Engineering bacterium for synthesizing d-borneol and construction method thereof

Abstract

The invention provides a method for producing borneol, the method comprising culturing the transgenic microorganism in a culture medium, and the microorganism comprises or expresses borneol diphosphate synthetase and phosphatase.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and fermentation engineering, and particularly relates to an engineered bacterium for synthesizing dextro-borneol and a construction method and application thereof. BACKGROUND

[0002] Borneol is divided into dextro-borneol and laevo-borneol according to optical activity. Dextro-borneol is a valuable aromatic monoterpene with multiple biological activities and has been used as a food flavoring and a component of medicine. Due to its analgesic, anti-inflammatory and antibacterial activities, it has been used in traditional medicine in China, Japan and India for a long time (Granger, R. E., Campbell, E. L. & Johnston, G. A. (+)-And (-)-borneol: efficacious positive modulators of GABA action at human recombinant alpha1beta2gamma2L GABA(A) receptors. Biochem Pharmacol 69, 1101-1111 (2005).; Chen, L., Su, J., Li, L., Li, B. & Li, W. A new source of natural D-borneol and its characteristic. Journal of Medicinal Plants Research 5, 3440-3447 (2011).) According to the Pharmacopoeia of the People's Republic of China (2020), (+)-Borneol is an important component of more than 60 kinds of Chinese patent medicines.

[0003] Borneol is mainly extracted from fresh leaves of Cinnamomum camphora by steam distillation, but this method has low yield and endangers the plant resources. Due to the limited natural borneol resources, chemically synthesized borneol is widely used as a substitute for natural borneol in clinical practice and occupies most of the market share. However, chemically synthesized borneol is a mixture of (±)-Borneol (61.39%) and iso-borneol (35.77%) (Zou, L. et al. Comparison of Chemical Profiles, Anti-Inflammatory Activity, and UPLC-Q-TOF / MS-Based Metabolomics in Endotoxic Fever Rats between Synthetic Borneol and Natural Borneol. Molecules 22 (2017).), in which iso-borneol has potential toxicity. Recently, emerging synthetic biology provides a promising alternative method for producing dextro-borneol by engineering cell factories.

[0004] In 2021, Ma et al. constructed a biosynthetic pathway of dextro-borneol in Saccharomyces cerevisiae, and obtained 2.89 mg / L of dextro-borneol in a 48 h shake flask after truncating the signal peptide and adding the Kozak sequence (Ma, R. et al. Bornyl Diphosphate Synthase From Cinnamomum burmanni and Its Application for (+)-Borneol Biosynthesis in Yeast. Front Bioeng Biotechnol 9, 631863 (2021).). However, the yield of dextro-borneol is still low. Therefore, further research is needed to develop an engineered strain for efficiently synthesizing dextro-borneol. SUMMARY

[0005] The present application provides a method for producing borneol, comprising culturing the transgenic microorganism in a culture medium, wherein the microorganism comprises or expresses bornyl diphosphate synthase and phosphatase, and the microorganism is a yeast. In the present application, borneol specifically refers to dextro-borneol.

[0006] Further, the microorganism comprises or expresses borneol diphosphate synthase from Salvia officinalis L., C. burmannii, Amomum villosum, and Lippia dulcis.

[0007] Preferably, the microorganism comprises or expresses a bornyl diphosphate synthase from Salvia officinalis L., from C. burmannii and from Amomum villosum.

[0008] Preferably, the microorganism comprises or expresses a bornyl diphosphate synthase from Salvia officinalis L. and from C. burmannii.

[0009] Preferably, the microorganism comprises or expresses a bornyl diphosphate synthase from Salvia officinalis L.

[0010] Further, the genes expressing bornyl diphosphate synthase from Salvia officinalis L., from C. burmannii, from Amomum villosum and from Lippia dulcis are codon-optimized and signal peptide-truncated, the present application is improved according to the codon bias of Saccharomyces cerevisiae, and the improved method is a routine technical means in the art, for example, the above-mentioned genes are artificially synthesized according to the sequences of related genes known in the art and with reference to the codon bias of Saccharomyces cerevisiae, and any of the above-mentioned genes designed according to the codon bias of Saccharomyces cerevisiae can be used in the present application.

[0011] In some embodiments, the bornyl diphosphate synthase which is codon-optimized and signal peptide-truncated is tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS, preferably tSoBPPS, tCbTPS1, tAvBPPS.

[0012] Further, the amino acid sequences of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown in SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 109 and SEQ ID NO: 110, respectively, and the encoding nucleic acids of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 35 and SEQ ID NO: 74, respectively.

[0013] Further, the method comprises introducing the nucleic acid coding sequence of the ibp into the yeast; and / or the introducing the nucleic acid coding sequence of the ibp into the yeast is introducing an expression cassette containing the nucleic acid coding sequence of the ibp into the yeast; and / or introducing the yeast by expressing a plasmid vector containing the nucleic acid coding sequence of the ibp expression cassette; preferably introducing the yeast by integration into the yeast chromosome. In certain embodiments, the expression cassette further comprises a promoter and a terminator. For the selection of the promoter and the terminator, one skilled in the art can properly select the promoter and the terminator suitable for the corresponding engineering bacteria according to the engineering bacteria. In further embodiments, the expression cassette comprises a promoter and a terminator, and in certain embodiments, the promoter is GAL2. In certain embodiments, the terminator is PYK1.

[0014] Further, the phosphatase comprises an endogenous phosphohydrolase and a heterologous phosphohydrolase, in certain embodiments, the phosphatase is an endogenous phosphohydrolase, in certain embodiments, the phosphatase is a heterologous phosphohydrolase, in certain embodiments, the phosphatase is an endogenous phosphohydrolase and a heterologous phosphohydrolase, preferably the microorganism overexpresses the phosphohydrolase.

[0015] In certain embodiments, the endogenous phosphohydrolase is a S. cerevisiae endogenous phosphatase, the S. cerevisiae endogenous phosphatase is LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1.

[0016] The amino acid sequences of the LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1 are respectively as SEQ ID NO: 87-98, further, the coding nucleic acids of the LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1 are as SEQ ID NO: 13-24.

[0017] Preferably, the S. cerevisiae endogenous phosphatase is LPP1, DPP1, PAH1, IPP1, NPP1, NPP2, APP1.

[0018] Preferably, the S. cerevisiae endogenous phosphatase is LPP1, DPP1, PAH1, IPP1, NPP1, NPP2.

[0019] Preferably, the S. cerevisiae endogenous phosphatase is LPP1, DPP1.

[0020] In some embodiments, the microorganism of the present application overexpresses 2 copies of LPP1, in some embodiments, the microorganism of the present application overexpresses 2 copies of LPP1 and 1 copy of DPP1. In some embodiments, the microorganism of the present application overexpresses 1 copy of LPP1 and 1 copy of DPP1. In some embodiments, the microorganism of the present application overexpresses 1 copy of LPP1 and 2 copies of DPP1.

[0021] Further, the heterologous phosphohydrolase comprises an E. coli-derived phosphohydrolase and a plant-derived phosphohydrolase, in some embodiments, the heterologous phosphohydrolase is an E. coli-derived phosphohydrolase, in some embodiments, the heterologous phosphohydrolase is a plant-derived phosphohydrolase, in some embodiments, the heterologous phosphohydrolase is an E. coli-derived phosphohydrolase and a plant-derived phosphohydrolase. The E. coli-derived phosphohydrolase is NudJ, NudF, NudC, mutT, cdH, phoA, ppa, lpxH, the amino acid sequence of NudJ, NudF, NudC, mutT, cdH, phoA, ppa, lpxH is SEQ ID NO:99-106, respectively, the nucleic acid encoding NudJ, NudF, NudC, mutT, cdH, phoA, ppa, lpxH is SEQ ID NO:25-32.

[0022] Preferably, the E. coli endogenous phosphatase is NudJ, NudF.

[0023] Further, the plant-derived phosphohydrolase comprises a Tanacetum cinerariifolium-derived phosphohydrolase, the Tanacetum cinerariifolium-derived (TcNudixl) phosphatase is TcNudixl, the amino acid sequence of the enzyme is SEQ ID NO: 107, preferably the nucleic acid encoding the enzyme is SEQ ID NO:33.

