Engineered yeast strain containing (-)-α-bisabolol synthase mutant and its application

Abstract

The present invention discloses a yeast engineering bacterium containing a (-)-α-bisabolol synthase mutant and its application. By designing (-)-α-bisabolol synthase, the present invention obtains a mutant with high expression level, co-expresses the mutant with ERG20, highly expresses the key rate-limiting enzymes in the metabolic pathway, deletes the promoter to reduce the side reaction pathway, and adopts a multi-copy strategy to improve the metabolic flux in the (-)-α-bisabolol biosynthesis pathway. The shake-flask fermentation yield of the yeast engineering bacterium constructed by the present invention is close to 70 mg / L, which is about 35 times higher than that of the starting strain, and has broad application prospects in the biological fermentation production of (-)-α-bisabolol.

Description

Technical Field

[0001] The present invention belongs to the field of microbial pharmaceuticals, and relates to a yeast engineering bacterium containing a (-)-α-bisabolol synthase mutant and its application. Background Art

[0002] (-)-α-Bisabolol is a monocyclic sesquiterpene alcohol isolated from chamomile, with the molecular formula C 15 H 26 O. (-)-α-Bisabolol has an anti-cancer effect on A549 NSCLC cells by inducing cell cycle arrest, mitochondrial death and inhibiting the PI3K / Akt signaling pathway, so it has anti-cancer activity. (-)-α-Bisabolol has low physiological toxicity and also has the properties of anti-inflammation, bactericidal, antibacterial, skin soothing and moisturizing, and has been used in pharmaceuticals and cosmetics. In addition, (-)-α-Bisabolol may be applied clinically due to its analgesic function. In summary, (-)-α-Bisabolol has good application value and market prospects.

[0003] Natural (-)-α-bisabolol is mainly extracted from chamomile and prepared by extraction methods such as organic reagent extraction and multiple vacuum distillations. Its extraction process is relatively complex and cumbersome. In addition, the yield of plant extraction is relatively low and the cycle is long. In recent years, with the development of synthetic biology technology, there have been studies on the heterologous biosynthesis of (-)-α-bisabolol using microbial cell factories. This method can improve efficiency, reduce costs and break through natural conditions, thus effectively alleviating the supply-demand contradiction of elemenene.

[0004] Currently, the biosynthesis of (-)-α-bisabolol mainly involves expressing (-)-α-bisabolol synthase to generate (-)-α-bisabolol. In addition, related synthetic genes have been reported in Escherichia coli and Saccharomyces cerevisiae, which has laid a good foundation for the heterologous synthesis of (-)-α-bisabolol. Pichia pastoris, as a recognized biosafety strain, has a fast growth rate, high environmental tolerance, simple fermentation conditions, and a strong ability to express proteins. Therefore, Pichia pastoris is expected to be an ideal host for synthesizing terpene products. Currently, there is no relevant report on the production of (-)-α-bisabolol using Pichia pastoris. Summary of the Invention

[0005] One object of the present invention is to provide a (-)-α-bisabolol synthase mutant Mut4 (F324Y), whose amino acid sequence is shown in SEQ ID NO.1.

[0006] Another object of the present invention is to provide the coding gene of the above (-)-α-bisabolol synthase mutant Mut4 (F324Y), whose nucleotide sequence is shown in SEQ ID NO.2.

[0007] A third object of the present invention is to provide a genetically engineered yeast strain containing a (-)-α-bisabolol synthase mutant, which is a genetically engineered yeast strain containing any one of the following sets of introduced genes:

[0008] (1) The coding gene of the (-)-α-bisabolol synthase mutant Mut4 (F324Y);

[0009] (2) The coding gene of the (-)-α-bisabolol synthase mutant Mut4 (F324Y) and the coding gene of the farnesyl pyrophosphate synthase ERG20;

[0010] (3) The coding gene of the (-)-α-bisabolol synthase mutant Mut4 (F324Y), the coding gene of the farnesyl pyrophosphate synthase ERG20, the coding gene of the isopentenyl pyrophosphate isomerase IDI1, the coding gene of the 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 tHMG1, the coding gene of the mevalonate kinase ERG12, and the coding gene of the mevalonate-5-diphosphate decarboxylase ERG19.

[0011] The yeast in the present invention is a yeast commonly used in genetically engineered bacteria, including but not limited to Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, etc. In a specific embodiment of the present invention, Pichia pastoris GS115 is taken as an example.

[0012] As a further preferred embodiment, the promoter of squalene synthase ERG9 is deleted in the genome of the genetically engineered yeast strain containing the (-)-α-bisabolol synthase mutant.

[0013] As a further preferred embodiment, among the introduced genes, the coding gene of the (-)-α-bisabolol synthase mutant Mut4 (F324Y) is in multiple copies, and in a specific embodiment of the present invention, 3 copies are taken as a representative example.

[0014] A fourth object of the present invention is to provide a method for constructing the above-mentioned genetically engineered yeast strain containing a (-)-α-bisabolol synthase mutant, including the following steps:

[0015] (1) Integrate the Cas9 nuclease coding gene into the genome of yeast to obtain a starting strain, and then integrate the coding gene of the (-)-α-bisabolol synthase mutant Mut4 (F324Y) into the genome of the starting strain to obtain a genetically engineered yeast strain 1;

[0016] Or (2) On the basis of the genetically engineered yeast strain 1, integrate the ERG20-linker fusion gene into the genome of the genetically engineered yeast strain 1 to obtain a genetically engineered yeast strain 2;

[0017] Or (3) based on the engineered yeast strain 2, integrate the coding genes of 3-hydroxy-3-methylglutaryl-CoA reductase, isopentenyl pyrophosphate isomerase IDI1, mevalonate kinase ERG12, and mevalonate-5-diphosphate decarboxylase ERG19 into the genome of the engineered yeast strain CCBOS-2 to obtain the engineered yeast strain 3;

[0018] Or (4) based on the engineered yeast strain 3, use gene editing technology to delete the promoter of squalene synthase ERG9 in the genome to obtain the engineered yeast strain 4;

[0019] Or (5) based on the engineered yeast strain 4, integrate the coding genes of more than one single copy of the (-)-α-bisabolol synthase mutant Mut4 (F324Y) into the engineered yeast strain 4 to obtain the engineered yeast strain 5.

