CN116478842A - Recombinant saccharomyces cerevisiae strain for producing (+) -phoropterygii and preparation method thereof - Google Patents

Abstract

The invention relates to a recombinant saccharomyces cerevisiae strain for producing sesquiterpene molecules (+) -phore, belonging to the field of metabolic engineering, and being used for the synthesis and preparation of spices, antimicrobial drugs and insect repellents. The recombinant strain for synthesizing (+) -phorbol in the present invention contains acetyl-CoA acylase gene ERG10, 3-hydroxy-3-methylglutaryl-CoA synthetase gene ERG13, 3-hydroxy-3-methylglutaryl-CoA reductase gene tHMGR, mevalonate kinase gene ERG12, mevalonate kinase gene ERG8, mevalonate diphosphate decarboxylase gene MVD1, isoprene pyrophosphate isomerase gene Idi, farnesyl pyrophosphate synthase gene ERG20, and (+) -phorbol alkene synthase gene ScAros. The recombinant strain is used for producing (+) -phorene, and is cultured in a 5L fermentation tank by adopting a batch-fed fermentation mode for 142h to achieve 8.7g/L.

Description

Recombinant saccharomyces cerevisiae strain for producing (+) -phoropterygii and preparation method thereof

Technical Field

The invention relates to a recombinant saccharomyces cerevisiae strain for synthesizing sesquiterpene molecules (+) -phorbol (eremophilene), belonging to the field of metabolic engineering and being used for biosynthesis application of the sesquiterpene molecules (+) -phorbol.

Background

Terpenes are an important group of natural products, and their structural diversity confers broad utility to such compounds, such as malaria-resistant artemisinin, breast cancer-resistant paclitaxel, anti-oxidant lycopene, natural rare spice sandalwood oil, aviation fuel precursor compounds farnesene, and the like. Sesquiterpenes are a class of terpenoid compounds of which the most abundant, and the reported sesquiterpene skeletons are more than 300, widely present in plants, fungi and bacteria, and are widely used in the industries of foods, medicines, fragrances, fuels, and the like. With the increasing market demand, the synthesis of sesquiterpenes from renewable raw materials by microbial fermentation is becoming more and more interesting, and the rapid development of synthetic biology and metabolic engineering provides a powerful motive force for its synthetic preparation.

The phore alkene (eremophilene) is a six-membered bicyclic sesquiterpene skeleton molecule, which theoretically contains at least 8 epimers and more kinds of terpene derivatives according to chiral combinations of skeleton carbon atoms. The skeleton molecule is the precursor of various sesquiterpenoidsThe body has wide biological activity, such as fragrance, can be used as essence and spice, has strong antioxidant, antimicrobial and insect repellent activities, and the like, and thus has great application value. At present, the microbial fermentation synthesis of the framework molecules is mainly concentrated on (+) -valene and (-) -eremophilene, the research on the microbial fermentation synthesis of (+) -eremophilene is less, and the fermentation yield in escherichia coli is only 19mg L at present ^-1 It is difficult to meet the demands of industrial production.

Disclosure of Invention

The invention aims to construct a recombinant saccharomyces cerevisiae strain for efficiently synthesizing (+) -phorbol, and solve the technical problem of low biosynthesis yield of (+) -phorbol.

The invention solves the problems by adopting the technical scheme that: the sesquiterpene synthase ScAroS from the myxobacteria Sorangium cellulosum is selected, and the efficient synthesis and preparation of (+) -eremophilene are realized in a Saccharomyces cerevisiae cell which is a high-yield terpenoid metabolic chassis through the optimized regulation and control of a combination of a biosynthesis technology and a metabolic engineering.

In a first aspect, the invention provides a recombinant s.cerevisiae strain producing (+) -phore comprising genes ERG10, ERG13, tggr, ERG12, ERG8, MVD1, idi, ERG20 and scAroS of the (+) -phore metabolic pathway, wherein:

