A Saccharomyces cerevisiae strain for producing sesquiterpene α-bisabolol using xylose and its application

Abstract

The present invention relates to a saccharomyces cerevisiae strain for producing sesquiterpene α-bisabolol by using xylose and an application thereof, belonging to the field of bioengineering. The saccharomyces cerevisiae strain for producing sesquiterpene α-bisabolol by using xylose, wherein the genotype of the saccharomyces cerevisiae strain is: XK , gre3::PPP , Cox4 Δ, AE,HO∷ P PGK1 ‑CCBOS‑ T CYC1 , ERG9∷ P CTR3 ‑ERG9 , YORWdelta22∷ P TEF1 ‑ tHMG1 ‑T ADH1 , DPP1∷ P HHF2 ‑ERG20‑ T TEF1 ,△ ROX1 , YPRCtua3 ∷ P PGK1 ‑ERG20‑ GGGS ‑CCBOS‑ T CYC1 ,pJX7‑XI,P TEF1 ‑ Ru‑xylA ‑T PGK1 The recombinant Saccharomyces cerevisiae strain that produces the sesquiterpene α-bisabolol using xylose according to the present invention has a yield of 186.308 mg / L. The technical strategy of the present invention lays a solid foundation for constructing Saccharomyces cerevisiae to produce and synthesize other high-value sesquiterpene compounds using xylose and realizing industrial production.

Description

Technical Field

[0001] The invention relates to a saccharomyces cerevisiae strain for producing sesquiterpene alpha-bisabolol by utilizing xylose and application thereof, belonging to the field of bioengineering. Background Art

[0002] Sesquiterpenoid α-bisabolol is a monocyclic sesquiterpenoid alcohol isolated and extracted from chamomile. It is used as an ingredient in medicines and cosmetics because of its bactericidal, anti-inflammatory, moisturizing and other functions. Most sesquiterpenes are obtained by chemical synthesis or plant extraction, which have obvious disadvantages such as high environmental cost, serious pollution, poor sustainable development, complicated process, and insufficient product purity. In order to solve the above problems, it is urgent to find a green, economical, efficient and sustainable method. The microbial cell factory strategy came into being, and its proposal perfectly solves the above problems. Saccharomyces cerevisiae with a complete endogenous mevalonate (MVA) pathway stands out among many microorganisms with its clear genetic background, high industrial tolerance, simple genetic manipulation, short growth cycle and many other advantages, becoming the dominant strain of sesquiterpene microbial cell factory. Therefore, the endogenous MVA pathway of Saccharomyces cerevisiae can be used to synthesize the sesquiterpene precursor FPP with acetyl-CoA as the key precursor, and then the sesquiterpene α-bisabolol synthase ( BOS ) converts FPP to synthesize sesquiterpene α-bisabolol. However, in Saccharomyces cerevisiae, there is a sesquiterpene α-bisabolol synthase ( BOS ) Low expression activity, insufficient precursor supply and cytotoxicity of sesquiterpenoids restrict the efficient synthesis of the sesquiterpene α-bisabolol.