[0020] The fifth object of the present invention is to provide the use of the above-mentioned engineered yeast strain containing the (-)-α-bisabolol synthase mutant in the production of (-)-α-bisabolol.

[0021] The present invention designed the (-)-α-bisabolol synthase, obtained 6 mutants, and screened out the mutant Mut4 (F324Y) with the (-)-α-bisabolol expression level increased by 46% compared with the original (-)-α-bisabolol synthase. On this basis, the present invention assembled the linker-promoted fusion expression of farnesyl pyrophosphate synthase ERG20 and bisabolol synthase mutant Mut4 (F324Y). Subsequently, isopentenyl pyrophosphate isomerase IDI1, 3-hydroxy-3-methylglutaryl-CoA reductase 1tHMG1, mevalonate kinase ERG12, and mevalonate-5-diphosphate decarboxylase ERG19 were highly expressed to further increase the metabolic flux. The present invention also down-regulated the expression of squalene synthase ERG9 by deleting the promoter to enhance the expression level of (-)-α-bisabolol. In addition, the present invention greatly increased the expression level of (-)-α-bisabolol by increasing the copy number of the (-)-α-bisabolol synthase mutant Mut4 (F324Y).

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention designed the (-)-α-bisabolol synthase, obtained mutants with high expression levels, co-expressed the mutants with ERG20, highly expressed the key rate-limiting enzymes in the metabolic pathway, deleted the promoter to reduce the side reaction pathway, and adopted the multi-copy strategy to increase the metabolic flux in the (-)-α-bisabolol biosynthesis pathway.

[0024] (2) The present invention uses yeast to efficiently produce (-)-α-bisabolol, which has the characteristics of short cycle, environmental protection and resource conservation. The shake-flask fermentation yield of the obtained recombinant strain is close to 70 mg / L, which is about 35 times higher than that of the starting strain. Moreover, the recombinant strain has stronger tolerance and has broad application prospects in the biological fermentation production of (-)-α-bisabolol. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the GC-MS detection mass spectrum of (-)-α-bisabolol;

[0026] Figure 2 It is the yield comparison chart of (-)-α-bisabolol obtained by shake-flask culture of the starting recombinant strain and different mutant recombinant strains in Example 1.

[0027] Figure 3 It is the yield comparison chart of (-)-α-bisabolol obtained by shake-flask culture of recombinant strains CCBOS-0, CCBOS-1, CCBOS-2, CCBOS-3, CCBOS-4, and CCBOS-5. DETAILED DESCRIPTION OF THE INVENTION

[0028] The technical solutions of the present invention will be described in detail below in conjunction with the drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the following embodiments in terms of specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications.

[0029] In the following embodiments, the auxiliary plasmids IntX pTEFtAOX1, IntX pGAPt0547, HZP-sgRNA, HHP-sgRNA, and HGP-sgRNA are all synthesized by the company. IntX pTEF tAOX1 and IntX pGAPt0547 mainly consist of an ampicillin resistance gene expression cassette, a replicon, pTEFtAOX1 or pGAP tAOX1, and homologous arms of 300-500 bp upstream and downstream of the insertion site. Among them, IntX refers to the position inserted into the genome of Pichia pastoris GS115. The auxiliary plasmids HZP-sgRNA, HHP-sgRNA, and HGP-sgRNA mainly consist of a universal backbone, a resistance gene expression cassette, the corresponding sgRNA of the site, and a BsaI restriction site. Z, H, and G represent bleomycin resistance gene, hygromycin resistance gene, and G418 resistance gene, respectively.

[0030] The initial Pichia pastoris strain used in the following examples is GS115-Cas9, which is obtained by transferring the plasmid pGAP-Cas9 into Pichia pastoris GS115 and integrating the Cas9 nuclease-encoding gene into the HIS4 locus of Pichia pastoris GS115. The Cas9 sequence was synthesized by the company.

[0031] The amino acid of (-)-α-bisabolol synthase CCBOS, the encoding gene thereof, the encoding gene of farnesyl pyrophosphate synthase ERG20, the encoding gene of geranyl diphosphate isomerase IDI1, the encoding gene of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 tHMG1, the encoding gene of mevalonate kinase ERG12, and the encoding gene of mevalonate-5-diphosphate decarboxylase ERG19 described in the present invention are all well-known in the art and have been fully disclosed in the existing literature.

[0032] The bisabolol synthase used in the present invention is from Cynara cardunculus var. Scolymus (CcBOS, GenBank XP_024994640.1). ((-)-α-Bisabolol Production in Engineered Escherichia coli Expressing a Novel (-)-α-Bisabolol Synthase from the Globe Artichoke Cynara cardunculus var. Scolymus. J Agric Food Chem. 2021 Aug 4;69(30):8492-8503.doi:10.1021 / acs.jafc.1c02759).

