CN117264792B - Engineering strain of yarrowia lipolytica for high yield sclareol, construction method and application thereof - Google Patents

Engineering strain of yarrowia lipolytica for high yield sclareol, construction method and application thereof Download PDF

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CN117264792B
CN117264792B CN202311260309.XA CN202311260309A CN117264792B CN 117264792 B CN117264792 B CN 117264792B CN 202311260309 A CN202311260309 A CN 202311260309A CN 117264792 B CN117264792 B CN 117264792B
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强珊
杨璐
郭建琦
牛永洁
孟永宏
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Shaanxi Healthful Biological Engineering Co ltd
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Abstract

The invention provides a recombinant yarrowia lipolytica with high sclareol yield, which is prepared by over-expressing a lysergic glycol ester synthase mutant gene calPPS in yarrowia lipolytica po1f R227Q The sclareol synthase gene SsTPS and the geranylgeranyl pyrophosphate synthase gene SaGGPP, and the 3-hydroxy-3-methylglutaryl-CoA reductase tHMGR and the isopentenyl pyrophosphate isomerase gene idi. The invention verifies that the yield of sclareol reaches 8510mg/L at the highest in a 5L fermentation tank, and confirms that the recombinant yarrowia lipolytica YLSC-19 can ferment and synthesize natural sclareol with high efficiency.

Description

Engineering strain of yarrowia lipolytica for high yield sclareol, construction method and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering. More specifically, the invention relates to a recombinant yarrowia lipolytica capable of producing sclareol in high yield, a construction method of the recombinant yarrowia lipolytica and application of the recombinant yarrowia lipolytica in fermentation production of sclareol.
Background
Sclareol, also called sclareol, is a diterpene di-tertiary alcohol compound of labdane, and is mainly found in plants such as sclareol (Salvia sclarea), and is commonly used as a spice component of cosmetics and perfumes and as a food seasoning material. It is the only practical raw material for chemical synthesis of ambrox, a substitute for ambrox, such as for synthesisTherefore, sclareol has higher economic valueValues. In addition, sclareol has cytotoxicity on human leukemia cells, tumor cells and colon cancer cells, and is a potential anti-tumor drug. At present, sclareol is mainly extracted from plants, however, the plant growth period is long, the yield is unstable, the sclareol content in the plants is low, and only 15kg of sclareol can be obtained in 1000kg of hay. Therefore, sclareol extracted from plants is far from meeting market demand. The method for synthesizing sclareol from glucose by utilizing a microbial fermentation method is an economic sustainable green environment-friendly method, has short production period and stable yield, and is expected to meet market demands.
Yarrowia lipolytica (Yarrowia lipolytica) is a well-established and safe strain (GRAS) and has been widely used for the synthesis of lipids, citric acid, succinic acid, and the like. The strains have high flux of acetyl-CoA and malonyl-CoA, wherein the acetyl-CoA is a precursor substance for synthesizing sclareol. Chinese patent application CN 114989997A discloses a recombinant yarrowia lipolytica strain with high sclareol yield, and a construction method and application thereof, but the maximum concentration of sclareol produced by the strain is only 0.919g/L, and industrial production is difficult to realize. Researchers in the Fenmei meaning of Swiss essence company have metabolically modified E.coli by introducing the genes for the Salmonella-derived lysyl glycol pyrophosphate synthase and sclareol synthase, and the yield of sclareol reaches 1.5g/L under high-density fermentation conditions (Schalk M., pasture L., mirata M.et al Toward a biosynthetic route to sclareol and amber odorants [ J ]. Journal of the American Chemical Society,2012, 134:18900-18903.).
Therefore, by taking yarrowia lipolytica as an original strain through a genetic engineering technology, the capacity of yarrowia lipolytica for producing sclareol is further improved through excavation and research on sclareol synthesis key genes, and the technical problem to be solved in the field is solved.
Disclosure of Invention
The invention aims to provide yarrowia lipolytica with high sclareol yield by genetic engineering technology.
The idea of the invention is to take yarrowia lipolytica as an initial strain, carry out gene mining on the key gene of sclareol synthesis, namely lysyl-behenyl-diol-pyrophosphate synthase (LPPS), select better genes from LPPS of different sources, then try to further improve the activity of lysyl-behenyl-diol-pyrophosphate synthase through mutation, and on the other hand, improve the yield of sclareol through over-expression of the key genes GGPP, tHMG and idi accumulated by geranylgeranyl-pyrophosphate as substrates of sclareol synthesis.
In view of the above, the present invention provides a recombinant yarrowia lipolytica producing sclareol with high yield, which is constructed by overexpressing lysyl alkylene glycol pyrophosphate synthase mutant gene CaLPPS, sclareol synthase gene SsTPS and geranylgeranyl pyrophosphate synthase gene samgpp, and expressing 3-hydroxy-3-methylglutaryl coa reductase tggr and isopentenyl pyrophosphate isomerase gene idi in yarrowia lipolytica po1 f.
Further, the recombinant yarrowia lipolytica is obtained by inserting an exogenous geranylgeranyl pyrophosphate synthase expression cassette, an exogenous lysyl alkene glycol pyrophosphate synthase mutant expression cassette, an exogenous sclareol synthase expression cassette, an endogenous 3-hydroxy-3-methylglutaryl coenzyme A reductase expression cassette and an endogenous isopentenyl pyrophosphate isomerase expression cassette into the genome of yarrowia lipolytica po1 f.
