the activated pichia guilliermondii is inoculated; preferably for 24 hours.

6. The method of claim 1, wherein the amount of the activated escherichia coli inoculated is 1-10% of the volume of the fermentation medium; the active season Pichia Mongolica is inoculated into an Escherichia coli fermentation system according to the inoculation amount of 10%.

7. The method of claim 1, wherein the E.coli fermentation conditions are: the fermentation temperature is 35-37 ℃, the fermentation pH is 6.0-7.0, and the rotation speed is 200-400 rpm.

8. The method of claim 1, wherein the fermentation medium formulation is: 3g/L MgSO₄，3 g/L KH₂PO₃，1 g/L NaCl，5 g/L (NH₄)₂SO₄，0.015 g/L CaCl₂·2H₂O，0.1125 g/L FeSO₄·7H₂O, 1 g/L trisodium citrate, 10 g/L yeast powder, 0.3 g/L tyrosine,0.15 g/L VB₁0.5 g/L YNB, 20 g/L glucose and water as solvent, and the pH is adjusted to 6.0-7.0.

9. The method of claim 1, wherein after inoculation with pichia guilliermondii, the fermentation conditions are: the fermentation temperature is 28-30 ℃, the fermentation time is 48-72h, the fermentation pH is 5.0-6.0, and the rotation speed is 500-600 rpm.

10. The method of claim 1, wherein after the escherichia coli is fermented for 12 hours, glucose is supplemented to the fermentation system at a glucose supplementation rate of 2-4 g/L/h; preferably 4 g/L/h.

Technical Field

The invention belongs to the field of microbial fermentation, and particularly relates to a method for producing 2-phenethyl alcohol by using mixed fermentation of escherichia coli and pichia guilliermondii.

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.

The mixed fermentation is a novel fermentation technology which adopts the synergistic effect of two or more microorganisms to jointly complete a certain fermentation process. Is a new development of pure fermentation technology, and is also a novel fermentation technology which can obtain similar effect without carrying out complicated DNA in vitro recombination. Has the advantages of improving the fermentation efficiency and even forming a new product. According to the binding mode between organisms, the following four types can be divided. (1) Combined fermentation: simultaneous inoculation with two or more microorganismsAnd culturing, for example, mixed fermentation in the process of converting sorbose into diketogulonic acid in the vitamin C production of our invention. (2) Sequential fermentation, in which conventional fermentation is carried out by using A bacteria, and then fermentation is carried out by using B bacteria and the like in sequence to jointly complete a plurality of biochemical reactions, such as rhizopus arrhizus (A), (B), (C)Rhizopus arhizus) Converting glucose into fumaric acid, and then preparing the fumaric acid by Enterobacter aerogenes (B), (B)Enterococcus aerogenes) Or Proteus vulgaris: (A)Proteus vulgaris) It is reduced to the fermentation product succinic acid. (3) Co-immobilized cell mixed fermentation: mixed fermentation in which two or more microbial cells are simultaneously embedded or adsorbed on the same carrier, e.g. Aspergillus niger (A. niger)Aspergillus niger) And Zymomonas mobilis: (Zymomonas mobilis) Converting starch into alcohol together; (4) mixed immobilized cell mixed fermentation: after two or more kinds of microbial cells are immobilized separately, they are mixed together for mixed fermentation. At present, no report of producing 2-phenethyl alcohol by using glucose mixed bacteria exists.

Disclosure of Invention

In order to solve the problems of long metabolic path, heavy load of single bacteria and low yield of the traditional 2-phenethyl alcohol fermentation method by taking glucose as a substrate, the invention provides a method for producing 2-phenethyl alcohol by mixed fermentation of escherichia coli and pichia guilliermondii.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

a method for producing 2-phenethyl alcohol by mixed fermentation comprises inoculating activated Escherichia coli into a glucose-containing fermentation culture medium, fermenting, inoculating activated Pichia guilliermondii into an Escherichia coli fermentation system, and fermenting to produce 2-phenethyl alcohol;

the Escherichia coli is a tyrosine defect type recombinant strain introduced into a key anti-feedback inhibition gene in phenylalanine synthesis.