[0033] Example 1: Construction of (-)-α-bisabolol yeast starting recombinant strain and mutant recombinant strain

[0034] Using the plasmid IntX-pTEF-tAOX1 as the starting plasmid, a recombinant vector Int1-pTEF-tAOX1 was constructed by seamless cloning. Then, the (-)-α-bisabolol synthase CCBOS gene was introduced into the recombinant vector Int1-pTEF-tAOX1 using the restriction enzyme site BamHI in the vector to obtain the recombinant vector Int1-pTEF-CCBOS-tAOX1. At the same time, different primers were designed to obtain 6 different mutants using the recombinant vector Int1-pTEF-CCBOS-tAOX1 as a template. Using the helper plasmid HZP-sgRNA as the starting vector, the sgRNA-Int1 was introduced using the restriction enzyme site BsaI in the vector to obtain the recombinant vector HZP-sgRNA-Int1. Then, donor primers were designed using the recombinant vector Int1-pTEF-CCBOS-tAOX1 and the 6 mutants as templates, and PCR was performed to obtain the corresponding Int1-donor, Int1-donor-1, Int1-donor-2, Int1-donor-3, Int1-donor-4, Int1-donor-5, Int1-donor-6. These were separately transformed together with the corresponding recombinant guide plasmid HZP-sgRNA-Int1 into the Pichia pastoris cell GS115-Cas9 to obtain the starting recombinant strain CCBOS-0 and 6 mutant recombinant strains, namely CCBOS1-1, CCBOS1-2, CCBOS1-3, CCBOS1-4 (also known as CCBOS-1), CCBOS1-5, CCBOS1-6. The specific steps are as follows:

[0035] 1. Construction of the Int1-pTEF-tAOX1 recombinant vector

[0036] Using the plasmid IntX-pTEF-tAOX1 as the starting plasmid, the backbone primers intX-gj-F / intX-gj-R (nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4) were designed, and PCR was performed to obtain the backbone fragment. At the same time, the antibiotic expression cassette primers amp-F / amp-R (nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6) were designed. Then, the homologous arms of int1-L (primer int1-L-F / int1-L-R (nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8) and int1-R (int1-R-F / int1-R-R (nucleotide sequences are shown in SEQ ID NO.9 and SEQ ID NO.10)) were designed respectively. The four fragments were subjected to seamless cloning and transformed into the Escherichia coli DH5α strain to obtain the recombinant vector Int1-pTEF-tAOX1.

[0037] 2. Construction of the recombinant plasmid Int1-pTEF-CCBOS-tAOX1

[0038] (-)-α-Bisabolol synthase CCBOS gene was synthesized by GenScript Biotech Corporation. Using the CCBOS gene as a template, primers CCBOS-F / CCBOS-R (nucleotide sequences are shown in SEQ ID NO.11 and SEQ ID NO.12) were designed to amplify the target gene CCBOS. It was assembled with the digested vector Int1-pTEF-tAOX1 by seamless cloning method, and transformed into Escherichia coli DH5α strain to obtain the recombinant vector Int1-pTEF-CCBOS-tAOX1.

[0039] 3. Construction of different mutant plasmids

[0040] Using Int1-pTEF-CCBOS-tAOX1 as a template, six mutants were designed, namely Mut1 (L57E) (i.e., the mutant formed by mutating leucine (L) at position 57 in (-)-α-bisabolol synthase CCBOS to glutamate (E); the naming method of the following mutants is the same), Mut2 (V318S), Mut3 (T323I), Mut4 (F324Y) (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2), Mut5 (L403A), and Mut6 (T421S). The corresponding primers are CCBOS-57-F / CCBOS-57-R (the nucleotide sequences are shown in SEQ ID NO.13 and SEQ ID NO.14), CCBOS-318-F / CCBOS-318-R (the nucleotide sequences are shown in SEQ ID NO.15 and SEQ ID NO.16), CCBOS-323-F / CCBOS-323-R (the nucleotide sequences are shown in SEQ ID NO.17 and SEQ ID NO.18), CCBOS-324-F / CCBOS-324-R (the nucleotide sequences are shown in SEQ ID NO.19 and SEQ ID NO.20), CCBOS-403-F / CCBOS-403-R (the nucleotide sequences are shown in SEQ ID NO.21 and SEQ ID NO.22), and CCBOS-421-F / CCBOS-421-R (the nucleotide sequences are shown in SEQ ID NO.23 and SEQ ID NO.24). Different mutant plasmids were obtained, namely Int1-pTEF-CCBOS(L57E)-tAOX1, Int1-pTEF-CCBOS(V318S)-tAOX1, Int1-pTEF-CCBOS(T323I)-tAOX1, Int1-pTEF-CCBOS(F324Y)-tAOX1, Int1-pTEF-CCBOS(L403A)-tAOX1, and Int1-pTEF-CCBOS(T421S)-tAOX1.

[0041] 4. Construction of the 4.sgRNA recombinant expression vector HZP-sgRNA-Int1

[0042] Using the plasmid HZP-sgRNA as a template, primers Int1-sgRNA-F (SEQ ID NO.25) and Int1-sgRNA-R (SEQ ID NO.26) were designed. Int1-sgRNA was introduced using the restriction enzyme site BsaI in the vector HZP-sgRNA, and the recombinant vector HZP-sgRNA-Int1 was obtained by ligation with T4 ligase.

[0043] 5. Construction of (-)-α-bisabolol yeast recombinant strain

[0044] Using the recombinant vector Int1-pTEF-CCBOS-tAOX1 and six mutant plasmids as templates, primers Int1-donor-F (SEQ ID NO.27) and Int1-donor-R (SEQ ID NO.28) were designed for PCR to obtain the corresponding fragments Int1-donor, Int1-donor-1, Int1-donor-2, Int1-donor-3, Int1-donor-4, Int1-donor-5, Int1-donor-6. They were respectively transformed into Pichia pastoris cell GS115-Cas9 together with the corresponding recombinant guide plasmid HZP-sgRNA-Int1 to obtain different recombinant strains, namely CCBOS-0, CCBOS1-1, CCBOS1-2, CCBOS1-3, CCBOS1-4 (also known as CCBOS-1), CCBOS1-5, CCBOS1-6. At the same time, the antibiotic resistance plasmids were lost after several passages.

