Engineered yeast strain with high yield of citronellol as well as preparation method and application of engineered yeast strain

Abstract

The invention relates to the technical field of biology, in particular to engineered yeast for high-yield citronellol as well as a preparation method and application of the engineered yeast. According to the invention, genetically engineered bacteria for high yield of citronellol are constructed by means of genetic engineering, SyBESc081300004 is taken as an original strain, Erg20ww, Erg8, tCrGES and CrIS are expressed in a peroxisome region in a room manner, and phosphopentose pathway genes TAL1 and TKL1 and endogenous transporter protein PDR1 are over-expressed, so that the genetically engineered bacteria are obtained; experimental results show that the shake flask fermentation yield of the genetically engineered bacterium reaches 3.38 g / L, the yield of a 100L fermentation tank reaches 10.556 g / L, and the yield of the genetically engineered bacterium is obviously higher than that of other existing citronellol production engineered bacterium.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a yeast engineering bacterium for high-yield citronellol, a preparation method thereof, and an application thereof. Background Art

[0002] Citronellol (3,7-dimethyl-6-octen-1-ol) is an acyclic monoterpenol. Due to its elegant aroma and the property of being easily esterified with other substances, it is widely used in the cosmetics and fragrance industries. At the same time, because citronellol has good antibacterial and biological activities, it also shows potential application values in the fields of skin care products, hygiene products, as well as medicine and pesticides. Citronellol naturally exists in rose, geranium, and citronella essential oil. Especially the Java citronella essential oil is one of the efficient sources of natural citronellol. The Java citronella essential oil extracted from the leaves can produce a natural citronellol content of up to 20%. Through fine separation and purification techniques, high-purity citronellol products can be extracted from these plant essential oils.

[0003] Currently, citronellol is mainly obtained through plant extraction and chemical synthesis. However, these methods are limited by the raw material yield and product purity, and the process of microbial synthesis of citronellol has received extensive attention in the market. In recent years, people have deeply analyzed the biosynthetic pathway of citronellol. Its biosynthetic pathway takes glucose as a substrate, generates geraniol via the MVA pathway, and geraniol is reduced by reductase to form citronellol. In 2021, Li et al. overexpressed the MVA pathway genes in Saccharomyces cerevisiae, fused ObGES-Erg20 ww , overexpressed the endogenous OYE2 under the control of the TEF1 promoter and replaced the natural promoter of Erg20 ww with Erg7, and achieved 360 mg / L of citronellol at the shake flask level and 6.37 g / L of citronellol at the 5 L bioreactor level.

[0004] So far, for the biosynthesis of citronellol, there are still many limitations in its in vivo metabolic pathway. Gerke et al. reported that 200 mg / L of geraniol would cause complete inhibition of cell growth, and citronellol also has a certain toxic effect on cells. However, the mechanism by which monoterpenoids cause toxicity to microbial cells is still being explored. Therefore, in view of the current cell toxicity and the need to further improve the yield of citronellol, it is particularly necessary to develop new engineering strains. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a yeast engineering bacterium for high-yield citronellol, a preparation method thereof, and an application thereof.

[0006] The present invention provides a genetically engineered bacterium, which is obtained by transforming a chassis bacterium. The transformation includes:

[0007] Overexpression of Erg20 ww and Erg8; and / or

[0008] Overexpression of IDI1 and tHMGR; and / or

[0009] Peroxisomal compartmentalized expression of Erg20 ww 、Erg8、tCrGES and / or CrIS; and / or

[0010] Overexpression of TAL1, TKL1, GND1, ZWF1, YEF1 and / or POS5Δ17; and / or

[0011] Overexpression of YMR275C F384C 、YBR074W T838N 、YBR172C K404Q 、YHR007C G466R and / or YMR275C-N714;

[0012] Overexpression of PDR1, PDR15 and / or AUS1.

[0013] Furthermore, the present invention provides a genetically engineered bacterium, which is obtained by modifying a chassis bacterium, and the modification includes:

[0014] Overexpression of Erg20 ww and Erg8; and / or

[0015] Peroxisomal compartmentalized expression of Erg20 ww 、Erg8、tCrGES and / or CrIS; and / or

[0016] Overexpression of TAL1 and TKL1; and / or

[0017] Overexpression of YMR275C F384C 、YBR074W T838N 、YBR172C K404Q 、YHR007C G466R and / or YMR275C-N714;

[0018] Overexpression of PDR1, PDR15 and / or AUS1.

[0019] Even further, the present invention provides a genetically engineered bacterium, which is obtained by modifying a chassis bacterium, and the modification includes:

[0020] Peroxisomal compartmentalized expression of Erg20 ww 、Erg8、tCrGES and / or CrIS; and / or

[0021] Overexpression of TAL1 and TKL1; and / or

[0022] Overexpress PDR1.

