Candida tropicalis engineering bacteria for high yield of (-)-alpha-bisabolol and construction method of candida tropicalis engineering bacteria

Abstract

The invention discloses candida tropicalis engineering bacteria for high yield of (-)-alpha-bisabolol and a construction method of the candida tropicalis engineering bacteria, and belongs to the technical field of microorganisms. According to the invention, chassis cell candida tropicalis DC04-WG04-2 is constructed, a gene Cat2 of catalase derived from saccharomyces cerevisiae and a gene ERG10 of 3-hydroxy-3-methylglutaryl coenzyme A reductase are expressed on the basis of the chassis cell candida tropicalis DC04-WG04-2, and a PEX15 motif is anchored on the surface of peroxisome for co-expression; the genes ADH1, GPD1 and GPD2 are further knocked out, so that the accumulation of ethanol is reduced, and the generation of glycerol is reduced. According to the method disclosed by the invention, the yield of the (-)-alpha-bisabolol of the recombinant candida tropicalis reaches 461.7 mg / L by optimizing fermentation conditions. The preparation method has the characteristics of low cost, high efficiency, simplicity in operation, wide prospect and the like.

Description

Technical Field

[0001] The invention relates to an engineering strain of Candida tropicalis capable of producing (-)-alpha-bisabolol in high yield and a construction method thereof, and belongs to the technical field of microorganisms. Background Art

[0002] Currently, most (-)-α-bisabolol is extracted from the Kandelia candel tree by steam distillation, which is unsustainable due to the shrinking natural habitat of the Kandelia candel tree. Although (-)-α-bisabolol can also be produced by semi-synthetic methods starting from natural products such as pinene, the resulting product usually contains two or more isomers that are difficult to separate. Therefore, in recent decades, many research groups have devoted themselves to developing suitable microorganisms into cell factories to produce bisabolol using microbial fermentation. At present, the yeast gene editing system is becoming more and more mature, and it has many excellent characteristics, such as short growth cycle and simple modification methods. Different yeasts have different advantages. For example, Candida tropicalis is a diploid unconventional yeast with the characteristics of fast cell growth, strong environmental tolerance, and a wide range of substrates. It has great potential for industrial application. Among them, the production of long-chain dibasic acids and xylitol using Candida tropicalis has been industrialized. Therefore, the characteristics of Candida tropicalis give Candida tropicalis a new generation of non-model strains for producing bisabolol.

[0003] The unsaturated monocyclic sesquiterpenoid (-)-α-bisabolol has anti-inflammatory and emollient properties, as well as a unique fragrance. It can not only be used in the fragrance industry, but also has high application value in medicine. Some sesquiterpenes have been shown to have anti-cancer, anti-malarial and hypoglycemic effects. Therefore, the synthesis of (-)-α-bisabolol using tropical Candida has broad prospects.

[0004] Since Candida tropicalis is an engineered strain, its metabolic pathway is still unclear and difficult to modify. Simply introducing the (-)-α-bisabolol synthase gene into the wild-type Candida tropicalis does not significantly increase the yield of (-)-α-bisabolol. Therefore, it is urgent to develop new strategies to improve the (-)-α-bisabolol production capacity of Candida tropicalis. Summary of the invention

[0005] The present invention provides a recombinant Candida tropicalis producing (-)-α-bisabolol. Compared with the starting strain, the recombinant Candida tropicalis expresses the catalase Cat2 derived from Saccharomyces cerevisiae, and anchors the PEX15 motif on the surface of the peroxisome.

[0006] In one embodiment, the catalase Cat2 is also co-expressed with 3-hydroxy-3-methylglutaryl-CoA reductase ERG10.

[0007] In one embodiment, the nucleotide sequence of the catalase Cat2 derived from Saccharomyces cerevisiae is shown as SEQ ID NO.7; the nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase ERG10 is shown as SEQ ID NO.8.

[0008] In one embodiment, the recombinant Candida tropicalis further has one or more of the following genes knocked out: the alcohol dehydrogenase gene ADH1, the glycerol-3-phosphate dehydrogenase gene GPD1, and the glycerol-3-phosphate dehydrogenase gene GPD2.