[0045] Example 2: Construction of (-)-α-bisabolol yeast recombinant strain CCBOS-2

[0046] Using the mutant plasmid Int1-pTEF-CCBOS(F324Y)-tAOX1 as the starting vector, the recombinant expression vector Int1-pTEF-ERG20-(PA)5-CCBOS(F324Y)-tAOX1 was constructed. Using Int1-pTEF-CCBOS(F324Y)-tAOX1 as the starting plasmid, backbone primers were designed for whole plasmid PCR to obtain the vector backbone, and then primers were designed to introduce ERG20. The recombinant vector Int1-pTEF-ERG20-(PA)5-CCBOS(F324Y)-tAOX1 was obtained by homologous recombination. The sgRNA used was HZP-sgRNA-Int1. Donor primers were designed using the recombinant vector Int1-pTEF-ERG20-(PA)5-CCBOS(F324Y)-tAOX1 as the template for PCR to obtain the corresponding Int1-ERG20-donor, which was transformed into Pichia pastoris cell GS115-Cas9 together with the corresponding recombinant guide plasmid HZP-sgRNA-Int1 to obtain the recombinant strain CCBOS-2. The specific steps are as follows:

[0047] 1. Construction of the recombinant vector Int1-pTEF-ERG20-(PA)5-CCBOS(F324Y)-tAOX1

[0048] Using the mutant plasmid Int1-pTEF-CCBOS(F324Y)-tAOX1 as a template, design the backbone primers int1-gujia-F / int1-gujia-R (nucleotide sequences are shown in SEQ ID NO.29 and SEQ ID NO.30) to perform whole plasmid PCR to obtain the vector backbone. Additionally, use the Pichia pastoris genome as a template to design primers ERG20-F / ERG20-R (nucleotide sequences are shown in SEQ ID NO.31 and SEQ ID NO.32) to amplify the target gene ERG20, and use the seamless cloning method to assemble it with the backbone vector Int1-pTEF-CCBOS(F324Y)-tAOX1, and transform the Escherichia coli DH5α strain to obtain the recombinant vector Int1-pTEF-ERG20-(PA)5-CCBOS(F324Y)-tAOX1.

[0049] 2. Construction of the sgRNA recombinant expression vector HZP-sgRNA-Int1

[0050] Same as the recombinant expression vector HZP-sgRNA-Int1 in Example 1.

[0051] 3. Construction of the (-)-α-bisabolol yeast recombinant strain CCBOS-2

[0052] Using the recombinant vector Int1-pTEF-ERG20-(PA)5-CCBOS(F324Y)-tAOX1 as a template, design primers Int1-ERG20-donor-F (SEQ ID NO.33) and Int1-ERG20-donor-R (SEQ ID NO.34), perform PCR to obtain the corresponding linearized fragment Int1-ERG20-donor, and co-transform it with the corresponding recombinant guide plasmid HZP-sgRNA-Int1 into the Pichia pastoris cell GS115-Cas9 to obtain the recombinant strain CCBOS-2. At the same time, passage several times to lose the antibiotic plasmid.

[0053] Example 3: Construction of the (-)-α-bisabolol yeast recombinant strain CCBOS-3-1

[0054] Using the plasmid IntX-pTEF-tAOX1 as the starting plasmid, the recombinant vector Int2-pTEF-tAOX1 was constructed by seamless cloning. Then, IDI1 was introduced into the vector at the restriction enzyme site BamHI to obtain the recombinant vector Int2-pTEF-IDI1-tAOX1. Using the plasmid IntX-pGAP-t0547 as the starting plasmid, the recombinant vector Int1-pGAP-t0547 was constructed by seamless cloning. Then, tHMG1 was introduced into the vector at the restriction enzyme site AatⅡ to obtain the recombinant vector Int1-pGAP-tHMG1-t0547. Using Int2-pTEF-IDI1-tAOX1 as a template, PCR was performed to obtain the backbone, and the fragment pGAP-tHMG1-t0547 was inserted by seamless cloning. Finally, the vector Int2-pTEF-IDI1-tAOX1-pGAP-tHMG1-t0547 was obtained. Using the auxiliary plasmid HZP-sgRNA as the starting vector, sgRNA-Int2 was introduced into the vector at the restriction enzyme site BsaI to obtain the recombinant vector HZP-sgRNA-Int2. Donor primers were designed using the recombinant vector int2-pTEF-IDI1-tAOX1-pGAP-tHMG1-t0547 as a template, and PCR was performed to obtain the corresponding Int2-donor. It was transformed together with the corresponding recombinant guide plasmid HZP-sgRNA-Int2 into the yeast CCBOS-2 in Example 2 to obtain the recombinant strain CCBOS-3-1. At the same time, it was passaged several times to discard the resistance plasmid. The specific steps are as follows:

[0055] 1. Construction of the recombinant vector Int2-pTEF-IDI1-tAOX1

[0056] (1) Construction of the recombinant vector Int2-pTEF-tAOX1: Using the plasmid IntX-pTEF-tAOX1 as the starting plasmid, the backbone primers intX-gj-F / intX-gj-R were designed to perform PCR to obtain the backbone fragment. At the same time, the antibiotic expression cassette primers amp-F / amp-R were designed. Then, the homologous arms of int2-L (primers int2-L-F / int2-L-R, nucleotide sequences as shown in SEQ ID NO.35, SEQ ID NO.36) and int2-R (primers int2-R-F / int2-R-R, nucleotide sequences as shown in SEQ ID NO.37, SEQ ID NO.38) were designed respectively. The four fragments were subjected to seamless cloning and transformed into the Escherichia coli DH5α strain to obtain the recombinant vector Int2-pTEF-tAOX1.