[0023] In specific embodiments of the present invention, the present invention provides a genetically engineered bacterium, which is obtained by modifying a chassis bacterium, and the modification is optimal when it includes the following:

[0024] Peroxisomal compartmentalized expression of Erg20 ww , Erg8, tCrGES, and CrIS; and

[0025] Overexpress TAL1 and TKL1; and

[0026] Overexpress PDR1.

[0027] Among them, the peroxisomal compartmentalized expression of Erg20 ww , Erg8, tCrGES, and / or CrIS is the fusion of the signal peptide SKL at the C-terminus of Erg20 ww , Erg8, tCrGES, and / or CrIS; the signal peptide SKL is a peroxisomal targeting signal peptide, and its main function is to guide proteins into the peroxisomes of cells to complete the peroxisomal compartmentalized expression of the above gene products.

[0028] In the present invention, the chassis bacterium includes yeast, algae, mold streptomyces, and / or bacteria;

[0029] The yeast includes Saccharomyces cerevisiae, Yarrowia lipolytica, and / or Kluyveromyces;

[0030] The bacteria include Escherichia coli and / or Bacillus subtilis.

[0031] Furthermore, the chassis bacterium is Saccharomyces cerevisiae; the Saccharomyces cerevisiae includes, but is not limited to, the CEN.PK series or the BY series.

[0032] Even further, the chassis bacterium is Saccharomyces cerevisiae optimized by ERG20 mutation, protein fusion, and protein scaffold strategies.

[0033] Specifically, the chassis bacterium is Saccharomyces cerevisiae that overexpresses IDI1, tHMGR, Erg10, Erg13, Erg12, and Erg19 and introduces GES and CrIS.

[0034] In a specific embodiment of the present invention, the chassis bacterium is one in which a T ADH1 -IDI1-P GAL1 / 10 -tHMGR-T TDH2 -His3 module is inserted at the Gal80 locus, the genomic Erg20 gene is singly mutated to Erg20 F96W , and a T GPM1-P GAL7 -GE-T GPD -P GAL10 -CrIS-GSG-PDZ-T FBA1 Module with T inserted at the Atf1 gene locus FBA1 -P GAL1 -IDI1-GSG-SH3-T TDH2 -P GAL7 -SF1-T GPD Module with T inserted at the Int11 gene locus FBA1 -Erg10-P GAL1 / 10 -Erg13-T TDH2 Module with T inserted at the Int14 gene locus FBA1 -Erg12-P GAL1 / 10 -Erg19-T TDH2 Module of Saccharomyces cerevisiae (SyBE_Sc08130004).

[0035] In the specific embodiments of the present invention, the genetically engineered bacteria are obtained through continuous attempts and optimizations; in the specific optimization process of the present invention, using SyBE_Sc08130004 as the chassis bacterium, through the following optimization comparisons, the optimal engineering strain is finally obtained:

[0036] (1) Overexpress Erg20 on the basis of SyBE_Sc08130004 ww and the combination of Erg8 (engineered bacterium Sc001). After overexpressing the combination of IDI1 and tHMGR on the basis of Sc001 (engineered bacterium Sc002), it was found that overexpressing IDI1 and tHMGR on the basis of Sc001 instead reduced the citronellol yield;

[0037] (2) On the basis of SyBE_Sc08130004, peroxisomal compartmentalized expression of Erg20 ww , Erg8, tCrGES and CrIS to obtain the engineered bacterium Sc003, and the citronellol yield was further improved compared with Sc001;

[0038] (3) On the basis of Sc003, explore the effects of TAL1, TKL1, GND1, ZWF1, NADH kinase YEF1 and POS5Δ17 on the citronellol yield. The results show that the engineered bacterium Sc004 with overexpressed TAL1 and TKL1 is the best;

[0039] (4) On the basis of Sc004, the effects of truncation of YMR275C, single-point mutation of YMR275C, and combined mutations of YBR074W, YBR172C, YHR007C, and YMR275C on citronellol production were investigated. The results showed that the above-mentioned modifications did not significantly increase citronellol production on the basis of Sc004.

[0040] (5) On the basis of Sc004, the effects of PDR1, PDR15, and AUS1 on citronellol production were investigated. The results showed that the strain Sc011 overexpressing PDR1 had the highest citronellol production. Among them, the yield in shake-flask fermentation reached 3.38 g / L, and the yield in a 100-L fermenter reached 10.556 g / L, which was significantly higher than that of other existing engineered citronellol-producing strains.

[0041] The present invention provides a method for preparing the above-mentioned genetically engineered bacterium, which comprises the following modifications in the chassis bacterium:

[0042] Overexpressing Erg20 ww and Erg8; and / or

[0043] Overexpressing IDI1 and tHMGR; and / or

[0044] Compartmentalized expression of Erg20 in peroxisomes ww 、Erg8、tCrGES and / or CrIS; and / or

[0045] Overexpressing TAL1, TKL1, GND1, ZWF1, YEF1 and / or POS5Δ17; and / or

[0046] Overexpressing YMR275C F384C 、YBR074W T838N 、YBR172C K404Q 、YHR007C G466R and / or YMR275C-N714;

[0047] Overexpressing PDR1, PDR15 and / or AUS1.