[0009] In one embodiment, the (-)-α-bisabolol synthase gene Cat2 is composed of P FBA1 The promoter initiates transcription; the P FBA1 The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

[0010] In one embodiment, the 3-hydroxy-3-methylglutaryl-CoA reductase gene ERG10 is driven by the promoter P GAPDH Start transcription; the promoter P GAPDH The nucleotide sequence is shown in SEQ ID NO.2.

[0011] In one embodiment, the nucleotide sequence of the PEX15 motif is shown as SEQ ID NO.3.

[0012] In one embodiment, a DNA fragment containing the catalase gene Cat2 and the 3-hydroxy-3-methylglutaryl-CoA reductase gene ERG10 is integrated into the DPP1 gene site of the starting strain.

[0013] In one embodiment, the nucleotide sequence of the diacylglycerol diphosphate phosphatase gene DPP1 is shown as SEQ ID NO.4.

[0014] In one embodiment, the amino acid sequence of alcohol dehydrogenase ADH1 is shown in Genbank accession number: AAA62378.1, and the encoding gene is shown in SEQ ID NO.10.

[0015] In one embodiment, the nucleotide sequence of the glycerol-3-phosphate dehydrogenase gene GPD1 is shown as SEQ ID NO.5, and the nucleotide sequence of the glycerol-3-phosphate dehydrogenase gene GPD2 is shown as SEQ ID NO.6.

[0016] In one embodiment, the starting strain is (a) or (b):

[0017] (a) Candida tropicalis DC-04, which has been disclosed in patent publication number CN114410631B;

[0018] (b) Based on (a), (-)-α-bisabolol synthase and farnesyl pyrophosphate synthase were co-expressed, and the (GS)9-linked protein scaffold genes WH1 and GBD, as well as the (GGGGS)3-linked genes ERG19 and IDI were integrated.

[0019] In one embodiment, the Genbank accession numbers of WH1 and GBD are EDL13116 and BAA21534, respectively; the Genbank accession numbers of genes ERG19 and IDI are AAC49252.1 and AF119715, respectively.

[0020] The present invention also provides a method for improving the production capacity of (-)-α-bisabolol of recombinant Candida tropicalis, wherein the method comprises performing any of the following improvements on the starting strain:

[0021] (1) Express the catalase gene Cat2 from Saccharomyces cerevisiae and anchor the PEX15 motif on the peroxisome surface to utilize the peroxisome's acetyl-CoA;

[0022] (2) Co-expression of the catalase gene Cat2 (containing PEX15) and the 3-hydroxy-3-methylglutaryl-CoA reductase gene ERG10 (containing PEX15) from Saccharomyces cerevisiae;

[0023] (3) The catalase gene Cat2 (containing PEX15) and the 3-hydroxy-3-methylglutaryl-CoA reductase gene ERG10 (containing PEX15) from Saccharomyces cerevisiae were integrated into the genome, and the single copy of the alcohol dehydrogenase gene ADH1 was knocked out;

[0024] (4) Knockout the genes GPD1 and GPD2 in the genome;

[0025] (5) Fermentation at 26°C to 30°C in a culture medium containing glucose, yeast powder and peptone.

[0026] In one embodiment, the method is to ferment in YPD medium at 28°C in a culture system with a liquid volume of 25 mL.

[0027] In one embodiment, the method is fermentation in 2×YPD medium at 28° C.

[0028] In one embodiment, the method is to ferment in YPD60 medium at 26°C to 30°C.

[0029] The present invention also provides a method for producing (-)-α-bisabolol, which comprises using the recombinant Candida tropicalis to ferment and produce (-)-α-bisabolol.

[0030] In one embodiment, the method comprises the following steps:

[0031] (1) culturing the recombinant Candida tropicalis in a seed culture medium to obtain a seed solution;

[0032] (2) inoculating the seed solution obtained in step (1) into a fermentation medium and fermenting to obtain (-)-α-bisabolol.

[0033] In one embodiment, the method comprises the following steps:

[0034] (1) Activating the recombinant tropical Candida, inoculating the activated recombinant tropical Candida into a seed culture medium, and culturing at 20° C. to 40° C. until OD 600 ≥8.0, seed solution was obtained;

[0035] (2) The seed solution obtained in step (1) is inoculated into the fermentation medium to make the initial OD of the fermentation medium 600 The temperature is 0.5 to 2, and (-)-α-bisabolol is obtained by fermentation at 20°C to 40°C; the fermentation medium contains n-dodecane.