[0057] (2) Construction of recombinant vector Int2-pTEF-IDI1-tAOX1: Using the Saccharomyces cerevisiae genome as a template, primers IDI1-F / IDI1-R (nucleotide sequences are shown in SEQ ID NO.39 and SEQ ID NO.40) were designed to amplify the target gene IDI1. It was assembled with the vector Int2-pTEF-tAOX1 digested by BamHI using seamless cloning method, and then transformed into Escherichia coli DH5α strain to obtain the recombinant vector Int2-pTEF-IDI1-tAOX1.

[0058] 2. Construction of recombinant vector Int1-pGAP-tHMG1-t0547

[0059] (1) Construction of recombinant vector Int1-pGAP-t0547: Using plasmid IntX-pGAP-t0547 as the starting plasmid, backbone primers int1-gj-F / int1-gj-R (nucleotide sequences are shown in SEQ ID NO.41 and SEQ ID NO.42) were designed for PCR to obtain the backbone fragment. At the same time, primers amp-F / amp-R for antibiotic expression cassette were designed, and then homologous arms Int1-L (primers Int1-L-F / Int1-L-R, nucleotide sequences are shown in SEQ ID NO.43 and SEQ ID NO.44) and Int1-R (Int1-R-F / Int1-R-R, nucleotide sequences are shown in SEQ ID NO.45 and SEQ ID NO.46) were designed respectively. The four fragments were assembled by seamless cloning and transformed into Escherichia coli DH5α strain to obtain the recombinant vector Int1-pGAP-t0547.

[0060] (2) Construction of recombinant vector Int1-pGAP-tHMG1-t0547t: Using the Saccharomyces cerevisiae genome as a template, primers tHMG1-F / tHMG1-R (nucleotide sequences are shown in SEQ ID NO.47 and SEQ ID NO.48) were designed to amplify the target gene tHMG1. It was assembled with the vector Int1-pGAP-t0547t digested by AatⅡ using seamless cloning method, and then transformed into Escherichia coli DH5α strain to obtain the recombinant vector Int1-pGAP-tHMG1-t0547t.

[0061] 3. Construction of recombinant vector Int2-pTEF-IDI1-tAOX1-pGAP-tHMG1-t0547

[0062] Using Int2-pTEF-IDI1-tAOX1 as a template, design the backbone primers int2-IDI1-gj-F / int2-IDI1-gj-R (nucleotide sequences are shown in SEQ ID NO.49 and SEQ ID NO.50), perform PCR to obtain the backbone, use seamless cloning to insert the fragment pGAP-tHMG1-t0547 (primers GAP-tHMG1-F / GAP-tHMG1-R, nucleotide sequences are shown in SEQ ID NO.51 and SEQ ID NO.52), and transform Escherichia coli DH5α strain to obtain the recombinant vector Int2-pTEF-IDI1-tAOX1-pGAP-tHMG1-t0547.

[0063] 4. Construction of the recombinant expression vector HZP-sgRNA-Int2 of sgRNA

[0064] Using the plasmid HZP-sgRNA as a template, design the primers Int2-sgRNA-F (SEQ ID NO.53) and Int2-sgRNA-R (SEQ ID NO.54), introduce Int2-sgRNA using the restriction enzyme site BsaI in the vector HZP-sgRNA, and obtain the recombinant vector HZP-sgRNA-Int2 by ligation with T4 enzyme.

[0065] 5. Construction of the recombinant Pichia pastoris strain CCBOS-3-1 of (-)-α-bisabolol

[0066] Using the recombinant vector Int2-pTEF-IDI1-tAOX1-pGAP-tHMG1-t0547 as a template, design the primers Int2-donor-F (SEQ ID NO.55) and Int2-donor-R (SEQ ID NO.56), perform PCR to obtain the corresponding linearized fragment Int2-donor, and co-transform it with the corresponding recombinant guide plasmid HZP-sgRNA-Int2 into the Pichia pastoris cell CCBOS-2 to obtain the recombinant strain CCBOS-3-1. At the same time, passage several times to discard the antibiotic plasmid.

[0067] Example 4: Construction of the recombinant yeast strain CCBOS-3 of (-)-α-bisabolol

[0068] Using the plasmid IntX-pTEF-tAOX1 as the starting plasmid, the recombinant vector Int3-pTEF-tAOX1 was constructed by seamless cloning. Then, ERG12 was introduced at the restriction enzyme site BamHI in the vector to obtain the recombinant vector Int3-pTEF-ERG12-tAOX1. Using the plasmid IntX-pGAP-t0547 as the starting plasmid, the recombinant vector Int1-pGAP-t0547 was constructed by seamless cloning. Then, ERG19 was introduced at the restriction enzyme site AatⅡ in the vector to obtain the recombinant vector Int1-pGAP-ERG19-t0547. The backbone was obtained by PCR using Int3-pTEF-ERG12-tAOX1 as the template, and the fragment pGAP-ERG19-t0547 was inserted by seamless cloning to finally obtain the vector Int3-pTEF-ERG12-tAOX1-pGAP-ERG19-t0547. Using the auxiliary plasmid HGP-sgRNA as the starting vector, sgRNA-Int3 was introduced at the restriction enzyme site BsaI in the vector to obtain the recombinant vector HGP-sgRNA-Int3. Donor primers were designed using the recombinant vector Int3-pTEF-ERG12-tAOX1-pGAP-ERG19-t0547 as the template, and PCR was performed to obtain the corresponding Int3-donor. It was transformed together with the corresponding recombinant guide plasmid HZP-sgRNA-Int3 into the yeast CCBOS-3-1 in Example 3 to obtain the recombinant strain CCBOS-3. At the same time, the antibiotic plasmid was lost after several passages.