In the present invention, the lysyl glycol pyrophosphate synthase gene is a mutant gene CaLPPS derived from CaLPPS of coffee (Coffea arabica) R227Q The nucleotide sequence is shown as SEQ ID NO. 1;
the sclareol synthase gene SsTPS is derived from sclareol (Salvia sclarea), and the nucleotide sequence of the sclareol synthase gene SsTPS is shown as SEQ ID NO. 2;
the geranylgeranyl pyrophosphate synthetase gene SaGGPP is derived from sulfolobus acidocaldarius (Sulfolobus acidocaldarius), and the nucleotide sequence of the geranylgeranyl pyrophosphate synthetase gene SaGGPP is shown as SEQ ID NO. 3;
the 3-hydroxy-3-methylglutaryl-CoA reductase tHMGR and the isopentenyl pyrophosphate isomerase gene idi are derived from yarrowia lipolytica.
According to a particularly preferred embodiment, the mutant gene CaLPPS R227Q Is obtained by saturation mutation of amino acid at 227 of CaLPPS derived from coffee (Coffea arabica) from original AGA to CAG.
In the present invention, preferably, lysyl glycol ester pyrophosphate synthase mutant gene CaLPPS and sclareol synthase gene SsTPS are fusion expressed by the connecting peptide GGGGS.
In the present invention, each expression cassette is composed of a promoter-gene-terminator, the promoter being P TEF 、P TEFin 、P EXP1 、P FBA 、P GPD Any one of the terminators is T xpr2 、T lip1 、T lip2 Any one of the following.
Based on this, the present invention also provides mutant CaLPPS R227Q Use of said mutant CaLPPS for increasing yield of sclareol produced by yarrowia lipolytica R227Q Is obtained by saturation mutation of amino acid at 227 of CaLPPS derived from coffee (Coffea arabica) from original AGA to CAG.
Another object of the present invention is to provide a method for constructing recombinant yarrowia lipolytica with high sclareol yield, which comprises the following steps:
(1)ura3-SaGGPP-CaL R227Q construction of SsT plasmid
Artificial Synthesis of Liepsenenediol pyrophosphate synthase mutant CapPPS derived from Coffea arabica (Coffea arabica) as shown in SEQ ID NO:1 R227Q Gene sequence, the synthesized gene fragment was inserted into HindIII and SmaI sites of plasmid pJN to give pJN-CaL R227Q The method comprises the steps of carrying out a first treatment on the surface of the The sclareol synthase SsTPS gene sequence from sclareol (Salvia sclarea) is artificially synthesized, and the synthesized gene fragment is inserted into HindIII and SmaI sites of plasmid pJN to obtain pJN-SsTPS; pJN44 to CaL R227Q As a template, the PCR amplification gives CaL R227Q A gene fragment; using pJN-SsTPS as a template, and amplifying SsTPS gene fragments by PCR; will CaL R227Q The gene fragment and the SsTPS gene fragment are connected to HindIII and SmaI sites of pJN by an Assembly kit to obtain fusion expression plasmids pJN-CaL R227Q SsT；
Artificially synthesizing a geranylgeranyl pyrophosphate synthetase SaGGPP gene derived from sulfolobus acidocaldarius (Sulfolobus acidocaldarius), and inserting the artificially synthesized gene fragment into HindIII and SmaI sites of a plasmid pJN to obtain pJN-SaGGPP; fusion expression plasmid pJN-CaL was digested with XbaI-SpeI R227Q CaL of SsT R227Q SsT fragment, digested with SpeI site ligated to pJN-SaGGPP plasmid to give pJN-SaGGPP-CaL R227Q SsT plasmid;
the ploxpura3loxp carrier is singly cut by using SpeI, and the ploxpura3loxp carrier fragment is obtained after gel recovery, pJN-SaGGPP-CaL R227Q SsT plasmid SaGGPP-CaL was digested with XbaI-SpeI R227Q SsT, ploxpura3loxp vector fragment and SaGGPP-CaL R227Q The SsT fragment was digested and ligated to give plasmid ura3-SaGGPP-CaL R227Q SsT；
(2) Construction of ura3-tHMGR-idi plasmid
Obtaining a tHMGR gene sequence and an idi gene sequence of yarrowia lipolytica source, extracting a genome of yarrowia lipolytica po1f, and carrying out PCR amplification on a tHMGR fragment by taking the po1f genome as a template; PCR amplifying the idi fragment by using the po1f genome as a template; the tHMGR fragment and the idi fragment are respectively connected to HindIII and SmaI sites of pJN44 by an Assembly kit to respectively obtain pJN-tHMGR and pJN44-idi;
PCR amplifying the TEF-tHMGR-XPR2 fragment by taking pJN-tHMGR as a template, and connecting the fragment to the SpeI and MluI sites of ploxpura3loxp by an Assembly kit to obtain ura3-tHMGR plasmid; PCR amplifying the TEF-idi-XPR2 fragment by taking pJN-idi as a template, and connecting the fragment to the MluI site of ura3-tHMGR through an Assembly kit to obtain ura3-tHMGR-idi plasmid;
(3) Construction of recombinant Strain YLSC-17
Subjecting ura3-SaGGPP-CaL of step (1) R227Q The SsT plasmid was digested with MluI, the linearized product was recovered with gel and ura3-SaGGPP-CaL was obtained by means of a yeast transformation kit R227Q Transferring the SsT linearization product into po1f, coating SD-ura solid culture medium, and culturing to obtain recombinant strain YLSC-17;
(4) Overexpression of SaGGPP and CaL R227Q Construction of recombinant strain YLSC-18 of SsT
Removing ura3 marks in the YLSC-17 strain by using a Cre/loxp system to obtain a YLSC-17D strain;
re-combining ura3-SaGGPP-CaL of step (1) R227Q Transferring the SsT linearization product into YLSC-17D, coating SD-ura solid culture medium, and culturing to obtain recombinant fungus YLSC-18;
removing ura3 marks in the YLSC-18 strain by using a Cre/loxp system to obtain a YLSC-18D strain;
cutting the ura3-tHMGR-idi plasmid in the step (2) by using NsiI, recovering the gel to obtain a linearization product, transferring the ura3-tHMGR-idi linearization product into YLSC-18D, coating an SD-ura solid culture medium, and culturing to obtain the recombinant yarrowia lipolytica with high sclareol yield.