ERG10 is the encoding acetyl CoA acyl transferase gene, ERG13 is the encoding 3-hydroxy-3-methyl glutaryl CoA synthase gene, tHMGR is the encoding 3-hydroxy-3-methyl glutaryl CoA reductase gene, ERG12 is the encoding mevalonate kinase gene, ERG8 is the encoding phosphate mevalonate kinase gene, MVD1 is encoding two phosphate mevalonate decarboxylase gene, idi is the encoding isoprene pyrophosphate isomerase gene, ERG20 is encoding farnesyl pyrophosphate synthase gene, the genes are all from Saccharomyces cerevisiae;

the scAroS is a gene encoding (+) -phorbol synthase, and the amino acid sequence encoded by the scAroS is SEQ ID NO.1: MPSERHSVVVSKGEVETFDYPFPASRNANVDIAERRTLQWIRRLQLAPEGRALSRLKATGFAQLAAWLLPWANMRTLELASDFTAALFLLDDAYDEGDLSMDPEAVERLNEKYLGELFGYVEPDMSDPLTRGLLDVRDRIKSSHPHFFLNRWLAHFQFYYEANLWEANNRRRARTPCVDEYLLMRRYSGAVYTYCDLLELLLERPLPLEVVQHPTIQCVRDICNDILCWTNDYFSLGKELRSGDVHNLILVLRDNHAITLEEAIARLKQMHDERIAEYQDVKEKVLALWDDEATRLYIGAADAMIAGNQRWALEARRYSGLESLIARAG.

Preferably, the (+) -phorene-producing recombinant strain is derived from a Saccharomyces cerevisiae CEN.PK2-1D strain.

In a second aspect, the invention provides a method for preparing the (+) -phorene-producing recombinant strain according to the first aspect, comprising the following specific steps:

s1, constructing a recombinant plasmid and a target fragment: amplifying a fragment erem containing (+) -phorbol synthase gene scarozizae by using a scaroS gene sequence as a template and connecting the fragment erem with a plasmid to assemble a recombinant plasmid pYeast-eremophilene;

s2, constructing a (+) -phorbol-producing synthetic strain: the plasmid pYeast-protoil-den is digested with restriction enzymes, the fragment with the scaroS gene is recovered, and transformed into a Saccharomyces cerevisiae strain to construct a recombinant expression strain EPS01.

Further, the operation in step S1 is specifically: amplifying primers pYeast-erem-F1 and pYH328-erem-R1 to obtain fragment erem, assembling erem and a general plasmid pYeast3939 into a recombinant plasmid by a Gibson connection method, and amplifying in escherichia coli to obtain a recombinant plasmid pYeast-eremophilene;

primer pYeast-erem-F1 sequence SEQ ID NO.2:

TCATAAGAAATTCGCTCAACCGGCTCTAGCGATGAGAGAT；

pYH328-erem-R1 sequence SEQ ID NO.3:

GAAAATTCAATATAAATGCCATCTGAAAGACACTCTGTTG。

further, the operation in the step 2 specifically includes: the plasmid pYeast-protoil luden is digested with restriction enzyme XhoI, a fragment with the scaroS gene is recovered, transformed into a Saccharomyces cerevisiae strain JCR27, integrated on a chromosome according to the principle of homologous recombination, and screened and marked as uracil auxotroph to construct a recombinant expression strain EPS01.

Further, the preparation method further comprises the following steps: the Saccharomyces cerevisiae CEN.PK2-1D genome is used as a template, and primers gal80-up-F1 (SEQ ID NO. 4) and gal80-up-R1 (SEQ ID NO. 5) are used for amplification to obtain an upstream homologous arm fragment gal80-up of a gal80 gene; the Saccharomyces cerevisiae CEN.PK2-1D genome is used as a template, and primers gal80-down-F1 (SEQ ID NO. 6) and gal80-down-R1 (SEQ ID NO. 7) are used for amplification to obtain a downstream homologous arm fragment gal80-down of a gal80 gene; the universal plasmid pRS424-Trp is used as a template, a screening marker is determined to be tryptophan auxotrophy, and a fragment pro-Trp containing a tryptophan synthesis essential gene and a promoter is obtained by utilizing primers Trp-F1 (SEQ ID NO. 8) and Trp-R1 (SEQ ID NO. 9) for amplification; the universal plasmid pRS423-His is used as a template to determine that the screening mark is histidine auxotrophy, and a fragment pro-His containing histidine synthesis essential genes and promoters is obtained by utilizing primers His-F1 (SEQ ID NO. 10) and His-R1 (SEQ ID NO. 11) for amplification; the general plasmid pYeast3939 is used as a template, and primers cyc1-F1 (SEQ ID NO. 12) and cyc1-R1 (SEQ ID NO. 13) are used for amplification to obtain a terminator sequence cyc1; the general plasmid pYeast3939 is used as a template, and primers adh1-F1 (SEQ ID NO. 14) and adh1-R1 (SEQ ID NO. 15) are used for amplification to obtain a terminator sequence adh1; ligating the above fragments gal80-up, gal80-down, pro-trp, pro-his, cyc1 and adh1 by OE-PCR to obtain recombinant fragment gal80-trp-his;

