Method for improving biosynthesis efficiency of sclareol

Abstract

The invention belongs to the field of application of microbial genetic engineering and metabolic engineering, and particularly relates to a method for improving the biosynthesis efficiency of sclareol. Specifically, in a host strain, gene knockout is realized through a CRISPR / Cas9 technology, or / and a DNA fragment is introduced, and a strain metabolic pathway is adjusted to obtain a corresponding engineering strain; carrying out biosynthesis by utilizing the obtained engineering strain to obtain sclareol; the gene knockout is realized by introducing donor DNA (Deoxyribose Nucleic Acid) containing upstream and downstream homologous arms of a coding frame; the DNA fragment sequentially comprises an upstream homologous arm, a promoter, a gene, a terminator and a downstream homologous arm from upstream to downstream, the host strain is selected from any one of saccharomyces cerevisiae. According to the method, the cell life is associated with product synthesis, and meanwhile, metabolic balance is coupled, so that the synthesis efficiency of a target product is improved, and a foundation is laid for industrial fed-batch fermentation.

Description

Technical Field

[0001] The invention belongs to the application field of microbial genetic engineering and metabolic engineering, and specifically relates to a method for improving the biosynthesis efficiency of sclareol. Technical Background

[0002] Sclareol is a tertiary alcohol of the hemiaran type diterpene. As a natural plant fragrance, it is widely used in the fields of flavors and fragrances, medicine, cosmetics, and health products. In particular, as a substitute for the precious spice ambergris, sclareol can be used to synthesize sclareol lactone and ambroxol, and has high economic value and market application prospects (Journal of Biotechnology, 2013, 29(08): 1185-1192). At present, the industrial production of sclareol mainly relies on plant extraction, but the yield is low, the growth cycle is long, and it is affected by factors such as geography, environment, and climate, which seriously hinders the industrial production and application of sclareol. Therefore, it is urgent to develop a new preparation process for sclareol that can achieve an efficient, clean, and renewable production process.

[0003] Microbial cell factories provide an economical and environmentally friendly way to produce high-value chemicals, including biofuels, fine chemicals, and pharmaceutical chemicals, from renewable and inexpensive raw materials. In recent years, with the development of functional genomics, metabolic engineering, and synthetic biology, yeast has become an ideal cell factory and is widely used in the biosynthesis of target products (Front. Bioeng. Biotechnol., 2020, 8: 594347). Therefore, yeast cell factories are expected to become a new technical route for the synthesis of sclareol. For example, in Saccharomyces cerevisiae, the production of sclareol was increased to 403 mg / L through the optimization of the mevalonate pathway system (Metab. Eng. 2015, 27, 65-75). Furthermore, through the transformation of global transcriptional regulation targets, the yield reached 750 mg / L (Microb. Cell Fact. 2015, 14, 60). Similarly, in Yarrowia lipolytica, with the help of system pathway engineering and optimization of two-phase fermentation conditions, the production of sclareol reached 12.9 g / L in batch fed-batch fermentation in a 5L fermenter (Green Chem., 2024, 26, 5202-5210). In the early stage, the applicant team integrated metabolic module switching, modular pathway engineering and regulatory target modification in Saccharomyces cerevisiae, and the production of sclareol was increased to 11.4 g / L (Metab. Eng., 2023, 75, 19-28; patent application: 202111539688.7).

[0004] Although the ability of microbial cell factories to synthesize sclareol can be greatly improved by conventional metabolic engineering, such as pathway engineering and enzyme engineering, the existing technology has neglected a key issue in the industrial production process, namely, the cell activity and sustained production capacity during long-term fermentation. Cell activity is usually determined by cell lifespan, including replication lifespan and chronological lifespan (Science, 2010, 328, 321–326). Therefore, by regulating cell lifespan, sclareol-producing cells can maintain high activity and high production capacity during long-term fed-batch fermentation. At the same time, coupled with pathway engineering optimization, it is expected to further break through the efficiency of sclareol synthesis and promote its industrial application process. Summary of the invention

[0005] The invention aims to improve the synthesis efficiency of the target product sclareol, and further proposes a method for improving the biosynthesis efficiency of sclareol.

