CN112760243B - Fermentation medium for saccharomyces cerevisiae and method for producing phenethyl alcohol by saccharomyces cerevisiae fermentation - Google Patents

Fermentation medium for saccharomyces cerevisiae and method for producing phenethyl alcohol by saccharomyces cerevisiae fermentation Download PDF

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CN112760243B
CN112760243B CN202110236693.4A CN202110236693A CN112760243B CN 112760243 B CN112760243 B CN 112760243B CN 202110236693 A CN202110236693 A CN 202110236693A CN 112760243 B CN112760243 B CN 112760243B
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saccharomyces cerevisiae
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glucose
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杭海峰
王春侠
杨承翰
郭美锦
储炬
庄英萍
刘吴一
邵博宁
李潇鑫
黄隆昊
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Qingdao Institute Of Innovation East China University Of Technology
East China University of Science and Technology
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Abstract

The application discloses fermentation medium for saccharomyces cerevisiae, fermentation medium contains: nitrogen source, carbon source, potassium dihydrogen phosphate; and the mass ratio of the nitrogen source to the carbon source is 3:4 to 8; the nitrogen source consists of phenylalanine and ammonium sulfate, and the mass percent of the phenylalanine in the nitrogen source is 26-100%; the carbon source is glucose; the fermentation medium also comprises metal ion mother liquor and vitamin mother liquor. The application also provides a method for producing the phenethyl alcohol by fermenting the saccharomyces cerevisiae. When the fermentation culture medium is used for fermentation production of phenethyl alcohol, the W303 strain has good respiratory metabolism effect, the utilization rate of a substrate is high, and the output of the phenethyl alcohol is up to 2.03g/L.

Description

Fermentation medium for saccharomyces cerevisiae and method for producing phenethyl alcohol by saccharomyces cerevisiae fermentation
Technical Field
The application relates to the technical field of microbial culture, in particular to the technical field of fermentation production of phenethyl alcohol by saccharomyces cerevisiae, and specifically relates to a fermentation culture medium for saccharomyces cerevisiae and a method for producing phenethyl alcohol by saccharomyces cerevisiae fermentation.
Background
Phenethyl alcohol (2-PE) is a high-value aromatic compound, and is widely used as a perfume in the fields of foods and cosmetics because it has a fragrance like rose. And also has bacteriostatic action, so that the bactericide can be widely applied to the field of pharmacy. The global market demand for 2-PE exceeds about ten thousand tons per year, and there are three main methods of extraction for 2-PE: natural extraction method, chemical synthesis method, and microbial transformation method. The natural 2-PE with high quality is mainly extracted from the volatile oil component of the rose, and the method has the defects of difficult extraction, high cost, market price of between about $ 150/kg and $ 200/kg, and incapability of meeting the market demand due to the shortage of raw materials and low extraction efficiency. Currently, most 2-PE is synthesized by chemical methods. Although this method is inexpensive and has high conversion, the formation of toxic by-products during chemical synthesis is prohibited in many industries due to safety and health concerns. Therefore, natural synthesis of 2-PE by using microorganisms has been a research focus in recent years.
Many yeasts, including Saccharomyces cerevisiae, yarrowia lipolytica, pichia pastoris and the like, have the ability to naturally synthesize 2-PE. Saccharomyces cerevisiae can produce 2-PE by two natural metabolic pathways, namely the synthesis of 2-PE by the Shikimate pathway (Shikimate pathway) using glucose and the synthesis of 2-PE by the Ehrlich pathway using L-phenylalanine. The synthesis of 2-PE by using glucose through a Shikimate pathway requires that phosphoenolpyruvate (PEP) and 4-erythrose phosphate (E4P) are provided for a central carbon metabolism library through a glycolysis process and a pentose phosphate pathway, then DAHP is formed under the catalysis of 2-keto-3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthase), and then the DAHP is converted into phenylpyruvic acid through a shikimic acid pathway, and the 2-PE is further produced through transamination, decarboxylation and reduction. In Saccharomyces cerevisiae this synthetic route is complex and tedious, often resulting in a low productivity of the desired product. This may be due to the complex biosynthesis that requires a coordinated supply of precursors, ATP and reducing equivalents (NADPH/NADH) to maintain the cellular metabolism and product formation processes. The synthesis of 2-PE via the Ehrlich pathway using phenylalanine (L-Phe) requires only relatively simple transamination, decarboxylation and reduction. The Ehrlich pathway is therefore the preferred bioconversion pathway for the synthesis of 2-PE, and when phenylalanine is provided as the sole nitrogen source in the medium, the Ehrlich pathway in Saccharomyces cerevisiae will dominate and 2-PE yield is significantly increased. In Saccharomyces cerevisiae, metabolites produced by Shikimate metabolic pathway and Ehrlich metabolic pathway of biotransformation to synthesize 2-PE flow to a central carbon metabolic pool to construct an interacting metabolic network. In the process of synthesizing 2-PE, PEP and E4P synthesized and accumulated by utilizing substrate glucose are crucial to product synthesis, and high-concentration L-Phe is a prerequisite for high yield of 2-PE. However, the high price of phenylalanine (L-Phe) has prevented its use on an industrial scale, and the development of inexpensive, economical substrates has led to extensive research.
Product synthesis by microorganisms requires a coordinated supply of precursors and cofactors to sustain cell growth and product formation. Different substrates have different entry points into the metabolic network and thus different energy and redox states, which adds degrees of freedom to the metabolic network system of the cell.