[0036] In one embodiment, when fermenting in a fermenter, step (1) is cultured until the seed liquid OD is 600 It is 12 to 15.

[0037] In one embodiment, the fermentation time is not less than 96 hours.

[0038] The present invention also provides the recombinant Candida tropicalis or the use of the method in preparing (-)-α-bisabolol or a product containing (-)-α-bisabolol.

[0039] Beneficial effects:

[0040] (1) The present invention provides a method for producing (-)-α-bisabolol with simple operation and high conversion efficiency. The present invention uses tropical Candida (C. tropicalis) DC04-WG04-2 as a host, has high tolerance to (-)-α-bisabolol, and the yield of (-)-α-bisabolol can reach 461.7 mg / L, which is conducive to the application of the strain in agriculture, food industry, medical and health fields and cosmetics.

[0041] (2) The recombinant Candida tropicalis strain constructed by the present invention is modified by metabolic engineering. The gene Cat2 of catalase and the gene ERG10 of -hydroxy-3-methylglutaryl-CoA reductase from Saccharomyces cerevisiae are anchored on the surface of peroxisomes with the PEX15 motif to increase the utilization rate of acetyl-CoA; the single copy of the gene ADH1 of alcohol dehydrogenase is knocked out to reduce the accumulation of ethanol; the genes GPD1 and GPD2 on the genome are knocked out to reduce the production of glycerol; the shake flask fermentation conditions are optimized, and the (-)-α-bisabolol yield of the recombinant strain C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔADH1 can be significantly increased. The (-)-α-bisabolol yield is increased from the starting strain C. tropicalis to The yield of DC04-WG04-2 increased from 250.9 mg / L to 461.7 mg / L, increasing the yield by 1.83 times. This increased yield provided ideas for the study of (-)-α-bisabolol producing strains.

[0042] (3) The present invention optimizes the fermentation conditions to increase the yield of strain C. tropicalis DC04-WG04-2-Cat2-ERG10-pex15-ΔADH1 to 461.7 mg / L after 96 h of fermentation, which is 31.7% higher than the yield of 331.2 mg / L before optimization, providing a reference for fermentation optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the pathway for metabolically engineering Candida tropicalis to synthesize (-)-α-bisabolol.

[0044] Figure 2 The catalase gene Cat2 from Saccharomyces cerevisiae is anchored on the peroxisome surface using the PEX15 motif, and the engineered bacteria co-expressing the catalase gene Cat2 from Saccharomyces cerevisiae and the 3-hydroxy-3-methylglutaryl-CoA reductase gene ERG10 anchored on the peroxisome surface using the PEX15 motif produce (-)-α-bisabolol.

[0045] Figure 3 The result diagram shows the knockout of a single copy of the alcohol dehydrogenase gene ADH1 to reduce ethanol accumulation, and the knockout of the genes GPD1 and GPD2 on the genome to reduce glycerol production.

[0046] Figure 4 This is a diagram showing the results of producing (-)-α-bisabolol under shake flask fermentation under different fermentation conditions. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0048] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0049] The Candida tropicalis DC-04 involved in the following examples is disclosed in the patent publication number CN114410631B.

[0050] The CRISPR-Cas9 gene editing vector and its construction method involved in the following embodiments are described in the patent publication number CN114410631B.

[0051] The gene expression cassette construction ideas and construction methods involved in the following examples are described in the paper "Systematic metabolic engineering of terpene natural products produced by Candida tropicalis." pMD19-T was purchased from Bao Biotechnology (Dalian) Co., Ltd.

[0052] (I) Culture medium:

[0053] YPD medium (g / L): 20% glucose, 10% yeast powder, and 20% peptone.

[0054] 2×YPD medium (g / L): 40% glucose, 20% yeast powder, and 40% peptone.

[0055] YPD60 medium (g / L): glucose 60, yeast powder 10, peptone 20.

[0056] MM medium (g / L): glucose 20, yeast nitrogen base (YNB) 6.7, ammonium sulfate 10.