[0069] The specific steps are as follows:

[0070] 1. Construction of the recombinant vector Int3-pTEF-ERG12-tAOX1

[0071] (1) Construction of the Int3-pTEF-tAOX1 recombinant vector: Using the plasmid IntX-pTEF-tAOX1 as the starting plasmid, the backbone primers int3-gj-F / int3-gj-R (the nucleotide sequences are shown in SEQ ID NO.57 and SEQ ID NO.58) were designed for PCR to obtain the backbone fragment. At the same time, the antibiotic expression cassette primers amp-F / amp-R were designed, and then the homologous arms int3-L (primers int3-L-F / int3-L-R, the nucleotide sequences are shown in SEQ ID NO.59 and SEQ ID NO.60) and int3-R (int3-R-F / int3-R-R, the nucleotide sequences are shown in SEQ ID NO.61 and SEQ ID NO.62) were designed respectively. The four fragments were subjected to seamless cloning and transformed into the Escherichia coli DH5α strain to obtain the recombinant vector Int3-pTEF-tAOX1.

[0072] (2) Construction of recombinant vector Int3-pTEF-ERG12-tAOX1: Using the Pichia pastoris genome as a template, primers ERG12-F / ERG12-R (nucleotide sequences are shown in SEQ ID NO.63 and SEQ ID NO.64) were designed to amplify the target gene ERG12, and it was assembled with the vector Int3-pTEF-tAOX1 digested with BamHI using seamless cloning method, then transformed into Escherichia coli DH5α strain to obtain the recombinant vector Int3-pTEF-ERG12-tAOX1.

[0073] 2. Construction of recombinant vector Int1-pGAP-ERG19-t0547

[0074] Using plasmid Int1-pGAP-t0547 as the starting plasmid and the Pichia pastoris genome as a template, primers ERG19-F / ERG19-R (nucleotide sequences are shown in SEQ ID NO.65 and SEQ ID NO.66) were designed to amplify the target gene ERG19, and it was assembled with the vector Int1-pGAP-t0547 digested with AatⅡ using seamless cloning method, then transformed into Escherichia coli DH5α strain to obtain the recombinant vector Int1-pGAP-ERG19-t0547.

[0075] 3. Construction of recombinant vector Int3-pTEF-ERG12-tAOX1-pGAP-ERG19-t0547

[0076] Using Int3-pTEF-ERG12-tAOX1 as a template, primers int3-ERG12-gj-F / int3-ERG12-gj-R (nucleotides are shown in SEQ ID NO.67 and SEQ ID NO.68) were designed to perform PCR to obtain the backbone, and the insert fragment pGAP-ERG19-t0547 (primers GAP-ERG19-F / GAP-ERG19-R, nucleotides are shown in SEQ ID NO.69 and SEQ ID NO.70) was inserted using seamless cloning method, then transformed into Escherichia coli DH5α strain to obtain the recombinant vector Int3-pTEF-ERG12-tAOX1-pGAP-ERG19-t0547.

[0077] 4. Construction of sgRNA recombinant expression vector HGP-sgRNA-Int3

[0078] Using plasmid HGP-sgRNA as a template, primers Int3-sgRNA-F (SEQ ID NO.71) and Int3-sgRNA-R (SEQ ID NO.72) were designed. Int3-sgRNA was introduced using the restriction enzyme site BsaI in vector HGP-sgRNA, and the recombinant vector HZP-sgRNA-Int3 was obtained by ligation with T4 ligase.

[0079] 5. Construction of Pichia pastoris recombinant strain CCBOS-3 of (-)-α-bisabolol

[0080] Using the recombinant vector Int3-pTEF-ERG12-tAOX1-pGAP-ERG19-t0547 as a template, primers Int3-donor-F (SEQ ID NO.73) and Int3-donor-R (SEQ ID NO.74) were designed. PCR was performed to obtain the corresponding linearized fragment Int3-donor, which was co-transformed with the corresponding recombinant guide plasmid HGP-sgRNA-Int3 into Pichia pastoris cells CCBOS-3-1 to obtain recombinant strain CCBOS-3. At the same time, the cells were passaged several times to lose the antibiotic plasmid.

[0081] Example 5: Construction of Pichia pastoris recombinant strain CCBOS-4 of (-)-α-bisabolol

[0082] Using the helper plasmid HHP-sgRNA as the starting vector, sgRNA-IntERG9 was introduced using the restriction enzyme site BsaI in the vector to obtain the recombinant vector HHP-sgRNA-IntERG9. Donor primers were designed using the Pichia pastoris genome as a template, and PCR was performed to obtain the corresponding IntERG9-donor, which was co-transformed with the corresponding recombinant guide plasmid HHP-sgRNA-IntERG9 into CCBOS-3 to obtain recombinant strain CCBOS-4. At the same time, the cells were passaged several times to lose the antibiotic plasmid. The specific steps are as follows:

[0083] 1. Construction of sgRNA recombinant expression vector HHP-sgRNA-IntERG9

[0084] Using plasmid HHP-sgRNA as a template, primers IntERG9-sgRNA-F (SEQ ID NO.75) and IntERG9-sgRNA-R (SEQ ID NO.76) were designed. IntERG9-sgRNA was introduced using the restriction enzyme site BsaI in vector HHP-sgRNA, and the recombinant vector HHP-sgRNA-IntERG9 was obtained by ligation with T4 ligase.

[0085] 2. Construction of Pichia pastoris recombinant strain CCBOS-4 of (-)-α-bisabolol

[0086] Using the Pichia pastoris genome as a template, primers IntERG9-donor-F (SEQ ID NO.77) and IntERG9-donor-R (SEQ ID NO.78) were designed, and PCR was performed to obtain the corresponding linearized fragment IntERG9-donor. It was co-transformed with the corresponding recombinant guide plasmid HHP-sgRNA-IntERG9 into Pichia pastoris cells CCBOS-3 to obtain the recombinant strain CCBOS-4. At the same time, it was passaged several times to lose the antibiotic plasmid.