The invention also provides application of the recombinant yarrowia lipolytica in sclareol production.
According to a preferred embodiment, the single colony of the recombinant yarrowia lipolytica is picked and inoculated into 25mL/250mL YPD liquid culture medium, the culture is carried out at the temperature of 30 ℃ and 220rpm for1 day, seed liquid is obtained, the seed liquid is inoculated into a 5L fermentation tank according to the inoculation amount of 2% of volume ratio, the fermentation tank contains 4L YPD fermentation culture medium, the fermentation culture is carried out at the temperature of 30 ℃, the stirring rotation speed is 800rpm, the aeration ratio is 1vvm, the dissolved oxygen is 10% -20%, the pH is adjusted to 5.5 by adding 5M sodium hydroxide, the glucose content is maintained to be 5g/L by feeding, and the sclareol is obtained after fermentation for 168-192 h.
Wherein, YPD liquid medium is: 20g/L peptone, 20g/L glucose, 10g/L yeast powder; YPD fermentation medium was 50g/L yeast extract, 100g/L peptone and 100g/L glucose.
The recombinant yarrowia lipolytica of the present invention is SaGGPP, caLPPS constructed by Non-homologous end-ligation mediated (Non-homologous End Joining-NHEJ) gene integration R227Q And SsTPS gene expression library, and verifying sclareol yield of each strain to construct a strain of fungus YLSC-17 with high sclareol yield, wherein the sclareol yield of the strain is 1208mg/L. The invention is realized by re-overexpression of SaGGPP, caLPPS R227Q And SsTPS gene, and overexpressing the tHMG and idi genes endogenous to yarrowia lipolytica to give YLSC-19 strain in a 5L fermenter,the highest yield of sclareol reaches 8510mg/L, and the recombinant yarrowia lipolytica YLSC-19 can be proved to be capable of efficiently fermenting and synthesizing natural sclareol.
Drawings
FIG. 1 is a diagram of sclareol biosynthesis pathway in yarrowia lipolytica;
FIG. 2 is a comparison of sclareol content in recombinant strain YLSC-00-YLSC 15;
FIG. 3 is ura3-SaGGPP-CaL R227Q SsT plasmid map;
FIG. 4 is a map of ura3-tHMGR-idi plasmid;
FIG. 5 shows the fermentation results of YLSC-19 strain in a 5L fermenter.
Detailed Description
The invention will be better understood by the following examples.
In the present invention, "%" used for describing the concentration is weight percent unless otherwise specified: "all are weight ratios.
The present invention relates to the following genes:
TABLE 1 lysyl alkene diol pyrophosphate synthase gene and its source
The present invention relates to the following synthetic primer sequences:
TABLE 2 primers used in the present invention
The invention relates to the following media:
LB medium: 10g/L peptone, 10g/L NaCl, 5g/L yeast powder, and 2% agar powder was added to the solid medium.
YPD medium: 20g/L peptone, 20g/L glucose, 10g/L yeast powder, and 2% agar powder was added to the solid medium.
SD-leu medium: 20g/L glucose, 1.7g/L YNB (yeast nitrogen source, free of amino acids and ammonium sulfate), 5g/L (NH) 4 ) 2 SO 4 2g/L Drop-out mix synthetic minus leucil w/o yeast nitrogen base, and 2% agar powder is added to the solid medium.
SD-leu fermentation medium: 40g/L glucose, 1.7g/L YNB (yeast nitrogen source, free of amino acids and ammonium sulfate), 5g/L (NH) 4 ) 2 SO 4 、4g/L Drop-out mix synthetic minus leucil w/o yeast nitrogen base。
SD-ura medium: 20g/L glucose, 1.7g/L YNB (yeast nitrogen source, free of amino acids and ammonium sulfate), 5g/L (NH) 4 ) 2 SO 4 2g/L Drop-out mix synthetic minus uracil w/o yeast nitrogen base, and 2% agar powder is added to the solid medium.
YPD fermentation medium: 50g/L yeast extract, 100g/L peptone and 100g/L glucose.
In the invention, the determination method of sclareol yield of each recombinant strain is as follows:
culturing of colonies: inoculating single colony in a test tube containing 2mL of SD-leu liquid culture medium, culturing at 30 ℃ for2 days at 220rpm to obtain seed liquid, and inoculating the seed liquid into 25mL of SD-leu fermentation culture medium according to the inoculum size of 2% by volume, and culturing at 30 ℃ for4 days at 220 rpm.
Extracting sclareol: extracting sclareol in the fermentation broth by using n-hexane as a solvent, adding 2mL of n-hexane into 4mL of the fermentation broth, stirring for1 hour at a rotation speed of 2000rpm by using a vortex oscillator, centrifuging at 1000 Xg to obtain 1mL of an upper organic phase, drying by using nitrogen, re-suspending by using 100 mu L of n-hexane, and filtering by using a 0.22 mu m organic pinhole filter head for detection by using GC-MS.
