CN116042425A - Yeast engineering bacteria for producing patchouli alcohol and application thereof - Google Patents
Yeast engineering bacteria for producing patchouli alcohol and application thereof Download PDFInfo
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- CN116042425A CN116042425A CN202211644975.9A CN202211644975A CN116042425A CN 116042425 A CN116042425 A CN 116042425A CN 202211644975 A CN202211644975 A CN 202211644975A CN 116042425 A CN116042425 A CN 116042425A
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- GGHMUJBZYLPWFD-UHFFFAOYSA-N patchoulialcohol Chemical compound C1CC2(C)C3(O)CCC(C)C2CC1C3(C)C GGHMUJBZYLPWFD-UHFFFAOYSA-N 0.000 title claims abstract description 201
- GGHMUJBZYLPWFD-MYYUVRNCSA-N Patchouli alcohol Natural products O[C@@]12C(C)(C)[C@H]3C[C@H]([C@H](C)CC1)[C@]2(C)CC3 GGHMUJBZYLPWFD-MYYUVRNCSA-N 0.000 title claims abstract description 100
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- KWFJIXPIFLVMPM-KHMAMNHCSA-N (+)-alpha-santalene Chemical compound CC(C)=CCC[C@]1(C)[C@@H]2C[C@H]3[C@@H](C2)[C@@]13C KWFJIXPIFLVMPM-KHMAMNHCSA-N 0.000 description 1
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Abstract
The invention discloses a yeast engineering bacterium for producing patchouli alcohol and application thereof, and relates to the field of microbial pharmacy. The invention can efficiently produce patchouli alcohol, the shake flask fermentation yield is close to 1.2mg/L, and the yield is improved by 10 times compared with the yield of the original strain. The invention utilizes pichia pastoris to efficiently generate patchouli alcohol, and has the characteristics of short period, environmental protection and resource conservation.
Description
Technical Field
The invention relates to the field of microbial pharmacy, in particular to a yeast engineering bacterium for producing patchouli alcohol and application thereof.
Background
Patchouli alcohol, also known as BaiAutumn plum alcohol with chemical formula C 15 H 26 O. Patchouli alcohol is a sesquiterpene alcohol, is widely applied to perfumes and cosmetics, is an important prodrug, has the characteristics of anti-inflammation, neuroprotection and the like, and is a sesquiterpene compound which is researched more at present. The main component of herba Pogostemonis is its volatile oil, and its content is about 1.5%. Patchouli alcohol is the main component of patchouli volatile oil, and is an index component for evaluating the quality of patchouli medicinal materials and patchouli oil specified in the pharmacopoeia of the people's republic of China. In addition, the natural patchouli essential oil has deep odor characteristics, is rich, strong and durable, is a common flavoring spice in perfumes, and about 1/3 of high-grade perfumes in the world can be used for patchouli alcohol. Therefore, the patchouli alcohol is used as a terpenoid product of a plant source, and has good application value and market prospect.
The existing patchouli alcohol production process is to extract the patchouli alcohol from plants, and the extraction process is relatively complex and cumbersome. In addition, the yield of plant extraction is relatively low and the period is long. In recent years, with the development of synthetic biology technology, if the microbial cell factory is adopted to carry out heterologous biosynthesis on patchouli alcohol, the efficiency can be improved, the cost can be reduced, the natural condition limitation can be broken, and the contradiction between supply and demand of patchouli alcohol can be effectively relieved. The biotransformation method has no limitation of various conditions and has better market prospect in green and environment-friendly. The biosynthetic pathway of patchouli alcohol has been analyzed at present, and in addition, related synthetic genes have been reported in E.coli and Saccharomyces cerevisiae. The pichia pastoris is taken as a recognized biological safe strain, has high growth speed, high environmental tolerance and simple fermentation condition, and the capability of expressing protein is more than ten times of that of the saccharomyces cerevisiae, so that the pichia pastoris cell factory is expected to improve the yield of patchouli alcohol. Although patchouli alcohol has important medical application prospect, the current low yield and complex preparation of plant extraction limit the application of patchouli alcohol in different fields. The synthesis of plant-derived natural products by microorganisms is a very environmentally friendly and efficient way.
The patent application with the publication number of CN113549562A discloses engineering bacteria for efficiently producing patchouli alcohol, and a construction method and application thereof, wherein the engineering bacteria are obtained by taking yarrowia lipolytica as an original strain and inserting optimized patchouli alcohol synthase coding genes PS1 and tHMGR coding genes into the genome of the yarrowia lipolytica; the engineering bacteria can efficiently synthesize patchouli alcohol, the construction method is efficient, the operation is simple, in addition, the method for collecting the product provided by the invention adopts a method of fermenting and extracting at the same time, and the produced patchouli alcohol is timely extracted into an organic phase, so that the pressure of intracellular dissolution of patchouli alcohol is relieved, the yield of patchouli alcohol is effectively improved, and a foundation is laid for synthesizing patchouli alcohol by artificial cells. The patent application with publication number of CN112175848A provides a patchouli alcohol producing yeast strain, a construction method and application thereof. The construction method comprises the steps of taking a saccharomyces cerevisiae strain as a starting strain, and integrating fusion enzyme FPTs of ERG20 and PTs in multiple copies in the starting strain to obtain the patchouli alcohol production yeast strain. In particular, when the patchouli alcohol producing yeast strain is a fusion enzyme FPTs which simultaneously integrates genes ERG20 and PTs in multiple copies, genes ROX1, YJL064w and YPL062w are deleted, and the capacity of the patchouli alcohol producing yeast strain for reinforcing genes ERG11 and CTT1 is greatly improved. There is no report on production of patchouli alcohol by pichia pastoris cell factories at present.