[0003] At present, there are three xylose decomposition pathways in natural xylose-utilizing microorganisms, including the xylose-to-glycolaldehyde (Dahms) or α-ketoglutarate oxidation (Weimberg) pathway, the xylose-1-phosphate (Xylulose-1-phosphate, X-1-P) or ribose-1-phosphate (Ribulose-1-phosphate, R-1-P) pathway, and the xylose reductase XR / xylitol dehydrogenase XDH (XR-XDH) or xylose isomerase (XI) pathway. Among them, the Dahms or Weimberg pathway and the X-1-P or R-1-P pathway have low enzyme gene activity, excessive accumulation of intermediate metabolites, and many limiting factors, resulting in poor xylose metabolism efficiency, making it difficult to effectively use in Saccharomyces cerevisiae. The XR-XDH or XI pathway is the most widely studied xylose metabolism pathway in Saccharomyces cerevisiae. These two pathways have the same goal. Although the paths are different, they both convert xylose into xylulose. Among them, the XR-XDH pathway mostly uses the XYL and XYL2The genes encode NAD(P)H-dependent XR and NADP + XDH, which depends on XR, can reduce xylose to xylitol, which is then oxidized to xylulose. In this pathway, XR prefers NADPH, while XDH only depends on NAD+. Different cofactor dependence can easily lead to insufficient NAD+ or excess NADPH, causing an imbalance in redox metabolism and the accumulation of intermediate metabolites such as xylitol, making it difficult to increase the conversion rate of sugar alcohols. Strategies such as adjusting the XR-XDH activity ratio, changing cofactor preference, or introducing heterologous NADH oxidation pathways can improve xylose utilization efficiency and reduce intermediate metabolite accumulation. In the previous work of this laboratory, a highly efficient xylose isomerase (Ru- xylA) , which can convert xylose to xylulose in one step in the XI pathway without causing cofactor imbalance; this breakthrough discovery successfully overcomes the weak upstream metabolic link of xylose conversion to xylulose in natural Saccharomyces cerevisiae, making the efficient use of xylose a reality and greatly improving the difficulties faced by Saccharomyces cerevisiae in the synthesis of bio-based products. Compared with glucose, there is a relative lack of research on the use of xylose in the synthesis of bio-based products by Saccharomyces cerevisiae. Therefore, it is particularly critical and urgent to construct an upstream xylose metabolic pathway in Saccharomyces cerevisiae to achieve the goal of efficiently using xylose to produce chemicals. Summary of the invention

[0004] With the progress of biorefining technology for lignocellulosic biomass and the greatly increased xylose metabolism capacity of engineered strains of Saccharomyces cerevisiae, the synthesis of chemicals using xylose as a carbon source has attracted more and more attention. In view of the limiting factors in the synthesis of sesquiterpenes, expanding the use of xylose as an efficient carbon source to synthesize products is extremely critical to achieving efficient and economical production of lignocellulose bioconversion. Based on the development of biorefining technology for lignocellulosic biomass, the present invention provides a recombinant Saccharomyces cerevisiae strain that uses xylose to produce the sesquiterpene α-bisabolol, which has a good application in the fermentation preparation of the sesquiterpene α-bisabolol.

[0005] The technical solution of this application is as follows: The first purpose of the present application is to protect a strain of Saccharomyces cerevisiae that utilizes xylose to produce the sesquiterpene α-bisabolol. The genotype of the Saccharomyces cerevisiae strain is as follows: XK , gre3::PPP , Cox4 Δ, AE,HO:: P PGK1 -CCBOS- T CYC1 , ERG9∷ P CTR3 -ERG9 , YORWdelta22 ∷ P TEF1 - tHMG1 -TADH1 , DPP1∷ P HHF2 -ERG20- T TEF1 ,△ ROX1 , YPRCtua3∷ P PGK1 -ERG20- GGGS -CCBOS- T CYC1 ,pJX7-XI,P TEF1 - Ru-xylA -T PGK1 .

[0006] Among them, genes CCBOS See SEQ ID No. 38 for the sequence, which is derived from artichoke and functions to catalyze the conversion of the sesquiterpene precursor farnesyl pyrophosphate (FPP) into the sesquiterpene α-bisabolol. CCBOS outside, tHMG1 , ERG20 , ROX1 , ERG9 These are genes that exist in Saccharomyces cerevisiae itself.

[0007] According to a specific embodiment of the present application, the method for constructing the strain includes: The Saccharomyces cerevisiae haploid BSPX051 ( XK , gre3::PPP , Cox4 Δ, AE ) was used as the starting strain, and the xylose isomerase gene Ru- xyA The expression plasmid pJX7-XI was transformed into strain 51H-XI, and then a promoter engineering strategy was adopted to use the copper ion responsive promoter P CTR3 Make it ERG9 The promoter of the gene was replaced in situ to obtain a promoter-replaced strain, and then the promoter-replaced strain 51H-XI was cloned into HO , YORWdelta2 , DPP1 , YPRC tua3 Insertion of sites CCBOS , tHMG1 , ERG20 , ERG20 -GGGS- CCBOS Knockout of regulatory factors ROX1 The recombinant Saccharomyces cerevisiae strain 51H6-XI was obtained, which is a recombinant Saccharomyces cerevisiae strain that produces the sesquiterpene α-bisabolol by utilizing xylose.