[0024] Further, preferably the plant-derived phosphohydrolase is codon-optimized and signal peptide-truncated tTcNudixl, the codon-optimized and signal peptide-truncated tTcNudixl, the amino acid sequence of the enzyme is SEQ ID NO: 108, preferably the nucleic acid encoding the enzyme is SEQ ID NO:34.

[0025] Further, the microorganism comprises a complemented amino acid, further, comprises a complemented HIS3, URA3 amino acid gene.

[0026] Furthermore, the in vivo expression of the phosphatase comprises introducing a nucleic acid sequence encoding the phosphatase into the yeast; and / or introducing a nucleic acid sequence encoding the phosphatase into the yeast comprises introducing an expression cassette containing the phosphatase-encoding nucleic acid into the yeast; and / or introducing the phosphatase into the yeast by expressing a plasmid vector containing the phosphatase-encoding nucleic acid expression cassette; preferably, introducing the phosphatase into the yeast by integration into the yeast chromosome. In certain embodiments, the expression cassette further comprises a promoter and a terminator. Regarding the selection of the promoter and terminator, those skilled in the art can appropriately select promoters and terminators suitable for the corresponding engineered bacteria based on the engineered bacteria. In further embodiments, the expression cassette comprises a promoter and a terminator. In certain embodiments, the promoter is selected from GAL7, GAL10, or GAL1. In certain embodiments, the terminator is selected from ENO2, CYC1, or IDP1.

[0027] Furthermore, the microorganism further comprises an optimized mevalonate pathway, including any one of (a) to (h):

[0028] (a) DNA fragment Donor 5 was introduced into yeast to replace the farnesyl pyrophosphate synthase gene ERG20 ww (the sequence of the protein encoded is SEQ ID NO: 77, preferably the encoding gene is SEQ ID NO: 3) introduced into yeast;

[0029] (b) DNA fragment Donor 5 was introduced into yeast to transform the hydroxymethylglutaryl-CoA reductase isozyme HMG2 mutant HMG2 K6R (The sequence of the protein encoded is SEQ ID NO: 81, preferably the encoding gene is SEQ ID NO: 7) gene introduced into yeast;

[0030] (c) Introducing the DNA fragment Donor 6 into yeast and replacing the promoter of the ERG20 gene on the yeast genome with P erg7 ,, where P erg7 The promoter of ERG7 is derived from Saccharomyces cerevisiae, and the promoter is replaced by P erg7 The sequence of the protein encoded by the endogenous ERG20 gene is SEQ ID NO: 78, and the nucleic acid encoding is preferably as shown in SEQ ID NO: 4;

[0031] (d) introducing the DNA fragment Donor 7 into yeast and the mevalonate kinase gene ERG12 (encoded protein sequence is SEQ ID NO: 83, encoding nucleic acid sequence is SEQ ID NO: 9) into the host strain;

[0032] (e) introducing DNA fragment Donor 8 into the yeast, and introducing EfmvaE and EfmvaS (the sequences of the encoded proteins are SEQ ID NO: 79, SEQ ID NO: 80, and the encoding nucleic acids are SEQ ID NO: 5, SEQ ID NO: 6) genes from Enterococcus faecalis into the yeast;

[0033] The (a)-(e) are not in a specific order.

[0034] Further, the method comprises the step of fermenting the microorganism to obtain dextro-borneol.

[0035] The second aspect of the present application provides an engineered bacterium, which comprises or expresses borneol-based bisphosphate synthetase from Salvia officinalis L., C. burmannii, Amomum villosum and Lippia dulcis, and the host bacterium is a yeast.

[0036] Preferably, the engineered bacterium comprises or expresses borneol-based bisphosphate synthetase from Salvia officinalis L., C. burmannii and Amomum villosum.

[0037] Preferably, the engineered bacterium comprises or expresses borneol-based bisphosphate synthetase from Salvia officinalis L. and C. burmannii.

[0038] Preferably, the engineered bacterium comprises or expresses borneol-based bisphosphate synthetase from Salvia officinalis L.

[0039] Further, the gene expressing borneol-based bisphosphate synthetase from Salvia officinalis L., C. burmannii, Amomum villosum and Lippia dulcis is codon-optimized and signal peptide-truncated, and the present application is improved according to the codon bias of Saccharomyces cerevisiae, and the improved method is a routine technical means in the art, for example, the above-mentioned gene is artificially synthesized according to the sequence of the related gene known in the art and with reference to the codon bias of Saccharomyces cerevisiae, and any of the above-mentioned genes designed according to the codon bias of Saccharomyces cerevisiae can be used in the present application.

[0040] In certain embodiments, the codon-optimized and signal peptide-truncated borneol diphosphate synthetase is tSoBPPS, tCbTPS1, tAvBPPS, and tLdBPPS, preferably tSoBPPS, tCbTPS1, and tAvBPPS.

[0041] Furthermore, the amino acid sequences of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown as SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:109 and SEQ ID NO:110, respectively, and the encoding nucleic acids of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:35 and SEQ ID NO:74, respectively.

[0042] In certain embodiments, the present invention includes or expresses borneol diphosphate synthase by introducing a nucleic acid sequence encoding the borneol diphosphate synthase into the yeast; and / or introducing a nucleic acid sequence encoding the borneol diphosphate synthase into the yeast by introducing an expression cassette containing a nucleic acid encoding the borneol diphosphate synthase into the yeast; and / or introducing the yeast by expressing a plasmid vector containing an expression cassette containing a nucleic acid encoding the borneol diphosphate synthase; preferably, introducing the yeast by integration into the yeast chromosome. In certain embodiments, the expression cassette further comprises a promoter and a terminator. Regarding the selection of the promoter and terminator, those skilled in the art will be able to appropriately select promoters and terminators suitable for the respective engineered bacteria based on the engineered bacteria. In further embodiments, the expression cassette comprises a promoter and a terminator, and in certain embodiments, the promoter is GAL2. In certain embodiments, the terminator is PYK1.

[0043] Furthermore, the engineered bacteria comprises a mutated codon-optimized farnesyl pyrophosphate synthase gene ERG20 ww .

[0044] Furthermore, the mutated codon-optimized farnesyl pyrophosphate synthase gene ERG20 ww The sequence of the protein it encodes is SEQ ID NO: 77, and preferably the encoding gene is shown in SEQ ID NO: 3.

[0045] Furthermore, the promoter of the endogenous ERG20 gene of the engineered bacteria was replaced with P erg7 , the promoter was replaced by P erg7 The sequence of the protein encoded by the endogenous ERG20 gene is SEQ ID NO: 78, and the preferred nucleic acid encoding is shown in SEQ ID NO: 4.

[0046] In certain embodiments, the present invention comprises or expresses a codon-optimized ERG20 ww To convert the codon-optimized ERG20 ww The nucleic acid encoding the gene is introduced into the yeast; and / or the codon-optimized ERG20 ww The nucleic acid encoding ERG20 is introduced into the yeast to ww The expression cassette encoding the nucleic acid is introduced into the yeast; and / or by expressing the ERG20 ww A plasmid vector encoding a nucleic acid expression cassette is introduced into the yeast; preferably, the introduction is carried out by integration into the yeast chromosome. In certain embodiments, the expression cassette further comprises a promoter and a terminator. Regarding the selection of the promoter and terminator, those skilled in the art will be able to appropriately select promoters and terminators suitable for the corresponding engineered bacteria based on the engineered bacteria. In further embodiments, the expression cassette comprises a promoter and a terminator, and in certain embodiments, the promoter is selected from GAL10. In certain embodiments, the terminator is selected from PRM9.