[0057] LB medium (g / L): peptone 10, yeast powder 5, sodium chloride 10.

[0058] (II) Detection method

[0059] Detection of (-)-α-bisabolol content:

[0060] 1. Extraction

[0061] In order to extract and analyze the target product (-)-α-bisabolol, 10% (v / v) dodecane was added to the fermentation broth (bacterial culture broth) to capture the product. After the fermentation was completed, all the fermentation broth was collected and centrifuged at 5000 rpm for 5 minutes in a refrigerated centrifuge to separate the organic phase. The organic phase was transferred to an EP tube, an appropriate amount of anhydrous sodium sulfate was added to remove water, shaken for 15 minutes, and allowed to stand for 2 hours. The organic phase was diluted with n-hexane and the (-)-α-bisabolol yield was detected after passing through an organic membrane.

[0062] 2. Detection

[0063] The (-)-α-bisabolol concentration obtained in step 1 was analyzed by performing gas chromatography (GC-MS). The GC (Agilent 8860) was equipped with a flame ionization detector (FID) and an HP-5 column (30 m×0.320 mm×0.25 m) with a flow rate of 1.0 mL / min. The method used for the detection of (-)-α-bisabolol was as follows: the injection volume was 1.0 L, the split ratio was 15, and the temperature of the injector and GC interface was 250°C. The initial temperature was 50°C, the holding time was 1 min, then it was increased to 210°C at a rate of 10°C / min, and the second time it was increased to 230°C at a rate of 20°C / min, the holding time was 8 min, and (-)-α-bisabolol was measured at 230°C for the construction of a standard curve.

[0064] (III) Sequence information

[0065] The nucleotide sequence encoding diacylglycerol diphosphate phosphatase DPP1 is shown in SEQ ID NO.4.

[0066] The nucleotide sequences encoding the catalase gene Cat2 and the 3-hydroxy-3-methylglutaryl-CoA reductase gene ERG10 from Saccharomyces cerevisiae are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0067] The amino acid sequence of alcohol dehydrogenase ADH1 is shown in Genbank accession number: AAA62378.1, and the nucleotide sequence of gene ADH1 is shown in SEQ ID NO.10.

[0068] The nucleotide sequences of the glycerol-3-phosphate dehydrogenase gene GPD1 and the glycerol-3-phosphate dehydrogenase gene GPD2 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0069] Promoter P FBA1 The nucleotide sequence of the promoter is shown in SEQ ID NO.1. GAPDH The nucleotide sequence is shown as SEQ ID NO.2.

[0070] The primer sequences involved in the following examples are shown in Table 1.

[0071] Table 1 Primer sequences

[0072]

[0073]

[0074] Example 1: Construction of chassis cell C. tropicalis DC04-WG04-2

[0075] The amino acid sequences of the scaffold proteins WH1, GBD, SH3, and PDZ are shown in Genbank accession numbers: AAH31149, EDL13116, BAA21534, and EDL06069, respectively.

[0076] By gene synthesis, plasmids PUC57-WH1 / PUC57-GBD containing the above protein WH1 and GBD coding genes were obtained. Using PUC57-WH1 / PUC57-GBD plasmids as templates, primers WH1-P FBA1 -FW and WH1-(GS)9-GBD-RS, GBD-(GS)9-WH1-FW, GBD-T ADH2 -RW was used for PCR, and the PCR product was identified by electrophoresis and recovered by gel recovery to obtain the WH1 and GBD gene fusion fragment connected by (GS)9, PMD19T-POX5-gda-URA3-P FBA1 -ERG20-T ADH2 (published in the paper "Systematic metabolic engineering of tropical Candida to produce terpenoid natural products") as a template, with P FBA1 -FW / T ADH2 -RS is the primer PCR amplified P FBA1 -POX5-PMD19T-GDA-URA3-T ADH2 vector fragment; the WH1 and GBD gene fusion fragment and the P FBA1 -POX5-PMD19T-GDA-URA3-T ADH2 The vector fragments were connected by one-step cloning and transformed into E. coli JM109 competent cells. The transformants were diluted and spread on LB solid medium supplemented with ampicillin and positive transformants were screened (using colony PCR method). The single colony with the correct colony PCR was picked and inoculated into liquid LB medium supplemented with ampicillin for 8-12 hours. The recombinant plasmid was extracted using a small amount of plasmid extraction kit, and the plasmid was sequenced and analyzed. The correct plasmid was named pMD19T-POX5-gda-URA3-P FBA1-WH1-(GS)9-GBD-T ADH2 , and the transformants were stored in a -80℃ refrigerator. FBA1 -WH1-(GS)9-GBD-T ADH2 The expression cassette encodes a fusion protein WH1-(GS)9-GBD, wherein the protein WH1 and the protein GBD are connected by a connecting peptide (GS)9; the recombinant plasmid pMD19T-DLD24-gda-URA3-P GAPDH -ERG20-T ADH2 -MrBBS-P FBA1 Constructed in the same way as above.