[0008] The second purpose of the present application is to protect the use of the above-mentioned Saccharomyces cerevisiae strain for producing the sesquiterpene α-bisabolol using xylose in the fermentation production of the sesquiterpene α-bisabolol.

[0009] The third purpose of the present application is to protect the use of the above-mentioned Saccharomyces cerevisiae strain for producing the sesquiterpene α-bisabolol using xylose in the fermentation production of sesquiterpene α-bisabolol derivatives.

[0010] BSPX051( XK , gre3::PPP , Cox4 Δ, AE ) Strain preparation: BSPX042 published in Journal of Bioscience and Bioengineering, VOL. 121 No. 2, 160-165, 2016 was used to knock out KanMX The gene can be used to obtain BSPX051. The more specific steps of the above technical solution are: The xylose isomerase expression plasmid pJX7-XI was transformed into the Saccharomyces cerevisiae haploid BSPX051 ( XK , gre3::PPP , Cox4 Δ, AE ), and successfully transformed transformants were obtained by screening with SX-Ura synthetic medium to obtain the cerevisiae yeast strain 51H-XI. The recombinant strain 51H6-XI was constructed and the fusion PCR method was used to transform ERG9 Promoter upstream and downstream homology arms, screening markers KanMX -URA and copper ion corresponding promoter P CTR3 The expression cassette was integrated into the Saccharomyces cerevisiae strain 51H-XI by lithium acetate / polyethylene glycol / single-stranded DNA co-transformation method, and finally 5-FOA was used to screen for non- KanMX -URA correct transformant; then use the same method to CCBOS With promoter P PGK1 and terminator T CYC1 , tHMG1 With promoter P TEF1 and terminator T ADH1 , ERG20 With promoter P HHF2 and terminator T TEF1 , ERG20 -GGGS- CCBOS With promoter P PGK1 and terminator T CYC1 Fusion is performed, and then the obtained fusion fragment is combined with the upstream and downstream homology arms and screening markers of the corresponding sites. KanMA -URA was used for another round of fusion, and the fusion gene fragment was transferred into the promoter-replaced strain 51H-XI by lithium acetate / polyethylene glycol / single-stranded DNA co-transformation method, and the strain without KanMXThe correct transformant of -URA is the recombinant Saccharomyces cerevisiae strain 51H6-XI, with the genotype: XK , gre3::PPP , Cox4 Δ, AE,HO∷ P PGK1 -CCBOS- T CYC1 , ERG9∷ P CTR3 - ERG9 , YORWdelta22∷ P TEF1 - tHMG1 -T ADH1 , DPP1∷ P HHF2 -ERG20- T TEF1 ,△ ROX1 , YPRCtua3∷ P PGK1 - ERG20- GGGS -CCBOS- T CYC1 ,pJX7-XI,P TEF1 - Ru-xylA -T PGK1 . (Gene CCBOS See SEQ ID No. 38 for the sequence, which is derived from artichoke and functions to catalyze the conversion of the sesquiterpene precursor farnesyl pyrophosphate (FPP) into the sesquiterpene α-bisabolol. CCBOS outside, tHMG1 , ERG20 , ROX1 , ERG9 These are genes that exist in Saccharomyces cerevisiae itself.) The yield of sesquiterpene α-bisabolol of the recombinant Saccharomyces cerevisiae strain 51H6-XI was analyzed by fermentation test, and the results showed that the yield of the recombinant Saccharomyces cerevisiae strain producing sesquiterpene α-bisabolol using xylose according to the present invention was 186.308 mg / L. Specifically, aerobic shake flask fermentation: 4 mL of dodecane was added to 20 mL of SX-Ura medium as an extractant, the initial inoculation OD value was 0.2, and the culture was carried out for 84 h, and the yield of sesquiterpene α-bisabolol reached a maximum of 186.308 mg / L.