using the general plasmid pYH328 as a template, and using the primers leu-up-F1 (SEQ ID NO. 16) and leu-up-R1 (SEQ ID NO. 17) to amplify to obtain a fragment leu-up containing a leucine synthesis essential gene and an upstream homology arm; the universal plasmid pYH328 is used as a template, and a downstream homologous arm fragment leu-down is obtained by amplification of a primer leu-down-F1 (SEQ ID NO. 18) and leu-down-R1 (SEQ ID NO. 19); connecting the two fragments leu-up and leu-down through OE-PCR to obtain a recombinant fragment leu-up-down;

the recombinant fragments gal80-trp-his and leu-up-down are utilized to transform the strain EPS01, the strain is integrated on a chromosome according to the homologous recombination principle, and the screening markers are tryptophan, histidine and leucine auxotrophs, and the sequencing verification proves that the fermentation strain EPS02 is constructed.

In a third aspect, the invention provides a method for producing (+) -phorbol of (+) -phorbol recombinant strain EPS01 according to the second aspect: inoculating the constructed recombinant strain EPS01 into YPD culture medium, fermenting, inducing expression and purifying to obtain (+) -phore.

Further, the method specifically comprises the following steps: the constructed recombinant strain EPS01 was inoculated into 5mL of YPD medium, medium formulation: 20g/L peptone, 10g/L yeast powder, 20g/L glucose, were cultured overnight at a shaking incubator of 30℃at 220r/min, then at an initial OD _600nm Inoculating 0.1-0.2 into 200mL YPD culture medium, culturing at 30deg.C in shake incubator at 220r/min, and obtaining OD _600nm When the value is 0.6-0.8, 20mL galactose is added for induction expression, and 20mL covering agent n-decane is added for extraction after 24h of culture.

A method for producing (+) -phorbol of (+) -phorbol recombinant strain EPS02 according to the second aspect: carrying out fed-batch fermentation on the constructed strain EPS02, and inoculating seed liquid into 2L of fermentation base material culture medium, wherein the formula of the fermentation base material culture medium is as follows: 40g/L glucose, 15g/L ammonium sulfate, 8g/L potassium dihydrogen phosphate, 6.2g/L magnesium sulfate, 12mL/L metal ion mother liquor, 15mL/L vitamin mother liquor, starting fermentation in a 5L fermentation tank, controlling the temperature of the fermentation process to be 30 ℃, controlling the dissolved oxygen to be more than 20%, controlling the rotating speed to be 250-500 r/min, adjusting the pH value to be 5.0 by ammonia water, controlling the ethanol concentration to be below 10g/L, and measuring the OD of the strain in real time _600nm Glucose and ethanol content; and (3) adding a feed 1 when the glucose concentration is reduced to below 5g/L in the fermentation process for 7-8 hours, wherein the formula of the feed 1 is as follows: 500g/L glucose, 9g/L potassium dihydrogen phosphate, 5.12g/L magnesium sulfate, 3.5g/L potassium sulfate, 0.28g/L sodium sulfate, 10mL/L metal ion mother liquor, 12mL/L vitamin mother liquor, and the adding speed is controlled between 10 and 20mL/h; after 24 hours, when the feed 1 is consumed, the feed 2 is added and the fermentation is finished, and the formula of the feed 2 is as follows: 800g/L sucrose, 9g/L potassium dihydrogen phosphate, 5.12g/L magnesium sulfate, 3.5g/L potassium sulfate, 0.28g/L sodium sulfate, 10mL/L metal ion mother liquor, 12mL/L vitamin mother liquor, and the feeding 2 flow acceleration is controlled at 10-20 mL/h; a covering agent is added at 24h of fermentation to realize in-situ extraction, wherein the covering agent can be n-dodecane, isopropyl myristate, isopropyl palmitate or liquid paraffin.

The invention has the beneficial effects that: the recombinant strain constructed by the invention is used for producing (+) -phorene, and is cultivated in a 5L fermentation tank by adopting a batch-fed fermentation mode, the yield reaches 8.7g/L, and the recombinant strain is the highest fermentation yield reported by the molecule at present.