Detection of sclareol: chromatographic column: DB-5MS (30 m.times.0.25 mm. Times.0.25 mm); chromatographic conditions: the temperature of the sample inlet is 250 ℃, the initial temperature of the column box is 100 ℃, the sample inlet volume is 1 mu L, no flow division is performed, the carrier gas is high-purity helium, and the flow is 1mL/min; programming temperature: the initial temperature was 150 ℃, raised to 200 ℃ at a rate of 20 ℃/min, raised to 260 ℃ at a rate of 5 ℃/min, and then raised to 300 ℃ at a rate of 20 ℃/min for 2min. Qualitative and quantitative analysis was performed with a standard of sclareol in n-hexane.
Example 1: selecting the source of the lysyl alkene glycol ester synthase gene LPPS and the connection mode with sclareol synthase gene TPS
According to the description of Table 1, LPPS gene sequences derived from sclareum sclarea (SsLPPS), nicotiana villosa (NtLPPS), cistus (CcLPPS), coffea arabica (CaLPPS) and Olea lutea (OeLPPS) were obtained from NCBI, and subjected to codon optimization and synthesis (as shown in SEQ ID Nos. 4 to 8), and the synthesized gene fragments were inserted into HindIII and SmaI sites of plasmid pJN to obtain recombinant plasmids pJN-SsLPPS, pJN44-NtLPPS, pJN44-CcLPPS, pJN44-CaLPPS and pJN44-OeLPPS, respectively.
Sclareol synthase gene (TPS) sequences (GenBank accession number: JN 133922.1) derived from sclareol were obtained from NCBI, and subjected to codon optimization for synthesis (as shown in SEQ ID No. 2), and the synthesized gene fragments were inserted into HindIII and SmaI sites of plasmid pJN to obtain recombinant plasmid pJN-SSTPS.
Respectively carrying out SpeI-MluI double digestion on 5 plasmids of pJN-SsLPPS, pJN44-NtLPPS, pJN44-CcLPPS, pJN44-CaLPPS and pJN44-OeLPPS, and recovering the glue to obtain 5 vector fragments; using pJN-SsTPS as a template, and using TPS-FOR/TPS-REV as a primer to amplify SsTPS gene fragments by PCR; TPS gene fragments and the 5 vector fragments are respectively passed through an Assembly kitBasic Seamless Cloning and Assembly Kit) to obtain plasmids pJN-SsLPPS-SsTPS, pJN44-NtLPPS-SsTPS, pJN44-CcLPPS-SsTPS, pJN 44-CAPPS-SsTPS, pJN44-OeLPPS-SsTPS, respectively.
These 5 plasmids were then transformed into yarrowia lipolytica polf strain by means of a yeast transformation kit (Frozen-EZ Yeast Transformation II Kit) and spread on SD-leu solid medium to give recombinant strains YLSC-01, YLSC-02, YLSC-03, YLSC-04, YLSC-05, which were confirmed to be correct by means of colony PCR.
PCR (polymerase chain reaction) amplification of SsLPPS gene fragments by using pJN-SsLPPS as a template and LPPS-FOR1/LPPS-REV1 as primers; amplifying the NtLPPS gene fragment by PCR with pJN-NtLPPS as a template and LPPS-FOR2/LPPS-REV2 as a primer; using pJN-CcLPPS as a template and LPPS-FOR3/LPPS-REV3 as a primer to amplify the CcLPPS gene fragment by PCR; PCR amplifying the CAPPS gene fragment by using pJN-CAPPS as a template and LPPS-FOR4/LPPS-REV4 as a primer; PCR amplifying the OeLPPS gene fragment by using pJN-OeLPPS as a template and LPPS-FOR5/LPPS-REV5 as a primer; using pJN-SsTPS as a template, and using TPS-FOR1/TPS-REV1 as a primer to amplify SsTPS gene fragments by PCR; the SsLPPS gene fragment, the NtLPPS gene fragment, the CcLPPS gene fragment, the CaLPPS gene fragment and the OeLPPS gene fragment are respectively connected with HindIII and SmaI sites of pJN through an Assembly kit to obtain fusion expression plasmids pJN-SsLSsT, pJN44-NtLSsT, pJN44-CcLSsT, pJN44-CaLSsT and pJN44-OeLSsT. Wherein the LPPS and TPS genes are fusion expressed via the connecting peptide GGGGS. The 5 fusion expression plasmids are then transformed into yarrowia lipolytica polf strain by a yeast transformation kit, and SD-leu solid culture medium is coated to obtain recombinant strains YLSC-06, YLSC-07, YLSC-08, YLSC-09 and YLSC-10, which are verified to be correct by colony PCR.