[0011] Beneficial Effects of the Invention The recombinant Saccharomyces cerevisiae strain of the present invention can produce sesquiterpene α-bisabolol using xylose as the sole carbon source, which has guiding significance for biorefining using lignocellulosic biomass as raw materials to produce sesquiterpene chemicals. The yield of sesquiterpene α-bisabolol of the recombinant Saccharomyces cerevisiae strain 51H6-XI was analyzed by fermentation test, and the results showed that the yield of the recombinant Saccharomyces cerevisiae strain producing sesquiterpene α-bisabolol using xylose according to the present invention was 186.308 mg / L. The technical strategy of the present invention lays a solid foundation for constructing Saccharomyces cerevisiae to produce and synthesize other high-value sesquiterpene compounds using xylose and realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of xylose isomerase expression plasmid pJX7-XI; Figure 2 Screening results of highly active sesquiterpene α-bisabolol synthase (the carbon source in Figure A is glucose, and the carbon source in Figure B is xylose); Figure 3 The effect of metabolic pathway modification on the production of α-bisabolol; Figure 4 GC spectra of sesquiterpene α-bisabolol standard (A), control strain BSPX051 (B) and recombinant Saccharomyces cerevisiae strain 51H6-XI (C) producing sesquiterpene α-bisabolol; Figure 5 Changes in OD value (A) and sesquiterpene α-bisabolol production (B) during aerobic shake flask fermentation of the recombinant Saccharomyces cerevisiae strain 51H6-XI. DETAILED DESCRIPTION

[0013] Example 1 Sources and culture of microbial materials and molecular biology techniques (1) Culture medium Escherichia coli ( Escherichia coli LB medium used for culture: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl; solid medium needs to be added with 20 g / L agar powder; sterilization conditions: 115 °C, 30 min; when used, add ampicillin (Amp) to a final concentration of 200 μg / mL for screening E. coli Transformant.

[0014] The culture medium used for culturing Saccharomyces cerevisiae includes: ① YEPD medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast powder; ② SX-Ura synthetic medium: 20 g / L xylose, 1.7 g / L yeast basal nitrogen source, 5 g / L ammonium sulfate, 0.77 g / L uracil-deficient amino acid mixture. Xylose needs to be sterilized separately before adding; solid medium needs to add 20 g / L agar powder; sterilization conditions: 115℃, 30 min. Shake flask culture needs to add 20% of the culture medium volume of dodecane to extract α-bisabolol (filter sterilization).

[0015] (2) Enzymes and reagents 2×MultiF Seamless Assembly Mix ligase (ABclonal Technology Co., Ltd.); Restriction Enzymes, FastDigest enzymes, GeneRuler 1 kb DNA Ladder (ThermoFisher Scientific Inc.); peptone, yeast extract (purchased from OXOID) and agar powder (purchased from Solarbio); xylose (Sinopharm Chemical Reagent Co., Ltd.); other chemical reagents were all domestically produced analytical grade.

[0016] EZNA® Plasmid Mini Kit I, EZNA® Cycle-Pure Kit, EZNA® Gel Extraction kit (Omega Bio-Tek Inc.); Yeast Plasmid Mini Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.); The corresponding experimental operations were carried out according to the product instructions; genomic DNA was extracted by the phenol-chloroform method (25:24:1).

[0017] Trans5α Chemically Competent Cell was prepared in the laboratory. 2×Rapid Taq Master Mix and Phanta Max Super-Fidelity DNA Polymerase used in PCR were purchased from Nanjing Novozyme Biotechnology Co., Ltd. The reaction system was added according to the instructions, and the PCR reaction program was set as follows: ① 95°C 3 min, 1 cycle; 95°C 15 s, Tm-5°C 15 s, 72°C 1 kb / 15s, 32 cycles; 72°C 5 min, 1 cycle; 4°C insulation. ② 95°C 5 min, 1 cycle; 95°C 15 s, 55-61°C 15s, 72°C 1 kb / 30s, 30-35 cycles; 72°C 5 min, 1 cycle; 4°C insulation.