Drawings

FIG. 1 is a schematic diagram of the metabolic pathway of (+) -phorbol for microbial synthesis;

FIG. 2 Mass Spectrometry (MS) method for identifying (+) -phorene molecular structure spectrogram;

in the figure: the ordinate is (+) -phorbol characteristic ion peak ion intensity in counts per second; the abscissa is the ratio of the mass number of fragment ions to the charge number, in daltons/proton charge;

FIG. 3 Nuclear Magnetic Resonance (NMR) method for identifying (+) -phorene molecular structure spectrum;

in the figure: a is the nuclear magnetic resonance hydrogen spectrum result; b is nuclear magnetic resonance carbon spectrum result;

FIG. 4 is a graph showing the results of shake flask fermentation of (+) -phorbol synthetic strain EPS01;

in the figure: a is the fermentation yield of the strain culture for 72 hours, b is the fermentation yield of the strain culture for 96 hours, c is the fermentation yield of the strain culture for 120 hours;

FIG. 5 is a graph showing the results of batch-fed fermentation production of (+) -phorene synthesis strain EPS 02;

in the figure: ■ Glucose, +.ethanol, +.t.and bacterial liquid OD _600nm -phorbol;

the abscissa is the incubation time, in hours; the left 1 on the ordinate is the residual glucose and ethanol content in g/L in the fermentation medium; the ordinate right 1 shows the strain growth curve OD _600nm Value, absorbance per unit; the right 2 on the ordinate is (+) -phorbol fermentation yield in mg/L.

Detailed Description

The working of the invention is further described below with reference to the accompanying drawings and examples.

EXAMPLE 1 construction of (+) -phorene synthetic plasmid and related recombinant fragment

The construction process of the recombinant plasmid pYeast-eremophilene comprises the following steps:

using the ScAroS gene sequence as a template, the primer pYeast-erem-F1 sequence was used: TCATAAGAAATTCGCTCAACCGGCTCTAGCGATGAGAGAT, pYH328-erem-R1 sequence: GAAAATTCAATATAAATGCCATCTGAAAGACACTCTGTTG amplifying to obtain fragment erem, assembling the fragment erem and a general plasmid pYeast3939 into a recombinant plasmid by a Gibson connection method, amplifying in escherichia coli, and obtaining the recombinant plasmid pYeast-eremophilene after sequencing correctly;

construction procedure of recombinant fragment gal 80-trp-his:

in order to release the inhibition of the metabolic pathway by the gal80 gene and thereby increase fermentation yield, the gal80 gene needs to be knocked out, first, the upstream and downstream homology arm sequences of the gal80 gene are amplified, the Saccharomyces cerevisiae CEN.PK2-1D genome is used as a template, and the primer gal80-up-F1 sequence (SEQ ID NO. 4) is used: CAATTTGGCACCTGCATACCCCATTTCCCCA and gal80-up-R1 sequence (SEQ ID NO. 5): ATATTATATATATAGTAATGTCGTTGACG GGAGTGGAAAGAACGGGAAACCAACT amplification to give the fragment gal80-up (upstream homology arm of gal80 gene); primer gal80-Down-F1 sequence (SEQ ID NO. 6):

CATAAAAAAATATAGAGTGTACTAGAAGCATCTTGCCCTGTGCTCCCAGT and gal80-Down-R1 sequence (SEQ ID NO. 7):

CCAGCAAAAATATGACCCCCAATATGAGAAATTAAGGCTA

amplifying to obtain a fragment gal80-Down (downstream homology arm of gal80 gene);

in order to enhance the growth advantage of the fermentation strain, it is necessary to supplement its auxotrophs Trp (tryptophan synthesis essential gene) and his (histidine synthesis essential gene) with the plasmid pRS424-Trp as a template and with the primer Trp-F1 sequence (SEQ ID NO. 8):

GTTTCCCGTTCTTTCCACTCCCGTCAACGACATTACTATATATATAATATAGGAA and trp-R1 sequence (SEQ ID NO. 9):

AAGCGTGACATAACTAATTACATGACTATTTCTTAGCATTTTTGACGAAATTTGC to obtain a fragment pro-trp (containing a gene essential for tryptophan synthesis and a promoter sequence thereof);

using plasmid pRS423-His as a template and primer His-F1 sequence (SEQ ID NO. 10):

AAATCATAAATCATAAGAAATTCGCCTACATAAGAACACCTTTGGTGGAGGGAAC and his-R1 sequence (SEQ ID NO. 11):