[0087] Example 6: Construction of the multi-copy (-)-α-bisabolol yeast recombinant strain CCBOS-5

[0088] The expression cassette of ERG20-(PA)5-CCBOS(F324Y) and the corresponding guide plasmid were constructed and successively transferred into the above-mentioned recombinant strain CCBOS-4. After transformation, the recombinant strain CCBOS-5 was finally obtained. The specific steps are as follows:

[0089] 1. Construction of the sgRNA recombinant expression vectors HZP-sgRNA-Int4 and HGP-sgRNA-Int5

[0090] Using the helper plasmid HZP-sgRNA as a template, primers Int4-sgRNA-F (SEQ ID NO.79) and Int4-sgRNA-R (SEQ ID NO.80) were designed. Int4-sgRNA was introduced using the restriction enzyme site BsaI in the vector HZP-sgRNA, and the recombinant vector HZP-sgRNA-Int4 was obtained by ligation with T4 ligase. Using the helper plasmid HGP-sgRNA as a template, primers Int5-sgRNA-F (SEQ ID NO.81) and Int5-sgRNA-R (SEQ ID NO.82) were designed. Int5-sgRNA was introduced using the restriction enzyme site BsaI in the vector HGP-sgRNA, and the recombinant vector HGP-sgRNA-Int5 was obtained by ligation with T4 ligase.

[0091] 2. Construction of the (-)-α-bisabolol yeast recombinant strain CCBOS-5

[0092] Primers Int4-donor-F (SEQ ID NO.83) and Int4-donor-R (SEQ ID NO.84) were designed using the recombinant vector Int1-pTEF-ERG20-(PA)5-CCBOS(F324Y)-tAOX1 as a template. PCR was performed to obtain the corresponding linearized fragment Int4-donor, which was co-transformed with the corresponding recombinant guide plasmid HZP-sgRNA-Int4 into the Pichia pastoris cell CCBOS-3 to obtain the recombinant strain CCBOS-5-1.

[0093] Primers Int5-donor-F (SEQ ID NO.85) and Int5-donor-R (SEQ ID NO.86) were designed using the recombinant vector Int1-pTEF-ERG20-(PA)5-CCBOS(F324Y)-tAOX1 as a template. PCR was performed to obtain the corresponding linearized fragment Int5-donor, which was co-transformed with the corresponding recombinant guide plasmid HGP-sgRNA-Int5 into the Pichia pastoris cell CCBOS-5-1 to obtain the recombinant strain CCBOS-5. At the same time, it was passaged several times to discard the antibiotic-resistant plasmids of two rounds of consecutive transformations.

[0094] Example 7: High-efficiency production of (-)-α-bisabolol by Pichia pastoris engineering strains

[0095] The recombinant strains constructed in Examples 1-6 were activated by streaking on solid YPD plates. Single colonies were picked and cultured in 5 mL test tubes, and then transferred to 50 mL shake flasks at an inoculation amount of 1% and cultured for 4 days. Then the samples were processed for product analysis. The specific steps are as follows:

[0096] 1. Cultivation of recombinant strains

[0097] Single colonies of GS115-Cas9, CCBOS-0, CCBOS-1, CCBOS-2, CCBOS-3, CCBOS-4, and CCBOS-5 were picked from the plates and inoculated into 5 mL YPD (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) test tubes and cultured at 30 °C and 220 rpm for 12 h. Then they were respectively inoculated into 50 mL YPD shake flask media at an inoculation amount of 1%, with three parallels for each strain. 10% n-dodecane was added on the upper layer of the medium, and cultured at 30 °C and 220 rpm for 4 days. 2% glucose was supplemented every 48 h.

[0098] 2. Detection method for (-)-α-bisabolol

[0099] (1) Sample preparation: Take 1 mL of the upper fermentation broth, centrifuge at 12,000 rpm for 10 min, take the upper organic phase and filter it through a 0.22 μm nylon filter membrane. The sample is diluted 1000-fold with ethyl acetate and then subjected to GC-MS detection.

[0100] (2) The GC-MS detection conditions are as follows:

[0101] The inlet temperature is 250 °C, the injection volume is 1 μL, and the injection is non-split; Chromatographic column: HP-5ms (30 m * 0.25 mM); Chromatographic conditions: The initial temperature is 60 °C, which is increased to 160 °C at a rate of 10 °C / min, then increased to 200 °C at a rate of 5 °C / min, and finally increased to 230 °C at a rate of 10 °C / min. Qualitative and quantitative analysis is performed using the standard of (-)-α-bisabolol.

[0102] 3. Comparison of (-)-α-bisabolol production of recombinant strains

[0103] The (-)-α-bisabolol production of each Pichia pastoris engineering strain is determined by the GC-MS detection method, and the mass spectrometry detection graph is as Figure 1 shown.

[0104] The yields of (-)-α-bisabolol obtained by flask culture of the parental recombinant strain and different mutant recombinant strains in Example 1 are as Figure 2 shown. The results show that the yields of mutants CCBOS-1-1 and CCBOS-1-4 are further increased compared with the parental recombinant strain CCBOS-0. Among them, the yield of recombinant strain CCBOS-1-4 is increased by 73% compared with the parental recombinant strain CCBOS-0.

[0105] The yields of (-)-α-bisabolol obtained by flask culture of recombinant strains CCBOS-0, CCBOS-1, CCBOS-2, CCBOS-3, CCBOS-4, and CCBOS-5 are as Figure 3 shown. It can be seen from the figure that the yield of recombinant strain CCBOS-5 reaches the highest, reaching 70 mg / L, which is 35 times higher than that of the parental recombinant strain CCBOS-0.