[0047] Furthermore, the present invention improves the yield of borneol by optimizing the MVA pathway. For example, acetoacetyl-CoA thiolase EfmvaE and mevalonate synthase EfmvaS from Enterococcus faecalis (protein sequences encoded by SEQ ID NO: 79, SEQ ID NO: 80, and nucleic acids encoded by SEQ ID NO: 5, SEQ ID NO: 6) are introduced, and a mutant HMG2 of the hydroxymethylglutaryl-CoA reductase isozyme is expressed. K6R(SEQ ID NO: 81, SEQ ID NO: 7), expressing N-terminally truncated 3-hydroxy-3- methylglutaryl-CoA reductase tHMG1 (the sequence of the encoded protein is SEQ ID NO: 82, the coding nucleic acid is SEQ ID NO: 8), overexpressing the yeast endogenous mevalonate kinase gene ERG12 (the sequence of the encoded protein is SEQ ID NO: 83, the coding nucleic acid is SEQ ID NO: 9), introducing the phosphomevalonate kinase gene ERG8 (the sequence of the encoded protein is SEQ ID NO: 84, the coding nucleic acid is SEQ ID NO: 10), the mevalonate pyrophosphate decarboxylase gene ERG19 (the sequence of the encoded protein is SEQ ID NO: 85, the coding nucleic acid is SEQ ID NO: 11), the isopentenyl pyrophosphate isomerase gene IDI1 (the sequence of the encoded protein is SEQ ID NO: 86, the coding nucleic acid is SEQ ID NO: 12), etc., preferably introducing EfmvaE and EfmvaS from Enterococcus faecalis, expressing the HMG2 mutant HMG2K6R, overexpressing the yeast endogenous mevalonate kinase gene ERG12. Among them, introducing or expressing represents providing increased activity or expression level of the relevant enzyme.

[0048] Further, the engineered bacteria comprise or express a phosphohydrolase, which comprises an endogenous phosphohydrolase and a heterologous phosphohydrolase, in some embodiments, the phosphohydrolase is an endogenous phosphohydrolase, in some embodiments, the phosphohydrolase is a heterologous phosphohydrolase, in some embodiments, the phosphohydrolase is an endogenous phosphohydrolase and a heterologous phosphohydrolase. Preferably, the engineered bacteria overexpress the phosphohydrolase.

[0049] The endogenous phosphohydrolase is a Saccharomyces cerevisiae endogenous phosphatase, which is LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1.

[0050] The amino acid sequences of the LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1 are respectively as SEQ ID NO: 87-98, further, the coding nucleic acids of the LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1 are as SEQ ID NO: 13-24.

[0051] Preferably, the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1, PAH1, IPP1, NPP1, NPP2, or APP1.

[0052] Preferably, the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1, PAH1, IPP1, NPP1, or NPP2.

[0053] Preferably, the endogenous phosphatase of Saccharomyces cerevisiae is LPP1 or DPP1.

[0054] In certain embodiments, the engineered bacteria of the present invention overexpress two copies of LPP1. In certain embodiments, the engineered bacteria of the present invention overexpress two copies of LPP1 and one copy of DPP1. In certain embodiments, the engineered bacteria of the present invention overexpress one copy of LPP1 and one copy of DPP1. In certain embodiments, the engineered bacteria of the present invention overexpress one copy of LPP1 and two copies of DPP1.

[0055] Furthermore, the heterologous phosphohydrolase comprises an Escherichia coli-derived phosphohydrolase and a plant-derived phosphohydrolase. In certain embodiments, the heterologous phosphohydrolase is an Escherichia coli-derived phosphohydrolase. In certain embodiments, the heterologous phosphohydrolase is a plant-derived phosphohydrolase. In certain embodiments, the heterologous phosphohydrolase is an Escherichia coli-derived phosphohydrolase and a plant-derived phosphohydrolase. The Escherichia coli-derived phosphatase is NudJ, NudF, NudC, mutT, cdH, phoA, ppa, and lpxH. The amino acid sequences of NudJ, NudF, NudC, mutT, cdH, phoA, ppa, and lpxH are as shown in SEQ ID NOs: 99-106, respectively. The encoding nucleic acids of NudJ, NudF, NudC, mutT, cdH, phoA, ppa, and lpxH are as shown in SEQ ID NOs: 25-32.

[0056] Preferably, the endogenous phosphatase of E. coli is NudJ or NudF.

[0057] Furthermore, the plant-derived phosphohydrolase includes a pyrethrum-derived (Tanacetumcinerariifolium) phosphohydrolase, the pyrethrum-derived phosphatase is TcNudix1, the amino acid sequence of the enzyme is SEQ ID NO: 107, and preferably the encoding nucleic acid of the enzyme is SEQ ID NO: 33; further, preferably, the plant-derived phosphohydrolase is tTcNudix1 after codon optimization and signal peptide truncation, the tTcNudix1 after codon optimization and signal peptide truncation, the amino acid sequence of the enzyme is SEQ ID NO: 108, and preferably the encoding nucleic acid of the enzyme is SEQ ID NO: 34.

[0058] Further, the engineering bacteria contain complementary amino acids, and further contain complementary HIS3 and URA3 amino acid genes.

[0059] Further, the application provides a method for producing dextro-borneol, which comprises using YPD medium or Delft medium as a fermentation medium and using the above engineering bacteria as a fermentation strain.

[0060] The Saccharomyces cerevisiae host is Saccharomyces cerevisiae CEN.PK.113-11C.

[0061] Further, the application provides a construction method for constructing the above engineering bacteria for producing dextro-borneol, which comprises introducing the nucleic acid coding sequences of the codon-optimized ERG20ww, borneol-based diphosphate synthetase, EfmvaE and EfmvaS, HMG2 K6R , ERG12, and phosphohydrolase into the yeast bacteria and replacing the endogenous ERG20 promoter with P erg7 , wherein P erg7 is the promoter of ERG7 and is derived from Saccharomyces cerevisiae; and / or introducing the expression cassette containing the nucleic acid coding sequences of the codon-optimized ERG20 ww , borneol-based diphosphate synthetase, EfmvaE and EfmvaS, HMG2 K6R , ERG12, and phosphohydrolase into the yeast bacteria; and / or introducing the plasmid vector containing the expression cassette of the nucleic acid coding sequences of the codon-optimized ERG20 ww , borneol-based diphosphate synthetase, EfmvaE and EfmvaS, HMG2 K6R , ERG12, and phosphohydrolase into the yeast bacteria; preferably introducing the yeast bacteria in the form of integration into the yeast chromosome. In some embodiments, the expression cassette further comprises a promoter and a terminator. For the selection of the promoter and the terminator, those skilled in the art can appropriately select the promoter and the terminator suitable for the corresponding engineering bacteria according to the engineering bacteria. In further embodiments, the expression cassette comprises a promoter and a terminator, and in some embodiments, the promoter is selected from GAL1, GAL2, GAL10, GAL7, etc. In some embodiments, the terminator is selected from PYK1, PRM9, IDP1, CYC1, ENO2, etc. Further, the construction method further comprises complementing the auxotrophy of the strain, including complementing the HIS3 and URA3 amino acid genes.

[0062] Further, the application provides the use of the above recombinant bacteria in the production of dextro-borneol.

[0063] Beneficial effects:

[0064] 1. The application constructs a method for efficiently producing d-borneol, which is conducive to obtaining high yield of d-borneol.

[0065] 2. The method can obtain high yield of d-borneol without loss of OD value, and is suitable for large-scale industrial production.

[0066] Specific implementation examples

[0067] The technical solutions of the application will be further described in detail below in combination with specific implementation examples, but the implementation of the application is not limited to this. Equivalent replacement, combination, improvement or modification of the technical solutions of the application by those skilled in the art according to the description of the application should be covered in the protection scope of the application.

[0068] The donor DNA fragment information constructed by the application is shown in Table 1, the primers used are shown in Table 2, and the reagents used and sources are shown in Table 3.

[0069] Table 1 shows the information of the constructed donor DNA and sgRNA

[0070]

[0071]

[0072] Table 2 shows the primer information

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] Table 3 shows the main reagents and sources

[0081]

[0082] Main culture medium and configuration:

[0083] (1) YPD liquid medium (1L): 10g yeast extract, 20g peptone and 20g glucose, dissolved and constant volume, sterilized by high pressure steam at 121℃ for 20 minutes.

[0084] (2) YPD solid medium (1 L) : 10 g of yeast extract, 20 g of peptone, 20 g of glucose and 2% (m / v) of agar powder, dissolved and aliquoted, sterilized by autoclaving at 121 °C for 20 minutes.