[0077] pMD19T-POX5-gda-URA3-P FBA1 -WH1-(GS)9-GBD-T ADH2 with pMD19T-DLD24-gda-URA3-P GAPDH -ERG20-T ADH2 -MrBBS-P FBA1 The strain was transformed into C. tropicalis DC-04 (disclosed in patent publication number CN114410631B) to construct the Candida tropicalis strain WGME.

[0078] According to the same strategy as above, the fusion expression cassettes of ACS1, ERG10, ERG13, tHMGR, ERG12, ERG8 (Genbank: AAA62378.1, RCK66100.1, AE014297, RCK63473.1, RCK59297.1), ERG19 (Genbank: AAC49252.1), and IDI gene (Genbank: AF119715) were linked with the linker peptide (GGGGS) 3 and pMD19T-GPP1-P FBA1 -ERG19-(GGGGS)3-IDI-T ADH2 -gda-URA3 was integrated into the GPP1 and DLD35 sites of the strain WGME genome to construct the strain C. tropicalis DC04-WG04-2.

[0079] Example 2: Construction of gene knockout cassette

[0080] 1. Construction of knockout cassette:

[0081] (1) Construction of ADH1 gene knockout cassette

[0082] The genome of tropical Candida DC04 was used as a template, and ADH1-FW and ADH1-RS were used as primers for amplification, and agarose gel electrophoresis was used for verification. After verification, gel recovery was performed, and the product was labeled ADH1. The fragment ADH1 was connected to the pM19Tsimple vector, and the successfully connected product fragment was transformed into E.coli JM109 competent cells. The transformants were diluted and spread on LB solid medium supplemented with ampicillin and positive transformants were screened (using the colony PCR method). The single colony with the correct colony PCR was picked and inoculated in liquid LB medium supplemented with ampicillin for 8 to 12 hours. The recombinant plasmid was extracted using a small amount of plasmid extraction kit, and the Mlu I enzyme was used for enzyme digestion verification. The plasmid was sequenced and analyzed, and the correct plasmid was named pMD19T-ADH1. Using the recombinant plasmid pMD19T-ADH1 as a template, reverse primers DOWN-ADH1-FW and UP-ADH1-RS (with built-in PstⅠ and XbaⅠ restriction sites), inverse PCR was used to obtain the fragment DPP1-PMD19T-ADH1 and the fragment was recovered by nucleic acid electrophoresis gel, and the PCR product was purified by a kit. The plasmid pMD19T-gda-URA3 (disclosed in "Establishment of a Genetic Operating System for Candida tropicalis and its application in dibasic acid synthesis") was digested with Pst I and XbaⅠ, and after gel recovery, the linear vector ADH1-pMD19T-ADH1 and the DNA fragment gda-URA3 were cloned and connected in one step using a one-step ligation kit to obtain the recombinant plasmid pMD19T-ADH1-gda-URA3.

[0083] (2) Construction of gene knockout cassettes for GPD1 and GPD2

[0084] Referring to the method of step (1) above, the difference is that the primers DPP1-FW / DPP1-RS and UP-DPP1-RS / DOWN-DPP1-FW are replaced with GPD1-FW / GPD1-RS, UP-GPD1-RS / DOWN-GPD1-FW, GPD2-FW / GPD2-RS, UP-GPD2-RS / DOWN-GPD2-FW, respectively, to construct plasmids pMD19T-GPD1-gda-URA3 and pMD19T-GPD2gda-URA3 containing the gene knockout cassettes of GPD1 and GPD2.