[0048] The present invention provides the use of the above-mentioned genetically engineered bacterium in the preparation of citronellol.

[0049] The present invention provides a product of citronellol, the production raw material of which comprises the genetically engineered bacterium described in the present invention.

[0050] The present invention provides a method for preparing citronellol, which uses the genetically engineered bacterium described in the present invention to obtain citronellol.

[0051] Furthermore, the preparation method comprises the following steps: fermenting the genetically engineered bacterium described in the present invention to obtain the citronellol.

[0052] The present invention constructs a genetically engineered bacterium with high-yield citronellol by genetic engineering means. The genetically engineered bacterium uses SyBE_Sc08130004 as the starting strain and expresses Erg20, Erg8, tCrGES, and CrIS through peroxisome compartmentalization, and overexpresses the pentose phosphate pathway genes TAL1, TKL1 and the endogenous transporter PDR1 transporter to obtain; the experimental results show that the yield of the genetically engineered bacterium in shake flask fermentation reaches 3.38 g / L, and the yield in a 100 L fermenter reaches 10.556 g / L, which is significantly higher than other existing citronellol-producing engineering bacteria. ww BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shows the plasmid map of pZK003;

[0054] Figure 2 Shows the effect of enhancing the MVA pathway on citronellol yield;

[0055] Figure 3 Shows the citronellol yield result map of peroxisome localization of some genes in the citronellol synthesis pathway;

[0056] Figure 4 Shows the result map of the effect of enhancing NADPH supply on citronellol yield;

[0057] Figure 5 Shows the result map of the effect of overexpressing monoterpene tolerance genes on citronellol yield;

[0058] Figure 6 Shows the result map of the effect of integrating endogenous transporters on citronellol yield;

[0059] Figure 7 Shows the result map of fed-batch fermentation in a 100 L fermenter. DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention provides a yeast engineering bacterium with high-yield citronellol, its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate changes and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0061] Table 1. Primers

[0062]

[0063] ​

[0064] The amino acid sequence of the signal peptide SKL is: SKL (SEQ ID NO: 42);

[0065] The GeneBank ID of the coding gene of Erg20 is: 853272;

[0066] Erg20 ww is Erg20 F96W-N127W specifically, the 96th F of Erg20 is mutated to W and the 127th N is mutated to W;

[0067] The GeneBank ID of the coding gene of Erg8 is: 855260;

[0068] The GeneBank ID of the coding gene of IDI1 is: 855986;

[0069] The GeneBank ID of the coding gene of tHMGR is: 854900;

[0070] The GeneBank ID of the coding gene of tCrGES is: KF561459.1;

[0071] The GeneBank ID of the coding gene of CrIS is: JX974564;

[0072] The GeneBank ID of the coding gene of TAL1 is: 851068;

[0073] The GeneBank ID of the coding gene of TKL1 is: 856188;

[0074] The GeneBank ID of the coding gene of GND1 is: 856589;

[0075] The GeneBank ID of the coding gene of ZWF1 is: 855480;

[0076] The GeneBank ID of the coding gene of the NADH kinase enzyme YEF1 is: 856670;

[0077] POS5Δ17 is the mitochondrial targeting sequence (amino acid sequence positions 2 - 17) deleted from POS5; the GeneBank ID of the coding gene of POS5 is: 855913;

[0078] The GeneBank ID of the coding gene of YMR275C is: 855318;

[0079] tBUL1 is a truncated form of YMR275C. Among them, the GeneBank ID of the coding gene of YMR275C is 855318, and the nucleotide sequence of the truncated form is the 1-2142 bp nucleotide region of the coding gene of YMR275C;

[0080] YMR275C F384C , also written as YMR275C mutated , is the F-to-C mutation at the 384th position of the product encoded by the YMR275C gene;

[0081] YBR074W T838N , also written as YBR074W mutated , is the T-to-N mutation at the 838th position of YBR074W. Among them, the GeneBank ID of the coding gene of YBR074W is: 852366;

[0082] YBR172C K404Q , also written as YBR172C mutated , is the K-to-Q mutation at the 404th position of YBR172C; Among them, the GeneBank ID of the coding gene of YBR172C is: 852470;

[0083] YHR007C G466R , also written as YHR007C mutated , is the G-to-R mutation at the 466th position of YHR007C. Among them, the GeneBank ID of the coding gene of YHR007C is: 856398;

[0084] The GeneBank ID of the coding gene of PDR1 is: 852871;

[0085] The GeneBank ID of the coding gene of PDR15 is: 852015;

[0086] The GeneBank ID of the coding gene of AUS1 is: 854175;

[0087] The LEU gene element refers to the gene related to leucine metabolism in yeast and its regulatory elements, and is used as a selection marker;

[0088] Peroxisome compartmentalized expression is usually achieved by fusing a peroxisome targeting signal peptide to the C-terminus or N-terminus of the target gene, enabling the protein to be transported into the peroxisome for compartmentalized or targeted expression.