GGGCCAAGCACAGGGCAAGATGCTTCTAGTACACTCTATATTTTTTTATGCCTCG to obtain a fragment pro-his (a gene essential for histidine synthesis and a promoter sequence thereof);

in order to regulate gene expression, it is necessary to add terminator sequences, using the universal plasmid pYeast3939 as a template and the primer cyc1-F1 sequence (SEQ ID NO. 12):

TTTCGTCAAAAATGCTAAGAAATAGTCATGTAATTAGTTATGTCACGCTTACATT and primer cyc1-R1 sequence (SEQ ID NO. 13):

CTCTTATTGACCACACCTCTACCGGGCAAATTAAAGCCTTCGAGCGTCCCAAAAC amplification to obtain terminator sequence cyc1;

the universal plasmid pYeast3939 was used as a template, and the primer adh1-F1 sequence (SEQ ID NO. 14):

GGGACGCTCGAAGGCTTTAATTTGCCCGGTAGAGGTGTGGTCAATAAGAGCGAC C and adh1-R1 sequence (SEQ ID NO. 15):

CTCCACCAAAGGTGTTCTTATGTAGGCGAATTTCTTATGATTTATGATTTTTATT amplification to obtain terminator sequence adh1;

the above fragments are connected by OE-PCR by using primers gal80-up-F1 and gal80-down-R1 to obtain recombinant fragment gal80-trp-his;

the construction process of the recombinant fragment leu-up-down:

similarly, in order to enhance the growth advantage of the fermentation strain, it is necessary to supplement its auxotroph gene leu (leucine synthesis essential gene) with the universal plasmid pYH328 as a template using the primer leu-up-F1 sequence (SEQ ID NO. 16): ATAACGAGAACACACAGGGGCGCTA and leu-up-R1 sequence (SEQ ID NO. 17): AAATATCATAAAAAAAGAGAATCTTTGCGAAAAGCCAATTAGTGTGATACTAAGTGCT, amplifying to obtain leu-up (homologous arm upstream of the insertion site of leucine-containing gene essential for synthesis); the universal plasmid pYH328 is used as a template, and a primer leu-down-F1 sequence (SEQ ID NO. 18) is used:

AGTATCACACTAATTGGCTTTTCGCAAAGATTCTCTTTTTTTATGATATTTGTACAT and leu-down-R1 sequence (SEQ ID NO. 19): GACAACGACCAAGCTCACATCAAAAGACTA, amplified to give leu-down (containing homology arms downstream of the insertion site); and then the two fragments are connected by using primers leu-up-F1 and leu-down-R1 through OE-PCR to obtain a recombinant fragment leu-up-down.

EXAMPLE 2 construction of (+) -phorene Synthesis Strain

Saccharomyces cerevisiae strain JCR27 which over-expresses mevalonate metabolic pathway is used as a chassis strain for synthesizing (+) -phore;

the plasmid pYeast-eremophilene is digested by restriction enzyme XhoI, a fragment with the scaroS gene is recovered, the fragment is transformed into a saccharomyces cerevisiae strain JCR27, the fragment is integrated on a chromosome according to the homologous recombination principle, a screening mark is uracil, and a recombinant expression strain EPS01 is constructed through sequencing verification;

the strain EPS01 is transformed by fragments gal80-trp-his and leu-up-down, integrated on chromosome according to the principle of homologous recombination, and screened for tryptophan, histidine and leucine, and verified by sequencing, so as to construct the fermentation strain EPS02.

EXAMPLE 3 shaking flask fermentation of (+) -phorene-synthesizing Strain

The strain EPS01 obtained in example 2 was cultivated by shake flask fermentation as follows: from the preservation plates, the single colonies were picked into a medium containing 5ml YPD, the medium formulation being: 20g/L peptone, 10g/L yeast powder and 20g/L glucose are subjected to shake culture for 24 hours at 220r/min in a constant temperature shaking table at 30 ℃, then are transferred to 200ml YPD culture medium, are placed in the constant temperature shaking table at 30 ℃ for shake culture for starting fermentation, 20ml galactose is added for 4 hours after transfer for induction expression, 20ml covering agent n-decane is added after culture for 24 hours, and upper organic phase n-decane is respectively sucked in different fermentation time of 72 hours, 96 hours and 120 hours for GC/MS detection analysis; the yield of the strain EPS01 after shaking flask fermentation for 120 hours reaches 438.9mg/L.