Further, leading a key feedback inhibition resistant gene in the synthesis of phenylalanine into escherichia coli with a key enzyme gene in a tyrosine synthesis pathway knocked out, and obtaining the recombinant bacterium; the key enzyme gene of the tyrosine synthesis pathway istyrA(Genbank:947115) Gene, the phenylalanine synthesisThe key anti-feedback inhibition gene isaroF(Genbank:947084) andpheA(Genbank:947081) gene.

Further, the construction method of the recombinant bacterium comprises the following steps:

(1) knock-out of E.coli K12 by Crispr gene editing technologytyrAObtaining tyrosine defect type Escherichia coli K12;

(2) the genearoFAndpheAintroducing pTrc99a plasmid, introducing the recombinant plasmid into tyrosine-deficient Escherichia coli K12, and expressing to obtain recombinant bacteria.

Further, the Pichia guilliermondii is Pichia guilliermondii (Pichia guilliermondii)) (Meyerozyma guilliermondii) YLG18 with the preservation number of CCTCC NO: m2020638.

Further, inoculating activated escherichia coli into a fermentation medium containing glucose, fermenting for 24-48 hours, and then inoculating activated pichia guilliermondii; preferably for 24 hours. At the moment, the fermentation liquor contains a certain amount of L-phenylalanine, the biomass of the escherichia coli is high, the L-phenylalanine can be synthesized by continuously utilizing glucose, and the Pichia guilliermondii can utilize the L-phenylalanine synthesized by the escherichia coli to produce the 2-phenylethyl alcohol. The inoculation time is too early (the fermentation of the escherichia coli is less than 24 hours), the biomass of the escherichia coli is not high, and the synthesis of the L-phenylalanine is not facilitated; too late inoculation time (48-72 h of escherichia coli fermentation) can result in too long culture time of the strain YLC20, limited increase of L-phenylalanine yield and too low nutrient content in fermentation liquor, thereby affecting the growth of Pichia guilliermondii and finally reducing the yield of 2-phenethyl alcohol. Therefore, the inoculation time of the pichia guilliermondii is a very critical step for producing 2-phenylethyl alcohol by mixed fermentation.

Further, the inoculation amount of the activated escherichia coli is 1-10% of the volume of the fermentation medium; the active season Pichia Mongolica is inoculated into an Escherichia coli fermentation system according to the inoculation amount of 10%.

Further, the fermentation conditions of the escherichia coli are as follows: the fermentation temperature is 35-37 ℃, the fermentation pH is 6.0-7.0, and the rotation speed is 200-400 rpm. The preferred fermentation conditions are a fermentation temperature of 37 ℃, a fermentation pH of 6.8 and a rotation speed of 300 rpm.

Further, the formula of the fermentation medium is as follows: 3g/L MgSO₄，3 g/L KH₂PO₃，1 g/L NaCl，5 g/L (NH₄)₂SO₄，0.015 g/L CaCl₂·2H₂O，0.1125 g/L FeSO₄·7H₂O, 1 g/L trisodium citrate, 10 g/L yeast powder, 0.3 g/L tyrosine, 0.15 g/L VB₁0.5 g/L YNB, 20 g/L glucose and water as solvent, adjusting pH to 6.0-7.0, and sterilizing at 115 deg.C for 20 min.

Further, after inoculating the pichia guilliermondii, the fermentation conditions are as follows: the fermentation temperature is 28-30 ℃, the fermentation time is 48-72h, the fermentation pH is 5.0-6.0, and the rotation speed is 500-600 rpm. Preferred fermentation conditions are: the fermentation temperature is 30 ℃, the fermentation time is 72h, the fermentation pH is 5.0, and the rotating speed is 500 rpm.

Further, after the escherichia coli is fermented for 12 hours, glucose is supplemented to the fermentation system according to a glucose supplementing rate of 2-4 g/L/h; preferably 4 g/L/h.