[0089] Starting from the recombinant Saccharomyces cerevisiae SyBE_Sc08130004 that produces citronellol, using systematic engineering strategies (enhancing the MVA pathway, peroxisomal targeting, regulating endogenous cofactor balance) and chassis cell optimization (tolerance engineering, transporter engineering) means, further optimize the synthesis of citronellol to obtain a Saccharomyces cerevisiae strain that stably and highly produces citronellol. The present invention determines the necessity of precursor GPP supply and NADPH balance for citronellol synthesis, as well as the feasible strategies to alleviate monoterpene cytotoxicity, which can provide a basis for the biosynthesis of other monoterpene compounds.

[0090] A method for optimizing the production of a recombinant Saccharomyces cerevisiae strain that produces citronellol, comprising the following steps: (1) Using a strain with high citronellol production in the laboratory as the starting strain, integrating Erg20 ww and Erg8 at the YPL062W locus to further enhance the utilization rate of acetyl-CoA in the cytoplasm, improve the GPP metabolic flux, and thus increase the citronellol yield; (2) After overexpressing the MVA pathway genes, it was found that the growth of the strain was inhibited, so ERG20 ww , ERG8, CrGES, and CrIS were all targeted to peroxisomes to alleviate cytotoxicity and restore cell growth; (3) Overexpressing pentose phosphate pathway genes and NADH kinases (YEF1, POS5) to enhance the intracellular NADPH supply; (4) To alleviate the cytotoxicity of monoterpenes, overexpress monoterpene tolerance-related genes and endogenous transporters, and it was found that the PDR1 transporter maximizes the citronellol yield; (5) Batch-fed fermentation of the high citronellol-producing strain in a 100 L fermenter;

[0091] All the test materials used in the present invention are ordinary commercially available products and can all be purchased in the market. The following further elaborates the present invention in conjunction with the examples:

[0092] Example 1 Construction of a strain integrating the enhanced MVA pathway - The first step of optimization

[0093] 1. Chassis bacteria

[0094] The strain number of the chassis bacteria is SyBE_Sc08130004, provided by the Yuan Yingjin research group. The genetic modifications included in this recombinant Saccharomyces cerevisiae are: inserting T ADH1 -IDI1-P GAL1 / 10 -tHMGR-T TDH2 -His3 module at the Gal80 locus, and the single mutation of the genomic Erg20 gene to Erg20 F96W , inserting T GPM1 -P GAL7 -GE-T GPD -P GAL10 -CrIS-GSG-PDZ-T FBA1Module, T inserted at the Atf1 gene locus FBA1 -P GAL1 -IDI1-GSG-SH3-T TDH2 -P GAL7 -SF1-T GPD Module, T inserted at the Int11 gene locus FBA1 -Erg10-P GAL1 / 10 -Erg13-T TDH2 Module, T inserted at the Int14 gene locus FBA1 -Erg12-P GAL1 / 10 -Erg19-T TDH2 Module.

[0095] 2. Construction of an enhanced MVA pathway integration strain

[0096] Erg20 ww and Erg8, IDI1 and tHMGR were divided into two groups and inserted into the tool expression cassette GQ416K-1 / 10 respectively. Then Erg20 ww and the two expression cassettes of Erg8, IDI1 and tHMGR were integrated into the genomic loci YPL062W and exg1 using the CRISPR / Cas9 technology.

[0097] Construction of the gRNA-Cas9 plasmid: Based on the plasmid pZK003( Figure 1 , also written as pZK003-BsaI-RFP-BsaI), a Cas9 plasmid containing gRNA was constructed. Primers were designed with the mutation site within the PAM sequence or 20 bp before it. Then the two primers were annealed and bonded to form double-stranded DNA. The annealed double-stranded DNA was ligated to pZK003 using Golden gate assembly to obtain the gRNA-Cas9 plasmid. The reaction system was transformed into TOP10 Escherichia coli competent cells and spread on an LB+Kan R plate and cultured overnight at 37°C. Single colonies that were not bright under the fluorescence imager were picked for sequencing to verify whether the gRNA was successfully inserted.