Example 4 fed-batch fermentation of (+) -phorene-producing strains

The strain EPS02 obtained in example 2 was subjected to fed-batch fermentation as follows: selecting monoclonal from a flat plate, adding into 5ml YPD culture medium, shake culturing at 30deg.C in 220r/min for 24 hr, transferring into 200ml YPD culture medium, shake culturing at 30deg.C in 220r/min overnight, transferring into 2L fermentation base material culture medium, fermenting in 5L fermentation tank, controlling fermentation temperature at 30deg.C, dissolving oxygen over 20%, rotating at 250-500 r/min, regulating pH to 5.0 with ammonia water, regulating ethanol concentration to below 10g/L, and measuring strain OD in real time _600nm Glucose and ethanol content; when the glucose concentration is reduced to below 5g/L in 7-8 h of fermentation, adding the feed 1, and controlling the adding speed to be equal to or lower than10-20 mL/h; the formula of the feed supplement 1 comprises the following components: 500g/L glucose, 9g/L potassium dihydrogen phosphate, 5.12g/L magnesium sulfate, 3.5g/L potassium sulfate, 0.28g/L sodium sulfate, 10mL/L metal ion mother liquor, 12mL/L vitamin mother liquor, and the adding speed is controlled between 10 and 20mL/h; after 24 hours, when the feed 1 is consumed, the feed 2 is added until the fermentation is finished, and the flow acceleration of the feed 2 is controlled to be 10-20 mL/h; the formula of the feed supplement 2 comprises the following components: 800g/L sucrose, 9g/L potassium dihydrogen phosphate, 5.12g/L magnesium sulfate, 3.5g/L potassium sulfate, 0.28g/L sodium sulfate, 10mL/L metal ion mother liquor, 12mL/L vitamin mother liquor, and the feeding 2 flow acceleration is controlled at 10-20 mL/h; after fermentation for 24 hours, 250ml of isopropyl myristate is added for covering so as to realize in-situ extraction and real-time measurement of products, an upper organic phase is collected for GC/MS detection analysis, and fermentation is finished when dissolved oxygen of the strain is not increased any more or the yield is not increased any more; finally, in a 5L fermenter, the strain EPS02 is fermented for 142 hours to realize that the (+) -phorbol yield reaches 8.7g/L, which is the highest level reported at present.

EXAMPLE 5 molecular structural identification of (+) -phorbol

The upper organic phase of the fermentation broth was collected, isopropyl myristate was removed by distillation under reduced pressure, and 50mg of (+) -phorbol alkene sample was diluted to CDCl ₃ Wherein the final concentration is 50mg/mL, and the mixture is placed in a nuclear magnetic resonance tube and is respectively carried out in a liquid nuclear magnetic resonance spectrometer (AVANCE III HD MHz) ¹ H NMR (Hydrogen Spectrometry) ¹³ C NMR (carbon spectrum) detection, and the data result is analyzed by nuclear magnetic analysis software MestReNova. NMR data are as follows:

100mg (+) -phorbol alkene sample is diluted into 10mL of chloroform, the final concentration is 10mg/mL, the diluted sample is placed into a detection tube, optical rotation test is carried out in an automatic polarimeter, the temperature is set to 25 ℃, and the test is repeated three times. The data are as follows:

sample of	α
		1	+154.84
2	+152.90
		3	+153.66
Literature values	+131.70

Claims (10)

1. A recombinant saccharomyces cerevisiae strain producing (+) -phore, characterized in that it comprises genes ERG10, ERG13, tgmggr, ERG12, ERG8, MVD1, idi, ERG20 and scAroS of the (+) -phore metabolic pathway, wherein:

ERG10 is the encoding acetyl CoA acyl transferase gene, ERG13 is the encoding 3-hydroxy-3-methyl glutaryl CoA synthase gene, tHMGR is the encoding 3-hydroxy-3-methyl glutaryl CoA reductase gene, ERG12 is the encoding mevalonate kinase gene, ERG8 is the encoding phosphate mevalonate kinase gene, MVD1 is encoding two phosphate mevalonate decarboxylase gene, idi is the encoding isoprene pyrophosphate isomerase gene, ERG20 is encoding farnesyl pyrophosphate synthase gene, the genes are all from Saccharomyces cerevisiae;