Therefore, it is highly desirable to provide a substrate co-feed synthetic medium and fermentation method for efficient fermentative production of 2-PE.
Disclosure of Invention
The embodiment of the application provides a fermentation medium for saccharomyces cerevisiae and a method for producing phenethyl alcohol (2-PE) by saccharomyces cerevisiae fermentation, which can promote the growth of saccharomyces cerevisiae and improve the yield of phenethyl alcohol.
The embodiment of the application provides a fermentation medium for saccharomyces cerevisiae, which comprises: nitrogen source, carbon source, potassium dihydrogen phosphate (KH) 2 PO 4 ) Metal ion mother liquor and vitamin mother liquor; and the mass ratio of the nitrogen source to the carbon source is 3:4 to 8; wherein, the first and the second end of the pipe are connected with each other,
the nitrogen source consists of phenylalanine (L-Phe) and ammonium sulfate ((NH) 4 ) 2 SO 4 ) The mass percentage of the phenylalanine in the nitrogen source is 26-100%; the carbon source is glucose.
In some embodiments, the mass percentage of phenylalanine in the nitrogen source is 50 to 67%.
In some embodiments, the fermentation medium comprises: 7.5g/L nitrogen source, 10-20 g/L carbon source, 14.4g/L potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.2-200 mL/L of metal ion mother liquor and 0.1-100 mL/L of vitamin mother liquor.
In some embodiments, the metal ion mother liquor comprises: feSO 4 ·7H 2 O、ZnSO 4 ·7H 2 O、CaCl 2 、MnCl 2 ·4H 2 O、CoCl 2 ·6H 2 O、CuSO 4 ·5H 2 O、Na 2 MoO 4 ·2H 2 O、H 3 BO 3 KI and EDTA.
In some embodiments, the metal ion mother liquor comprises: 3.0g/L FeSO 4 ·7H 2 O, 4.5g/L ZnSO 4 ·7H 2 O, 4.5g/L CaCl 2 1.0g/L of MnCl 2 ·4H 2 O, 0.3g/L CoCl 2 ·6H 2 O, 0.3g/L CuSO 4 ·5H 2 O, 0.4g/L of Na 2 MoO 4 ·2H 2 O, 1g/L H 3 BO 3 0.1g/L KI and 19g/L EDTA.
In some embodiments, the pH of the metal ion mother liquor is 4.0.
In some embodiments, the vitamin mother liquor comprises: biotin, pantothenic acid hemicalcium salt, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, p-aminobenzoic acid and inositol.
In some embodiments, the vitamin mother liquor comprises: 50mg/L biotin, 1000mg/L pantothenic acid hemicalcium salt, 1000mg/L thiamine hydrochloride, 1000mg/L pyridoxine hydrochloride, 1000mg/L nicotinic acid, 200mg/L p-aminobenzoic acid, and 25000mg/L inositol.
In some embodiments, the pH of the vitamin mother liquor is 6.5.
In some embodiments, the fermentation medium is sterilized at a temperature of 121 ℃ for a period of 30min.
In some embodiments, the saccharomyces cerevisiae is saccharomyces cerevisiae W303, from the china industrial collection of microorganisms. The fermentation medium can be applied to fermentation of saccharomyces cerevisiae W303 from China industrial microorganism strain preservation management center for producing phenethyl alcohol by high-efficiency fermentation.
The application also provides a method for producing phenethyl alcohol by saccharomyces cerevisiae fermentation of the fermentation medium, which comprises the following steps: adding the fermentation medium to a bioreactor; centrifuging, washing and resuspending seed liquid of the saccharomyces cerevisiae strain, inoculating the seed liquid into a bioreactor, wherein the inoculation amount is 10% of the fermentation volume, stirring and ventilating, and fermenting at the temperature of 27-33 ℃ until the fermentation is finished; wherein, the dissolved oxygen of the fermentation liquor is controlled to be more than or equal to 30 percent in the fermentation process.
In some embodiments, the rotational speed of the agitation does not exceed 700rpm.
In some embodiments, the ventilation has a ventilation volume of no more than 500mL/min.
In some embodiments, the fermentation medium comprises: 7.5g/L nitrogen source, 10-20 g/L carbon source, 14.4g/L potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.2-200 mL/L of metal ion mother liquor and 0.1-100 mL/L of vitamin mother liquor.
In some embodiments, the preparation of the seed liquid of the saccharomyces cerevisiae strain comprises:
taking out the strains preserved in a glycerol tube, dipping a proper amount of bacterial liquid, coating the bacterial liquid on a slant culture medium, and culturing at the constant temperature of 30 ℃ for 72 hours;
step two, eluting the bacterial colony cultured in the step one by using distilled water, performing gradient dilution on the eluted bacterial liquid, coating the diluted bacterial liquid on a slant culture medium, and culturing the bacterial colony for 72 hours in a constant-temperature incubator at the temperature of 30 ℃;
and step three, selecting the single colony obtained in the step two to be added into 5mL of seed culture medium added with metal ion mother liquor and vitamin mother liquor, and culturing for 12h at 30 ℃ and 220rpm to obtain the seed liquor.
In some embodiments, in step one and step two, the slant medium comprises: 3g/L of yeast extract, 3g/L of malt extract, 5g/L of peptone, 10g/L of glucose and 20g/L of agar.