[0098] First, the tool expression cassette GQ416K-1 / 10 was digested with NotI enzyme to obtain a nick in the middle to obtain a linearized vector. Homologous arms of 20 bp each at the two nicks of the vector were added to Erg20 gal1 -T TDH1 through the upstream and downstream primers. ww (ERG20 ww -F and ERG20 ww-R), both ends of tHMGR (tHMGR-F and tHMGR-R) were obtained with the help of a PCR instrument, connected in vitro by seamless cloning method, the ligation system was introduced into TOP10 competent cells together, transformants were selected for amplification culture to extract plasmids for sequencing. After the plasmid with correct sequencing was digested with EcoRI enzyme, a cut was obtained in the middle to obtain a linearized vector. Homologous arms of 20 bp at each of the two cut sites of the vector were added to both ends of Erg8 (ERG8-F and ERG8-R), IDI1 (IDI1-F and IDI1-R) by upstream and downstream primers obtained with the help of a PCR instrument. Two final expression cassettes were obtained by the same method. Using the CRISPR / Cas9 technology, the expression cassette was overlapped with 500 bp homologous arms upstream and downstream of YPL062W and exg1 to obtain the fusion fragment YPL062Wup-P gal10 -T FBA1 -Erg20 gal1 -Erg20 ww -T TDH1 -P gal10 -Erg8-T FBA1 -YPL062Wdown and exg1up-P gal1 -tHMGR-T TDH1 -Pg al10 -IDI1-T FBA1 -exg1down, the fusion fragment YPL062Wup-P gal1 -Erg20 ww -T TDH1 -P gal10 -Erg8-T FBA1 -YPL062Wdown and the cas9 plasmid at the corresponding site were transformed into SyBE_Sc08130004 to obtain Sc001. Subsequently, the fusion fragment exg1up-P gal1 -tHMGR-T TDH1 -Pg al10 -IDI1-T FBA1-exg1down and the corresponding site cas9 plasmid were transformed into Sc001 to obtain Sc002. After transformation, SC-URA solid plates (synthetic yeast nitrogen source YNB 6.7 g / L, glucose 20 g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2 g / L, 2% agar powder, 0.02 g / L tryptophan, 0.1 g / L leucine, 0.02 g / L histidine) were used for screening. The obtained transformants were subjected to PCR verification by boiling bacteria with 20 mM NaOH solution. The correct single colonies were used to lose plasmids on 5-FOA solid plates (glucose 20 g / L, YNB 6.7 g / L, lacking amino acid powder mixture 2 g / L, leucine 0.1 g / L, histidine 0.02 g / L, tryptophan 0.02 g / L, uracil 0.05 g / L, 5-FOA 1 mg / mL, 2% agar powder). Finally, the bacteria with successful plasmid loss were isolated and purified for fermentation detection.

[0099] The control strain number is SyBE_Sc08130004, and the experimental bacteria are numbered as Sc001 (Erg20 ww and Erg8) and Sc002 (Erg20 ww and Erg8, IDI1 and tHMGR).

[0100] 3. Geraniol fermentation detection

[0101] Test materials: strains SyBE_Sc08130004, Sc001, Sc002

[0102] Test method:

[0103] Seed medium: YPD liquid medium (yeast extract powder 10 g / L, peptone 20 g / L, glucose·H2O 22 g / L);

[0104] Fermentation medium: YPD liquid medium (yeast extract powder 10 g / L, peptone 20 g / L, glucose·H2O 22 g / L).

[0105] The above strains were inoculated into 3 mL of seed medium and cultured at 30 °C and 250 rpm for 24 h. The primary seeds were inoculated into 3 mL of seed medium at an initial cell concentration OD 600 = 0.2 and cultured at 22 °C and 250 rpm for 12 - 15 h. Then the secondary seeds were inoculated into 5 mL of YPD fermentation medium at OD 600 = 0.2, and 20% volume of IPM was added. The culture was carried out at 30 °C and 200 rpm for 120 h. Anhydrous ethanol was added once at 24 h and 48 h of fermentation. The cell density (OD 600 ) at the end of fermentation and the yield of geraniol were monitored.

[0106] Quantitative determination of citronellol: After fermentation, pour the fermentation broth into a 10 mL centrifuge tube and centrifuge at 12,000 rpm for 5 minutes. Remove the upper organic phase using a pipette and transfer it to a 2 mL EP tube. Add an appropriate amount of anhydrous sodium sulfate to remove water and let it stand for approximately 20 minutes. Some of the anhydrous sodium sulfate in the EP tube should be powdery. After dehydration, dilute the mixture 21-fold with n-hexane. Remove the upper organic phase using a 1 mL syringe and filter through a 0.22 μm organic filter membrane into a clean EP tube. This completes the product treatment process. The product was analyzed by GC-MS using a DB-5MS (30 m × 0.25 μm × 0.25 μm) capillary column for separation. The GC conditions were as follows: column oven temperature 70°C, inlet temperature 260°C, injection split ratio 50, and column flow rate 1 mL / min. The temperature program was as follows: 70°C for 1 minute; 30°C / min to 180°C, hold for 1 minute; 50°C / min to 265°C, hold for 6 minutes. Mass spectrometry conditions were as follows: ion source temperature of 230°C, interface temperature of 270°C, solvent delay time of 2 minutes, and scan range of m / z 50-500. Citronellol's characteristic ion was 138, while reference ions were 55 and 69. The peak elution time was around 4.89.

[0107] The test results are as follows Figure 2 As shown, overexpression of Erg20 ww and Erg8 can increase citronellol production (Sc001). In addition, overexpression of IDI1 and tHMGR on the basis of Sc001 instead reduces citronellol production (Sc002).