[0085] (3) Delft medium containing histidine and uracil (1 L) : 5 g of (NH4)2SO4, 14.4 g of KH2PO4, 0.5 g of MgSO4-7H2O, 40 mg of histidine and 60 mg of uracil were added to 800 mL of deionized water, and the pH was adjusted to 5.6 with a 2 M potassium hydroxide solution, and then sterilized by autoclaving at 121 °C for 20 minutes. After the sterilized medium was cooled, 1 mL of vitamin, 2 mL of trace metal and 50 mL of a 400 g / L glucose solution were added thereto.

[0086] (4) Delft medium containing histidine (1 L) : 5 g of (NH4)2SO4, 14.4 g of KH2PO4, 0.5 g of MgSO4-7H2O and 40 mg of histidine were added to 800 mL of deionized water, and the pH was adjusted to 5.6 with a 2 M potassium hydroxide solution, and then sterilized by autoclaving at 121 °C for 20 minutes. After the sterilized medium was cooled, 1 mL of vitamin, 2 mL of trace metal and 50 mL of a 400 g / L glucose solution were added thereto.

[0087] (5) Delft medium (1 L) : 5 g of (NH4)2SO4, 14.4 g of KH2PO4, 0.5 g of MgSO4-7H2O were added to 800 mL of deionized water, and the pH was adjusted to 5.6 with a 2 M potassium hydroxide solution, and then sterilized by autoclaving at 121 °C for 20 minutes. After the sterilized medium was cooled, 1 mL of vitamin, 2 mL of trace metal and 50 mL of a 400 g / L glucose solution were added thereto.

[0088] (6) SD solid medium containing histidine (1 L) : 6.7 g of amino acid-free yeast nitrogen base, 20 g of glucose and 2% (m / v) of agar powder, 40 mg of histidine, dissolved and aliquoted, sterilized by autoclaving at 121 °C for 20 minutes.

[0089] (7) SD solid medium containing pentafluorouracil : 100 mg of pentafluorouracil was weighed, immediately dissolved by shaking in 1 mL of dimethyl sulfoxide, and the dissolved solution was sterilized by membrane filtration. The entire filtered solution was added to 100 mL of liquid SD solid medium, and then 0.1 mL of a sterilized histidine solution (20 g / L) and 0.1 mL of a sterilized uracil solution (20 g / L) were added thereto.

[0090] Example 1

[0091] The gene expression cassettes were integrated into the yeast chromosome one by one by CRISPR-Cas9 technology. The donor DNA fragment construction process refers to Zhou, Y. J. et al. Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J Am Chem Soc 134, 3234-3241 (2012). The site of gene integration refers to Kong, S., Yu, W., Gao, N., Zhai, X. & Zhou, Y. J. Expanding the neutral sites for integrated gene expression in Saccharomyces cerevisiae. FEMS Microbiol Lett 369 (2022).

[0092] 1. Strain construction

[0093] The initial strain of the application is CEN.PK.113-11C from Saccharomyces cerevisiae (MATa; MAL2-8c; SUC2; his3D1; ura3-52, source University of Frankfurt, Germany).

[0094] Construction of HMG2 K6R and ERG20 ww The sequences of the proteins encoded by HMG2 K6R and ERG20 ww are SEQ ID NO: 81 and SEQ ID NO: 77, and the encoding genes are SEQ ID NO: 7 and SEQ ID NO: 3): The donor DNA fragment includes 500 bp of homologous arms upstream and downstream of the NSXI-6 site amplified with CEN.PK.113-11C as a template, the promoter P GAP1,10 , the terminator T PRM9 , T PYK1 and the structural gene (Table 2). The donor DNA fragment Donor 5 (XI-6::(P GAL1 -HMG2 K6R -T PYK1 )+(P GAL10 -ERG20 ww -T PRM9) and sgRNA plasmid XI-6 were transformed into the starting strain XC1 (MATa; MAL2-8c; SUC2; his3D1; ura3-52D; XI-5::Cas9; gal80D, from X. Cao et al. 2023) by lithium acetate transformation method, spread on SD+HIS selection plates, and incubated at 30°C for 3-4 days. The correct strains were verified by colony PCR and spread on plates containing 5-fluoroorotic acid for plasmid loss. Strain ZHY1 was obtained.

[0095] Construction of donor DNA expression cassette: The donor DNA fragment includes a 500bp homologous arm upstream of the ERG20 promoter, the ERG7 promoter and the ERG20 structural gene amplified from the template of CEN.PK.113-11C (the ERG20 structural gene and the ERG7 promoter are derived from the genomic DNA of CEN.PK.113-11C). The donor DNA fragment Donor 6 (P ERG7 -ERG20) and sgRNA plasmid ERG20p were transformed into the starting strain ZHY1 by lithium acetate transformation method, spread on SD+HIS selection plates, and incubated at 30°C for 3-4 days. The correct strains were verified by colony PCR and spread on plates containing 5-fluoroorotic acid for plasmid loss. Strain ZHY2 was obtained.

[0096] The donor DNA fragment Donor 1 (expression cassette IX-1::P GAL2 -tSoBPPS-T PYK1 ) and sgRNA plasmid IX-1 were transformed into strain ZHY1 by lithium acetate transformation method, spread on SD+HIS selection plates, and incubated at 30°C for 3-4 days. The correct strains were verified by colony PCR and spread on plates containing 5-fluoroorotic acid for plasmid loss. Strain ZHY8 was obtained.

[0097] Integration of borneol diphosphate synthase into yeast chromosome. Four donor DNA expression cassettes for separately expressing four borneol diphosphate synthases were constructed. The donor DNA fragment includes a 500bp homologous arm upstream and downstream of the NSIX-1 site amplified from the template of CEN.PK.113-11C, the promoter P GAP2 , the terminator T PYK1 and the borneol diphosphate synthases tSoBPPS, tCbTPS3, tAvBPPS or LdBPPS S488T(tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are respectively shown in SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 109, SEQ ID NO: 110, and the encoding nucleic acids of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are respectively shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 35 and SEQ ID NO: 74). The donor DNA fragments Donor 1 (IX-1::P GAL2 -tSoBPPS-T PYK1 ), Donor 2 (IX-1::P GAL2 -tCbBPPS-T PYK1 ), Donor 3 (IX-1::P GAL2 -tAvBPPS-T PYK1 ), Donor 4 (IX-1::P GAL2 -tLdBPPS S488T -T PYK1 ) and sgRNA plasmid IX-1 were transformed into the engineering strain ZHY2 by lithium acetate transformation method, and the strains ZHY3-tSo, ZHY3-tCb, ZHY3-tAv and ZHY3-tLd were obtained respectively.

[0098] The genotypes of the above engineering bacteria are shown in Table 4:

[0099] Table 4

[0100]

[0101] 2. Fermentation

[0102] The obtained engineering bacteria ZHY2, ZHY8, ZHY3-tSo, ZHY3-tCb, ZHY3-tAv and ZHY3-tLd were fermented. The fermentation conditions were as follows: the glycerol bacteria stored at -80°C were streaked on YPD solid medium, and the strain was activated for 3-4 days; on the first day at 15-17 points, a single colony was picked up in a 15 ml centrifuge tube containing 3 mL Delft+HIS+URA liquid medium, and incubated overnight to the logarithmic growth phase; the next morning, the seed liquid OD 600 was measured, and the bacteria were inoculated at an initial OD of 0.1, and the fermentation medium was 20 ml of Delft+HIS+URA liquid medium. After inoculation, 10% isopropyl myristate was added as an extraction solvent; the fermentation was carried out at 30°C, 220 rpm on a shaking table for 72 h.

[0103] 3. Fermentation product processing

[0104] The upper isopropyl myristate was taken at 72 h, centrifuged, and 10 ul of the supernatant was diluted to 100 ul for GC-MS detection;

[0105] 50 ul of the culture solution was taken and diluted to 2 ml (40-fold dilution) for OD measurement 600 The final growth condition of the cells was detected.