PCR (polymerase chain reaction) amplification of SsTPS gene fragments by taking recombinant plasmid pJN-SsTPS as a template and TPS-FOR2/TPS-REV2 as a primer; PCR (polymerase chain reaction) amplification of SsLPPS gene fragments by using pJN-SsLPPS as a template and LPPS-FOR6/LPPS-REV6 as primers; the SsTPS gene fragment and the SsLPPS gene fragment were ligated to HindIII and SmaI sites of pJN by means of an Assemble kit to obtain fusion expression plasmid pJN-SsTSsL. Similarly, using pJN-SsTPS as a template and TPS-FOR2/TPS-REV3 as a primer to amplify SsTPS gene fragments by PCR; amplifying the NtLPPS gene fragment by PCR with pJN-NtLPPS as a template and LPPS-FOR7/LPPS-REV7 as a primer; the SsTPS gene fragment and the NtLPPS gene fragment were ligated to HindIII and SmaI sites of pJN by means of an Assemble kit to obtain fusion expression plasmid pJN-SsTNtL. Using pJN-SsTPS as a template, and using TPS-FOR2/TPS-REV4 as a primer to amplify SsTPS gene fragments by PCR; using pJN-CcLPPS as a template and LPPS-FOR8/LPPS-REV8 as a primer to amplify the CcLPPS gene fragment by PCR; the SsTPS gene fragment and the CcLPPS gene fragment were ligated to the HindIII and SmaI sites of pJN by means of an Assemble kit to obtain fusion expression plasmid pJN-SsTCcL. Using pJN-SsTPS as a template, and using TPS-FOR2/TPS-REV5 as a primer to amplify SsTPS gene fragments by PCR; PCR amplifying the CAPPS gene fragment by using pJN-CAPPS as a template and LPPS-FOR9/LPPS-REV9 as a primer; the SsTPS gene fragment and the calpPS gene fragment were ligated to HindIII and SmaI sites of pJN by means of an Assembly kit to obtain fusion expression plasmid pJN-SsTCaL. Using pJN-SsTPS as a template, and using TPS-FOR2/TPS-REV6 as a primer to amplify SsTPS gene fragments by PCR; PCR amplifying the OeLPPS gene fragment by using pJN-OeLPPS as a template and LPPS-FOR10/LPPS-REV10 as a primer; the SsTPS gene fragment and the OeLPPS gene fragment were ligated to HindIII and SmaI sites of pJN by means of an Assemble kit to obtain fusion expression plasmid pJN-SsTOeL. Wherein TPS and LPPS genes are fusion expressed by the connecting peptide GGGGS. The 5 fusion expression plasmids of pJN-SstsL, pJN44-SstNTL, pJN 44-SstcL, pJN44-SstcaL and pJN 44-SstonL are transformed into yarrowia lipolytica polf strain through a yeast transformation kit, and SD-leu solid culture medium is coated to obtain recombinant strains YLSC-11, YLSC-12, YLSC-13, YLSC-14 and YLSC-15, and the correctness is verified by colony PCR.
The genotype of the recombinant strain YLSC00-YLSC15 is:
the content of sclareol in the fermentation broth is detected to screen recombinant strain of sclareol with high yield, and the fermentation result is shown in figure 2.
According to experimental results, the sclareol content of each recombinant strain is compared, and the sclareol yield of the recombinant strain obtained by adopting different connection modes of the LPPS and TPS genes is different, wherein the highest sclareol content of YLSC-09 is 25.56mg/L, the LPPS gene is derived from coffee (Coffea arabica), and the CAPPS and SsTPS genes are fusion expressed through the connecting peptide GGGGS. The LPPS gene derived from espresso coffee (cofea arabica) and the sclareolide derived SsTPS gene were thus selected for subsequent study.
Example 2: construction of mutant plasmid of lysyl-glycol-pyrophosphate synthase (CaLPPS)
1. Expression vector construction
(1) CaLPPS and substrate GGPP are molecularly docked: three-dimensional model of CaLPPS was downloaded on UniProt (A0 A6P6TX 94), GGPP substrate model was searched for downloads from NCBI (CID: 5497105). Taking a CaLPPS enzyme molecular model as a receptor and a GGPP substrate as a ligand, and respectively hydrogenating and balancing charges to CaLPPS and GGPP by using Autodock 4.0; the grid box firstly covers all the CAPPS structures, molecular docking is completed according to a docking algorithm of the Lacarckian GA, a region with the highest frequency of active sites in the CAPPS is found from different docking results, and the grid box is accurately determined; based on the precisely determined CaLPPS enzyme grid box, the CaLPPS and GGPP are docked again, and an optimal model is determined. Based on the docking result, pyMOL was used to find the length and position (length less than 3.0) of the hydrogen bond between the GGPP and the vicinity of the active site of the protein (residues in the 6A range), and based on the ranking, the binding position R227 was found with a higher probability of lower binding energy.
(2) The partial overlap primer (R227-FOR/R227-REV) was designed by using the site-directed mutagenesis kit and was synthesized by Shanghai Biotechnology Co. The nucleotide at the R227 position was changed to NNK by designing a primer, and CaL was amplified by PCR using the pJN-CaLSsT plasmid obtained in example 1 as a template and using a site-directed mutagenesis kit 227NNK SsT, after amplification, 10. Mu.L of the PCR product was collected and detected by 1% agarose gel electrophoresis. The remaining PCR liquid was added with 1. Mu.L DMT enzyme to digest the methylated template in the PCR product, mixed well and incubated at 37℃for 1h. Then the plasmid was transformed into E.coli DMT, uniformly spread on LB solid medium containing 100. Mu.g/mL ampicillin, cultured overnight at 37℃and single colony was picked up to LB liquid medium containing 100. Mu.g/mL ampicillin, cultured overnight at 37℃and 200rpm, and the plasmid was extracted with plasmid extraction kit to obtain plasmid pJN-CaL having mutated to NNK at position 227 227NNK SsT。
2. Construction of free screening strains
pJN44, 44-CaL 227NNK SsT transferring into yarrowia lipolytica polf strain by yeast transfer kit, uniformly coating on SD-leu solid medium, culturing at 30deg.C for4 days, culturing single colony Zadoff in SD-leu liquid medium at 30deg.C for 48 hr at 200rpm, 2% passaging in new SD-leu liquid medium at 30deg.C for 48 hr at 200rpm, continuously passaging for4 times to fully saturate mutation, diluting and coating SD-leu solid plate, culturing at 30deg.C for4 days to obtain strain YLSC-16 (po 1f, pJN-CaL) 227NNK SsT)。
3. Free screening strain fermentation and CaLPPS mutation screening
Fermentation experiments of YLSC-16 strain: 40 single colonies were picked up and respectively inoculated into test tubes containing 2mL of SD-leu liquid medium, streaked on SD-leu solid plates, cultured at 30℃for2 days at 220rpm to obtain seed liquid, and the seed liquid was inoculated into 25mL of SD-leu fermentation medium at 2% by volume and cultured at 220rpm for4 days at 30 ℃. Detecting sclareol in the fermentation liquid.