In some embodiments, in step three, the seed medium comprises: (NH) 4 ) 2 SO 4 7.5g/L、KH 2 PO 4 14.4g/L、MgSO 4 ·7H 2 0.5g/L of O, 10g/L of glucose, 200 mu L/L of metal ion mother liquor and 100 mu L/L of vitamin mother liquor. The sterilization temperature of the seed culture medium is 115 ℃, and the sterilization time is 20min.
In some embodiments, the Saccharomyces cerevisiae (Saccharomyces cerevisiae) may be Saccharomyces cerevisiae W303. The Saccharomyces cerevisiae can be Saccharomyces cerevisiae S288C, a model strain, and is from Beijing Beinanna institute of Biotechnology. The Saccharomyces cerevisiae may be Saccharomyces cerevisiae W101.
For example, from the China center for Industrial culture Collection of microorganisms, accession number CICC 32451. For example, the strain is from China center for Industrial culture Collection of microorganisms, and the collection number is CICC 33319.
In order to prevent the components of the culture medium from being damaged by the Maillard reaction, the glucose solution in the culture medium can be independently prepared and sterilized. Sterilizing at 115 deg.C for 20min.
Phenylalanine as used herein is L-phenylalanine (L-Phe).
The beneficial effect of this application lies in:
according to the fermentation medium provided by the embodiment of the application, phenylalanine is used as an organic nitrogen source, ammonium sulfate is used as an inorganic nitrogen source, glucose is used as a carbon source, and phenylalanine, ammonium sulfate and glucose are jointly used as co-feeding substrates according to a certain proportion, and the growth of saccharomyces cerevisiae can be promoted and the yield of phenethyl alcohol can be improved by performing mixed fermentation in the medium. The application adopts phenylalanine and ammonium sulfate as nitrogen sources, has better metabolic advantages than a single phenylalanine substrate, can relieve highly controlled natural metabolic limitation, overcomes the limitation of an internal path, improves the carbon conversion rate and reduces the economic cost.
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The technical solution and other advantages of the present application will become apparent from the detailed description of the embodiments of the present application with reference to the accompanying drawings.
FIG. 1 is a graph showing fermentation characteristics of Saccharomyces cerevisiae W303 in test example 1 of the present application.
FIG. 2 is a graph showing fermentation characteristics of Saccharomyces cerevisiae W303 in test example 2 of the present application.
FIG. 3 is a graph showing fermentation characteristics of Saccharomyces cerevisiae S288C and Saccharomyces cerevisiae W303 in Experimental example 3 of the present application.
FIG. 4 is a bar graph showing the change in the concentration of phenethyl alcohol during fermentation of Saccharomyces cerevisiae S288C, saccharomyces cerevisiae W303 and Saccharomyces cerevisiae W101 in Experimental example 4 of the present application.
FIG. 5 is a line graph showing the growth of Saccharomyces cerevisiae S288C, saccharomyces cerevisiae W303 and Saccharomyces cerevisiae W101 in the fermentation process in Experimental example 4 of the present application.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. It should be apparent that the described embodiments are only a few embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The present application is further described with reference to the following drawings and specific examples:
the embodiment of the application provides a fermentation medium for saccharomyces cerevisiae, which comprises: nitrogen source, carbon source, potassium dihydrogen phosphate, metal ion mother liquor and vitamin mother liquor. The mass ratio of the nitrogen source to the carbon source is 3:4 to 8. The carbon source is glucose. The nitrogen source is composed of phenylalanine (L-Phe) and ammonium sulfate ((NH) 4 ) 2 SO 4 ) The mass percentage of the phenylalanine in the nitrogen source is 26-100%. Further, styrene acrylicThe mass percentage of the amino acid in the nitrogen source is preferably 50 to 67%.
In one embodiment, the fermentation medium comprises: 7.5g/L of nitrogen source, 10-20 g/L of carbon source and 14.4g/L of monopotassium phosphate (KH) 2 PO 4 ) 0.2-200 mL/L of metal ion mother liquor and 0.1-100 mL/L of vitamin mother liquor. Further, the sterilization temperature of the fermentation medium is 121 ℃, and the time is 30min. Further, the glucose is separately prepared and sterilized; for example, sterilization is carried out at 115 ℃ for 20min. Further, in the fermentation medium, the concentration of the carbon source is preferably 20g/L.
The metal ion mother liquor comprises: feSO 4 ·7H 2 O、ZnSO 4 ·7H 2 O、CaCl 2 、MnCl 2 ·4H 2 O、CoCl 2 ·6H 2 O、CuSO 4 ·5H 2 O、Na 2 MoO 4 ·2H 2 O、H 3 BO 3 KI and EDTA. Further, the pH of the metal ion mother liquor is 4.0. The pH of the metal ion mother liquor can be adjusted, for example, with 2mol/L HCl.
The vitamin mother liquor comprises: biotin, pantothenic acid hemicalcium salt, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, p-aminobenzoic acid and inositol. The pH of the vitamin mother liquor is 6.5. The pH of the vitamin mother liquor can be adjusted, for example, with 2mol/L NaOH.
The fermentation medium provided by the application can be applied to fermentation of saccharomyces cerevisiae for producing phenethyl alcohol by high-efficiency fermentation.