[0006] To achieve the above purpose, the present invention adopts the technical solution as follows:

[0007] A method for improving the biosynthesis efficiency of sclareol, characterized in that: in a host strain, gene knockout is achieved by CRISPR / Cas9 technology, and / or DNA fragments are introduced to adjust the metabolic pathway of the strain to obtain a corresponding engineered strain; and then the obtained engineered strain is used to perform biosynthesis to obtain sclareol;

[0008] The gene knockout is achieved by introducing donor DNA containing upstream and downstream homologous arms of the coding frame;

[0009] The DNA fragment includes, from upstream to downstream, an upstream homology arm, a promoter, a gene, a terminator, and a downstream homology arm;

[0010] The host strain is selected from any one of Saccharomyces cerevisiae.

[0011] Knock out at least one of the following lifespan regulator genes using CRISPR / Cas9 technology;

[0012] The lifespan regulatory factor genes include Tor1, Sch9, Gpa2, Gpr1, Ras1, Ras2, Bcy1, Tpk1, Tpk3, Hxt1, Hxk2, Vhs1, Reg1, Mig1, and Ppg1.

[0013] The above design uses the CRISPR / Cas9 system to seamlessly knock out the target gene. First, donor DNA is obtained by fusion PCR, including 500bp upstream homology arms and 500bp downstream homology arms; secondly, a gRNA plasmid targeting the target gene is constructed (for specific methods, please refer to the inventor's previous authorized patent ZL 202010428088.2); then, 500ng of gRNA plasmid and donor DNA are transformed into the sclarea alcohol synthesis yeast strain SCX42 (Metab. Eng., 2023, 75, 19-28). The genomic DNA of the obtained transformants is extracted for PCR verification to verify the correct strain for sclarea alcohol fermentation.

[0014] The strain obtained above was then fermented and cultured in a basic component culture medium containing 20 g / L glucose at 30° C. and 220 rpm for 96 h. The sclareol yield was determined by gas chromatography.

[0015] Furthermore, at least one DNA fragment in (a) to (e) is introduced into the strain obtained by regulating the lifespan regulatory factor gene as the host strain by CRISPR / Cas9 technology, and the metabolic pathway of the strain is adjusted to obtain the corresponding engineered strain; and then the obtained engineered strain is used for biosynthesis to obtain sclareol; wherein at least one of (a) to (e):

[0016] (a) Introducing the ERG8 gene into the host strain genome;

[0017] (b) introducing the ERG19 gene into the host strain gene;

[0018] (c) introducing the IDI1 gene into the host strain genome;

[0019] (d) introducing the ERG12 gene into the host strain genome;

[0020] (e) The EfmvaE and EfmvaS genes were simultaneously introduced into the host strain genome.

[0021] Furthermore, at least one DNA fragment in (a) to (c) is introduced into a host strain by CRISPR / Cas9 technology, and the metabolic pathway of the strain is adjusted to obtain a corresponding engineered strain; and then the obtained engineered strain is used to biosynthesize sclareol; wherein at least one of (a) to (c):

[0022] (a) Introducing ACS1,2 genes into the host genome;

[0023] (b) introducing the ALD6 gene into the host gene;

[0024] (c) Introducing the ADH2 gene into the host gene.

[0025] As described above, (a) the ACS1,2 genes are introduced into the X-3 site of the host genome;

[0026] (b) introducing the ALD6 gene into the IX-2 site of the host genome;

[0027] (c) introducing the ADH2 gene into the IX-2 site of the host genome;

[0028] The (a) engineering strain is obtained using a strong constitutive promoter P TEF1 , wherein (b) the engineered strain is obtained using a strong constitutive promoter P TEF1 The (c) engineering strain is obtained by using any of the following promoters, the promoter being a strong constitutive promoter P TDH3 , ethanol-inducible promoter P SSA1 , low glucose responsive promoter P HXT7 .