[0106] 4. GC-MS detection of fermentation products

[0107] The qualitative detection parameters of GC-MS were TG-5MS column (0.25 mm, 0.25 pm), ISQ LT single quadrupole mass spectrometer, and thermoTRACE 1300 series GC. The injection port temperature of GC-MS was 250 °C, the detector temperature was 300 °C, 1 pL of aliquot was injected at a helium flow rate of 1 mL / min in split mode (split ratio 50:1). The GC-MS temperature program was initial temperature 80 °C for 2 min, 30 °C / min to 138 °C (5 min hold), 5 °C / min to 150 °C (2 min hold), 50 °C / min to 300 °C (15 min hold). (+)-borneol standard was used as a control to make a standard curve, and the content of (+)-borneol in the sample was determined.

[0108] The yield of (+)-borneol was detected as shown in Table 5:

[0109] Table 5

[0110] Strain Yield (mg / L) ZHY2 0.0 ZHY8 0.5±0.0 ZHY3-tSo 2.4±0.3 ZHY3-tCb 1.5±0.1 ZHY3-tAv 1.2±0.1 ZHY3-tLd 0.0±0.0

[0111] Example 2: Optimization of the mevalonate pathway

[0112] 1. Strain construction

[0113] On the basis of engineering strain ZHY3-tSo, the MVA pathway was further strengthened. The endogenous genes of the MVA pathway, ERG12, tHMG1, ERG8, ERG19, IDI1, and ERG20, were from the genomic DNA of CEN.PK.113-11C. The sources of EfmvaE and EfmvaS were Enterococcus faecalis (Dusséaux S, Wajn WT, Liu Y, Ignea C, Kampranis SC. Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids. Proc Natl Acad Sci USA. 2020 Dec 15; 117(50): 31789-31799.). The specific steps of related gene expression cassette construction and genome integration are as follows:

[0114] With the help of CRISPR-Cas9 technology, donor DNA fragment Donor 7 (expression cassette X-3::P GAL2 -ERG12-T ENO2 ) and sgRNA plasmid X-3 were transformed into strain ZHY3-tSo by lithium acetate transformation, or donor DNA fragment Donor 8 (expression cassette VIII-1:: (P GAL10 -EfmvaS-T PRM9 ) + (P GAL1 +EfmvaE-T IDP1 )) and sgRNA plasmid VIII-1 were transformed into strain ZHY3-tSo by lithium acetate transformation. They were respectively coated on SD+HIS selection plates and incubated at 30°C for 3-4 days. The correct ones were verified by colony PCR, coated on plates containing 5-fluoroorotic acid for plasmid loss, and strains ZHY5 and ZHY6 were obtained, respectively.

[0115] With the help of CRISPR-Cas9 technology, donor DNA fragment Donor 8 (expression cassette VIII-1:: (P GAL10 -EfmvaS-T PRM9 ) + (P GAL1 +EfmvaE-T IDP1 )) and sgRNA plasmid VIII-1 were transformed into strain ZHY5 engineering bacteria by lithium acetate transformation, respectively coated on SD+HIS selection plates and incubated at 30°C for 3-4 days. The correct ones were verified by colony PCR, coated on plates containing 5-fluoroorotic acid for plasmid loss, and strain ZHY7 was obtained.

[0116] Using CRISPR-Cas9 technology, the donor DNA fragment Donor 9 (expression cassette XVI-2::P GAL2 -HMG2 K6R -T IDP1 ) and sgRNA plasmid XVI-2 were transformed into strain ZHY7 by lithium acetate transformation, or the donor DNA fragment Donor 10 (expression cassette XVI-2::P GAL2 -tHMG1-T IDP1 ) and sgRNA plasmid XVI-2 were transformed into strain ZHY7 via the lithium acetate method. The plasmids were plated onto SD+HIS screening plates and incubated at 30°C for 3–4 days. Colony PCR was performed to verify the plasmids, and plasmids were plated onto plates containing 5-fluoroorotic acid for plasmid loss, resulting in strains ZHY9 and ZHY10, respectively.

[0117] Using CRISPR-Cas9 technology, the donor DNA fragment Donor 11 (expression cassette VI-1::P GAL1 -ERG8-T CYC1 ) and sgRNA plasmid VI-1 were transformed into strain ZHY7 by lithium acetate transformation; or the donor DNA fragment Donor 13 (expression cassette VI-1::P GAL7 -IDI1-T ENO2 ) and sgRNA plasmid VI-1 were transformed into strain ZHY7 by lithium acetate transformation; or the donor DNA fragment Donor 14 (expression cassette VI-1::(P GAL1 -ERG8-T CYC1 )+(P GAL10 -ERG19-T IDP1 )) and sgRNA plasmid VI-1 were transformed into strain ZHY7 by lithium acetate transformation; or the donor DNA fragment Donor 16 (expression cassette VI-1::(P GAL1 -ERG19-T IDP1 )+(P GAL7 -IDI1-T ENO2 )) and sgRNA plasmid VI-1 were transformed into strain ZHY7 via the lithium acetate method. The strains were plated onto SD+HIS screening plates and incubated at 30°C for 3-4 days. Colony PCR was performed to verify the correctness of the plasmids. Plasmids were then plated onto plates containing 5-fluoroorotic acid for plasmid loss, resulting in strains ZHY12, ZHY14, ZHY15, and ZHY17, respectively.

[0118] The genotypes of the above-mentioned engineered bacteria are shown in Table 6:

[0119] Table 6

[0120]

[0121]

[0122] 2. Fermentation

[0123] The engineered bacteria ZHY3-tSo, ZHY5, ZHY6, ZHY7, ZHY9, ZHY10, ZHY12, ZHY14, ZHY15, ZHY17 were fermented. The fermentation process was the same as Example 1.

[0124] 3. Fermentation product processing

[0125] The sample processing was performed according to the method of Example 1.

[0126] 4. Fermentation product GC-MS detection

[0127] The detection was performed according to the method of Example 1. Through detection, the production of (+)-borneol was as shown in Table 7:

[0128] Table 7

[0129] Strain Yield (mg / L) ZHY3-tSo 2.4±0.3 ZHY5 2.8±0.0 ZHY6 2.2±0.4 ZHY7 2.8±0.1 ZHY9 3.1±0.4 ZHY10 2.3±0.4 ZHY12 1.9±0.1 ZHY14 1.6±0.3 ZHY15 1.5±0.3 ZHY17 1.5±0.1

[0130] Example 3 Expression of phosphatase

[0131] 1. Strain construction

[0132] Further, on the basis of the engineered bacteria ZHY7, the endogenous dephosphorylation reaction in yeast was strengthened. The phosphatases LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, GPP1, GPP2, YPP1 were from the genomic DNA of CEN.PK.113-11C. With the help of CRISPR-Cas9 technology, donor DNA fragments Donor 18 (expression cassette XV-2::P GAL7 -DPP1-T ENO2 ) and sgRNA plasmid XV-2 were transformed into strain ZHY7 by lithium acetate transformation method; or donor DNA fragments Donor 19 (expression cassette XV-2::P GAL7 -LPP1-T ENO2 ) and sgRNA plasmid XV-2 were transformed into strain ZHY7 by lithium acetate transformation method; or donor DNA fragments Donor 30 (expression cassette XV-2::P GAL7 -PAH1-T ENO2 ) and sgRNA plasmid XV-2 were transformed into strain ZHY7 by lithium acetate transformation method; or donor DNA fragments Donor 31 (expression cassette XV-2::P GAL7 -APP1-T ENO2 ) and sgRNA plasmid XV-2 were transformed into strain ZHY7 by lithium acetate transformation method; or donor DNA fragments Donor 32 (expression cassette XV-2::PGAL7 -IPP1-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 33 (expression cassette XV-2::P GAL7 -NPP1-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 34 (expression cassette XV-2::P GAL7 -NPP2-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 35 (expression cassette XV-2::P GAL7 -PHO8-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 36 (expression cassette XV-2::P GAL7 -t 62 PHO8-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 37 (expression cassette XV-2::P GAL7 -GPP1-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 38 (expression cassette XV-2::P GAL7 -GPP2-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 39 (expression cassette XV-2::P GAL7 -YPP1-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method. Respectively spread on SD+HIS selection plate, incubated at 30°C for 3-4 days, verified by colony PCR, spread on plate containing 5-fluorouracil for plasmid loss, and obtained strains ZHY19, ZHY20 and ZHY32-41, respectively.