the scAroS is a gene encoding (+) -phorbol synthase, and the amino acid sequence encoded by the scAroS is SEQ ID NO.1: MPSERHSVVVSKGEVETFDYPFPASRNANVDIAERRTLQWIRRLQLAPEGRALSRLKATGFAQLAAWLLPWANMRTLELASDFTAALFLLDDAYDEGDLSMDPEAVERLNEKYLGELFGYVEPDMSDPLTRGLLDVRDRIKSSHPHFFLNRWLAHFQFYYEANLWEANNRRRARTPCVDEYLLMRRYSGAVYTYCDLLELLLERPLPLEVVQHPTIQCVRDICNDILCWTNDYFSLGKELRSGDVHNLILVLRDNHAITLEEAIARLKQMHDERIAEYQDVKEKVLALWDDEATRLYIGAADAMIAGNQRWALEARRYSGLESLIARAG.

2. The (+) -phore-producing recombinant s.cerevisiae strain according to claim 1, wherein the (+) -phore-producing recombinant strain is derived from s.cerevisiae cen.pk2-1D strain.

3. A method for preparing a (+) -phore-producing recombinant strain according to claim 1 or 2, characterized by the specific steps of:

s1, constructing a recombinant plasmid and a target fragment: amplifying a fragment erem containing (+) -phorbol synthase gene scarozizae by using a scaroS gene sequence as a template and connecting the fragment erem with a plasmid to assemble a recombinant plasmid pYeast-eremophilene;

s2, constructing a (+) -phorbol-producing synthetic strain: the plasmid pYeast-protoil-den is digested with restriction enzymes, the fragment with the scaroS gene is recovered, and transformed into a Saccharomyces cerevisiae strain to construct a recombinant expression strain EPS01.

4. The method for preparing a (+) -phore-producing recombinant strain according to claim 3, wherein the operation in step S1 is specifically: amplifying primers pYeast-erem-F1 and pYH328-erem-R1 to obtain fragment erem, assembling erem and a general plasmid pYeast3939 into a recombinant plasmid by a Gibson connection method, and amplifying in escherichia coli to obtain a recombinant plasmid pYeast-eremophilene;

primer pYeast-erem-F1 sequence SEQ ID NO.2:

TCATAAGAAATTCGCTCAACCGGCTCTAGCGATGAGAGAT；

pYH328-erem-R1 sequence SEQ ID NO.3:

GAAAATTCAATATAAATGCCATCTGAAAGACACTCTGTTG。

5. the method for preparing a (+) -phorene-producing recombinant strain according to claim 4, wherein the operation in the step 2 is specifically: the plasmid pYeast-protoil luden is digested with restriction enzyme XhoI, a fragment with the scaroS gene is recovered, transformed into a Saccharomyces cerevisiae strain JCR27, integrated on a chromosome according to the principle of homologous recombination, and screened and marked as uracil auxotroph to construct a recombinant expression strain EPS01.

6. The method for preparing a (+) -phore-producing recombinant strain according to claim 5, wherein the steps of said preparation method further comprise: the Saccharomyces cerevisiae CEN.PK2-1D genome is used as a template, and primers gal80-up-F1 (SEQ ID NO. 4) and gal80-up-R1 (SEQ ID NO. 5) are used for amplification to obtain an upstream homologous arm fragment gal80-up of a gal80 gene; the Saccharomyces cerevisiae CEN.PK2-1D genome is used as a template, and primers gal80-down-F1 (SEQ ID NO. 6) and gal80-down-R1 (SEQ ID NO. 7) are used for amplification to obtain a downstream homologous arm fragment gal80-down of a gal80 gene; the universal plasmid pRS424-Trp is used as a template, a screening marker is determined to be tryptophan auxotrophy, and a fragment pro-Trp containing a tryptophan synthesis essential gene and a promoter is obtained by utilizing primers Trp-F1 (SEQ ID NO. 8) and Trp-R1 (SEQ ID NO. 9) for amplification; the universal plasmid pRS423-His is used as a template to determine that the screening mark is histidine auxotrophy, and a fragment pro-His containing histidine synthesis essential genes and promoters is obtained by utilizing primers His-F1 (SEQ ID NO. 10) and His-R1 (SEQ ID NO. 11) for amplification; the general plasmid pYeast3939 is used as a template, and primers cyc1-F1 (SEQ ID NO. 12) and cyc1-R1 (SEQ ID NO. 13) are used for amplification to obtain a terminator sequence cyc1; the general plasmid pYeast3939 is used as a template, and primers adh1-F1 (SEQ ID NO. 14) and adh1-R1 (SEQ ID NO. 15) are used for amplification to obtain a terminator sequence adh1; ligating the above fragments gal80-up, gal80-down, pro-trp, pro-his, cyc1 and adh1 by OE-PCR to obtain recombinant fragment gal80-trp-his;