Further, the formula of the activating culture medium of the escherichia coli is 3g/L MgSO₄, 3 g/L KH₂PO₃, 1 g/L NaCl, 5 g/L (NH₄)₂SO₄, 0.015 g/L CaCl₂·2H₂O, 0.1125 g/L FeSO₄·7H₂O, 1 g/L trisodium citrate, 10 g/L yeast powder, 0.3 g/L tyrosine, 0.15 g/L VB₁0.5 g/L YNB, 20 g/L glucose and water as solvent, and the pH is adjusted to 6.0-7.0. The activation conditions were: escherichia coli was inoculated into an activation medium at an inoculum size of 5% v/v, and activated at 35-37 ℃ for 24-48 h at 200-400 rpm.

Further, the formula of the activating medium of the pichia guilliermondii is 20 g/L peptone, 10 g/L yeast powder, 20 g/L glucose and the solvent is water; the activation conditions were as follows: inoculating the Pichia guilliermondii with an inoculum size of 5% v/v into an activation medium, and activating at 30 ℃ and 200 rpm for 12-24 h to obtain an activated Pichia guilliermondii culture solution.

Has the advantages that: compared with the prior art, the invention has the following technical advantages:

(1) according to the invention, the escherichia coli YLC20 capable of growing by taking glucose as a unique carbon source is cultured firstly, the glucose is converted into the L-phenylalanine under the optimal condition, the yield of the 2-phenethyl alcohol synthesized by the pichia guilliermondii is extremely low by directly taking the glucose as the pichia guilliermondii, but the yield of the 2-phenethyl alcohol synthesized by taking the L-phenylalanine as a substrate is higher; at present, the yield of 2-phenethyl alcohol directly synthesized by microorganisms by taking glucose as a substrate is very low. The invention adopts a sequential fermentation method in mixed fermentation, glucose is gradually converted into L-phenylalanine through the fermentation of escherichia coli YLC20, Pichia guilliermondii is inoculated when the L-phenylalanine reaches a certain amount, and the converted L-phenylalanine and glucose are utilized for fermentation to produce the 2-phenethyl alcohol.

(2) When Escherichia coli YLC20 is cultured for 24 h, the culture solution of Pichia guilliermondii is added, the yield of 2-phenethyl alcohol is the highest and reaches 2.3 g/L, and the highest 2-phenethyl alcohol yield is obtained by mixed fermentation when glucose is used as the only substrate at present. When the YLC20 is cultured for 24 hours, the biomass of escherichia coli in the fermentation liquid is higher, the glucose is effectively utilized, and about 9 g/L of L-phenylalanine is obtained; thereafter, in a mixed bacteria system, the biomass of the escherichia coli is still continuously improved, glucose can be continuously converted into L-phenylalanine, and the pichia guilliermondii can utilize the converted L-phenylalanine to produce 2-phenylethyl alcohol.

(3) The method is a novel fermentation technology which can obtain similar effect without carrying out complicated DNA in vitro recombination. The method reduces the cost of industrial production of the 2-phenethyl alcohol and has important application value.

Drawings

FIG. 1 shows the inoculation time of Pichia guilliermondii under optimized mixed fermentation conditions.

FIG. 2 shows the optimized inoculation amount of Pichia guilliermondii under mixed fermentation conditions.

The biological material of the invention is classified and named as Pichia guilliermondii (A)Meyerozyma guilliermondii) YLG18, which is preserved in China Center for Type Culture Collection (CCTCC) with a preservation date of 26/10/2020 and a preservation number of CCTCC NO: m2020638, accession number: wuhan, Wuhan university.

Detailed Description

The Pichia guilliermondii used in the examples is Pichia guilliermondii (R.guilliermondii) (Meyerozyma guilliermondii) YLG18 with the preservation number of CCTCC NO: m2020638.

The Escherichia coli is a self-constructed genetic engineering bacterium, and the gene is expressedaroFAndpheAthe plasmid pTrc99a was introduced, and the recombinant plasmid was expressed in a host bacterium, tyrosine-deficient Escherichia coli K12, which was named Escherichia coli YLC20 in the examples.