Claims

1. (-)-α-Bisabolol synthase mutant Mut4, characterized in that, The amino acid sequence is as shown in SEQ ID NO.

1.

2. The coding gene of (-)-α-bisabolol synthase mutant Mut4, characterized in that, The nucleotide sequence is as shown in SEQ ID NO.

2.

3. Engineered yeast strains containing (-)-α-bisabolol synthase mutants, characterized in that, It is a genetically engineered yeast strain containing any one of the following imported genes: (1) The coding gene of the (-)-α-bisabolol synthase mutant Mut4 described in claim 1; (2) The coding gene of the (-)-α-bisabolol synthase mutant Mut4 described in claim 1 and the coding gene of farnesyl pyrophosphate synthase ERG20; (3) The coding gene of the (-)-α-bisabolol synthase mutant Mut4 described in claim 1, the coding gene of farnesyl pyrophosphate synthase ERG20, the coding gene of isopentenyl pyrophosphate isomerase IDI1, the coding gene of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1tHMG1, the coding gene of mevalonate kinase ERG12, and the coding gene of mevalonate-5-diphosphate decarboxylase ERG19.

4. The engineered yeast strain according to claim 3, wherein The yeast is Pichia pastoris, Saccharomyces cerevisiae or Yarrowia lipolytica.

5. The engineered yeast strain according to claim 3, wherein The yeast is Pichia pastoris GS115.

6. The engineered yeast strain according to claim 3, wherein The promoter of squalene synthase ERG9 is deleted in the genome of the genetically engineered yeast strain.

7. The engineered yeast strain according to claim 3, wherein Among the imported genes, the coding gene of the (-)-α-bisabolol synthase mutant Mut4 described in claim 1 is in 3 copies.

8. The method for constructing a genetically engineered yeast strain containing the (-)-α-bisabolol synthase mutant according to claim 3, comprising the following steps: Integrate the Cas9 nuclease coding gene into the genome of the yeast to obtain a starting strain, and then integrate the coding gene of the (-)-α-bisabolol synthase mutant Mut4 into the genome of the starting strain to obtain genetically engineered yeast strain 1; Or integrate the Cas9 nuclease coding gene into the genome of the yeast to obtain a starting strain, and then integrate the coding gene of the (-)-α-bisabolol synthase mutant Mut4 into the genome of the starting strain to obtain genetically engineered yeast strain 1. On the basis of genetically engineered yeast strain 1, integrate the ERG20-linker fusion gene into the genome of genetically engineered yeast strain 1 to obtain genetically engineered yeast strain 2; Or integrate the Cas9 nuclease coding gene into the genome of the yeast to obtain a starting strain, and then integrate the coding gene of the (-)-α-bisabolol synthase mutant Mut4 into the genome of the starting strain to obtain genetically engineered yeast strain 1. On the basis of genetically engineered yeast strain 1, integrate the ERG20-linker fusion gene into the genome of genetically engineered yeast strain 1 to obtain genetically engineered yeast strain 2. On the basis of genetically engineered yeast strain 2, integrate the coding genes of isopentenyl pyrophosphate isomerase IDI1, 3-hydroxy-3-methylglutaryl coenzyme A reductase 1tHMG1, mevalonate kinase ERG12, and mevalonate-5-diphosphate decarboxylase ERG19 into the genome of genetically engineered yeast strain 2 to obtain genetically engineered yeast strain 3; Alternatively, the Cas9 nuclease-encoding gene is integrated into the genome of yeast to obtain a starting strain. Then, the (-)-α-bisabolol synthase mutant Mut4-encoding gene is integrated into the genome of the starting strain to obtain yeast engineering strain 1. On the basis of yeast engineering strain 1, the ERG20-linker fusion gene is integrated into the genome of yeast engineering strain 1 to obtain yeast engineering strain 2. On the basis of yeast engineering strain 2, the isopentenyl pyrophosphate isomerase IDI1-encoding gene, 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 tHMG1-encoding gene, mevalonate kinase ERG12-encoding gene, and mevalonate-5-diphosphate decarboxylase ERG19-encoding gene are integrated into the genome of yeast engineering strain 2 to obtain yeast engineering strain 3. On the basis of yeast engineering strain 3, the promoter of squalene synthase ERG9 in the genome is deleted by gene editing technology to obtain yeast engineering strain 4; Alternatively, the Cas9 nuclease-encoding gene is integrated into the genome of yeast to obtain a starting strain. Then, the (-)-α-bisabolol synthase mutant Mut4-encoding gene is integrated into the genome of the starting strain to obtain yeast engineering strain 1. On the basis of yeast engineering strain 1, the ERG20-linker fusion gene is integrated into the genome of yeast engineering strain 1 to obtain yeast engineering strain 2. On the basis of yeast engineering strain 2, the isopentenyl pyrophosphate isomerase IDI1-encoding gene, 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 tHMG1-encoding gene, mevalonate kinase ERG12-encoding gene, and mevalonate-5-diphosphate decarboxylase ERG19-encoding gene are integrated into the genome of yeast engineering strain 2 to obtain yeast engineering strain 3. On the basis of yeast engineering strain 3, the promoter of squalene synthase ERG9 in the genome is deleted by gene editing technology to obtain yeast engineering strain 4. On the basis of yeast engineering strain 4, the (-)-α-bisabolol synthase mutant Mut4-encoding gene with 2 copies is integrated into yeast engineering strain 4 to obtain yeast engineering strain 5.

9. Use of the yeast engineering strain containing the (-)-α-bisabolol synthase mutant according to claim 3 in the production of (-)-α-bisabolol.

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