[0108] Example 2: Investigating the effect of peroxisome localization on citronellol production

[0109] The biomass of strain Sc001 was found to be reduced by 33%. To solve this problem, Erg20 ww , Erg8, tCrGES and CrIS are localized to peroxisomes.

[0110] 1. Construction steps:

[0111] The present invention uses the C-terminal signal peptide SKL to ww , Erg8, tCrGES, and CrIS are localized to peroxisomes, and the signal peptide SKL is added to Erg20 by designing specific primers. ww (ERG20 ww -F and ERG20 wwSKL-R), Erg8 (ERG8-F and ERG8SKL-R), tCrGES (tCrGESSKL-F and tCrGESSKL-R), the C-terminus of CrIS (CrISSKL-F and CrISSKL-R). By using the seamless cloning method, Erg20 ww SKL and Erg8 SKL , tCrGES SKL and CrIS SKL were divided into two groups and inserted into the tool expression cassette GQ416K-1 / 10 to obtain plasmid 416K-T FBA1 -Erg20 ww SKL -P GAL1 / 10 -Erg8 SKL -T TDH2 and 416K-T FBA1 -tCrGES SKL -P GAL1 / 10 -CrIS SKL -T TDH2 . Using the above two plasmids as templates, the homologous arms of YPL062W and DPP1 were introduced into the left and right ends of the above two plasmid elements by PCR respectively to obtain the expression cassettes YPL062Wup-T FBA1 -Erg20 ww SKL -P GAL1 / 10 -Erg8 SKL -T TDH2 -YPL062Wdown, DPP1up-T FBA1 -tCrGES SKL -P GAL1 / 10 -CrIS SKL -DPP1down. The expression element fragments and the corresponding site cas9 plasmid were transformed into SyBE_Sc08130004 together by the lithium acetate method. After transformation, SC-URA solid plates were used for screening. The obtained transformants were subjected to boiling bacteria PCR verification with 20 mM NaOH solution. The correct single colonies were plasmid-lost on 5-FOA solid plates, and finally strain Sc003 was obtained.

[0112] II. Geraniol fermentation detection

[0113] Geraniol fermentation detection was carried out on Sc001 and Sc003, and the detection steps were as in Example 1. The test results were as Figure 3 shown. Locating some genes in the geraniol synthesis pathway to the peroxisome could effectively restore cell growth, increasing the biomass of the strain by 20% and raising the geraniol yield to 2.45 g / L.

[0114] Example 3 explores the effects of TAL1, TKL1, GND1, ZWF1, NADH kinase YEF1, and POS5Δ17 on citronellol production

[0115] I. Construction steps:

[0116] Using Sc003 as the starting strain for transformation. Insert TAL1 (TAL1-F and TAL1-R), TKL1 (TKL1-F and TKL1-R), GND1 (GND1-F and GND1-R), ZWF1 (ZWF1-F and ZWF1-R), NADH kinase YEF1 (YEF1-F and YEF1-R), and POS5Δ17 (POS5Δ17-F and POS5Δ17-R) into the tool expression cassette GQ416K-1 / 10, T HXT7 -P TDH3 -T TDH1 ,P GAL10 -T GPD . Connect them in vitro using seamless cloning method, and import the ligation system into TOP10 together to obtain plasmid GQ416K-T FBA1 -TAL1-P GAL1 / 10 -TKL1-T TDH2 ,GQ416K-T FBA1 -GND1-P GAL1 / 10 -ZWF1-T TDH2 ,pRS416-T HXT7 -P TDH3 -YEF1-T TDH1 and pRS416-P GAL10 -POS5Δ17-T GPD . Using the above four plasmids as templates, introduce DAK2#ZNF1, EXG1, and delta22 homologous arms into the left and right ends of the above four plasmid elements respectively by PCR, to obtain expression cassettes DAK2#ZNF1up-T FBA1 -TAL1-P GAL1 / 10 -TKL1-T TDH2 -DAK2#ZNF1down, EXG1up-T FBA1 -GND1-P GAL1 / 10 -ZWF1-T TDH2 -EXG1down, EXG1up-T FBA1 -GND1-P GAL1 / 10 -ZWF1-T TDH2 -EXG1down, delta22up-T HXT7 -P TDH3 -YEF1-T TDH1 -delta22down and delta22up-PGAL10 -POS5Δ17-T GPD -delta22down. The expression element fragment and the corresponding site cas9 plasmid were co-transformed into Sc003 by the lithium acetate method. After transformation, SC-URA solid plates were used for screening. The obtained transformants were boiled with 20 mM NaOH solution for PCR verification. The correct single colonies were plasmid-lost on 5-FOA solid plates. Finally, strains Sc004 (TAL1 and TKL1), Sc005 (GND1 and ZWF1), Sc006 (NADH kinase YEF1), and Sc007 (POS5Δ17) were obtained.