[0133] On the basis of engineering bacteria ZHY7, NudJ, NudF, NudC, mutT, cdH, phoA, ppa, lpxH (from DH5a genomic DNA) were introduced. With the help of CRISPR-Cas9 technology, donor DNA fragment Donor21 (expression cassette XV-2::P GAL7 -ECNudJ-T ENO2) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 22 (expression cassette XV-2::P GAL7 - ECNudF-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 23 (expression cassette XV-2::P GAL7 - ECNudC-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 24 (expression cassette XV-2::P GAL7 - ECmutT-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 25 (expression cassette XV-2::P GAL7 - ECcdH-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 26 (expression cassette XV-2::P GAL7 - ECphoA-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 27 (expression cassette XV-2::P GAL7 - ECppa-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method; or donor DNA fragment Donor 28 (expression cassette XV-2::P GAL7 - EC1pxH-T ENO2 ) and sgRNA plasmid XV-2 into strain ZHY7 by lithium acetate transformation method. Respective spread on SD+HIS screening plate, at 30°C static culture for 3-4 days, correct by colony PCR verification, spread on plate containing 5-fluorouracil for plasmid loss, obtain engineering strain ZHY22-29.

[0134] The above engineering bacteria genotype is shown in Table 8:

[0135] Table 8

[0136]

[0137]

[0138]

[0139] 2. Fermentation

[0140] The engineered bacteria ZHY19, ZHY20, ZHY32-41, and ZHY22-29 were fermented in the same manner as in Example 1.

[0141] 3. Fermentation product processing

[0142] The samples were processed according to the method of Example 1.

[0143] 4. GC-MS Detection of Fermentation Products

[0144] The detection was carried out according to the method of Example 1. After detection, the yield of (+)-borneol is shown in Table 9:

[0145] Table 9

[0146] Strain Yield (mg / L) ZHY19 7.30±0.3 ZHY20 13.80±1.0 ZHY22 4.6±0.3 ZHY23 3.7±0.5 ZHY24 2.7±0.1 ZHY25 2.5±0.3 ZHY26 1.9±0.3 ZHY27 2.1±0.3 ZHY28 2.1±0.2 ZHY29 1.7±0.1 ZHY32 3.3±0.3 ZHY33 3.0±0.2 ZHY34 3.0±0.2 ZHY35 3.2±0.3 ZHY36 3.1±0.1 ZHY37 2.6±0.1 ZHY38 2.3±0.1 ZHY39 2.6±0.1 ZHY40 2.5±0.1 ZHY41 2.1±0.1

[0147] Example 4 Increasing the copy number of endogenous phosphatase

[0148] 1. Strain Construction

[0149] Furthermore, the CRISPR-Cas9 technology was used to increase the copy numbers of LPP1 and DPP1 in the engineered strain ZHY20. GAL2 -LPP1-T IDP1t ) and sgRNA plasmid X-3 were transformed into strain ZHY20 by lithium acetate transformation; or the donor DNA fragment Donor41 (IX-2::P GAL7 -DPP1-T ENO2 ) and sgRNA plasmid X-3 were transformed into strain ZHY20 via the lithium acetate method. The plasmids were plated onto SD+HIS screening plates and incubated at 30°C for 3–4 days. Colony PCR was performed to verify the plasmids, and plasmids were plated onto plates containing 5-fluoroorotic acid for plasmid loss, resulting in strains ZHY61 and ZHY69, respectively.

[0150] Furthermore, by using CRISPR-Cas9 technology, one copy of LPP1 and one copy of DPP1 were simultaneously added to the engineered bacteria ZHY20, and the donor DNA fragment Donor 42 (expression cassette IX-2::(P GAL10 -DPP1-T CYC1 )+(P GAL1 -LPP1-T IDP1 )) and sgRNA plasmid IX-2 were transformed into strain ZHY20 via the lithium acetate method. The strain was plated onto SD+HIS selection plates and incubated at 30°C for 3-4 days. Colony PCR was performed to verify the correctness of the plasmid. Plasmid loss was then performed by plating onto plates containing 5-fluoroorotic acid to obtain engineered strain ZHY73.

[0151] The genotype of the above-mentioned engineering bacteria is shown in Table 10:

[0152] Table 10

[0153]

[0154] 2. Fermentation

[0155] ZHY20, ZHY61, ZHY69 and ZHY73 were subjected to fermentation verification. The detailed fermentation process was the same as that in Example 1.

[0156] 3. Fermentation product processing

[0157] The sample processing was performed according to the method of Example 1.

[0158] 4. Fermentation product GC-MS detection

[0159] The detection was performed according to the method of Example 1. The yield of (+)-borneol was detected as shown in Table 11:

[0160] Table 11

[0161] Strain Yield (mg / L) ZHY20 13.8±1.0 ZHY61 19.1±1.6 ZHY69 19.0±1.5 ZHY73 19.1±0.8

[0162] Example 5 Construction of engineering bacteria

[0163] 1. Strain construction

[0164] Further, by means of CRISPR-Cas9 technology, a heterologous phosphatase tTcNudix1 (the amino acid sequence of the enzyme is SEQ ID NO: 108, and preferably the nucleic acid encoding the enzyme is SEQ ID NO: 34) was introduced into engineering bacteria ZHY20 and ZHY73, respectively. The donor DNA fragment Donor 46 (expression cassette XIV-2::P GAL7 -tTcNudix1-T ENO ) and sgRNA plasmid XVI-2 were transformed into strains ZHY20 and ZHY73, respectively, by lithium acetate transformation method. They were coated on SD+HIS selection plates and incubated at 30°C for 3-4 days, and then verified by colony PCR. The plasmid was lost by coating on plates containing 5-fluoroorotic acid, and engineering strains ZHY72 and ZHY74 were obtained.

[0165] The genotype of the above-mentioned engineering bacteria is shown in Table 12:

[0166] Table 12

[0167]

[0168] 2. Fermentation

[0169] The engineering bacteria ZHY72 and ZHY74 were fermented. The fermentation process was the same as that in Example 1.

[0170] 3. Fermentation product processing

[0171] The sample was processed according to the method of Example 1.

[0172] 4. Fermentation product GC-MS detection

[0173] The detection was performed according to the method of Example 1. The (+)-borneol yield was detected as shown in Table 13

[0174] Table 13

[0175] Strain Yield (mg / L) ZHY72 20.1±1.2 ZHY74 27.3±1.5

[0176] Example 6 Engineering bacteria construction

[0177] 1. Strain construction

[0178] On the basis of ZHY74, H IS3 and URA3 were back-supplemented, the S288C genome of Saccharomyces cerevisiae was used as a template to amplify the HIS3 and URA3 gene expression frame (the gene and its upstream and downstream 500 bp are the upstream and downstream homologous arms), which was transformed into Saccharomyces cerevisiae by lithium acetate method, coated on the SD screening plate and incubated at 30°C for 3-4 days. After verification, the transformants were stored for later use and named as engineering bacteria ZHY74UH (MATa; MAL2-8c; SUC2; his3Δ1; ura3-52Δ; XI-5::Cas9; gal80Δ, XI-6::(P GAL1 -HMG2 K6R -T PYK1 )+(P GAL10 -ERG20 ww -T PRM9 ), P ERG7 -ERG20, the native ERG20 promoter was replaced by PERG7, IX-1::P GAL2 -tSoBPPS-T PYK1 , X-3::P GAL2 -ERG12-T ENO2 , VIII-1::(P GAL10 -EfmvaS-T PRM9 )+(P GAL1 +EfmvaE-T IDP1 ), XV-2::P GAL7 -LPP1-T ENO2 , IX-2::(P GAL10 -DPP1-T CYC1 )+(PGAL1 - LPP1-T IDP1 ), XTV-2::P GAL7 - t 56 TcNudixl-T ENO2 , insitu URA3, insitu HIS3).