As a result of the detection, the yield of sclareol of the YLSC-16 strain was up to 65.43mg/L, and the yield was improved by 156% as compared with the YLSC-09 strain of example 1. The colony is verified by PCR and sent to Shanghai biological limited company for sequencing, and the result shows that the amino acid at 227 position is saturated mutated from the original AGA in YLSC-16 strain to CAG, namely R227Q, and the sequence is shown as SEQ ID No. 1.
That is, a mutant CaLPPS obtained by saturation mutation of 227 th amino acid of CaLPPS derived from coffee (Coffea arabica) from the original AGA to CAG R227Q Can improve the yield of sclareol produced by yarrowia lipolytica, and has better effect than other mutants at the mutation point.
Example 3: construction of recombinant yarrowia lipolytica of the present invention
1. Construction of an Integrated expression vector
(1)ura3-SaGGPP-CaL R227Q Construction of SsT plasmid
Artificially synthesizing a gene fragment with a sequence shown as SEQ ID No.1, and inserting the synthesized gene fragment into HindIII and SmaI sites of a plasmid pJN to obtainpJN44-CaL R227Q . pJN44 to CaL R227Q As a template, LPPS-FOR4/LPPS-REV4 was used as a primer FOR PCR amplification CaL R227Q A gene fragment; using pJN-SsTPS as a template, and using TPS-FOR1/TPS-REV1 as a primer to amplify SsTPS gene fragments by PCR; will CaL R227Q The gene fragment and the SsTPS gene fragment are connected to HindIII and SmaI sites of pJN by an Assembly kit to obtain fusion expression plasmids pJN-CaL R227Q SsT. The gene sequence of geranylgeranyl pyrophosphate synthetase SaGGPP (accession number D28748.1) derived from sulfolobus acidocaldarius (Sulfolobus acidocaldarius) was obtained on NCBI, synthesized after codon optimization (sequence shown as SEQ ID No. 3), and the synthesized gene fragment was inserted into HindIII and SmaI sites of plasmid pJN to obtain pJN-SaGGPP. Fusion expression plasmid pJN-CaL was digested with XbaI-SpeI R227Q CaL of SsT R227Q SsT fragment, digested with SpeI site ligated to pJN-SaGGPP plasmid to give pJN-SaGGPP-CaL R227Q SsT plasmid.
The ploxpura3loxp carrier is singly cut by using SpeI, and the ploxpura3loxp carrier fragment is obtained after gel recovery, pJN-SaGGPP-CaL R227Q SsT plasmid SaGGPP-CaL was digested with XbaI-SpeI R227Q SsT, ploxpura3loxp vector fragment and SaGGPP-CaL R227Q The SsT fragment is subjected to enzyme digestion and connection to obtain ura3-SaGGPP-CaL R227Q SsT plasmid.
(2) Construction of ura3-tHMGR-idi plasmid
Obtaining the tHMGR gene sequence (accession number: YALI0E04807 g) and idi gene sequence (accession number: YALI0F04015 g) derived from yarrowia lipolytica from NCBI, extracting the genome of yarrowia lipolytica po1F, and PCR amplifying tHMGR fragments using po1F genome as a template and tHMGR-FOR and tHMGR-REV as primers; PCR amplifying the idi fragment by using the po1f genome as a template and the idi-FOR and idi-REV as primers; the tHMGR fragment and idi fragment were ligated to HindIII and SmaI sites of pJN, respectively, by means of an Assembly kit to give pJN-tHMGR and pJN44-idi.
PCR amplifying the TEF-tHMGR-XPR2 fragment by taking pJN-tHMGR as a template and taking TEF-1-FOR and XPR-1-REV as primers, and connecting the TEF-tHMGR-XPR2 fragment to the SpeI and MluI sites of ploxpura3loxp by using an Assembly kit to obtain ura3-tHMGR plasmid; the TEF-idi-XPR2 fragment was amplified by PCR using pJN-idi as template and TEF-2-FOR and XPR-2-REV as primers, and ligated to the MluI site of ura3-tHMGR by means of an Assemble kit to obtain ura3-tHMGR-idi plasmid.
2. Construction of recombinant Strain YLSC-17
ura3-SaGGPP-CaL R227Q The SsT plasmid was digested with MluI, the linearized product was recovered with gel and ura3-SaGGPP-CaL was obtained by means of a yeast transformation kit R227Q SsT linearized products were transferred to po1f, plated with SD-ura solid medium and incubated at 30℃for4 days until single colonies developed. Single colonies were picked up and inoculated into a test tube containing 2mL of YPD liquid medium, cultured at 30℃for1 day at 220rpm to obtain a seed solution, and the seed solution was inoculated into 25mL of YPD liquid medium at 2% of the inoculum size, and cultured at 30℃for4 days at 220 rpm. Detecting sclareol content of the culture solution.
As a result, sclareol yield was 1208mg/L, and the strain was designated as YLSC-17.
3. Overexpression of SaGGPP and CaL R227Q Construction of recombinant strain YLSC-18 of SsT
The ura3 marker in YLSC-17 strain is removed by using the Cre/loxp system, and the specific method is as follows: the pJN-Cre plasmid is transformed into YLSC-17 strain, cre protein is expressed, SD-leu solid culture medium is coated, screening is carried out on SD-leu and SD-ura plates respectively, the SD-ura plate is not long, the SD-leu long strain is the correct strain without ura3 mark, pJN-Cre plasmid is removed after 3 passages in YPD liquid culture medium, and YLSC-17D strain is obtained.