Disclosure of Invention
The invention provides a yeast engineering bacterium for producing patchouli alcohol, a construction method and application thereof, which are obtained by modifying pichia pastoris by a genetic engineering technology. The specific technical scheme is as follows:
the invention provides a yeast engineering bacterium for producing patchouli alcohol, which is obtained by taking pichia pastoris as an original strain and constructing an introduced gene, wherein the introduced gene comprises any one of the following groups:
(1) A gene encoding patchouli alcohol synthase PTS, a gene encoding farnesyl pyrophosphate synthase ERG20, a gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase 1 tHMG1, and a gene encoding isopentenyl pyrophosphate isomerase IDI 1; (2) Encoding gene of patchouli alcohol synthase PTS and encoding gene of farnesyl pyrophosphoric acid synthase ERG 20; (3) Pogostemon cablin synthase PTS coding gene.
Preferably, the encoding gene of patchouli alcohol synthase PTS is connected with the encoding gene of farnesyl pyrophosphoric acid synthase ERG20 so that the patchouli alcohol synthase PTS and the ERG20 are fused and expressed. More preferably, the copy number of the coding gene expressed by fusion of Ji Maxi A synthase GAS and farnesyl pyrophosphate synthetase ERG20 is 1-4. Multiple copies may further increase yield.
The encoding gene of patchouli alcohol synthase PTS is shown as SEQ ID NO.1, the encoding gene of farnesyl pyrophosphate synthase ERG20 is shown as SEQ ID NO.2, the encoding gene of isopentenyl pyrophosphate isomerase IDI1 is shown as SEQ ID NO.3, and the encoding gene of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 tHMG1 is shown as SEQ ID NO. 4.
The invention also provides a construction method of the yeast engineering bacteria for producing patchouli alcohol, and the introduced genes comprise: the construction method comprises the following steps of:
(1) Introducing a Cas9 protein coding gene from a Pichia pastoris GS115 strain genome to obtain Pichia pastoris GS115-Cas9; (2) Integrating a coding gene sequence containing patchouli alcohol synthase PTS into Pichia pastoris GS115-Cas9 to obtain a strain PTS-1; (3) Integrating a coding gene sequence of patchouli alcohol synthase PTS and a coding gene sequence of farnesyl pyrophosphate synthase ERG20 into pichia pastoris GS115-Cas9 to obtain a strain PTS-2; (4) 3 single copies of the coding gene sequence of patchouli alcohol synthase PTS and the coding gene sequence of farnesyl pyrophosphate synthetase ERG20 are integrated into the strain PTS-2 to obtain the strain PTS-3. (5) Integrating the coding gene sequence of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 tHMG1 and the coding gene sequence of isopentenyl pyrophosphate isomerase IDI1 into the strain PTS-3 to obtain a strain PTS-4; namely the yeast engineering bacteria for producing patchouli alcohol.
The method comprises the following specific steps:
(1) Through literature research, constructing patchouli alcohol synthase PTS recombinant expression vectors and sgRNA expression vectors corresponding to the patchouli alcohol synthase PTS recombinant expression vectors; (2) Constructing a patchouli alcohol synthase PTS and ERG20 fusion expression vector, and constructing an sgRNA expression vector corresponding to the patchouli alcohol synthase PTS and ERG20 fusion expression vector; (3) 3 copies of recombinant plasmids of patchouli alcohol synthase PTS and ERG20 fusion expression and corresponding sgRNA expression vectors are constructed; (4) Respectively constructing key speed limiting enzymes IDI1 and tHMG1 in a biosynthesis pathway and sgRNA expression vectors corresponding to the key speed limiting enzymes IDI1 and tHMG 1; (5) The patchouli alcohol synthase PTS recombinant expression vector and the sgRNA expression vector corresponding to the same in the step (1) are electrically transformed into competent cells of Pichia pastoris GS115-Cas9 to obtain a Pichia pastoris recombinant strain PTS-1 for producing patchouli alcohol; (6) The recombinant expression vector of the patchouli alcohol synthase PTS and ERG20 fusion expression vector in the step (2) and the sgRNA expression vector corresponding to the recombinant expression vector are electrically transformed into pichia pastoris GS115-Cas9 competent cells to obtain recombinant strain PTS-2 for efficiently producing patchouli alcohol; (7) And (3) converting 3 single copy recombinant plasmids of the patchouli alcohol synthase PTS and ERG20 fusion expression vector in the step (3) and the sgRNA expression vector corresponding to the 3 single copy recombinant plasmids into the step (6) to obtain competent cells of the pichia pastoris recombinant strain PTS-2, thereby obtaining the recombinant strain PTS-3 for efficiently producing patchouli alcohol. (8) Converting the recombinant expression vectors of the key speed limiting enzymes IDI1 and tHMG1 and the corresponding sgRNA expression vectors in the step (4) into competent cells of the pichia pastoris recombinant strain PTS-3 obtained in the step (7) to obtain a recombinant strain PTS-4 for efficiently producing patchouli alcohol;
the construction of the patchouli alcohol synthase recombinant expression vector in the step (1) and the sgRNA expression vector corresponding to the patchouli alcohol synthase recombinant expression vector are obtained by the following method: constructing an Int1-pTEF-PTS-tAOX1 recombinant expression vector by taking the laboratory-stored Int1-pTEF-tAOX1 as a starting vector; and constructing an HZP-sgRNA-Int1 expression vector by taking the HZP-sgRNA as a starting vector.