[0179] 2. Fermentation and yield detection

[0180] ZHY74UH was fermented in YPD medium, and the yield was detected at 24h, 48h, 72h and 96h, respectively. The detailed fermentation process was as follows: the glycerol bacteria stored at -80°C was streaked on YPD solid medium, and the strain was activated for 3-4 days; on the first morning, a single colony was picked up in a 15ml centrifuge tube containing 3ml Delft liquid medium, and the overnight culture was incubated to the logarithmic growth phase; the next morning, the seed liquid OD 600 was measured, and the bacteria were inoculated at an initial OD of 0.1, and the fermentation medium was 20ml of YPD liquid medium. After inoculation, 10% isopropyl myristate was added as an extraction solvent; the fermentation was carried out at 30°C, 220rpm for 72h. The upper isopropyl myristate was taken out at 48h and 72h, centrifuged, and 10ul of the supernatant was diluted to 100ul for GC-MS detection; the detection was carried out according to the method of Example 1. The detection results showed that the yield of d-borneol was 31.6±1.9mg / L when ZHY74UH was fermented for 48h, and the yield of d-borneol increased to 69.5±2.4mg / L when ZHY74UH was fermented for 72h.

[0181] Example 7 Batch fermentation of the engineering bacteria

[0182] The engineering bacteria ZHY74UH was fermented at pH 5.9, 50mL / 250mL volume, 20% isopropyl myristate coverage, and initial OD600=0.4 (YPD) inoculation. 5x liquid YPD was used for feeding, and 5x YPD liquid medium was added when the glucose was almost consumed, and the pH was adjusted to 6 with 4M potassium hydroxide every 24h. The fermentation was carried out at 30°C, 220rpm for 168h, and the highest yield was YPD initial, and YPD medium was added. The yield of d-borneol was 753mg / L when ZHY74UH was fermented for 168h, and the OD600 reached 66, which reached the highest yield reported so far without loss of OD value.

Claims

1. A method for producing borneol, characterized in that The method comprises culturing the transgenic microorganism in a culture medium, wherein the microorganism comprises or expresses borneol diphosphate synthase and phosphatase, and the microorganism is a yeast.

2. The method according to claim 1, characterized in that The microorganism contains or expresses borneol diphosphate synthase derived from Salvia officinalis L., C. burmannii, Amomum villosum and Lippia dulcis. Preferably, the microorganism contains or expresses borneol diphosphate synthase derived from Salvia officinalis L., C. burmannii and Amomum villosum. Preferably, the microorganism contains or expresses borneol diphosphate synthase derived from Salvia officinalis L. and C. burmannii. Preferably, the microorganism contains or expresses borneol diphosphate synthase derived from Salvia officinalis L.

3. The method according to claim 2, characterized in that The borneol diphosphate synthase is tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS, preferably tSoBPPS, tCbTPS1, tAvBPPS, and the amino acid sequences of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown in SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 109, and SEQ ID NO: 110, respectively. Preferably, the encoding nucleic acids of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 35, and SEQ ID NO: 110, respectively. NO:74; preferably, the borneol diphosphate synthase is codon-optimized and the signal peptide is truncated, and the borneol diphosphate synthases that have been codon-optimized and signal peptide-truncated are tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS, preferably tSoBPPS, tCbTPS1, tAvBPPS, and the amino acid sequences of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown in SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:109, and SEQ ID NO:110, respectively. Preferably, the encoding nucleic acids of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:35 and SEQ ID NO:74, respectively.

4. The method according to claim 1, characterized in that The phosphatase comprises an endogenous phosphohydrolase and / or a heterologous phosphohydrolase.

5. The method according to claim 4, characterized in that The endogenous phosphohydrolase is an endogenous phosphatase of Saccharomyces cerevisiae, and the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1. Preferably, the amino acid sequences of LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, and YPP1 are as shown in SEQ ID NOs: 87-98, respectively. Preferably, the nucleic acid encoding the nucleic acid of LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, and YPP1 is as shown in SEQ ID NOs: 87-98, respectively. NO:13-24, preferably the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1, PAH1, IPP1, NPP1, NPP2, APP1, preferably the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1, PAH1, IPP1, NPP1, NPP2, preferably the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1.

6. The method according to claim 4, characterized in that The heterologous phosphohydrolase comprises an Escherichia coli-derived phosphohydrolase and a plant-derived phosphohydrolase, wherein the Escherichia coli-derived phosphohydrolase is NudJ, NudF, NudC, mutT, cdH, phoA, ppa, and lpxH, and the amino acid sequences of the NudJ, NudF, NudC, mutT, cdH, phoA, ppa, and lpxH are as shown in SEQ ID NOs: 99-106, respectively. Preferably, the encoding nucleic acids of the NudJ, NudF, NudC, mutT, cdH, phoA, ppa, and lpxH are as shown in SEQ ID NOs: 25-32, and the endogenous phosphatase of Escherichia coli is NudJ or NudF; preferably, the plant-derived phosphohydrolase comprises a pyrethrum-derived phosphohydrolase, and the pyrethrum-derived phosphohydrolase is TcNudix1, and the amino acid sequence of the enzyme is as shown in SEQ ID NO: 107, and the encoding nucleic acid of the enzyme is as shown in SEQ ID NO: 25-32. NO:33, preferably, the plant-derived phosphohydrolase is tTcNudix1 with codon optimization and signal peptide truncated, the amino acid sequence of the tTcNudix1 with codon optimization and signal peptide truncated is SEQ ID NO:108, and preferably the encoding nucleic acid of the enzyme is SEQ ID NO:

34.

7. The method according to claim 6, characterized in that The method comprises supplementing amino acids to the yeast, preferably comprising supplementing HIS3 and URA3 amino acid genes to the yeast.

8. The method according to any one of claims 1 to 7 is characterized in that Further including any one of (a) to (h): (a) DNA fragment Donor 5 was introduced into yeast to replace the farnesyl pyrophosphate synthase gene ERG20 ww Introduced into yeast, the ERG20 ww The sequence of the encoded protein is SEQ ID NO: 77, preferably the encoding gene is SEQ ID NO: 3; (b) DNA fragment Donor 5 was introduced into yeast to transform the hydroxymethylglutaryl-CoA reductase isozyme HMG2 mutant HMG2 K6R Gene introduced into yeast, preferably HMG2 K6R The sequence of the encoded protein is SEQ ID NO: 81, preferably the encoding gene is SEQ ID NO: 7; (c) Introducing the DNA fragment Donor 6 into yeast and replacing the promoter of the ERG20 gene on the yeast genome with P erg7 , preferably the promoter is replaced by P erg7 The sequence of the protein encoded by the endogenous ERG20 gene is SEQ ID NO: 78, and the nucleic acid encoding is preferably as shown in SEQ ID NO: 4; (d) introducing the DNA fragment Donor 7 into the yeast and introducing the mevalonate kinase gene ERG12 into the host strain, preferably the protein sequence encoded by ERG12 is SEQ ID NO: 83 and the encoding nucleic acid is SEQ ID NO: 9; (e) introducing the DNA fragment Donor 8 into yeast, and introducing the EfmvaE and EfmvaS genes from Enterococcus faecalis into yeast, preferably, the protein sequences encoded by the EfmvaE and EfmvaS genes from Enterococcus faecalis are SEQ ID NO:79 and SEQ ID NO:80, and the encoding nucleic acids are SEQ ID NO:5 and SEQ ID NO:6; (a) to (e) are not in any particular order.

9. An engineered bacterium, characterized in that The engineered bacteria contain or express borneol diphosphate synthases derived from Salvia officinalis L., C. burmannii, Amomum villosum and Lippia dulcis. The host bacteria is a yeast. Preferably, the engineered bacteria contain or express borneol diphosphate synthases derived from Salvia officinalis L., C. burmannii and Amomum villosum. Preferably, the engineered bacteria contain or express borneol diphosphate synthases derived from Salvia officinalis L. and C. burmannii. Preferably, the engineered bacteria contain or express borneol diphosphate synthases derived from Salvia officinalis L.

10. The engineered bacteria according to claim 9, characterized in that The borneol diphosphate synthase is tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS, preferably tSoBPPS, tCbTPS1, tAvBPPS, and preferably the amino acid sequences of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown in SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 109, and SEQ ID NO: 110, respectively. Preferably, the encoding nucleic acids of tSoBPPS, tCbTPS1, tAvBPPS and tLdBPPS are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 35, and SEQ ID NO: 74, respectively.