[0029] The host strain is any one of the Saccharomyces cerevisiae or a strain obtained from at least one DNA fragment described above.

[0030] Furthermore, at least one DNA fragment in (a) to (e) is introduced into a host strain by CRISPR / Cas9 technology, and the metabolic pathway of the strain is adjusted to obtain a corresponding engineered strain; and then the obtained engineered strain is used to biosynthesize sclareol; wherein at least one of (a) to (e):

[0031] (a) Using high glucose responsive promoter P HXT1 Initiate ERG9 gene expression;

[0032] (b) Using endogenous ERG9 promoter P ERG9 Initiate ERG9 gene expression;

[0033] (c) The endogenous ERG9 promoter was expressed with the upstream activation sequence removed. ERG9-ΔUAS Initiate ERG9 gene expression;

[0034] (d) Using methionine-repressible promoter P MET3 Initiate ERG9 gene expression;

[0035] (e) The ERG9 gene was fused with the protein degradation tag protein CLN2 for expression.

[0036] The host strain is any one of the Saccharomyces cerevisiae or a strain obtained from at least one DNA fragment described above.

[0037] An engineered bacterium constructed by the method described above.

[0038] An application of the engineered bacteria, and an application of the engineered bacteria in the preparation of sclareol.

[0039] The beneficial effects of this application include:

[0040] The method of the present invention for regulating cell lifespan and metabolic pathway modification to improve the biosynthesis efficiency of the target product is the first to link cell lifespan with product synthesis. On this basis, by coordinating the intensity of metabolic pathways, adapting endogenous metabolic pathways, and exogenous sclareol synthesis pathways, product synthesis is maximized, promoting the industrial production of products represented by sclareol. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Diagram of the mechanism of nutrient sensing and mitochondrial function in Saccharomyces cerevisiae

[0042] Figure 2 Superposition and coordination of lifespan regulation strategies to improve sclareol production

[0043] Figure 3 Coordinating the strength of the MVA pathway based on lifespan regulation strains to further improve production

[0044] Figure 4 Enhanced ethanol utilization coupled with MVA pathway optimization to synergistically increase sclareol production

[0045] Figure 5 Optimization of ERG9 attenuation strategy to promote product fed-batch fermentation

[0046] Figure 6 Efficient synthesis of sclareol by fed-batch fermentation DETAILED DESCRIPTION

[0047] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical companies.

[0048] The embodiment of the present invention is based on the modification of the clarysol synthesizing strain SCX42, which is reported in the paper Metab. Eng., 2023, 75, 19-28, but the present invention is not specifically limited thereto. The method for improving the synthesis efficiency of clarysol provided by the present invention is applicable to any Saccharomyces cerevisiae strain.

[0049] Example 1 Regulating cell lifespan to improve product synthesis efficiency

[0050] In order to explore the effect of regulating cell lifespan on sclareol production, genes related to nutrient sensing signaling pathway and mitochondrial autophagy pathway were selected for verification ( Figure 1, Table 3). In strain SCX42, the above genes were knocked out according to the CRISPR / Cas9-based gene editing method described above to obtain the corresponding engineered strains. The fermentation method of sclareol in the engineered strain is as follows:

[0051] (1) Culture medium

[0052] YPD medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast powder;

[0053] Fermentation medium (basic medium): glucose 20 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 14.4 g / L, MgSO4·7H2O 0.5 g / L, vitamins (Table 1), trace metals (Table 2). The initial pH was adjusted to 5.6 with KOH, and 20 mg / L uracil and 60 mg / L histidine were added as needed during use.