Re-combining ura3-SaGGPP-CaL R227Q SsT linearized products were transferred to YLSC-17D, plated with SD-ura solid medium, and incubated at 30℃for4 days until single colonies developed. Single colonies were picked up and inoculated into a test tube containing 2mL of YPD liquid medium, cultured at 30℃for1 day at 220rpm to obtain a seed solution, and the seed solution was inoculated into 25mL of YPD liquid medium at 2% inoculum size and cultured at 30℃for4 days at 220 rpm. Detecting sclareol content of the fermentation liquid.
As a result, sclareol was produced at 2073mg/L, and the strain was designated as YLSC-18.
4. Construction of recombinant Strain YLSC-19 with removal of ura3 markers
The ura3 mark in the YLSC-18 strain is removed by using a Cre/loxp system, and the YLSC-18D strain is obtained. The ura3-tHMGR-idi plasmid was digested with NsiI, the linearized product was recovered by gel recovery, the ura3-tHMGR-idi linearized product was transferred to YLSC-18D, SD-ura solid medium was spread, and incubated at 30℃for4 days until single colonies developed. Single colonies were picked up and inoculated into a test tube containing 2mL of YPD liquid medium, cultured at 30℃for1 day at 220rpm to obtain a seed solution, and the seed solution was inoculated into 25mL of YPD liquid medium at 2% of the inoculum size, and cultured at 30℃for4 days at 220 rpm. Detecting sclareol content of the fermentation liquid.
As a result, sclareol was produced at 2649mg/L, and the strain was designated as YLSC-19.
The genotype of the recombinant strain YLSC17-YLSC19 is:
example 4: fermentation experiment of recombinant Strain YLSC-19 in 5L fermentor
Single colony of YLSC-19 strain is selected and inoculated into 25mL/250mL YPD liquid culture medium, the culture is carried out at 30 ℃ for1 day at 220rpm, seed liquid is obtained, the seed liquid is inoculated into a 5L fermentation tank (containing 4L YPD fermentation culture medium) according to the inoculum size of 2 percent, the fermentation culture is carried out at 30 ℃, the stirring rotation speed is 800rpm, the aeration ratio is 1vvm, the dissolved oxygen is 10% -20%, the PH is adjusted to 5.5 by adding 5M sodium hydroxide, the glucose content is maintained to be 5g/L by feeding, and the fermentation is carried out for 168-192 h. Samples were taken every 24h to detect OD, sclareol content and glucose content.
Wherein, sclareol is extracted: when the yield of sclareol is between 50 and 100mg/L, n-hexane and fermentation broth are mixed according to the ratio (V/V) of 1:1; when the sclareol yield is more than 100mg/L, the fermentation broth is diluted 10-to 20-fold with water, and other steps are the same as the detection method.
As shown in FIG. 5, the YLSC-19 strain had a sclareol content of 8510mg/L at 192h of fermentation.
The invention constructs SaGGPP, caLPPS by Non-homologous end-linked mediated (Non-homologous End Joining-NHEJ) gene integration R227Q And SsTPS gene expression library, and successfully constructing a recombinant yarrowia lipolytica YLSC-17 with high sclareol yield from the gene library by detecting sclareol yield of each colony, wherein the sclareol yield of the strain is 1208mg/L. On this basis, the present invention is achieved by re-overexpressing SaGGPP, caLPPS R227Q And SsTPS gene, and overexpression of the tHMG and idi genes endogenous to yarrowia lipolytica, resulting in YLSC-19 strain. The recombinant strain realizes the yield of sclareol 2649mg/L in a shake flask, and the yield of sclareol in a 5L fermentation tank reaches 8510mg/L, which is obviously higher than the prior art. The recombinant yarrowia lipolytica YLSC-19 can be fermented and synthesized into natural sclareol with high efficiency.

Claims (6)

1. A recombinant yarrowia lipolytica with high sclareol yield, which is characterized in that the recombinant yarrowia lipolytica is constructed by over-expressing lysyl glycol pyrophosphate synthase mutant gene CaLPPS, sclareol synthase gene SsTPS and geranylgeranyl pyrophosphate synthase gene samgpp in yarrowia lipolytica po1f and expressing 3-hydroxy-3-methylglutaryl-coa reductase tggr and isopentenyl pyrophosphate isomerase gene idi;
the lysyl alkene glycol ester synthase gene is derived from small particle coffeeCoffea arabica) Mutant gene CaLPPS of (a) R227Q The nucleotide sequence is shown as SEQ ID NO. 1;
the sclareol synthase gene SsTPS is derived from sclareolSalvia sclarea) The nucleotide sequence is shown as SEQ ID NO. 2;
the geranylgeranyl pyrophosphate synthetase gene SaGGPP is derived from acidophilic sulfolobus spSulfolobus acidocaldarius) The nucleotide sequence is shown as SEQ ID NO. 3;
the 3-hydroxy-3-methylglutaryl-coa reductase tggr and isopentenyl pyrophosphate isomerase gene idi is derived from yarrowia lipolytica;
the lysbaidecne pyrophosphateAlcohol ester synthase mutant gene CaLPPS R227Q The sclareol synthase gene SsTPS and the sclareol synthase gene SsTPS are expressed in a fusion way through a connecting peptide GGGGS.
2. Recombinant yarrowia lipolytica with high sclareol yield according to claim 1, characterized in that the mutant gene CaLPPS R227Q Is derived from small-grain coffeeCoffea arabica) Amino acid 227 of CaLPPS is obtained by saturation mutation of the original AGA into CAG.