The construction of the patchouli alcohol synthase PTS and ERG20 fusion expression vector and the corresponding sgRNA expression vector in the step (2) is obtained by the following method: constructing an Int1-pTEF-ERG20-PTS-tAOX1 recombinant expression vector by taking the Int1-pTEF-PTS-tAOX1 constructed in the step (1) as a starting vector; the expression sgRNA vector is identical to the expression vector HZP-sgRNA-Int1 in the step (1).
In the step (3), constructing 3 copies of recombinant plasmids of ERG20-PTS and the sgRNA expression vector corresponding to the recombinant plasmids are obtained by the following method: constructing Int32-pTEF-ERG20-PTS-tAOX1, int33-pGAP-ERG20-PTS-tAOX1 and Int34-pTEF-ERG20-PTS-tAOX1 recombinant expression vectors by taking Int32-pTEF-tAOX1, int33-pGAP-tAOX1 and Int34-pTEF-ERG20-PTS-tAOX1 as starting vectors respectively; HGP-sgRNA-Int32, HHP-sgRNA-Int33 and HZP-sgRNA-Int34 recombinant expression vectors are constructed by taking HGP-sgRNA, HHP-sgRNA and HZP-sgRNA as starting vectors respectively.
The recombinant expression vectors of the key enzymes IDI1 and tHMG1 in the biosynthesis pathway and the sgRNA expression vector corresponding to the recombinant expression vectors are constructed in the step (4) and are obtained by the following method: constructing an Int11-pGAP-tHMG1-tAOX1 recombinant expression vector by taking the Int11-pGAP-tAOX1 as a starting vector; constructing an Int12-pTEF-IDI1-tAOX1 recombinant expression vector by taking the Int12-pTEF-tAOX1 as a starting vector; constructing an HZP-sgRNA-Int11 recombinant expression vector by taking HZP-sgRNA as a starting vector, and constructing an HZP-sgRNA-Int12 recombinant expression vector by taking HHP-sgRNA as a starting vector.
Two promoters used in the above steps: pTEF and pGAP, one terminator tAOX1 nucleotide sequence is: SEQ ID No.5, SEQ ID No.6, SEQ ID No.7.
The Pichia pastoris transformation strategy in the above procedure is as follows: the method can simultaneously transform the donor corresponding to the target expression cassette and the guide plasmid corresponding to the donor into the pichia pastoris by utilizing a Cas9 system in the pichia pastoris so as to obtain a recombinant yeast strain, wherein the starting strain is pichia pastoris GS115-Cas9.
The invention also provides application of the yeast engineering bacteria for producing patchouli alcohol in the production of patchouli alcohol.
The invention also provides a method for producing patchouli alcohol, which comprises the steps of fermenting and culturing the yeast engineering bacteria for producing patchouli alcohol, and extracting to obtain patchouli alcohol.
The fermentation culture method comprises the following steps: inoculating the genetically engineered bacteria into a liquid fermentation culture medium in an inoculum size of 1-2%, and culturing for 4 days at 30 ℃ and 220rpm, wherein glucose with mass fraction of 2% is added every 48 hours.
Compared with the prior art, the invention has the following advantages:
1. the invention can produce patchouli alcohol, the shake flask fermentation yield is close to 1.2mg/L, and the yield is improved by 10 times compared with the yield of the strain PTS-1.
2. The invention utilizes pichia pastoris to efficiently generate patchouli alcohol, and has the characteristics of short period, environmental protection and resource conservation.
Drawings
FIG. 1 is a metabolic scheme for the biosynthesis of patchouli alcohol.
FIG. 2 is a schematic representation of key enzyme expression cassettes in the synthetic pathway.
FIG. 3 is a map of plasmid pGAP-tAOX 1.
FIG. 4 is a GC-MS detection mass spectrum of patchouli alcohol.
FIG. 5 is a graph showing comparison of yields of patchouli alcohol obtained by shake flask culture of different strains in examples.
Detailed Description
The metabolic flow chart of the biosynthesis of patchouli alcohol is shown in figure 1, the pathway modification key enzyme expression cassette is shown in figure 2, and the detailed synthesis pathway is shown in the examples. The Int1-pTEF-tAOX1, helper plasmids HZP-sgRNA, HHP-sgRNA and HGP-sgRNA are shown in detail in the prior application of patent publication No. CN114507613A by the applicant and the invention is named as "a yeast engineering bacterium for producing alpha-santalene by fermentation", and application thereof.