[0018] (3) Primers and sequencing Primer synthesis, plasmid and DNA fragment sequencing were completed by Genewise Biotechnology Co., Ltd., Qingdao Weilai Biotechnology Co., Ltd., Beijing Qingke Biotechnology Co., Ltd. and other companies.

[0019] (4) Yeast transformation Saccharomyces cerevisiae transformation was performed by the lithium acetate / polyethylene glycol / ssDNA co-transformation method (Daniel Gietz & Woods, 2002).

[0020] Example 2 Screening of highly active sesquiterpene α-bisabolol synthase Two plant-derived sesquiterpene α-bisabolol synthase genes, one from Matricaria MRBBS (AIG92846.1, sequence as shown in SEQ ID No.37) gene and from artichoke CCBOS (XP_024994640.1, sequence as shown in SEQ ID No.38) gene, and in the Saccharomyces cerevisiae strain BSPC039 ( XK , gre3::PPP, Journal ofBioscience and Bioengineering, VOL. 121 No. 2, 160-165, 2016), BSPC042 ( XK , gre3::PPP , Cox4 Δ, Journal of Bioscience and Bioengineering, VOL. 121 No. 2, 160-165, 2016) and BSPX051 ( XK , gre3::PPP , Cox4 Δ, AE ) to further test its activity. CCBOS After the gene, only BSPX051 (C51 explanation: C represents the gene CCBOS, and the number is the last two digits of the experimental strain, the same below) obtained a higher yield of 8.991 mg / L of sesquiterpene α-bisabolol than the other two strains BSPC039 (C39) and BSPC042 (C42) ( Figure 2 A) and heterologous expression MRBBS After the gene was added, BSPX051 (M51) obtained a higher yield of 3.715 mg / L of sesquiterpene α-bisabolol than BSPC039 (M39) and BSPC042 (M42). Figure 2 A); Heterologous expression under xylose carbon source CCBOSAfter the gene was added, BSPX051 (C51) produced 21.635 mg / L of sesquiterpene α-bisabolol, which was significantly higher than the other two strains (C39 and C42). Figure 2 B) The same heterologous expression MRBBS After the gene was added, BSPX051 (M51) also obtained the highest yield of 6.718 mg / L ( Figure 2 B). Found from the screening results CCBOS With tradition MRBBS Compared with the control group, it showed higher activity and better xylose adaptability.

[0021] Example 3 Construction of recombinant Saccharomyces cerevisiae strain 51H6-XI The xylose isomerase expression plasmid pJX7-XI (Ura-deficient, see schematic diagram) was inserted into Figure 1 ) were transformed into the Saccharomyces cerevisiae haploid BSPX051 ( XK , gre3::PPP , Cox4 Δ, AE ), the correct transformant 51H-XI was screened by SX-Ura solid medium, and then two single colonies were picked in 5 mL SX-Ura liquid medium, cultured at 30℃, 200 rpm for 24 h, the yeast genome was extracted, and the presence of plasmid pJX7-XI was verified by PCR using primers 1 and 2. The correct transformant was streaked on SX-Ura solid medium plates and cultured at 30℃ for two days. After obvious single colonies were formed, they were placed in a 4℃ refrigerator for later use.