[0117] II. Detection of Citronellol Fermentation

[0118] The detection steps were as in Example 1. The test results were as Figure 4 shown. Overexpression of pentose phosphate pathway genes (TAL1 and TKL1, GND1 and ZWF1), NADH kinase YEF1, and POS5Δ17 could all increase the citronellol yield. Among them, the strain overexpressing the downstream genes TAL1 and TKL1 of the pentose phosphate pathway had the highest citronellol yield (Sc004), which was 2.77 g / L.

[0119] Example 4 explored the effects of truncation of YMR275C, single-point mutation of YMR275C, and combined mutations of YBR074W, YBR172C, YHR007C, and YMR275C on citronellol yield

[0120] I. Construction steps:

[0121] Further optimization was carried out based on Sc004. It was designed to truncate YMR275C (denoted as tBUL1, primers tBUL1-F and tBUL1-R), single-point mutated YMR275C (YMR275C mutated , tBUL1-F and BUL1 mutated -R), and the combination of four mutant genes (YBR074W mutated (YBR074W mutated -F and YBR074W mutated -R), YBR172C mutated (YBR172C mutated -F and YBR172C mutated -R), YHR007C mutated (YHR007C mutated -F and YHR007C mutated -R), and YMR275C mutated (YMR275C mutated -F and YMR275C mutated-R)) is inserted into the tool expression cassette T GPM1 -P GAL10 -T GPD In GQ416K-1 / 10, seamless cloning is used for ligation in vitro, and the ligation system is co-introduced into TOP10 to obtain plasmid pRS416-T GPM1 -P GAL10 -tBUL1-T GPD , pRS416-T GPM1 -P GAL10 -YMR275C mutated -T GPD , pRS416-T GPM1 -P GAL10 -YBR172C mutated -T GPD , GQ416K-T FBA1 -YBR074W mutated -P GAL1 / 10 -YHR007C mutated -T TDH2 . Using the above four plasmids as templates, the homologous arms of TKL2#TEF2, YJL064W, and LPP1 are introduced into the left and right ends of the above four plasmid elements by PCR, respectively, to obtain the expression cassette TKL2#TEF2up-T GPM1 -P GAL10 -tBUL1-T GPD -TKL2#TEF2down(①), TKL2#TEF2up-T GPM1 -P GAL10 -YMR275C mutated -T GPD -TKL2#TEF2down(②), YJL064Wup-T GPM1 -P GAL10 -YMR275C mutated -T GPD -YJL064Wdown(③) and LPP1up-T GPM1 -P GAL10 -YBR172C mutated -T GPD -LPP1down(④). The LEU gene element is obtained by PCR using the Sc001 strain genome as a template, and it is subjected to OE-PCR reaction with the XI homologous arm and the gene fragment T FBA1 -YBR074W mutated -P GAL1 / 10 -YHR007C mutated -T TDH2 to obtain the yeast transformation fragment XIup-T TDH2 -YBR074Wmutated -P GAL1 / 10 -YHR007C mutated -T FBA1 -P LEU2 -LEU2-T LEU2 XIdown(⑤).

[0122] The expression element fragments were combined into Sc004 in different combinations by the lithium acetate method. After transformation, screening was carried out using SC-URA solid plates. The obtained transformants were subjected to PCR verification by boiling bacteria with 20 mM NaOH solution. The correct single colonies were plasmid-lost on 5-FOA solid plates. Finally, strains Sc008 (containing fragment ①), Sc009 (containing fragment ②), and Sc010 (containing fragments ② + ③ + ④ + ⑤) were obtained.

[0123] II. Citronellol fermentation detection

[0124] The detection steps were as in Example 1.

[0125] Test results: As Figure 5 shown, after integrating the geraniol tolerance gene and the linalool tolerance gene into the strain genome, the citronellol yield did not increase significantly, and no obvious difference in the growth of the strain was observed by dilution spotting. Therefore, the tolerance mechanism of yeast strains to citronellol still needs to be further explored.

[0126] Example 5 explored the effects of PDR1, PDR15, and AUS1 on citronellol production.

[0127] I. Construction steps:

[0128] Based on Sc004, further optimization was carried out. Attempts were made to integrate PDR1 (PDR1-F and PDR1-R), PDR15 (PDR15-F and PDR15-R), and AUS1 (AUS1-F and AUS1-R) into the multi-copy sites of the yeast genome. The insertion cassette pRS415K-T for PDR1, PDR15, and AUS1 was designed HXT7 -P TDH3 -T TDH1 into which, and in vitro ligation was carried out by seamless cloning. The ligation system was co-introduced into TOP10 to obtain plasmids pRS415K-T HXT7 -P TDH3 -PDR1-T TDH1 、pRS415K-T HXT7 -P TDH3 -PDR15-T TDH1 、pRS415K-T HXT7 -P TDH3 -AUS1-T TDH1Using the above three plasmids as templates, the delta22 homologous arms were introduced into the left and right ends of the above three plasmid components by PCR to obtain the yeast integration fragment components delta22up-T HXT7 -P TDH3 -PDR1-T TDH1 -delta22down, delta22up-T HXT7 -P TDH3 -PDR15-T TDH1 -delta22down, delta22up-T HXT7 -P TDH3 -AUS1-T TDH1 -delta22down. The expression element fragment and the csa9 plasmid of delta22 were transferred into Sc004 by the lithium acetate method. After transformation, SC-URA solid plates were used for screening. The obtained transformants were subjected to boiling bacteria PCR verification with 20 mM NaOH solution. The correct single colonies were plasmid-lost on 5-FOA solid plates. Finally, the strains Sc011 (PDR1), Sc012 (PDR15), and Sc013 (AUS1) were obtained.