11. The engineered bacteria according to claim 10, characterized in that Said inclusion or expression of borneol diphosphate synthase is to introduce the nucleic acid coding sequence of borneol diphosphate synthase into the yeast; and / or said introduction of the nucleic acid coding sequence of borneol diphosphate synthase into the yeast is to introduce an expression cassette containing the nucleic acid encoding borneol diphosphate synthase into the yeast; and / or by expressing a plasmid vector containing the nucleic acid encoding cassette of borneol diphosphate synthase into the yeast; preferably, the introduction into the yeast is carried out by integration into the yeast chromosome.

12. The engineered bacteria according to claim 11, characterized in that The engineered bacteria comprises a mutated codon-optimized farnesyl pyrophosphate synthase gene ERG20 ww , preferably ERG20 ww The sequence of the protein encoded by it is SEQ ID NO: 77, preferably the coding gene is shown in SEQ ID NO: 3, and preferably the promoter of the endogenous ERG20 gene of the engineered bacteria is replaced with P erg7 , the promoter was replaced by P erg7 The sequence of the protein encoded by the endogenous ERG20 gene is SEQ ID NO: 78, and the preferred nucleic acid encoding is shown in SEQ ID NO:

4.

13. The engineered bacteria according to claim 12, characterized in that The protein comprises or expresses a codon-optimized ERG20 ww To convert the codon-optimized ERG20 ww The nucleic acid encoding the gene is introduced into the yeast; and / or the codon-optimized ERG20 ww The nucleic acid encoding ERG20 is introduced into the yeast to ww The expression cassette encoding the nucleic acid is introduced into the yeast; and / or by expressing the ERG20 ww The plasmid vector encoding the nucleic acid expression cassette is introduced into the yeast; preferably, it is introduced into the yeast by integration into the yeast chromosome.

14. The engineered bacteria according to claim 13, characterized in that The engineered bacteria comprises one or more enzymes selected from the following: 1) Introducing acetoacetyl-CoA thiolase EfmvaE and mevalonate synthase EfmvaS from Enterococcus faecalis, preferably the protein sequences encoded by EfmvaE and EfmvaS are SEQ ID NO: 79 and SEQ ID NO: 80, and preferably the encoding nucleic acids are SEQ ID NO: 5 and SEQ ID NO: 6; 2) Expression of hydroxymethylglutaryl-CoA reductase isozyme HMG2 mutant HMG2 K6R , preferably HMG2 K6R The sequence of the encoded protein is SEQ ID NO: 81, and the encoding nucleic acid is SEQ ID NO: 7; 3) expressing an N-terminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, wherein the protein sequence encoded by the tHMG1 is SEQ ID NO: 82 and the encoding nucleic acid is SEQ ID NO: 8 4) Overexpressing the yeast endogenous mevalonate kinase gene ERG12, preferably the protein sequence encoded by ERG12 is SEQ ID NO: 83, and the encoding nucleic acid is SEQ ID NO: 9; 5) introducing the phosphomevalonate kinase gene ERG8, preferably the protein sequence encoded by ERG8 is SEQ ID NO: 84, and the encoding nucleic acid is SEQ ID NO: 10; 6) introducing the mevalonate pyrophosphate decarboxylase gene ERG19, preferably the protein sequence encoded by ERG19 is SEQ ID NO: 85, and the encoding nucleic acid is SEQ ID NO: 11; 7) introducing the isopentenyl pyrophosphate isomerase gene IDI1, preferably the protein sequence encoded by IDI1 is SEQ ID NO: 86, and the encoding nucleic acid is SEQ ID NO: 12; Preferably, the engineered bacteria comprises EfmvaE and EfmvaS introduced from Enterococcus faecalis, expresses the HMG2 mutant HMG2K6R, and overexpresses the yeast endogenous mevalonate kinase gene ERG12.

15. The engineered bacteria according to claim 14, characterized in that The engineered bacteria contains or expresses phosphohydrolase, and the phosphohydrolase contains endogenous phosphohydrolase and / or heterologous phosphohydrolase. Preferably, the engineered bacteria overexpresses phosphohydrolase.

16. The engineered bacteria according to claim 15, characterized in that The endogenous phosphohydrolase is an endogenous phosphatase of Saccharomyces cerevisiae, preferably the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1, preferably the amino acid sequences of LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1 are as SEQ ID NO: 87-98, respectively, preferably the encoding nucleic acid of LPP1, DPP1, PAH1, APP1, IPP1, NPP1, NPP2, PHO8, t62PHO8, GPP1, GPP2, YPP1 is as SEQ ID NO: 87-98. NO:13-24, preferably the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1, PAH1, IPP1, NPP1, NPP2, APPI, preferably the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1, PAH1, IPP1, NPP1, NPP2, preferably the endogenous phosphatase of Saccharomyces cerevisiae is LPP1, DPP1.

17. The engineered bacteria according to claim 16, characterized in that The heterologous phosphohydrolase comprises an Escherichia coli-derived phosphohydrolase and a plant-derived phosphohydrolase. Preferably, the heterologous phosphohydrolase is an Escherichia coli-derived phosphohydrolase. Preferably, the Escherichia coli-derived phosphatase is NudJ, NudF, NudC, mutT, cdH, phoA, ppa, and lpxH. The amino acid sequences of NudJ, NudF, NudC, mutT, cdH, phoA, ppa, and lpxH are respectively as SEQ ID NOs: 99-106. Preferably, the encoding nucleic acids of NudJ, NudF, NudC, mutT, cdH, phoA, ppa, and lpxH are SEQ ID NOs: 25-32. Preferably, the endogenous phosphatases of Escherichia coli are NudJ and NudF.

18. The engineered bacteria according to claim 17, characterized in that The heterologous phosphohydrolase comprises a plant-derived phosphohydrolase, preferably the plant-derived phosphohydrolase comprises a pyrethrum-derived (Tanacetum cinerariifolium) phosphohydrolase, preferably the pyrethrum-derived phosphohydrolase is TcNudix1, preferably the amino acid sequence of the enzyme is SEQ ID NO: 107, preferably the encoding nucleic acid of the enzyme is SEQ ID NO: 33; preferably the plant-derived phosphohydrolase is tTcNudix1 after codon optimization and signal peptide truncation, preferably the tTcNudix1 after codon optimization and signal peptide truncation, preferably the amino acid sequence of the enzyme is SEQ ID NO: 108, preferably the encoding nucleic acid of the enzyme is SEQ ID NO:

34.

19. The engineered bacteria according to claim 18, characterized in that The engineered bacteria contain anaplerotic amino acids, preferably anaplerotic HIS3 and URA3 amino acid genes.

20. A method for producing d-borneol, characterized in that The method comprises using YPD medium or Delft medium as a fermentation medium and using the engineered bacteria according to any one of claims 9 to 19 as a fermentation strain.

21. A method for constructing an engineered bacterium according to any one of claims 9 to 19 for producing d-borneol, characterized in that The method comprises the step of: ww , borneol diphosphate synthase, EfmvaE and EfmvaS, HMG2 K6R , ERG12, phosphohydrolase nucleic acid coding sequence is introduced into the yeast and replaces the endogenous ERG20 promoter with P erg7 , where P erg7 is the promoter of ERG7, which is derived from Saccharomyces cerevisiae; and / or contains the codon-optimized ERG20 ww , borneol diphosphate synthase, EfmvaE and EfmvaS, HMG2 K6R , ERG12, phosphohydrolase encoding nucleic acid expression cassette into the yeast; and / or by expressing the codon-optimized ERG20 ww , borneol diphosphate synthase, EfmvaE and EfmvaS, HMG2 K6R , ERG12, and a plasmid vector containing a nucleic acid expression cassette encoding phosphohydrolase is introduced into the yeast; preferably, it is introduced into the yeast by integration into the yeast chromosome. Preferably, the construction method also includes complementing the nutritional deficiency of the strain, including complementing the HIS3 and URA3 amino acid genes.

22. Use of the engineered bacteria according to any one of claims 9 to 19 in the production of borneol.

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