[0022] By PCR, primers 31 and 32 were used first, and then primers 33 and 34 were used to amplify P ERG9 Gene upstream and downstream homology arms; amplified using primers 23 and 24 KanMX -URA screening marker fragment; using primers 35 and 36 to amplify the copper ion inducible promoter P CTR3 The sequence is shown in SEQ ID No.39. Then, primers 31 and 34 were used to convert P ERG9 Gene upstream and downstream homology arms, screening marker gene fragments and copper ion inducible promoter P CTR3 Then, the P-containing CTR3 The expression cassette was transformed into Saccharomyces cerevisiae 51H-XI. Finally, the gene that successfully replaced P was screened by YPD solid medium containing G418. CTR3 Then, two single colonies were selected and placed in 5 mL YPD liquid medium containing G418 at 30°C and 200 rpm for 24 h. The yeast genome was extracted and verified by PCR.CTR3 The presence of the gene will successfully replace P CTR3 A single colony of 51H-XI was streaked onto a YPD solid culture medium plate containing G418 and cultured at 30°C for two days. When a single strain was clearly formed, it was placed in a 4°C refrigerator for later use.

[0023] Then, PCR was performed using primers 3 and 4, primers 5 and 6, primers 23 and 24, and primers 25 and 26 to obtain the HO site integration frame, which was then transformed into the successfully replaced P by the lithium acetate / polyethylene glycol / single-stranded DNA co-transformation method. CTR3 Finally, the correct transformant 51H1-KanMX was obtained by screening on YPD solid medium containing G418. Then, two single colonies were picked and placed in 5 mL YPD liquid medium containing G418 at 30°C and 200 rpm for 24 h. The yeast genome was extracted and verified by PCR. PGK1 - CCBOS -T CYC1 The correct transformant 51H1-KanMX was spread on the 5-FOA selection plate to remove the resistance gene KanMX. When a single colony grew out, it was the recombinant Saccharomyces cerevisiae strain 51H1. It was streaked on the YPD solid culture medium plate and cultured at 30℃ for two days. When a single strain was clearly formed, it was placed in a 4℃ refrigerator for later use.

[0024] Next, primer 7 and primer 8 were used first, then primer 9 and primer 10, and finally primer 27 and primer 28 were used for amplification to obtain YORW delta22 Site integration frame; first use primers 11 and 12, then use primers 13 and 14, and finally use primers 29 and 30 to amplify and obtain DPP1 Site integration frame; first use primers 19 and primers 20, and then use primers 21 and primers 22 to amplify ROX1 Site knockout cassette; first use primer 15 and primer 16, then use primer 17 and primer 18, and finally use primer 25 and primer 26 to amplify and obtain YPRC tua3 Then insert the YORW delta22 The site integration frame was introduced into the yeast strain 51H1, and the recombinant Saccharomyces cerevisiae strain 51H3 was obtained after screening and verification. DPP1 Site integration box and ROX1 The knockout cassettes were sequentially introduced into the recombinant Saccharomyces cerevisiae strain 51H3 and screened to obtain the recombinant Saccharomyces cerevisiae strain 51H5. YPRC tua3 The site integration frame was introduced into the recombinant Saccharomyces cerevisiae strain 51H5, and the final recombinant Saccharomyces cerevisiae strain 51H6-XI was obtained after screening and verification.

[0025] The genotype of the Saccharomyces cerevisiae strain is as follows: XK , gre3::PPP , Cox4 Δ, AE,HO:: P PGK1 -CCBOS- T CYC1 , ERG9∷ P CTR3 -ERG9 , YORWdelta22 ∷ P TEF1 - tHMG1 -T ADH1 , DPP1∷ P HHF2 -ERG20- T TEF1 ,△ ROX1 , YPRCtua3∷ P PGK1 -ERG20- GGGS -CCBOS- T CYC1 ,pJX7-XI,P TEF1 - Ru-xylA -T PGK1 .

[0026] The genes CCBOS See SEQ ID No. 38 for the sequence, which is derived from artichoke and functions to catalyze the conversion of the sesquiterpene precursor farnesyl pyrophosphate (FPP) into the sesquiterpene α-bisabolol. CCBOS outside, tHMG1 , ERG20 , ROX1 , ERG9 These are genes that exist in Saccharomyces cerevisiae itself.