the universal plasmid pYH328 is used as a template, the screening mark is determined to be leucine auxotrophy, and the primers leu-up-F1 (SEQ ID NO. 16) and leu-up-R1 (SEQ ID NO. 17) are used for amplification to obtain a fragment leu-up containing a leucine synthesis essential gene and an upstream homology arm; the universal plasmid pYH328 is used as a template, and a downstream homologous arm fragment leu-down is obtained by amplification of a primer leu-down-F1 (SEQ ID NO. 18) and leu-down-R1 (SEQ ID NO. 19); connecting the two fragments leu-up and leu-down through OE-PCR to obtain a recombinant fragment leu-up-down;

the recombinant fragments gal80-trp-his and leu-up-down are utilized to transform the strain EPS01, the strain EPS01 is integrated on a chromosome according to the homologous recombination principle, and the strain EPS02 is constructed by screening the nutritional defects of tryptophan, histidine and leucine.

7. A method for producing (+) -phorbol by using the recombinant strain EPS01 for producing (+) -phorbol according to claim 6, wherein the constructed recombinant strain EPS01 is inoculated in YPD medium for fermentation, induced expression and purification to obtain (+) -phorbol.

8. The method for producing (+) -phorbol according to claim 7, characterized in that it comprises the following steps: the constructed recombinant strain EPS01 was inoculated into 5mL of YPD medium, medium formulation: 20g/L peptone, 10g/L yeast powder, 20g/L glucose, were cultured overnight at a shaking incubator of 30℃at 220r/min, then at an initial OD _600nm Inoculating 0.1-0.2 into 200mL YPD culture medium, culturing at 30deg.C in shake incubator at 220r/min, and obtaining OD _600nm When the value is 0.6-0.8, 20mL galactose is added for induction expression, and 20mL covering agent n-decane is added for extraction after 24h of culture.

9. A method for producing (+) -phorene using (+) -phorene producing recombinant strain EPS02 according to claim 6, characterized in that the constructed strain EPS02 is fed-batch fermented, the strain EPS02 seed liquid is inoculated into a fermentation substrate medium, the fermentation substrate medium formula is: 40g/L glucose, 15g/L ammonium sulfate, 8g/L potassium dihydrogen phosphate, 6.2g/L magnesium sulfate, 12mL/L metal ion mother liquor and 15mL/L vitamin mother liquor.

10. (+) -phorbol alkene-producing weight according to claim 9A method for producing (+) -phore for group strain EPS02, characterized in that it comprises in particular the following steps: inoculating the strain EPS02 seed solution into 2L of fermentation bed charge culture medium, and starting fermentation in a 5L fermentation tank; controlling the temperature of the fermentation process to 30 ℃, controlling the dissolved oxygen to be more than 20%, controlling the rotating speed to be between 250 and 500r/min, adjusting the pH value to 5.0 by ammonia water, controlling the concentration of ethanol to be less than 10g/L, and measuring the OD of the strain in real time _600nm Glucose and ethanol content; and (3) adding a feed 1 when the glucose concentration is reduced to below 5g/L in the fermentation process for about 7-8 hours, wherein the formula of the feed 1 is as follows: 500g/L glucose, 9g/L potassium dihydrogen phosphate, 5.12g/L magnesium sulfate, 3.5g/L potassium sulfate, 0.28g/L sodium sulfate, 10mL/L metal ion mother liquor, 12mL/L vitamin mother liquor, and the adding speed is controlled between 10 and 20mL/h; after 24 hours, when the addition of feed 2 was started and until the fermentation ended, feed 2 formulation: 800g/L sucrose, 9g/L potassium dihydrogen phosphate, 5.12g/L magnesium sulfate, 3.5g/L potassium sulfate, 0.28g/L sodium sulfate, 10mL/L metal ion mother liquor, 12mL/L vitamin mother liquor, and the feeding 2 flow acceleration is controlled at 10-20 mL/h; and adding a covering agent for in-situ extraction during fermentation for 24 hours, wherein the covering agent is any one of n-dodecane, isopropyl myristate, isopropyl palmitate or liquid paraffin.