Example 1: construction of Pichia pastoris recombinant strain GS115-Cas9
Taking the plasmid pGAP-tAOX1 as a starting plasmid (mainly comprising a promoter GAP, a terminator tAOX1 and a screening mark His), and introducing Cas9 by using a restriction enzyme cutting site BamHI in the vector to obtain a recombinant vector pGAP-Cas9-tAOX1, and transferring the recombinant vector pGAP-Cas9-tAOX1 into a pichia pastoris cell GS115 preserved in a laboratory after the restriction enzyme StuI is used for cutting the vector into a linearization fragment to obtain pichia pastoris GS115-Cas9, wherein the preparation method comprises the following specific steps:
1. construction of Pichia pastoris recombinant strain GS115-Cas9
Cas9 gene (NCBI gene number ID: 6IFO_A) is synthesized by Kirschner biotechnology Co., ltd, a primer Cas9-F/Cas9-R is designed to amplify target gene Cas9 by taking Cas9 gene as a template, and the target gene Cas9 is assembled with a vector pGAP-tAOX1 after enzyme digestion (enzyme digestion site BamHI) by using a seamless cloning method, and an escherichia coli DH5 alpha strain is transformed to obtain a recombinant vector pGAP-Cas9-tAOX1. The primer sequences used were as follows:
Cas9-F：tcaatcaattgaacaactatATGCCAAAGAAGAAAAGAAAAGTTG；
Cas9-R：ttgaactgagcgagaagagaTCAGCTACCCACCTTCCTC。
2. construction of Pichia pastoris recombinant strain GS115-Cas9
And (3) cutting the recombinant vector pGAP-Cas9-tAOX1 into a linearization fragment by using a restriction enzyme StuI, and transferring the linearization fragment into a Pichia pastoris cell GS115 preserved in a laboratory to obtain Pichia pastoris GS115-Cas9.
Example 2: construction of Pichia pastoris recombinant strain PTS-1
Taking plasmid Int1-pTEF-tAOX1 as an initial plasmid, and introducing patchouli alcohol synthase gene PTS by using enzyme cutting site BamHI existing in the vector to obtain recombinant vector Int1-pTEF-PTS-tAOX1; and taking auxiliary plasmid HZP-sgRNA stored in a laboratory as a starting vector, and introducing sgRNA-Int1 by using enzyme cutting sites BsaI in the vector to obtain a recombinant vector HZP-sgRNA-Int1. Then, a donor primer is designed by taking a recombinant vector Int1-pTEF-PTS-tAOX1 as a template, PCR is carried out to obtain a corresponding Int1-donor, the corresponding Int1-donor and a corresponding recombinant guide plasmid HZP-sgRNA-Int1 are transformed into Pichia pastoris cells GS115-Cas9 stored in a laboratory to obtain a recombinant strain PTS-1, and the implementation is as follows:
1. construction of Pichia pastoris recombinant strain PTS-1
The patchouli alcohol synthase PTS gene PTS (the nucleotide sequence is shown as SEQ ID NO. 1) is synthesized by Jinsri biotechnology limited company, the PTS gene is taken as a template, a primer PTS-F/PTS-R is designed to amplify a target gene PTS, the target gene PTS is assembled with an enzyme-digested vector Int1-pTEF-tAOX1 by a seamless cloning method, and an escherichia coli DH5a strain is transformed to obtain a recombinant vector Int1-pTEF-PTS-tAOX1. The primer sequences used were as follows:
PTS-F：cattttagttattcgccaacATGGAATTGTATGCACAGTCTGTC；
PTS-R：tcctcttgattagaatctagtCAATAAGGTACCGGGTGTAGATAAAG。
2. construction of sgRNA recombinant expression vector HZP-sgRNA-Int1
The plasmid HZP-sgRNA is used as a template, primers Int1-sgRNA-F and Int1-sgRNA-R are designed, the cutting site BsaI in the vector HZP-sgRNA is utilized to introduce the Int1-sgRNA, and the recombinant vector HZP-sgRNA-Int1 is obtained through T4 enzyme connection, wherein the sgRNA sequence is 5'-TATCTGAAGTATTTACTGGG-3'. The primer sequences used were as follows:
Int1-sgRNA-F：acgcTATCTGAAGTATTTACTGGG；
Int1-sgRNA-R：aaacCCCAGTAAATACTTCAGATA。
3. construction of Pichia pastoris recombinant strain PTS-1
The recombinant vector Int1-pTEF-PTS-tAOX1 is used as a template to design primers Int1-donor-F and Int1-donor-R, PCR is carried out to obtain a corresponding linearized fragment Int1-donor (the left side gene PAS_FragB_0066 and the right side gene PAS_FragB_0067) which is simultaneously transformed with a corresponding recombinant guide plasmid HZP-sgRNA-Int1 into pichia pastoris cells GS115-Cas9 obtained in the example 1 to obtain the recombinant strain PTS-1. The resistant plasmid was discarded at the same time as passaging several times. The primer sequences used were as follows:
Int1-donor-F：CTGGGCAGTAGTGAATTGGTTG；
Int1-donor-R：ACATTGTTCGTGAGGCTAATCC。
example 3: construction of Pichia pastoris recombinant strain PTS-2
And constructing an Int1-pTEF-ERG20-PTS-tAOX1 recombinant expression vector by taking the Int1-pTEF-PTS-tAOX1 as a starting vector. Taking Int1-pTEF-PTS-tAOX1 as a starting plasmid, designing a skeleton primer to carry out full plasmid PCR to obtain a vector skeleton, and then designing the primer to introduce ERG20 to obtain a recombinant vector Int1-pTEF-ERG20-PTS-tAOX1 through homologous recombination. In addition, this part of the sgRNA is HZP-sgRNA-Int1. Then, a donor primer is designed by taking a recombinant vector Int1-pTEF-PTS-tAOX1 as a template, PCR is carried out to obtain a corresponding Int1-ERG20-donor, the corresponding Int1-ERG20-donor and a corresponding recombinant guide plasmid HZP-sgRNA-Int1 are transformed into a pichia pastoris cell GS115-Cas9 obtained in the example 1 to obtain a recombinant strain PTS-2, and the specific implementation is as follows:
1. construction of Pichia pastoris recombinant strain PTS-2
The starting vector of Int1-pTEF-PTS-tAOX1 is used as a template, and a skeleton primer Int 1-guia-F/Int 1-guia-R is designed to carry out full plasmid so as to obtain a vector skeleton. In addition, a genome of Pichia pastoris GS115 (the strain with the accession number of ATCC 20864) preserved in a laboratory is used as a template to design a primer ERG20-F/ERG20-R to amplify a target gene ERG20 (the nucleotide sequence is shown as SEQ ID NO. 2), and the target gene ERG20 is assembled with a framework vector Int1-pTEF-PTS-tAOX1 by a seamless cloning method to transform an escherichia coli DH5a strain so as to obtain a recombinant vector Int1-pTEF-ERG20-PTS-tAOX1.
The primer sequences used were as follows:
Int1-gujia-F：GGCGGCAGCATGGAATTG；
Int1-gujia-R：GTTGGCGAATAACTAAAATGTATG；
ERG20-F：cattttagttattcgccaacATGTCCAAAGAAGTAGCAGC；
ERG20-R：gactgtgcatacaattccatgctgccgcctttgGTTCTCTTGTAGATCTTGTC。
2. construction of sgRNA recombinant expression vector HZP-sgRNA-Int1
The sgRNA used in this section was identical to the recombinant expression vector HZP-sgRNA-Int1 of example 2.
3. Construction of Pichia pastoris recombinant strain PTS-2
The recombinant vector Int1-pTEF-ERG20-PTS-tAOX1 is used as a template to design primers Int1-ERG20-donor-F and Int1-ERG20-donor-R, corresponding linearized fragments Int1-ERG20-donor (preserved in a laboratory, the left side group of Int20 is caused by PAS_chr4_0465 and the right side gene PAS_chr4_ 0467) are obtained by PCR, and the primers Int1-ERG20-donor-F and Int1-ERG20-donor-R are simultaneously transformed into Pichia pastoris cells GS115-Cas9 obtained in the example 1 to obtain the recombinant strain PTS-2. The resistant plasmid was discarded at the same time as passaging several times. The primer sequences used were as follows:
Int1-ERG20-donor-F：CTGGGCAGTAGTGAATTGGTTG；
Int1-ERG20-donor-F：ACATTGTTCGTGAGGCTAATCC。
example 4: construction of multi-copy patchouli alcohol pichia pastoris recombinant strain PTS-3
An expression cassette of ERG20-PTS and a corresponding guide plasmid are constructed, and are sequentially transferred into the recombinant strain PTS-2, and the recombinant strain PTS-3 is finally obtained through transformation, and the method is implemented as follows:
1. construction of PTS recombinant expression vector
The recombinant vector Int1-pTEF-ERG20-PTS-tAOX1 is used as a template, a primer ERG20-PTS-F/ERG20-PTS-R is designed to obtain an ERG20-PTS fusion gene, the ERG20-PTS fusion gene is assembled with the BamHI digested vectors Int32-pTEF-tAOX1, int33-pGAP-tAOX1 and Int34-pTEF-tAOX1 respectively through a seamless cloning method, and the recombinant vectors Int32-pTEF-ERG20-PTS-tAOX1, int33-pGAP-ERG20-PTS-tAOX1 and Int34-pTEF-ERG20-PTS-tAOX1 are obtained after transformation of E.coli DH5 alpha. The primer sequences used were the same as in example 2.