[0027] The above strains were subjected to shake flask fermentation tests to detect their sesquiterpene α-bisabolol production capacity. The results showed that the sesquiterpene α-bisabolol production of strains 51H1, 51H3, 51H5, and 51H6-XI was 52.789, 107.172, 147.729, and 186.308 mg / L, respectively (e.g. Figure 3 as shown).

[0028] Table 1 List of primers used in PCR Example 4 Detection of the yield of sesquiterpene α-bisabolol A single colony of the strain 51H6-XI to be tested was inoculated into 5 mL of SX-Ura liquid medium, cultured at 30°C and 200 rpm for 24 h, and then transferred to 10 mL of SX-Ura liquid medium for secondary activation for 12 h. The activated strain was inoculated into a 100 mL Erlenmeyer flask containing 20 mL of SX-Ura liquid medium, and 20% volume (4 mL) of dodecane was added. The initial inoculation OD value was 0.2, and the flask was sealed with a cotton plug. The flask was cultured at 30°C and 200 rpm for 60 h / 72 h / 84 h. The dodecane layer was centrifuged at 13,000 rpm for 10 min, transferred to a gas phase vial, and stored at -20°C. Sesquiterpene α-bisabolol was detected by gas chromatography (GC), and the sesquiterpene α-bisabolol content of the sample was calculated according to the standard curve.

[0029] 1. Preparation of standard curve: Add 4 mL of dodecane to a 5 mL volumetric flask, then accurately weigh 6 mg of sesquiterpene α-bisabolol and dissolve it in dodecane. Make the volume of dodecane to 5 mL and mix thoroughly to prepare a 1.20 mg / mL α-bisabolol standard stock solution. Then, perform a 5-fold gradient dilution to obtain sesquiterpene α-bisabolol standard solutions with concentrations of 0.24, 0.048, 0.0096, and 0.00192 mg / mL, respectively. Perform GC-MS or GC / FID detection and draw a standard curve based on the peak area of ​​different concentrations of the standard and the corresponding concentration. R 2 ≥0.99.

[0030] 2. GC detection method: Agilent GC system was used to qualitatively detect the sesquiterpene α-bisabolol standards and samples, and to quantitatively detect the experimental samples; GC chromatographic conditions: a DB-FFAP column (specifications: 30 m×0.25 mm×0.25 µm) was used; the sample injection volume was 1 µL, the flow rate was 0.78 mL / min, and the split ratio was set to 10:1; the initial temperature of the column oven was 60°C, the injection port temperature was set to 260°C, and the detector temperature was set to 280°C; programmed temperature conditions: the instrument was maintained at an initial temperature of 60°C for 2 min, then heated to 150°C at a rate of 20°C / min, maintained for 3 min, and then heated to 250°C at a rate of 10°C / min, maintained for 3 min, and the carrier gas was helium.

[0031] The results are as follows Figure 4 As shown, Figure 4 A is the GC spectrum of the sesquiterpene α-bisabolol standard. Figure 4 B is the GC spectrum of the control strain BSPX051, Figure 4 C is the GC spectrum of strain 51H6-XI, Figure 4 A shows that the peak time of the sesquiterpene α-bisabolol product is around 14.478 min. Figure 4C determined that strain 51H6-XI could produce the sesquiterpene α-bisabolol; Figure 4 B shows that the starting strain does not have the ability to produce the sesquiterpene α-bisabolol; by drawing a standard curve and integrating the peak area, further quantitative analysis was performed to determine the sesquiterpene α-bisabolol production of the strain.