The YLC20 is constructed in the following manner:

(1) knock-out of E.coli K12 by Crispr gene editing technologytyrAObtaining tyrosine defect type Escherichia coli K12; crispr Gene Editing technology is a current method, and reference is made to Duan CL, Yang, S, Chen, B, et al, Multigene Editing in theEscherichia coliGenome via the CRISPR-Cas9 System, Applied and Environmental microbiology 2015, 81(7): 2506-E. Coli Chromosome by CRISPR/Cas9. Biotechnology and bioengineering. 2017, 114(1), 172-183；

(2) The genearoFAndpheAintroducing pTrc99a plasmid, introducing the recombinant plasmid into tyrosine-deficient Escherichia coli K12, and expressing to obtain recombinant bacteria. The construction and expression of recombinant plasmids are substantially the same as those of the prior art, and only the genes are distinguished, and reference is made to Wang J, Wang HY, Yang L, et al, A novel plasmid switch system of gene expression for enhanced microbial expression of general acid, Journal of Industrial Microbiology&Biotechnology, 2018, 45(4), 253-269, and Wang W, Li ZM, Xie JL, et al, Production of suberate by apflBldhA double mutant of Escherichia coli overexpressing malate dehydrogenase. Bioprocess and biosystems engineering. 2009, 32(6), 737-745.

EXAMPLE 1 Effect of different sugar supplementation rates on the Final 2-phenylethyl alcohol production

(1) Inoculating Escherichia coli into activation medium with inoculum size of 5% v/v, and activating at 37 deg.C and 300 rpm for 24 hr;

the formula of the activation medium is as follows: 10 g/L NaCl, 5 g/L yeast powder, 10 g/L peptone and water as a solvent, and adjusting the pH value to 6.8;

(2) inoculating activated Escherichia coli YLC20 into a fermentation medium at an inoculation amount of 5% v/v, and fermenting at 37 ℃ for 24 h;

the formula of the fermentation medium is as follows: 3g/L MgSO₄，3 g/L KH₂PO₃，1 g/L NaCl，5 g/L (NH₄)₂SO₄， 0.015 g/L CaCl₂·2H₂O，0.1125 g/L FeSO₄·7H₂O, 1 g/L trisodium citrate, 10 g/L yeast powder, 0.3 g/L tyrosine, 0.15 g/L VB₁0.5 g/L YNB, 20 g/L glucose and water as solvent, adjusting pH to 6.0-7.0, sterilizing at 115 deg.C for 20 min; three groups of fermentation experiments are set, wherein sugar supplement rates are respectively 2 g/L/h, 4g/L/h and 6 g/L/h;

(3) inoculating the Pichia guilliermondii into an activation culture medium of the Pichia guilliermondii at an inoculation amount of 5% v/v, and activating at 30 ℃ and 200 rpm for 12-24 h;

the formula of the activation medium is as follows: 20 g/L of peptone, 10 g/L of yeast powder, 20 g/L of glucose and water as a solvent;

(4) taking the mixing time as an example of 72h, the activated Pichia guilliermondii is inoculated into the fermentation system of the step (2) at 5% v/v, and the fermentation is carried out for 72h at 30 ℃ and 500 rpm.

During the culture process, samples are taken every 12h to determine the 2-phenethyl alcohol yield. When the sugar supplement rate is 4g/L/h, the concentration of the 2-phenethyl alcohol obtained by mixed fermentation reaches 1.5 g/L, and when the sugar supplement rate is 2 g/L/h, the concentration of the 2-phenethyl alcohol obtained by mixed fermentation is only 1.1 g/L; the concentration of 2-phenylethyl alcohol was 1.53 g/L when the sugar-replenishing rate was 6 g/L/h, but a sugar-replenishing rate of 4g/L/h was finally selected from the economical viewpoint.

Example 2 Effect of different inoculation times for Pichia guilliermondii on Final 2-phenylethyl alcohol production

The method is the same as example 1, wherein the sugar supplement rate is 4 g/L/h; except that 4 groups of experiments are set in the step (2), and the fermentation time is 12h, 24 h, 36 h and 48 h respectively.