The embodiment of the application also provides a method for producing phenethyl alcohol (2-PE) by saccharomyces cerevisiae fermentation, which adopts the fermentation culture medium, and specifically, the fermentation method comprises the following steps:
adding the fermentation medium to a bioreactor; centrifuging, washing and resuspending seed liquid of the saccharomyces cerevisiae strain, inoculating the seed liquid into a bioreactor, wherein the inoculation amount is 10% of the fermentation volume, stirring and ventilating, and fermenting at the temperature of 27-33 ℃ until the fermentation is finished; wherein, the dissolved oxygen of the fermentation liquor is controlled to be more than or equal to 30 percent in the fermentation process. Specifically, the rotation speed of the stirring does not exceed 700rpm. The ventilation volume does not exceed 500mL/min.
The fermentation medium is as described above.
Further, at the later stage of fermentation, the dissolved oxygen may be lower than 30% due to the limitation of reactor performance, and the ventilation amount may be increased or the rotation speed may be increased appropriately. Specifically, the whole fermentation process can acquire dissolved oxygen, pH, OUR, CER, ventilation, rotation speed, temperature and other on-line parameters through biostar software.
Further, the preparation step of the seed liquid of the saccharomyces cerevisiae strain comprises the following steps:
taking out the strains preserved in a glycerol tube, dipping a proper amount of bacterial liquid, coating the bacterial liquid on a slant culture medium, and culturing at the constant temperature of 30 ℃ for 72 hours;
step two, eluting the bacterial colony cultured in the step one by using distilled water, performing gradient dilution on the eluted bacterial liquid, coating the diluted bacterial liquid on a slant culture medium, and culturing for 72 hours in a constant-temperature incubator at 30 ℃;
and step three, selecting the single colony obtained in the step two to be added into 5mL of seed culture medium added with metal ion mother liquor and vitamin mother liquor, and culturing for 12h at 30 ℃ and 220rpm to obtain the seed liquor.
In the first step and the second step, the slant culture medium comprises: 3g/L of yeast extract, 3g/L of malt extract, 5g/L of peptone, 10g/L of glucose and 20g/L of agar.
In step three, the seed culture medium comprises: (NH) 4 ) 2 SO 4 7.5g/L、KH 2 PO 4 14.4g/L、MgSO 4 ·7H 2 0.5g/L of O, 10g/L of glucose, 200 mu L/L of metal ion mother liquor and 100 mu L/L of vitamin mother liquor. The sterilization temperature of the seed culture medium is 115 ℃, and the sterilization time is 20min.
The saccharomyces cerevisiae strains used in the examples of the present application were derived from: saccharomyces cerevisiae S288C, a model strain, purchased from the institute of biotechnology, chuanglian, beijing; saccharomyces cerevisiae W101 purchased from China center for culture Collection of Industrial microorganisms; saccharomyces cerevisiae W303, purchased from China center for Industrial culture Collection of microorganisms.
The detection categories and detection methods according to the present application are as follows:
(1) dry weight of the cells: the absorbance was measured at a wavelength of 600, and the dry weight of the cells was characterized by the absorbance.
(2) Glucose concentration in the fermentation broth: the measurement was carried out using an SGD-IV reducing sugar meter. (the sample is diluted before quantification, and the glucose measurement range of the instrument is 0-0.5 g/L)
(3) And (3) measuring the contents of ethanol, glycerol, acetic acid and other components in the fermentation liquor: detection was performed by High Performance Liquid Chromatography (HPLC). The sample pretreatment method comprises the following steps: the fermentation broth sample was centrifuged at 4000rpm for 5min. And after the centrifugation is finished, taking the supernatant, and filtering by using a water system filter membrane to obtain the sample to be detected. Detection conditions are as follows: the chromatographic column was a Hi-Plex H column, 300x7.7 mm, (Agilent technologies); dilute sulfuric acid with the mobile phase of 0.01 mol/L; the measurement of each component of the fermentation liquid is carried out under the conditions that the flow rate is 0.4mL/min, the column temperature is 50 ℃, the detector is a differential refraction detector, and the sample injection amount is 10 mu L. The standard curves for the 3 fermentation broth components involved in the experiment are shown in Table 1.
TABLE 1 ethanol, acetic acid and glycerol standard curves
Detecting substance Time to peak (min) Standard curve R 2 Standard curve range (g/L)
Ethanol 35.661 y＝0.0014x-1.0533 R 2 ＝0.9974 0.5～50
Acetic acid 26.478 y＝0.0024x-5.2425 R 2 ＝0.9997 0.1～10
Glycerol 23.362 y＝0.0004x-1.1888 R 2 ＝0.9999 0.1～10
(4) Measuring the contents of phenylalanine, phenethyl alcohol and other components in the fermentation liquor: detection was performed by High Performance Liquid Chromatography (HPLC). The sample pretreatment method comprises the following steps: the fermentation broth sample was centrifuged at 4000rpm for 5min. And after the centrifugation is finished, taking the supernatant, and filtering by using a water system filter membrane to obtain the sample to be detected. Detection conditions are as follows: the column used was a Unitry C18 column, 4.6X250 mm,5 μm (Water Corporation, ireland); the mobile phase is 50% methanol water solution; the flow rate is 0.8mL/min, the column temperature is 30 ℃, the detector is a variable wavelength ultraviolet detector, and the sample injection amount is 10 mu L. The standard curves for the 2 fermentation broth components involved in the experiment are shown in Table 2.