[0085] Example 3: Construction of an expression cassette containing the PEX15 motif

[0086] (1) Cat2 gene expression cassette:

[0087] The nucleotide sequence of the (-)-α-bisabolol synthase encoding gene Cat2 is shown in SEQ ID NO. 1. Using the genome of Saccharomyces cerevisiae ATCC 204508 as a template, Cat2-GGGS-PEX15-FW and Cat2-RS as primers, PCR amplification and purification were performed to obtain the Cat2-GGGS-PEX15 gene fragment. The Cat2-GGGS-PEX15 gene fragment was combined with the fragment P FBA1 -PMD19T-T ADH2 (shown in SEQ ID NO.9) was connected by a one-step cloning kit to obtain an expression cassette P FBA1 -Cat2-GGGS-PEX15-T ADH2 The recombinant plasmid pMD19T-P FBA1 -Cat2-GGGS-PEX15-T ADH2 . Recombinant plasmid pMD19T-P FBA1 -Cat2-GGGS-PEX15-T ADH2 As template, primer P FBA1 -FW and T ADH2 -RS, P obtained by PCR FBA1 -Cat2-GGGS-PEX15-T ADH2 Finally, the expression fragment was ligated with the pMDT19-POX5-gda-URA3 vector fragment (disclosed in "Systematic Metabolic Engineering of Natural Terpene Products Produced by Candida tropicalis") digested with PstⅠ and XbaⅠ to obtain the recombinant plasmid pMD19T-DPP1-gda-URA3-P FBA1 -Cat2-GGGS-PEX15-T ADH2 Among them, the expression cassette P FBA1 -Cat2-GGGS-PEX15-T ADH2 In the promoter P FBA1 Drives the expression of the gene Cat2.

[0088] (2) Co-expression cassette of gene Cat2 and gene ERG10:

[0089] Using the Saccharomyces cerevisiae genome as a template, Cat2-GGGS-PEX15-FW and Cat2-RS as primers, PCR amplification and purification were performed to obtain the Cat2-GGGS-PEX15 gene fragment, which was then cloned and cloned into the fragment P FAB1 -pMD19T-P GAPDH -ERG20-T ADH2 (Published in "Systematic Metabolic Engineering of Natural Terpenoid Products Produced by Candida Tropicalis") One-step cloning and ligation were performed to obtain plasmid pMD19T-PGAPDH -ERG20-T ADH2 -Cat2-GGGS-PEX15-P FBA1 . With plasmid pMD19T-P GAPDH -ERG20-T ADH2 -Cat2-GGGS-PEX15-P FBA1 As a template, GAPDH -FW and T ADH2 -RS primer, PCR to obtain P GAPDH -pMD19T-P FBA1 -Cat2-GGGS-PEX15-T ADH2 The expression fragment was obtained by PCR using the genome of tropical Candida DC-04 as a template, using primers ERG10-GGGS-PEX15-FW and ERG10-RS, and the ERG10-GGGS-PEX15 expression fragment was connected with the pMD19T-DPP1-gda-URA3 vector fragment digested with PstⅠ and XbaⅠ enzymes to obtain the recombinant plasmid pMD19T-DPP1-gda-URA3-P GAPDH -ERG10-GGGS-PEX15-T ADH2 -PEX15-GGGS-Cat2-P FBA1 , which contains the expression cassette P GAPDH -ERG10-GGGS-PEX15-T ADH2 -PEX15-GGGS-Cat2-P FBA1 In this expression cassette, P GAPDH The promoter drives the expression of ERG10 gene, with P FBA1 The promoter drives the expression of the gene Cat2.