During the culture, samples were taken every 12 hours and the 2-phenylethyl alcohol yield was measured. When Escherichia coli YLC20 is cultured for 24 h, Pichia guilliermondii is added, and the yield of 2-phenethyl alcohol is the highest and reaches 1.75 g/L. In the system, the strain YLC20 effectively utilizes glucose to grow and obtain about 9 g/L of L-phenylalanine, and then in a mixed strain system, Escherichia coli can still utilize glucose to continuously grow, and can continuously convert glucose into L-phenylalanine, and Pichia guilliermondii can utilize the converted L-phenylalanine to produce 2-phenylethyl alcohol. The inoculation time is too early (12 h), the biomass of escherichia coli is not high, the glucose utilization capacity is limited, and the inoculation time is too late (36-48 h), so that the culture time of the strain YLC20 is too long, the nutrient substances in the fermentation liquor are low, the pichia mondii is difficult to achieve the required biomass, and finally the yield of 2-phenethyl alcohol is reduced.

Example 3 Pichia guilliermondiiMeyerozyma guilliermondiiInfluence of different inoculum sizes of YLG18 on 2-phenylethyl alcohol yield

The procedure is as in example 1, wherein the rate of sugar supplementation is 4g/L/h and the inoculation time is 24 h, except that 4 sets of experiments were set up in step (3), and the inoculation amounts of Pichia guilliermondii in each set were 5% v/v, 10% v/v, 15% v/v and 20% v/v, respectively.

During the culture, samples were taken every 12 hours and the 2-phenylethyl alcohol yield was measured. When the inoculation amount is 10% v/v, the yield of the 2-phenethyl alcohol is the highest and is 2.0 g/L; when the inoculation amount is 5% v/v, the strain grows slowly, and the yield of the 2-phenethyl alcohol is influenced; when the inoculation amount is 15-20% v/v, the L-phenylalanine synthesis of escherichia coli can be influenced due to excessive inoculation amount, so that the 10% v/v inoculation amount is adopted as the optimal inoculation amount.

EXAMPLE 4 Effect of different pH values in culture Medium of Mixed culture System on the Final 2-Phenylethanol production

The procedure is as in example 3, except that 6 sets of experiments were carried out in step (3) with pH values of 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 for each set, with a sugar supplementation rate of 4g/L/h, an inoculation time of 24 h, and an inoculation amount of 200 mL.

During the culture, samples were taken every 12 hours and the 2-phenylethyl alcohol yield was measured. When the pH value is 5.0, the yield of the 2-phenethyl alcohol is the highest and reaches 2.3 g/L. When the pH value is increased, the fermentation conditions are more favorable for the bioconversion of escherichia coli to generate L-phenylalanine and are unfavorable for the pichia guilliermondii to synthesize the 2-phenethyl alcohol by utilizing the L-phenylalanine. The most suitable pH value of the escherichia coli is 6.8, the most suitable pH value of the pichia guilliermondii is 5.0, and the yield of the 2-phenethyl alcohol can be improved to a certain extent by optimizing the pH value. Since Escherichia coli can be converted into L-phenylalanine by using glucose all the time, it can not grow after mixed bacteria by lowering pH (Escherichia coli can hardly grow at low pH) and lowering fermentation temperature (fermentation temperature is 30 ℃ after inoculating Mongolia Pichia pastoris), and L-phenylalanine secreted from Escherichia coli still can stably exist in fermentation liquor and can be used by Mongolia Pichia pastoris to synthesize 2-phenylethyl alcohol in enough seasons.

Experiments prove that the Pichia guilliermondii is fermented directly by glucose, the yield of 2-phenethyl alcohol is very low and is only less than 0.1 g/L; when the mixed bacteria system is used, 2.3 g/L2-phenethyl alcohol can be produced. The mixed bacteria system is a novel fermentation technology which can obtain similar effect without complex DNA in vitro recombination, not only reduces the substrate cost of industrial production of 2-phenethyl alcohol, but also can effectively improve the yield of the 2-phenethyl alcohol.