[0054] Table 1 Vitamin formula table (1000×)

[0055] Reagents Concentration (g / L) D-Biotin 0.05 D-Calcium Pantothenate 1.0 Ammonium Sulfate 1.0 Pyridoxine 1.0 niacin 1.0 p-Aminobenzoic acid 0.2 Inositol 25.0

[0056] Table 2 Trace metal formula table (500×)

[0057] Reagents Concentration (g / L) <![CDATA[FeSO4·7H2O]]> 3.0 <![CDATA[ZnSO4·7H2O]]> 4.5 <![CDATA[CaCl2·2H2O]]> 4.5 <![CDATA[MnCl2·4H2O]]> 1.0 <![CDATA[CoCl2·6H2O]]> 0.3 <![CDATA[CuSO4·5H2O]]> 0.3 <![CDATA[Na2MoO4·2H2O]]> 0.4 <![CDATA[H3BO3]]> 1.0 KI 0.1 <![CDATA[Na2EDTA·2H2O]]> 19.0

[0058] (2) Experimental process and conditions

[0059] The engineered strain was activated by streaking on a YPD plate, and single colonies were picked and cultured in 3 / 15 mL YPD liquid medium at 30 °C and 220 rpm for 16 h; inoculation, the seed liquid was washed twice with fermentation medium, and the initial OD 600 =0.1 was inoculated into the fermentation medium, the liquid volume was 20mL / 100mL conical flask, and the fermentation was carried out at 30°C and 220rpm for 72 to 96h. Fixed-point sampling or endpoint sampling was used for biomass (expressed as absorbance at 600nm) and sclareol yield analysis

[0060] (3) Synthesis of Sclareol

[0061] The results showed that compared with the starting strain SCX42 (0.67 g / L), the loss of nutrient sensing pathway-related and mitochondrial autophagy negative regulatory factors had different degrees of influence on the yield of sclareol (Table 3). Therefore, the above results show that regulating cell lifespan can indeed improve the synthesis efficiency of the target product, which can be used as a new metabolic engineering transformation strategy for efficient biosynthesis and subsequent industrialization.

[0062] Table 3 Effects of knockout of factors regulating cell life span on sclareol production

[0063]

[0064] Example 2: Superposition of strategies to synergistically increase the yield of sclareol

[0065] The above results show that knocking out the negative regulator of mitochondrial autophagy Ppg1, the nutrient sensing factor Tor1, and Ras2 can increase the yield of sclareol. Therefore, this study further combined these three factors in pairs according to the above records to verify whether they can coordinate to increase the yield of sclareol. The results showed that the combined deletion of tor1Δppg1Δ and ras2Δppg1Δ can indeed further increase the yield, up to 1.3g / L and 1.2g / L ( Figure 2 ).

[0066] Example 3 Optimization of mevalonate pathway to promote sclareol synthesis

[0067] In the present invention, the starting strain SCX42 for sclareol synthesis only enhances the expression of the key rate-limiting genes ERG10 and HMG1,2 in the mevalonate (MVA) pathway (Metab. Eng., 2023, 75, 19-28). However, considering that the above-mentioned embodiment regulates the cell lifespan and significantly improves the product synthesis efficiency, it may lead to insufficient precursors for sclareol synthesis. In order to maximize product synthesis, the MVA pathway is further optimized to coordinate its expression intensity with lifespan optimization. The engineering strain SZL37 (tor1Δppg1Δ) with the highest yield in Example 1 is used as the starting strain, and IDI1, ERG8, ERG19, ERG12, EfmvaE and EfmvaS are expressed separately or in combination, and the sequence information is shown in Tables 4 and 5. The genes shown in Table 4 are integrated at the neutral sites respectively. For the integration site gRNA sequence and site sequence information, please refer to the previous article FEMS Microbiol. Lett., 2022, 369, 1-5 published by the applicant's research group.

[0068] The correct strain was verified and fermented according to the culture conditions of Example 1. Figure 3 As shown in the figure, overexpression alone increased the yield of sclareol to varying degrees, which was 4% to 11.2% higher than the starting strain. Similarly, overexpression of the genes EfmvaE-EfmvaS also slightly promoted the yield of sclareol. The above results indicate that the efficiency of sclareol synthesis is increased by regulating cell lifespan, resulting in insufficient synthesis of precursor substances, and the product yield can be further increased by coordinating the MVA pathway.