[0090] Example 4: Construction of recombinant Candida tropicalis

[0091] (1) Construction of strain C. tropicalis DC04-WG04-2-Cat2-PEX15:

[0092] The recombinant plasmid pMD19T-DPP1-gda-URA3-P constructed in Example 3 was converted into FBA1 -Cat2-GGGS-PEX15-T ADH2 Digestion and purification were performed to obtain DPP1-gda-URA3-P FBA1 -Cat2-GGGS-PEX15-T ADH2-DPP1 linearized fragment. Using the LiCl transformation method, the strain C. tropicalis DC04-WG04-2 constructed in Example 1 was used as the starting strain, and the URA3 gene was used as the screening marker. FBA1 -Cat2-GGGS-PEX15-T ADH2 -DPP1 linearized fragment was transferred into the host, and a recombinant bacterium expressing the peroxidase gene Cat2 from Saccharomyces cerevisiae shown in SEQ ID NO.7 was constructed at the DPP1 site. The transformants were spread on MM solid medium and cultured in a 30°C incubator for 3 days. The transformants on the MM solid medium were picked, the genome was extracted, PCR verification and sequencing were performed, and the correct recombinant transformants were identified and named C. tropicalis DC04-WG04-2-Cat2-PEX15.

[0093] (2) Construction of strain C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15:

[0094] Construction of reference strain C. tropicalis DC04-WG04-2-Cat2-PEX15. The plasmid pMD19T-DPP1-gda-URA3-P was digested with Mlu I. GAPDH -ERG10-GGGS-PEX15-T ADH2 -PEX15-GGGS-Cat2-P FBA1 , get the linearized fragment

[0095] DPP1-GDA-URA3-P GAPDH -ERG10-GGGS-PEX15-T ADH2 -PEX15-GGGS-Cat2-P FBA1 -DPP1, which was transformed into the strain C. tropicalis DC04-WG04-2 to obtain the strain C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15.

[0096] (3) Construction of strains C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔADH1, C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔGPD1, and C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔGPD2 The plasmid pMD19T-ADH1-gda-URA3 obtained in Example 2 was linearized to obtain a linearized fragment, which was transformed into the strain C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15. The ADH1 gene was knocked out by the CRISPR-Cas9 method. After the transformant was verified to be correct by PCR, a recombinant strain with the ADH1 gene knocked out was obtained, which was named C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔADH1.

[0097] The knockout of genes GPD1 and GPD2 was the same as above. After the transformants were verified to be correct by PCR, recombinant strains with knockout genes GPD1 and GPD2 were obtained. The recombinant strains were named C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔGPD1 and C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔGPD2.

[0098] Example 5: Construction of recombinant Candida tropicalis and fermentation production of (-)-α-bisabolol

[0099] The recombinant strains C. tropicalis DC04-WG04-2-Cat2-PEX15, C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15, C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔADH1, C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔGPD1, and C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔGPD2 constructed in Example 4 were streaked on YPD plates, and single colonies were picked from the solid plates and transferred to 10 mL YPD liquid culture medium and cultured at 200 r / min and 30° C. until OD 600 When the initial OD value reaches 8.0 or above, it is used as seed solution, and then the seed solution is transferred to a 250 mL shake flask containing 30 mL YPD60 medium at a 1% inoculum volume. 600The concentration of n-dodecane was 1.0, and 10% n-dodecane was added during the initial period. The cells were placed in a 30°C shaker at 200 r / min and cultured for 96 h. Three biological parallels were set for each single colony.

[0100] The changes in biomass during fermentation were measured by UV-visible spectrophotometer at 600 nm. 600 To characterize, the measurement range is kept between 0.2 and 0.8. When the bacterial concentration is too high, it is diluted and then measured and calculated.

[0101] The results showed that the accumulated yields of the high-yield recombinant strains C. tropicalis DC04-WG04-2-Cat2-PEX15, C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15, and C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔADH1 after 96 hours of culture were 271.8 mg / L, 290.1 ​​mg / L, and 360.2 mg / L, respectively.

[0102] Example 6: Optimizing the fermentation conditions of recombinant Candida tropicalis to produce (-)-α-bisabolol

[0103] The C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔADH1 strain constructed in Example 4 and the starting strain DC04-WG04-2 in Example 1 were fermented according to the fermentation conditions of Example 5, except that:

[0104] (1) In a 250 mL shake flask system, YPD60 medium with a liquid volume of 25 mL, 30 mL, and 35 mL was used for fermentation at a temperature of 30°C. The results showed that the yield of C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔADH1 strain could reach 344.7 mg / L at a liquid volume of 25 mL.

[0105] (2) In a 250 mL shake flask system, the strain was fermented at fermentation temperatures of 26°C, 28°C, and 30°C, respectively, with a liquid volume of 25 mL YPD60 medium. The results showed that at 28°C, the yield of C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔADH1 strain could reach 366.9 mg / L.