[0129] The test method and the quantitative detection method of citronellol are the same as above.

[0130] Test results: As Figure 6 shown, PDR1 increased the citronellol yield by 23%, reaching 3.38 g / L (Sc011).

[0131] Example 6 Fed-batch fermentation of strain Sc011 in a 100 L fermenter

[0132] Test materials: Strain Sc011

[0133] To explore the potential production capacity of citronellol, fed-batch fermentation culture was carried out in a 100 L bioreactor. Since the GAL promoter was used to control the expression of enzymes involved in the citronellol biosynthesis pathway, the fermentation process could be divided into a cell growth stage and a citronellol production stage. During fed-batch fermentation, the initial glucose concentration was 20 g / L, and 20 vt% isopropyl myristate was added after 6 h of fermentation to collect citronellol. After the glucose was exhausted, a glucose stock solution at 500 g / L was injected into the bioreactor at a rate of 4 - 6 g / L / h to keep the glucose concentration below 1 g / L. When the OD 600 reached 84.6 at 46 h, the cells entered the stationary phase and the remaining glucose was exhausted. Then the carbon source was changed to ethanol, and ethanol was added at a rate of 0.5 - 1 L / h to keep the concentration below 10 g / L. The OD 600 value gradually increased to 174.4 until harvest.

[0134] Test results: As Figure 7 shown, through the above fermentation conditions, the yield of citronellol reached 10.556 g / L, which is the highest titer reported in eukaryotic cells so far.

[0135] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Genetically engineered bacteria, characterized in that, Obtained by modifying chassis bacteria, and the modifications include: Overexpression of Erg20 ww and Erg8; and / or Overexpressing IDI1 and tHMGR; and / or Peroxisomal compartmentalized overexpression of Erg20 ww , Erg8, tCrGES, and / or CrIS; and / or Overexpressing TAL1, TKL1, GND1, ZWF1, YEF1 and / or POS5Δ17; and / or Overexpression of YMR275C F384C 、YBR074W T838N 、YBR172C K404Q 、YHR007C G466R and / or YMR275C-N714; and / or Overexpressing PDR1, PDR15 and / or AUS1.

2. The genetically engineered bacterium according to claim 1, characterized in that, The chassis bacteria include yeast, algae, mold Streptomyces and / or bacteria; The yeast includes Saccharomyces cerevisiae, Yarrowia lipolytica and / or Kluyveromyces; The bacteria include Escherichia coli and / or Bacillus subtilis.

3. The genetically engineered bacterium according to claim 2, characterized in that, The chassis bacteria is Saccharomyces cerevisiae.

4. The genetically engineered bacterium according to claim 3, wherein The chassis bacteria is Saccharomyces cerevisiae optimized by ERG20 mutation, protein fusion and protein scaffold strategies.

5. The genetically engineered bacterium according to claim 4, characterized in that, The chassis bacteria is Saccharomyces cerevisiae overexpressing IDI1, tHMGR, Erg10, Erg13, Erg12 and Erg19, and introducing GES and CrIS.

6. The preparation method of the genetically engineered bacterium according to any one of claims 1 to 5, characterized in that, For the following modifications in the chassis bacteria: Overexpression of Erg20 ww and Erg8; and / or Overexpressing IDI1 and tHMGR; and / or Compartmentalized expression of Erg20 in peroxisomes ww , Erg8, tCrGES, and / or CrIS; and / or Overexpressing TAL1, TKL1, GND1, ZWF1, YEF1 and / or POS5Δ17; and / or Overexpression of YMR275C F384C , YBR074W T838N , YBR172C K404Q , YHR007C G466R and / or YMR275C-N714; Overexpressing PDR1, PDR15 and / or AUS1.

7. Use of the genetically engineered bacteria according to any one of claims 1 to 6 in the preparation of citronellol.

8. A product of citronellol, characterized in that, The production raw materials include the genetically engineered bacteria according to any one of claims 1 to 6.

9. A method for preparing citronellol, characterized in that, Obtaining citronellol using the genetically engineered bacteria according to any one of claims 1 to 6.

10. The preparation method according to claim 9, characterized in that, Including the following steps: fermenting the genetically engineered bacteria according to any one of claims 1 to 6 to obtain the citronellol.

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