TABLE 2 phenylalanine (L-Phe) and phenethyl alcohol (2-PE) standard curves
Detecting substance Time to peak (min) Standard curve R 2 Standard curve range (g/L)
L-Phe 3.915 y＝1E-06x+0.0356 R 2 ＝0.9998 0.5～10
2-PE 8.903 y＝9E-07x-0.0315 R 2 ＝0.9993 0.1～10
(5) Measuring the content of oxygen and carbon dioxide components in the tail gas in the fermentation process: the volume fractions of oxygen and carbon dioxide in the exhaust gas were determined using a process mass spectrometer (MAX 300-LG, extrel, USA). The Oxygen Uptake Rate (OUR) and carbon dioxide release rate (CER) were calculated on-line using the method described by Parekh et al.
Example 1:
this example provides a fermentation medium for saccharomyces cerevisiae, comprising: 3.75g/L phenylalanine (L-Phe), 3.75g/L ammonium sulfate ((NH) 4 ) 2 SO 4 ) 20g/L glucose and 14.4g/L potassium dihydrogen phosphate (KH) 2 PO 4 ) And, 200mL/L of the metal ion mother liquor and 100mL/L of the vitamin mother liquor.
In some embodimentsWherein the metal ion mother liquor comprises: 3.0g/L FeSO 4 ·7H 2 O, 4.5g/L ZnSO 4 ·7H 2 O, 4.5g/L CaCl 2 1.0g/L of MnCl 2 ·4H 2 O, 0.3g/L CoCl 2 ·6H 2 O, 0.3g/L CuSO 4 ·5H 2 O, 0.4g/L Na 2 MoO 4 ·2H 2 O, 1g/L H 3 BO 3 0.1g/L KI, 19g/L EDTA. The pH of the metal ion mother liquor is 4.0.
In some embodiments, the vitamin mother liquor comprises: 50mg/L biotin, 1000mg/L pantothenic acid hemicalcium salt, 1000mg/L thiamine hydrochloride, 1000mg/L pyridoxine hydrochloride, 1000mg/L nicotinic acid, 200mg/L p-aminobenzoic acid, and 25000mg/L inositol. The pH of the vitamin mother liquor is 6.5.
Example 2:
this example provides a fermentation medium for saccharomyces cerevisiae, the fermentation medium comprising: phenylalanine (L-Phe) at 5.0g/L, ammonium sulfate ((NH) at 2.5g/L 4 ) 2 SO 4 ) 20g/L glucose and 14.4g/L potassium dihydrogen phosphate (KH) 2 PO 4 ) And, 200mL/L of the metal ion mother liquor and 100mL/L of the vitamin mother liquor.
Other conditions were the same as in example 1.
Example 3:
this example provides a fermentation medium for saccharomyces cerevisiae, comprising: 3.75g/L phenylalanine (L-Phe), 3.75g/L ammonium sulfate ((NH) 4 ) 2 SO 4 ) 10g/L glucose and 14.4g/L potassium dihydrogen phosphate (KH) 2 PO 4 ) And, 200mL/L of the metal ion mother liquor and 100mL/L of the vitamin mother liquor.
Other conditions were the same as in example 1.
Example 4:
this example provides a fermentation medium for saccharomyces cerevisiae, comprising: phenylalanine (L-Phe) at 5.0g/L, ammonium sulfate ((NH) at 2.5g/L 4 ) 2 SO 4 ) 10g/L glucose and 14.4g/L potassium dihydrogen phosphate (KH) 2 PO 4 ) And, 200mL/L metal ionMother-child liquid and 100mL/L vitamin mother liquid.
Other conditions were the same as in example 3.
Example 5:
the embodiment provides a method for producing phenethyl alcohol (2-PE) by saccharomyces cerevisiae fermentation, which comprises the following steps:
taking out a saccharomyces cerevisiae strain preserved in a glycerol tube, dipping a proper amount of bacterial liquid, coating the bacterial liquid on a slant culture medium, and culturing for 72 hours at a constant temperature of 30 ℃;
step two, eluting the bacterial colony cultured in the step one by using distilled water, performing gradient dilution on the eluted bacterial liquid, coating the diluted bacterial liquid on a slant culture medium, and culturing the bacterial colony for 72 hours in a constant-temperature incubator at the temperature of 30 ℃;
step three, selecting the single colony obtained by the culture in the step two to 5mL of seed culture medium added with metal ion mother liquor and vitamin mother liquor, and culturing for 12h at 30 ℃ and 220rpm to obtain seed liquor;
step four, adding a fermentation medium into the bioreactor; centrifuging, washing and resuspending the seed solution obtained in the third step, inoculating the seed solution into a bioreactor, wherein the inoculation amount is 10% of the fermentation volume, stirring and ventilating, and fermenting at 30 ℃ until the fermentation is finished; wherein, the dissolved oxygen of the fermentation liquor is controlled to be more than or equal to 30 percent in the fermentation process.
In the embodiment, the saccharomyces cerevisiae is saccharomyces cerevisiae W303.
In the first step and the second step, the slant culture medium comprises: 3g/L of yeast extract, 3g/L of malt extract, 5g/L of peptone, 10g/L of glucose and 20g/L of agar.