2. Construction of the sgRNA recombinant expression vectors HGP-sgRNA-Int32, HHP-sgRNA-Int33 and HP-sgRNA-Int34
The helper plasmid HGP-sgRNA is used as a template, primers Int32-sgRNA-F and Int32-sgRNA-R are designed, the Int32-sgRNA is introduced by using an enzyme cutting site BsaI in the vector HGP-sgRNA, and the recombinant vector HGP-sgRNA-Int32 is obtained through T4 enzyme connection; the helper plasmid HHP-sgRNA stored in a laboratory is used as a template, primers Int33-sgRNA-F and Int33-sgRNA-R are designed, the Int33-sgRNA is introduced by using an enzyme cutting site BsaI in the vector HHP-sgRNA, and the recombinant vector HHP-sgRNA-Int33 is obtained through T4 enzyme connection. The helper plasmid HZP-sgRNA stored in a laboratory is used as a template, primers Int34-sgRNA-F and Int34-sgRNA-R are designed, the Int34-sgRNA is introduced by using the cleavage site BsaI in the vector HZP-sgRNA, and the recombinant vector HZP-sgRNA-Int34 is obtained through T4 enzyme connection. The primer sequences used were as follows:
Int32-sgRNA-F：acgcGTGACGAAAGAGATGAGGTG；
Int32-sgRNA-R：aaacCACCTCATCTCTTTCGtcac；
Int33-sgRNA-F：acgcCCGTCACTATGAGGACAAAG；
Int33-sgRNA-R：aaacCTTTGTCCTCATAGTGacgg；
Int34-sgRNA-F：acgcCCGTCACTATGAGGACAAAG；
Int34-sgRNA-R：aaacTGTCTATCAATGAACTGATC。
3. construction of Pichia pastoris recombinant strain PTS-3
The recombinant vector Int32-pTEF-ERG20-PTS-tAOX1 is used as a template to design primers Int32-donor-F and Int32-donor-R, corresponding linearized fragments Int32-donor (saved in a laboratory, the left side group of Int32 is caused by PAS_chr2-2_0143 and the right side gene PAS_chr2-2_0142) are obtained by PCR, and the recombinant fragments and corresponding recombinant guide plasmid HHP-sgRNA-Int32 are simultaneously transformed into pichia pastoris cells PTS-2 to obtain recombinant strains PTS-3-1. The recombinant vector Int33-pTEF-ERG20-PTS-tAOX1 is used as a template to design primers Int33-donor-F and Int33-donor-R, corresponding linearized fragments Int33-donor (saved in a laboratory, the left side group of Int33 is caused by PAS_chr1-1_0053 and the right side group of the Int33 is caused by PAS_chr1-1_0054), and the recombinant vector and the corresponding recombinant guide plasmid HHP-sgRNA-Int33 are simultaneously transformed into pichia pastoris cells PTS-3-1 to obtain recombinant strains PTS-3-2. The recombinant vector Int34-pTEF-ERG20-PTS-tAOX1 is used as a template to design primers Int34-donor-F and Int34-donor-R, PCR is carried out to obtain a corresponding linearized fragment Int34-donor, and the linearized fragment Int34-donor and a corresponding recombinant guide plasmid HHP-sgRNA-Int34 (stored in a laboratory, PAS_chr3_0053 is taken as a left side gene PAS_chr3_0054 of the Int 34) are simultaneously transformed into pichia pastoris cells PTS-3-2 to obtain a recombinant strain PTS-3. The two rounds of serially transformed resistant plasmid were discarded at the same time as passaging several times. The primer sequences used were as follows:
Int32-donor-F：GACTGGTGCCTTGATTTTCG；
Int32-donor-R：TCGTAAGAAGGGTCTGTGATAG；
Int33-donor-F：AAAGAGAGAGTCTAAAAGTGGTG；
Int33-donor-R：ATTATAATTAGCACGGTGTTGC；
Int34-donor-F：AATTATAAGACAGGACTGTTGTGG；
Int34-donor-R：TCGGAATCCTGTGTCTGGAAG。
example 5: construction of Pichia pastoris recombinant strain PTS-4
Respectively constructing expression cassettes of high-expression IDI1 and tHMG1 and corresponding guide plasmids, sequentially transferring the expression cassettes into the recombinant strain PTS-3, and respectively obtaining pichia pastoris recombinant strains PTS-4-1 and PTS-4 through round-by-round transformation, wherein the method is specifically implemented as follows:
1. construction of IDI1 recombinant expression vector
The genome of Saccharomyces cerevisiae BY4741 (strain deposit number ATCC 4040002) is used as a template, primers IDI1-F/IDI1-R are designed to amplify a target gene IDI1 (the nucleotide sequence is shown as SEQ ID NO. 3), the target gene IDI is assembled with a BamHI digested vector Int12-pTEF-tAOX1 BY a seamless cloning method, and an escherichia coli DH5 alpha strain is transformed to obtain a recombinant vector Int12-pTEF-IDI1-tAOX1. The primer sequences used were as follows:
IDI1-F：catacattttagttattcgccaacGATGACTGCCGACAACAATAGTATG；
IDI1-R：aaatggcattctgacatcctcttgagTTATAGCATTCTATGAATTTGCC。
2. construction of the guide RNA recombinant expression vector HHP-sgRNA-Int12
The plasmid HHP-sgRNA stored in a laboratory is used as a template, primers Int12-sgRNA-F and Int12-sgRNA-R are designed, the Int12-sgRNA is introduced by using an enzyme cutting site BsaI in the vector HHP-sgRNA, and the recombinant vector HHP-sgRNA-Int12 is obtained through T4 enzyme connection. The primer sequences used were as follows:
Int12-sgRNA-F：acgcGGGGTTTGAATAACAGACAC；
Int12-sgRNA-R：aaacGTGTCTGTTATTCAAACCCC。
3. construction of Pichia pastoris recombinant strain PTS-4-1
The recombinant vector Int12-pTEF-IDI1-tAOX1 is used as a template to design primers Int12-donor-F and Int12-donor-R, PCR is carried out to obtain a corresponding linearized fragment Int12-donor (stored in a laboratory, the left side group of Int12 is caused by PAS_chr4_0465 and the right side group of PAS_chr4_0465), and the linearized fragment Int12-donor-R and the corresponding recombinant guide plasmid HHP-sgRNA-Int12 are simultaneously transformed into Pichia pastoris cells PTS-3 to obtain a recombinant strain PTS-4-1. The resistant plasmid was discarded at the same time as passaging several times. The primer sequences used were as follows:
Int12-donor-F：ATACTACAAGAAAGGTTGTTGATGTAG；
Int12-donor-R：TGTTTCTTTACTATTGAATCTTCAGAGATAG。
4. construction of tHMG1 recombinant expression vector
The saccharomyces cerevisiae BY4741 genome is used as a template, primers tHMG1-F/tHMG1-R are designed to amplify a target gene tHMG1 (the nucleotide sequence is shown as SEQ ID NO. 4), the target gene tHMG1 is assembled with a vector Int11-pGAP-tAOX1 subjected to AatII enzyme digestion BY using a seamless cloning method, and an escherichia coli DH5a strain is transformed to obtain a recombinant vector Int11-pGAP-tHMG1-tAOX1. The primer sequences used were as follows:
tHMG1-F：aatcaattgaacaactatcaaaacacaGATGGCTGCAGACCAATTGGTG；
tHMG1-R：aggcaaatggcattctgacatCCTCTTGAGTTAGGATTTAATGCAGGTG。
5. construction of vector HZP-sgRNA-Int11 for recombinant expression of guide RNA
The plasmid HZP-sgRNA is used as a template, primers Int11-sgRNA-F and Int11-sgRNA-R are designed, the cutting site BsaI in the vector HZP-sgRNA is utilized to introduce Int11-sgRNA, and the recombinant vector HZP-sgRNA-Int11 is obtained through T4 enzyme connection. The primer sequences used were as follows:
Int11-sgRNA-F：tcttGTATGCAAACATTTCGTCC；
Int11-sgRNA-R：aaacCGGGATCGTCTTTTTTAATA。
6. construction of Pichia pastoris recombinant strain PTS-4
The recombinant vector Int11-pGAP-tHMG1-tAOX1 is used as a template to design primers Int11-donor-F and Int11-donor-R, PCR is carried out to obtain a corresponding linearized fragment Int11-donor (stored in a laboratory, the left side group of Int11 is caused by PAS_chr3_1156, and the right side group of the In 11 is caused by PAS_chr3_0154), and the fragment Int11 and a corresponding recombinant guide plasmid HZP-sgRNA-Int11 are simultaneously transformed into Pichia pastoris cells PTS-4-1 to obtain a recombinant strain PTS-4. The resistant plasmid was discarded at the same time as passaging several times. The primer sequences used were as follows:
Int11-donor-F：CTGGGCAGTAGTGAATTGGTTG；
Int11-donor-R：TGGTAAATGGTGAAGTTGGCTG。
example 6: efficient production of patchouli alcohol by pichia pastoris engineering strain
The recombinant strains in examples 2-5 were streaked on solid YPD plates, and single colonies were picked up and cultured in 5mL test tubes, and transferred to 50mL shake flasks at 1% inoculum size for 4 days, and then treated for product analysis. The specific implementation is as follows:
1. cultivation of recombinant strains
GS115-Cas9, PTS-1, PTS-2, PTS-3 and PTS-4 single colonies were picked from the plates, inoculated into 5mL YPD (glucose 20g/L, peptone 20g/L, yeast extract 10 g/L) tubes, cultured at 30℃and 220rpm for 12 hours, and then inoculated into 50mL YPD shake flask medium at 1% inoculum size, three strains each in parallel, 10% n-dodecane was added to the upper layer of the medium to cover, and cultured at 30℃and 220rpm for 4 days, with 2% glucose added every 48 hours.
2. Detection method of patchouli alcohol
Sample treatment: the upper layer fermentation broth was centrifuged at 12000rpm for 10min, the upper layer organic phase was filtered through a 0.22 μm nylon filter, and the sample was diluted 1000-fold with ethyl acetate and subjected to GC-MS detection.
The GC-MS detection conditions were as follows:
chromatographic separation conditions: the initial column temperature is 50 ℃, and the temperature is kept constant for 1min; at 10 ℃ min -1 The temperature rise speed of the furnace is increased to 200 ℃; then at 20 ℃ min -1 The temperature rise rate of (2) is increased to 280 ℃, and the constant temperature is maintained for 3min.
Mass spectrometry conditions: selecting 35-300 m/z ion scan, setting the temperature of the sample inlet at 280 deg.C and the flow rate at 1.2mL min -1 The ion source temperature is 280 ℃, the ionization mode is electron ionization (60 EV), the split-flow sample injection mode is selected, the split-flow ratio is 4:2, the sample injection amount is 1 mu L, and the quantitative analysis is carried out by using selective reaction monitoring.