3. Mutant CaLPPS R227Q Use of a mutant CaLPPS for increasing yield of sclareol produced by yarrowia lipolytica, characterized in that the mutant CaLPPS R227Q Is derived from small-grain coffeeCoffea arabica) Amino acid 227 of CaLPPS is obtained by saturation mutation of the original AGA into CAG.
4. A method for constructing recombinant yarrowia lipolytica with high sclareol yield, which comprises the following steps:
（1）ura3-SaGGPP-CaL R227Q construction of SsT plasmid
Artificial synthesis of small particle coffee as shown in SEQ ID No.1Coffea arabica) Is characterized by comprising a mutant CaLPPS of lysyl pyrophosphate R227Q Gene sequence, the synthesized gene fragment was inserted into HindIII and SmaI sites of plasmid pJN to give pJN-CaL R227Q The method comprises the steps of carrying out a first treatment on the surface of the Is derived from sclareumSalvia sclarea) Artificially synthesizing a sclareol synthase SsTPS gene sequence, and inserting the synthesized gene fragment into HindIII and SmaI sites of a plasmid pJN to obtain pJN-SsTPS; pJN44 to CaL R227Q As a template, the PCR amplification gives CaL R227Q A gene fragment; using pJN-SsTPS as a template, and amplifying SsTPS gene fragments by PCR; will CaL R227Q The gene fragment and the SsTPS gene fragment are connected to HindIII and SmaI sites of pJN by an Assembly kit to obtain fusion expression plasmids pJN-CaL R227Q SsT；
Artificial synthesis of acidophilic sulfolobusSulfolobus acidocaldarius) A kind of electronic deviceThe gene sequence of geranylgeranyl pyrophosphate synthetase SaGGPP, and the artificially synthesized gene fragment is inserted into HindIII and SmaI sites of plasmid pJN to obtain pJN-SaGGPP; fusion expression plasmid pJN-CaL was digested with XbaI-SpeI R227Q CaL of SsT R227Q SsT fragment, digested with SpeI site ligated to pJN-SaGGPP plasmid to give pJN-SaGGPP-CaL R227Q SsT plasmid;
the ploxpura3loxp carrier is singly cut by using SpeI, and the ploxpura3loxp carrier fragment is obtained after gel recovery, pJN-SaGGPP-CaL R227Q SsT plasmid SaGGPP-CaL was digested with XbaI-SpeI R227Q SsT, ploxpura3loxp vector fragment and SaGGPP-CaL R227Q The SsT fragment was digested and ligated to give plasmid ura3-SaGGPP-CaL R227Q SsT；
(2) Construction of ura3-tHMGR-idi plasmid
Obtaining a tHMGR gene sequence and an idi gene sequence of yarrowia lipolytica source, extracting a genome of yarrowia lipolytica po1f, and carrying out PCR amplification on a tHMGR fragment by taking the po1f genome as a template; PCR amplifying the idi fragment by using the po1f genome as a template; the tHMGR fragment and the idi fragment are respectively connected to HindIII and SmaI sites of pJN44 by an Assembly kit to respectively obtain pJN-tHMGR and pJN44-idi;
PCR amplifying the TEF-tHMGR-XPR2 fragment by taking pJN-tHMGR as a template, and connecting the fragment to the SpeI and MluI sites of ploxpura3loxp by an Assembly kit to obtain ura3-tHMGR plasmid; PCR amplifying the TEF-idi-XPR2 fragment by taking pJN-idi as a template, and connecting the fragment to the MluI site of ura3-tHMGR through an Assembly kit to obtain ura3-tHMGR-idi plasmid;
(3) Construction of recombinant Strain YLSC-17
Subjecting ura3-SaGGPP-CaL of step (1) R227Q The SsT plasmid was digested with MluI, the linearized product was recovered with gel and ura3-SaGGPP-CaL was obtained by means of a yeast transformation kit R227Q Transferring the SsT linearization product into po1f, coating SD-ura solid culture medium, and culturing to obtain recombinant strain YLSC-17;
(4) Overexpression of SaGGPP and CaL R227Q Construction of recombinant strain YLSC-18 of SsT
Removing ura3 marks in the YLSC-17 strain by using a Cre/loxp system to obtain a YLSC-17D strain;
re-combining ura3-SaGGPP-CaL of step (1) R227Q Transferring the SsT linearization product into YLSC-17D, coating SD-ura solid culture medium, and culturing to obtain recombinant fungus YLSC-18;
removing ura3 marks in the YLSC-18 strain by using a Cre/loxp system to obtain a YLSC-18D strain;
cutting the ura3-tHMGR-idi plasmid in the step (2) by using NsiI, recovering the gel to obtain a linearization product, transferring the ura3-tHMGR-idi linearization product into YLSC-18D, coating an SD-ura solid culture medium, and culturing to obtain the recombinant yarrowia lipolytica with high sclareol yield.
5. Use of the recombinant yarrowia lipolytica of any one of claims 1-2 for sclareol production.
6. The method according to claim 5, wherein the recombinant yarrowia lipolytica is selected and inoculated into 25mL/250mL of YPD liquid medium, cultured at 30 ℃ for1 day at 220rpm to obtain seed liquid, the seed liquid is inoculated into a 5L fermentation tank containing 4L of YPD fermentation medium according to an inoculum size of 2% by volume, the fermentation tank is fermented and cultured at 30 ℃ at a stirring speed of 800rpm, a ventilation ratio of 1vvm and dissolved oxygen of 10% -20%, pH is adjusted to 5.5 by adding 5M sodium hydroxide, glucose content is maintained to 5g/L by feeding, and fermentation is carried out for 168-192 h to obtain sclareol.
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