In step three, the seed culture medium comprises: (NH) 4 ) 2 SO 4 7.5g/L、KH 2 PO 4 14.4g/L、MgSO 4 ·7H 2 0.5g/L of O, 10g/L of glucose, 200 mu L/L of metal ion mother liquor and 100 mu L/L of vitamin mother liquor. The sterilization temperature of the seed culture medium is 115 ℃, and the sterilization time is 20min.
In the fourth step, the fermentation medium is the fermentation medium in any of the above embodiments of the present application.
Test example 1:
this test example used the fermentation medium of example 1 to carry out the method for the fermentative production of phenethyl alcohol (2-PE) by using Saccharomyces cerevisiae as described in example 5. In addition, in this test example, fermentation characteristics such as glucose, phenylalanine (L-Phe), phenethyl alcohol (2-PE), dry Cell Weight (DCW), oxygen Uptake Rate (OUR), carbon dioxide release rate (CER), and ethanol were monitored during the course of the fermentation process to examine the fermentation characteristics, as shown in FIG. 1. FIG. 1 shows the fermentation results of Saccharomyces cerevisiae W303 with 20g/L glucose substrate co-feed.
Test example 2:
this test example used the fermentation medium of example 3 to carry out the process for the fermentative production of phenethyl alcohol (2-PE) by Saccharomyces cerevisiae as described in example 5. In addition, in this test example, fermentation characteristics such as glucose, phenylalanine (L-Phe), phenethyl alcohol (2-PE), dry Cell Weight (DCW), oxygen Uptake Rate (OUR), carbon dioxide release rate (CER), and ethanol were monitored during the course of the fermentation process to examine the fermentation characteristics, as shown in FIG. 2. FIG. 2 shows the results of fermentation of Saccharomyces cerevisiae W303 with a co-feed of 10g/L glucose substrate.
As is clear from the above test examples 1 and 2, the whole fermentation stage of Saccharomyces cerevisiae W303 can be divided into a stage of co-use of glucose and phenylalanine and a stage of co-use of ethanol and phenylalanine. When glucose exists, the spawn is in an exponential growth stage by taking the glucose as a substrate. At this time, glucose is continuously consumed, and metabolites such as ethanol, acetic acid, acetaldehyde and the like are gradually synthesized and accumulated. Meanwhile, 2-PE is synthesized in a large amount through a shikimic acid pathway taking glucose as a substrate and an ehrlich pathway taking phenylalanine as a substrate. When the glucose in the fermentation liquor is exhausted, the thalli gradually show autolysis phenomenon, so that the thalli amount is slightly reduced, and the adaptation period is that the thalli uses ethanol as a substrate to carry out secondary fermentation. When the glucose in the substrate is exhausted, the thallus is gradually fermented by using ethanol, acetaldehyde, acetic acid and the like as substrates, and the fermentation process provides a precursor and reducing power required by the synthesis of 2-PE. In this stage, the cells synthesize 2-PE mainly through the Ehrlich pathway, and the required precursors and energy cofactors are supplied through the precursor metabolic pathway during the fermentation process, so that the synthetic ability of 2-PE in this stage is weaker than that in the fermentation stage in the presence of glucose.
By combining the determination of the concentration of the substrate in the fermentation liquid, the observation of the thallus morphology and the quantitative analysis of related parameters, the change of the related metabolic parameters presents a linear relation with the thallus growth along with the growth of the thallus in the exponential growth phase. Under the condition of not changing the ventilation capacity in the whole fermentation process, the W303 is in the first exponential growth stage, the dissolved oxygen in the fermentation liquor is gradually reduced along with the consumption and utilization of thalli, the W303 utilizes oxygen and generates carbon dioxide through intracellular metabolism, and the increasing synchronicity of OUR and CER is shown in figures 2 and 1. When the glucose substrate in the fermentation liquid is exhausted, the primary fermentation is finished, the dissolved oxygen content in the fermentation liquid slightly rises along with the death of a small part of the thalli, and the CER also sharply decreases at the moment. After the secondary fermentation with ethanol as the substrate begins, the change of the related metabolic parameters also presents a linear relation with the growth of the thalli along with the growth of the thalli. The secondary increase of OUR and CER is shown, but the fermentation enters a slow growth phase mainly for maintaining thalli, the carbon source provided at the time is mainly used for maintaining thalli, and actually, the conversion from primary metabolism to secondary metabolism is represented at the time.
As can be seen from the comparative analysis of FIG. 2 and FIG. 1, the culture medium is more suitable for the growth of the W303 strain and the transformation production of 2-PE when the glucose concentration in the substrate is 20g/L, and the respiratory metabolic capability of the thalli at the carbon source concentration is stronger than that at the glucose concentration of 10g/L in the substrate; at the same time, the conversion of L-Phe increased from 47.2% to 83.6% and the product concentration of 2-PE increased from 1.24g/L to 2.03g/L.
Test example 3:
this test example used the fermentation media of example 3 and example 4, respectively, to perform the method for the production of phenethyl alcohol (2-PE) by fermentation of Saccharomyces cerevisiae as described in example 5; and Saccharomyces cerevisiae S288C was used as a control experiment. In this test example, fermentation characteristics such as glucose, phenylalanine (L-Phe), phenethyl alcohol (2-PE), dry Cell Weight (DCW), oxygen Uptake Rate (OUR), carbon dioxide release rate (CER), and ethanol were monitored during the fermentation process to investigate the fermentation characteristics under the effect of co-feeding substrates of different concentrations, and the results are shown in FIG. 3.