3. Recombinant strain PTS-4 fermentation tank culture
Starting from the recombinant strain PTS-4 of Pichia pastoris, firstly inoculating single colony of PTS-4 activated on a YPD plate into a 5mL YPD test tube for culturing for 12 hours, then transferring the single colony to a 50mL YPD liquid culture medium in an inoculum size of 1%, culturing for 12 hours under the conditions of 30 ℃ and 220rpm of a shaking table rotation speed as seed liquid, transferring the seed liquid into a 1L fermentation tank (liquid filling amount of 600 mL) in an inoculum size of 10%, culturing at a temperature of 30 ℃ and correcting dissolved oxygen DO to 100%, wherein the tank pressure is 0.8 atm, and adding 10% (v/v) of sterile filtered n-dodecane after fermenting for 24 hours. When the mass fraction of glucose in the fermentation tank is reduced to 1%, feeding glucose solution with the mass fraction of 40% (v/v), controlling the stirring rotation speed to be 200-600rpm, keeping dissolved oxygen to be more than 20%, feeding ammonia water with the volume fraction of 25% and acetic acid with the volume fraction of 18% in the later fermentation period, adjusting the pH value to be between 5.0 and 5.5, and finishing fermentation after 96 hours.
4. Comparison of patchouli alcohol yield of recombinant strains
Culturing Pichia pastoris engineering strains according to the culture method in the step 1, measuring patchouli alcohol yield by adopting the detection method in the step 2, wherein a detection mass spectrum is shown in figure 4, the yield in shake flask is shown in figure 5, and the shake flask fermentation yield is close to 1.2mg/L. The pichia pastoris engineering strain PTS-4 is subjected to fed-batch fermentation according to the method shown in the step 3, and the highest yield of patchouli alcohol in a 1L fermentation tank after 96 hours is about 22mg/L, so that a foundation is laid for green synthesis of patchouli alcohol and high added value derivatives thereof in pichia pastoris, and reference can be provided for efficient green biological production of other terpene natural products.
Claims (8)
1. The yeast engineering bacteria for producing patchouli alcohol is characterized in that pichia pastoris is taken as an original strain, the yeast engineering bacteria are obtained through construction of introduced genes, and the introduced genes comprise any one of the following groups:
(1) A gene encoding patchouli alcohol synthase PTS, a gene encoding farnesyl pyrophosphate synthase ERG20, a gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase 1 tHMG1, and a gene encoding isopentenyl pyrophosphate isomerase IDI 1;
(2) Encoding gene of patchouli alcohol synthase PTS and encoding gene of farnesyl pyrophosphoric acid synthase ERG 20;
(3) A gene encoding patchouli alcohol synthase PTS.
2. The yeast engineering bacterium for producing patchouli alcohol according to claim 1, wherein the coding gene of patchouli alcohol synthase PTS is linked with the coding gene of farnesyl pyrophosphate synthase ERG20 to make patchouli alcohol synthase PTS and ERG20 fusion expressed.
3. The yeast engineering bacterium for producing patchouli alcohol according to claim 2, wherein the copy number of the encoding gene expressed by fusion of Ji Maxi a synthase GAS and farnesyl pyrophosphate synthase ERG20 is l-4.
4. The yeast engineering bacterium for producing patchouli alcohol according to claim 1, wherein the coding gene of patchouli alcohol synthase PTS is shown in SEQ ID NO.1, the coding gene of farnesyl pyrophosphate synthase ERG20 is shown in SEQ ID NO.2, the coding gene of isopentenyl pyrophosphate isomerase IDI1 is shown in SEQ ID NO.3, and the coding gene of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 tHMG1 is shown in SEQ ID NO. 4.
5. The method for constructing a yeast engineering bacterium for producing patchouli alcohol according to any one of claims 1 to 4, wherein the introduced gene comprises: a gene encoding patchouli alcohol synthase PTS, a gene encoding farnesyl pyrophosphate synthase ERG20, a gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase 1 tHMG1, a gene encoding isopentenyl pyrophosphate isomerase IDI1,
the construction method comprises the following steps:
(1) Introducing a Cas9 protein coding gene from a Pichia pastoris GS115 strain genome to obtain Pichia pastoris GS115-Cas9;
(2) Integrating a coding gene sequence containing patchouli alcohol synthase PTS into Pichia pastoris GS115-Cas9 to obtain a strain PTS-1;
(3) Integrating a coding gene sequence of patchouli alcohol synthase PTS and a coding gene sequence of farnesyl pyrophosphate synthase ERG20 into pichia pastoris GS115-Cas9 to obtain a strain PTS-2;
(4) 3 single copies of the coding gene sequence of patchouli alcohol synthase PTS and the coding gene sequence of farnesyl pyrophosphate synthetase ERG20 are integrated into the strain PTS-2 to obtain the strain PTS-3.
(5) Integrating the coding gene sequence of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 tHMG1 and the coding gene sequence of isopentenyl pyrophosphate isomerase IDI1 into the strain PTS-3 to obtain a strain PTS-4; namely the yeast engineering bacteria for producing patchouli alcohol.
6. Use of the yeast engineering bacteria for producing patchouli alcohol according to any one of claims 1 to 4 for producing patchouli alcohol.
7. A method for producing patchouli alcohol, which is characterized in that the yeast engineering bacteria for producing patchouli alcohol according to any one of claims 1 to 4 are cultivated by fermentation, and patchouli alcohol is obtained by extraction.
8. The method for producing patchouli alcohol according to claim 7, wherein the conditions of fermentation culture are: culturing at 30deg.C and 220rpm for 4 days, and supplementing 2% glucose every 48 hr.
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