[0069] Table 4 Construction of optimized expression cassette for MVA pathway

[0070] Serial number Gene NCBI No. Promoter Terminator Location 1 ERG8 QHB10950.1 <![CDATA[P GAL1,10 ]]> <![CDATA[T CYC1 ]]> VI-1 2 ERG19 QHB11421.1 <![CDATA[P GAL1,10 ]]> <![CDATA[T IDP1 ]]> VI-1 3 IDI1 QHB12144.1 <![CDATA[P GAL7 ]]> <![CDATA[T ENO2 ]]> VI-1 4 ERG8,IDI1 / <![CDATA[P GAL1,10 ,P GAL7 ]]> <![CDATA[T CYC1 ,T ENO2 ]]> VI-1 5 ERG19,IDI1 / <![CDATA[P GAL1,10 ,P GAL7 ]]> <![CDATA[T IDP1 ,T ENO2 ]]> VI-1 6 ERG8-ERG19 / <![CDATA[P GAL1,10 ]]> <![CDATA[T CYC1 ,T IDP1 ]]> VI-1 7 ERG8-ERG19-IDI1 / <![CDATA[P GAL1,10 ,P GAL7 ]]> <![CDATA[T CYC1 ,T IDP1 ,T ENO2 ]]> VI-1 8 ERG12 QHB10938.1 <![CDATA[P GAL2 ]]> <![CDATA[T ENO2 ]]> X-3 9 EfmvaE,EfmvaS As shown in Table 5 <![CDATA[P GAL1,10 ]]> <![CDATA[T IDP1 ,T PRM9 ]]> VIII-1

[0071] Table 5 Some genes, promoters and terminator sequences

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] Example 4 Enhanced ethanol utilization coupled with MVA pathway optimization to synergistically increase sclareol production

[0078] Ethanol is the main byproduct of Saccharomyces cerevisiae fermentation, which will affect the efficiency of carbon source utilization, and high ethanol accumulation may cause stress to cell survival, affecting the role of lifespan regulatory factors. Therefore, in order to improve the ethanol utilization ability of the strain, the ethanol utilization pathway is further strengthened on the basis of the above-mentioned SZL37 strain (tor1Δppg1Δ), including alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (ACS1,2). The gene sequence information is shown in Tables 6 and 7. For the integration site gRNA sequence and site sequence information, please refer to the previous article FEMS Microbiol. Lett., 2022, 369, 1-5 published by the applicant's research group.

[0079] Table 6 Construction of expression cassettes for optimizing ethanol utilization pathway

[0080]

[0081] Table 7 Promoter and terminator sequences required for optimization of ethanol utilization pathway

[0082]

[0083]

[0084] The experimental results are as follows Figure 4 As shown in Figure 2, when only ACS1 and ACS2 were expressed, the sclareol production increased by 6%. On this basis, when ALD6 and ADH2 were expressed in sequence, the sclareol production decreased significantly. TDH3 , ethanol-inducible promoter P SSA1 , and the low glucose concentration responsive promoter P HXT7The gene ADH2 was expressed, but the sclareol production did not further increase, indicating that in the strain constructed by the present invention, it is the rate-limiting step and the expression levels of other genes are sufficient.

[0085] On this basis, increasing the supply of acetyl-CoA by strengthening the ethanol utilization pathway may lead to insufficient strength of the MVA pathway. Therefore, referring to the results of Example 3, the key genes ERG8, ERG19 and EfmvaE-EfmvaS were overexpressed in strains SGN01 and SGN04, respectively. Figure 4 As shown, when strain SGN01 was used as the host, the genes EfmvaE-EfmvaS were overexpressed to obtain strain SGN10, which could significantly increase the sclareol production to 1.7 g / L, while the expression of other genes had no obvious effect.