[0106] (3) In a 250 mL shake flask system, the strain was fermented at 28°C in 1×YPD medium, 2×YPD medium, and YPD60 medium with a volume of 25 mL. The results showed that the yield of C. tropicalis DC04-WG04-2-Cat2-ERG10-PEX15-ΔADH1 strain could reach 461.7 mg / L in 2×YPD, which was 31.7% higher than the fermentation conditions before optimization. Compared with the control strain C. tropicalis DC04-WG04-2 (250.6 mg / L), the yield of (-)-α-bisabolol was significantly increased, which provided a reference for fermentation optimization.

[0107] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Candida tropicalis producing (-)-α-bisabolol, characterized in that: Compared with the starting strain, the catalase Cat2 from Saccharomyces cerevisiae is expressed, and the PEX15 motif is anchored on the surface of the peroxisome; the nucleotide sequence of the PEX15 motif is shown in SEQ ID NO.

3.

2. The recombinant Candida tropicalis according to claim 1, characterized in that The catalase Cat2 is also co-expressed with 3-hydroxy-3-methylglutaryl-CoA reductase ERG10; the nucleotide sequence of the gene Cat2 encoding the catalase is shown in SEQ ID NO.7; the nucleotide sequence of the gene ERG10 encoding the 3-hydroxy-3-methylglutaryl-CoA reductase is shown in SEQ ID NO.

8.

3. The recombinant Candida tropicalis according to claim 1 or 2, characterized in that One or more of the following genes were also knocked out: the alcohol dehydrogenase gene ADH1, the glycerol-3-phosphate dehydrogenase gene GPD1, and the glycerol-3-phosphate dehydrogenase gene GPD2.

4. The recombinant Candida tropicalis according to any one of claims 1 to 3, characterized in that Gene Cat2 is expressed by P FBA1 The promoter initiates transcription; the P FBA1 The nucleotide sequence of the promoter is shown in SEQ ID NO.1; the gene ERG10 is driven by the promoter P GAPDH Start transcription; the promoter P GAPDH The nucleotide sequence is shown in SEQ ID NO.

2.

5. The recombinant Candida tropicalis according to any one of claims 1 to 4, characterized in that The starting strain is (a) or (b): (a) Candida tropicalis DC-04; (b) Based on (a), (-)-α-bisabolol synthase and farnesyl pyrophosphate synthase were co-expressed, and the (GS)9-linked protein scaffold genes: WH1 and GBD, and the (GGGGS)3-linked genes ERG19 and IDI were integrated.

6. A method for improving the production capacity of (-)-α-bisabolol of recombinant Candida tropicalis, characterized in that: Including at least one improvement from (a) to (e): (1) Express the catalase gene Cat2 from Saccharomyces cerevisiae and anchor the PEX15 motif on the peroxisome surface; (2) co-expression of the gene for catalase containing PEX15 and the gene for 3-hydroxy-3-methylglutaryl-CoA reductase containing PEX15, ERG10; (3) The genes of catalase containing PEX15 and 3-hydroxy-3-methylglutaryl-CoA reductase gene ERG10 containing PEX15 were integrated into the genome, and the single copy of alcohol dehydrogenase gene ADH1 was knocked out; (4) Knock out the GPD1 and GPD2 genes in the genome.

7. The method according to claim 6, characterized in that The method also includes fermenting the recombinant tropical Candida in a culture medium containing glucose, yeast powder and peptone at 26°C to 30°C.

8. A method for preparing (-)-α-bisabolol, characterized in that: The recombinant Candida tropicalis according to any one of claims 1 to 5 is used to ferment and produce (-)-α-bisabolol.

9. The method according to claim 8, characterized in that include: Fermentation in YPD medium at 28°C in a 25 mL culture system; or Fermentation at 28°C in 2×YPD medium; or Fermentation was carried out at 26°C to 30°C in YPD60 medium.

10. Use of the recombinant Candida tropicalis according to any one of claims 1 to 5, or the method according to any one of claims 6 to 9 in the preparation of (-)-α-bisabolol or a product containing (-)-α-bisabolol.

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