[0032] Example 5 Aerobic shake flask fermentation of recombinant Saccharomyces cerevisiae strain 51H6-XI The ability of the recombinant Saccharomyces cerevisiae strain 51H6-XI to produce the sesquiterpene α-bisabolol was tested under aerobic conditions in a shake flask using the SX-Ura medium mentioned in Example 1, and a strain transformed with the pJFE3 plasmid was used as a control. The recombinant Saccharomyces cerevisiae strain 51H6-XI was activated in 10 mL SX-Ura liquid medium for 24 h before fermentation experiments. The fermentation conditions were as follows: the culture temperature was 30°C, 20 mL SX-Ura medium was placed in a 100 mL shake flask, the shaker speed was 200 rpm, the flask was sealed with a cotton plug, the initial inoculation OD value was 0.2, 20% volume of the fermentation broth was added with dodecane to extract the sesquiterpene α-bisabolol, and the culture was continued for 84 h ( Figure 5 A). During the fermentation process, 200 μL of the dodecane layer was taken at 60 h, 72 h, and 84 h, and the yield of sesquiterpene α-bisabolol was quantified according to the GC method described in Example 4. The fermentation experiment was repeated three times, and the data were averaged for calculation. The changes in the accumulation of sesquiterpene α-bisabolol and the growth of the strain during the fermentation of strain 51H6-XI are shown in Figure 2. Figure 5 As shown in Figure 2, when fermented with 20 g / L xylose as the carbon source, no sesquiterpene α-bisabolol was detected in the fermentation metabolites of the control strain ( Figure 4 B), while the presence of sesquiterpene α-bisabolol was detected in strain 51H6-XI ( Figure 4 C), and the yield increases with the extension of the culture time ( Figure 5 B). Strain 51H6-XI reached the maximum growth point at 72 h, and the sesquiterpene α-bisabolol production also reached a maximum of 186.308 mg / L at 72 h. After 72 h, the production of sesquiterpene α-bisabolol began to decline. This proves that in the starting strain BSPX051 ( XK , gre3::PPP , Cox4 Δ, AE Heterologous expression of the sesquiterpene α-bisabolol synthase gene in CCBOS and xylose isomerase gene Ru- xyA The 51H6-XI strain obtained after metabolic pathway optimization has good xylose metabolism ability and can synthesize the sesquiterpene α-bisabolol using xylose as the sole carbon source.

Claims

1. A Saccharomyces cerevisiae strain for producing the sesquiterpene α-bisabolol from xylose, characterized in that: The genotype of the Saccharomyces cerevisiae strain is as follows: XK , gre3::PPP , Cox4 Δ, AE,HO:: P PGK1 -CCBOS- T CYC1 , ERG9∷ P CTR3 -ERG9 , YORWdelta22∷ P TEF1 - tHMG1 -T ADH1 , DPP1∷ P HHF2 -ERG20- T TEF1 ,△ ROX1 , YPRCtua3∷ P PGK1 -ERG20- GGGS -CCBOS- T CYC1 ,pJX7-XI,P TEF1 - Ru-xylA -T PGK1 。 2. The Saccharomyces cerevisiae strain for producing the sesquiterpene α-bisabolol using xylose according to claim 1, characterized in that: The construction method of the strain comprises: The Saccharomyces cerevisiae haploid BSPX051 ( XK , gre3::PPP , Cox4 Δ, AE ) was used as the starting strain, and the xylose isomerase gene Ru- xyA The expression plasmid pJX7-XI was transformed into strain 51H-XI, and then a promoter engineering strategy was adopted to use the copper ion responsive promoter P CTR3 Put it ERG9 The promoter of the gene was replaced in situ to obtain a promoter-replaced strain, and then the promoter-replaced strain 51H-XI was cloned into HO , YORWdelta2 , DPP1 , YPRC tua3 Insertion of sites CCBOS , tHMG1 , ERG20 , ERG20 -GGGS- CCBOS , and knocking out regulatory factors ROX1 The recombinant Saccharomyces cerevisiae strain 51H6-XI was obtained, namely, the recombinant Saccharomyces cerevisiae strain capable of producing the sesquiterpene α-bisabolol by utilizing xylose was obtained.

3. Use of the Saccharomyces cerevisiae strain for producing the sesquiterpene α-bisabolol by utilizing xylose as claimed in claim 1 in fermentation production of the sesquiterpene α-bisabolol.

4. Use of the Saccharomyces cerevisiae strain for producing the sesquiterpene α-bisabolol by utilizing xylose as claimed in claim 1 in fermentation production of sesquiterpene α-bisabolol derivative products.

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