FIG. 3 is a comparison of fermentation characteristics of S288C (A, B) and W303 (C, D) Saccharomyces cerevisiae co-fed with different concentrations of substrate; in FIG. 3, A and C represent fermentation media used in example 3, and B and D represent fermentation media used in example 4.
The whole process is a substrate co-utilization stage, and a carbon source and a nitrogen source are synchronously utilized in the fermentation process. As can be seen by comparative analysis of the fermentation parameters in FIG. 3, the rate of glucose consumption by the W303 strain is faster at the same nitrogen source substrate concentration. The conversion rate of glucose of the W303 strain reaches 99.7 percent and 99.9 percent respectively when the strain is fermented for about eight hours. Whereas the glucose conversion rate of the S288C strain is only 52.4% and 54.1% respectively at around eight hours of fermentation. Meanwhile, under the same nitrogen source substrate concentration, the utilization rate of phenylalanine in the substrate by the saccharomyces cerevisiae strain W303 is higher. The L-Phe utilization by the W303 strain was 47.2% and 36.7%, respectively, when the L-Phe concentration in the substrate was 3.75g/L and 5.0g/L, respectively. The utilization rate of the S288C strain is only 37.8 percent and 30.8 percent respectively, obviously, the L-Phe utilization rate of the strain W303 is higher, and the fermentation characteristic is better.
From this result, it was found that W303 can efficiently utilize both substrates glucose and phenylalanine in the mixed fermentation by the substrate co-feeding action. The utilization capacity of the W303 strain to glucose and phenylalanine is obviously better than that of S288C.
Test example 4:
in this test example, three strains of Saccharomyces cerevisiae (Saccharomyces cerevisiae S288C, saccharomyces cerevisiae W101 and Saccharomyces cerevisiae W303) were fermented under nitrogen sources consisting of phenylalanine and ammonium sulfate at different ratios to produce phenethyl alcohol, and the 2-PE yield and the bacterial concentration were compared, and the test results were recorded, as shown in FIGS. 4 and 5.
The method comprises the following steps: shake flask fermentation culture
Shake flask fermentation medium (g/L): phenylalanine, (NH) 4 ) 2 SO 4 ，KH 2 PO 4 14.4，MgSO 4 ·7H 2 O0.5, glucose 10; 200 mu L/L of metal ion mother liquor and 100 mu L/L of vitamin mother liquor. Sterilization temperature of 115 deg.CThe time is 20min; moreover, different phenylalanine concentration and ammonium sulfate concentration ratios are set in the shake flask fermentation medium to respectively be (7.5 + 0) g/L, (6.0 + 1.5) g/L, (5.0 + 2.5) g/L, (3.75 + 3.75) g/L, (3.0 + 4.5) g/L, and (2.0 + 5.5) g/L.
Respectively inoculating the cultured test tube seeds into sterilized shake flask fermentation culture medium, performing fermentation culture, and culturing at 30 deg.C and 220rpm for 60h. And (3) comparing and inspecting the utilization conditions of the three saccharomyces cerevisiae strains on different nitrogen sources, and analyzing the fermentation characteristic changes of the three saccharomyces cerevisiae strains. The results are shown in FIGS. 4 and 5.
FIG. 4 shows the change in the concentration of the product phenethyl alcohol during fermentation by Saccharomyces cerevisiae S288C, saccharomyces cerevisiae W303, and Saccharomyces cerevisiae W101. FIG. 5 shows the growth of Saccharomyces cerevisiae S288C, saccharomyces cerevisiae W303, and Saccharomyces cerevisiae W101 during fermentation at different nitrogen source ratios.
As shown in FIG. 4, the effect of utilizing L-Phe nitrogen source by three Saccharomyces cerevisiae strains under different concentrations of phenylalanine and ammonium sulfate nitrogen source ratios compares the yield change of 2-PE synthesized by three Saccharomyces cerevisiae strains fermented for 60h in shake flask. As can be seen from FIG. 4, the W303 strain synthesized 2-PE with a significantly higher yield than the other two strains, and when the phenylalanine concentration was 7.5, 6.0, 5.0, 3.75, 3.0, 2.0g/L, the yield of 2-PE after 60h fermentation was 1.26 + -0.023, 1.52 + -0.021, 1.45 + -0.015, 1.57 + -0.020, 1.34 + -0.013, 1.18 + -0.018 g/L, respectively.
By comparing and analyzing the overall fermentation trend of the three strains of saccharomyces cerevisiae in fig. 4, the yield of 2-PE can be gradually increased along with the increase of the concentration of the phenylalanine substrate. However, when the concentration of L-Phe is higher than 5.0g/L, the production of 2-PE is rather reduced with increasing proportion of phenylalanine concentration in the substrate.
FIG. 5 is a graph showing the growth curves of three strains of Saccharomyces cerevisiae at different nitrogen source ratios of phenylalanine and ammonium sulfate. As can be seen from FIG. 5, the substrate has obvious inhibitory effect on the growth of three strains of Saccharomyces cerevisiae when the concentration of phenylalanine in the culture medium is 7.5 g/L. When substrates with different nitrogen source ratios are fed together and fermented, the W303 can efficiently utilize L-Phe in the substrates to synthesize 2-PE, and the effect is obvious when the concentration of phenylalanine nitrogen source substrates is 3.75g/L and 5.0 g/L.
From FIGS. 4 and 5, it is understood that the ratio of phenylalanine to ammonium sulfate in the nitrogen source of the present application, and the amount and concentration of synthesized phenethyl alcohol were higher than those when phenylalanine was used as the sole nitrogen source. Therefore, the culture medium which is claimed by the application and adopts the phenylalanine and the ammonium sulfate as the nitrogen source has the effect of promoting fermentation, and has positive beneficial effects on the production of the phenethyl alcohol by fermentation.
In conclusion, it is known that the microbial production of phenethyl alcohol (2-PE) is limited to the industrial production due to the economic fermentation of substrates and the restriction of strains, and the past research mainly focuses on the optimization of the components of the complete fermentation medium and the metabolic regulation of the Ehrlich pathway. The innovation of the application is that a synthetic fermentation medium with clear components is optimized in a system for the first time, a mode of co-feeding glucose, phenylalanine and ammonium sulfate substrates is adopted, a proper nitrogen source proportion of phenylalanine and ammonium sulfate is provided, and the influence of the carbon source concentration of glucose on the fermentation metabolic characteristics of the shikimic acid pathway and the Ehrlich pathway is provided. In addition, the fermentation medium can meet the fermentation requirement of the saccharomyces cerevisiae strain W303, and can realize the high yield of 2-PE.
In addition, through comparison of fermentation characteristics of the saccharomyces cerevisiae strains under the co-feeding action of different substrates, the fermentation method of the fermentation medium has high utilization rate of the substrates, and shows high respiratory metabolism rate, high glucose utilization rate and strong phenethyl alcohol (2-PE) synthesis capacity.
When the components of a fermentation medium in the substrate are 3.75g/L of phenylalanine, 3.75g/L of ammonium sulfate and 20g/L of glucose, the respiratory metabolism effect of the W303 strain is obviously enhanced under the co-feeding action of the substrate, the utilization rate of the substrate is obviously improved, and the yield of 2-PE is up to 2.03g/L.
The application provides a synthetic culture medium with definite components for efficiently fermenting and producing 2-PE, which can be used for subsequent production 13 C metabolic flux analysis strain high-yield metabolic mechanism lays a foundation.
The fermentation medium and the method for producing phenethyl alcohol by fermentation provided by the embodiments of the present application are described in detail above, and the principle and the embodiments of the present application are explained by applying specific examples herein, and the description of the above embodiments is only used to help understanding the technical scheme and the core concept of the present application; those of ordinary skill in the art will understand that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications or substitutions do not depart from the spirit and scope of the present disclosure as defined by the appended claims.

Claims (4)

1. A fermentation medium for Saccharomyces cerevisiae, comprising: nitrogen source, carbon source, potassium dihydrogen phosphate, metal ion mother liquor and vitamin mother liquor; wherein the content of the first and second substances,
the nitrogen source consists of phenylalanine and ammonium sulfate; the carbon source is glucose;
wherein the fermentation medium comprises: 3.75g/L of phenylalanine, 3.75g/L of ammonium sulfate, 20g/L of glucose and 14.4g/L of monopotassium phosphate, as well as 200mL/L of metal ion mother liquor and 100mL/L of vitamin mother liquor;
the metal ion mother liquor comprises: 3.0g/L FeSO 4 ·7H 2 O, 4.5g/L ZnSO 4 ·7H 2 O, 4.5g/L CaCl 2 1.0g/L of MnCl 2 ·4H 2 O, 0.3g/L CoCl 2 ·6H 2 O, 0.3g/L CuSO 4 ·5H 2 O, 0.4g/L Na 2 MoO 4 ·2H 2 O, 1g/L H 3 BO 3 0.1g/L KI and 19g/L EDTA; the pH of the metal ion mother liquor is 4.0;
the vitamin mother liquor comprises: 50mg/L biotin, 1000mg/L pantothenic acid hemicalcium salt, 1000mg/L thiamine hydrochloride, 1000mg/L pyridoxine hydrochloride, 1000mg/L nicotinic acid, 200mg/L p-aminobenzoic acid, 25000mg/L inositol; the pH of the vitamin mother liquor is 6.5;
the saccharomyces cerevisiae is saccharomyces cerevisiae W303 and comes from China center for the preservation and management of industrial microbial strains.
2. A method for producing phenylethyl alcohol by saccharomyces cerevisiae fermentation using the fermentation medium of claim 1, wherein the fermentation method comprises:
adding the fermentation medium to a bioreactor; centrifuging, washing and resuspending a seed solution of the saccharomyces cerevisiae strain, inoculating the seed solution into a bioreactor, wherein the inoculation amount is 10% of the fermentation volume, stirring and ventilating, and fermenting at the temperature of 27 to 33 ℃ until the fermentation is finished;
wherein, the dissolved oxygen of the fermentation liquor is controlled to be more than or equal to 30 percent in the fermentation process;
the saccharomyces cerevisiae is saccharomyces cerevisiae W303 and comes from China center for preservation and management of industrial microbial strains.
3. The method for producing phenylethyl alcohol by fermentation of saccharomyces cerevisiae as claimed in claim 2 wherein the rotation speed of said stirring is not more than 700rpm.
4. The method for producing phenylethyl alcohol by fermentation of saccharomyces cerevisiae according to claim 3 wherein said aeration is provided with an aeration rate of not more than 500mL/min.
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