[0086] Example 5 Optimization of ERG9 Weakening Strategy to Promote Product Batch Fed-Batch Fermentation

[0087] The strain SCX42 was used as the starting strain and the high glucose concentration responsive promoter P was used to generate HXT1 Attenuation of the gene ERG9 is essential for the production of clarysol (Metab. Eng., 2023, 75, 19-28). However, considering that the weakening effect may be unstable due to the continuous addition of glucose during the subsequent fed-batch fermentation process, and if the glucose level is controlled at a low level for a long time, it is not conducive to cell growth. Therefore, different ERG9 weakening strategies are optimized to make it more conducive to the fed-batch fermentation process.

[0088] Based on the best strain SGN10 in Example 4, the present invention further modified its original P HXT1 -ERG9 were replaced by the endogenous promoter P ERG9 , remove the endogenous promoter P of the upstream activation sequence ERG9-ΔUAS , methionine-repressed promoter P MET3 and ERG9-CLN2 fused with a degradation tag ( Figure 5 ).

[0089] (1) Strain construction

[0090] Using SGN10 as the starting strain, the endogenous ERG9 gene promoter P ERG9 The complement was performed and G418 was used as a resistance screening (kanMX gene). The integration DNA fragment was constructed, containing P ERG9 Fragment (upstream homology arm), fragment kanMX, fragment P HXT1 , fragment ERG9 (downstream homology arm), PCR amplified the above DNA fragments in sequence, and then assembled the above fragments using fusion PCR to obtain a complete integrated DNA fragment P ERG9 -kanMX-PHXT1 -ERG9, 500 ng was transformed into the SGN10 strain, and cultured and screened on YPD+G418 plates at 30°C to obtain the correct transformant SGN10-kan.

[0091] On this basis, while recovering kanMX resistance, the corresponding donor DNA fragments obtained by using different promoters were integrated into the genome. Specifically, the donor DNA was constructed in sequence: P ERG9 -ERG9 (1018 bp), P ERG9-ΔUAS -ERG9 (972 bp), P MET3 -ERG9 (1623 bp), P ERG9 -ERG9~CLN2 (2917bp), all donor DNA fragments were amplified and assembled using fusion PCR technology. The gRNA plasmid targeting kanMX and 500ng of the above donor DNA fragments were transformed into SGN10-kan, cultured and screened on SD+His plates at 30°C, and the obtained transformants were identified by PCR and named SJQ01, SJQ02, SJQ03, and SJQ04, respectively. In order to verify the fed-batch fermentation, the nutritional deficiency genes URA3 and HIS3 were in situ complemented to the above transformants (i.e., SGN10-kan, SJQ01, SJQ02, SJQ03, SJQ04), and strains SGN10UH, SJQ01UH, SJQ02UH, SJQ03UH, and SJQ04UH were obtained, respectively.

[0092] (2) Shake flask fed-batch fermentation

[0093] The shake flask batch fermentation used a 50 mL fermentation broth / 250 mL conical flask culture system, and selected engineered bacteria SGN10UH, SJQ01UH, SJQ02UH, SJQ03UH and SJQ04UH. The basic component culture medium Delft (same as Example 1) was used for batch fermentation, with a volume of 50 mL and an inoculation OD of 600 =0.2, pH 5.6. 5ⅹDelft medium (containing 500 g / L glucose) was used for fed-batch culture. When glucose was consumed in batch fermentation, 1 mL of 5ⅹDelft medium was added, and 4 mol / L potassium hydroxide was used to adjust the pH to 5-6 every 24 h. Fermentation was carried out at 30°C and 220 rpm for 213 h, and the biomass and sclareol yield were measured.

[0094] (3) Fermentation results analysis

[0095] The fermentation results are as follows Figure 5As shown, there is no significant difference in the growth of the five strains, and the final OD600 is maintained at about 30-35. In the late fermentation, the strain SJQ04 is slightly higher than the other strains. The yield of sclareol products at 93h, 137h, 161h and 213h was measured. There was no significant difference in the early fermentation period, but in the late fermentation period, the yield of strain SJQ04 was significantly higher than that of the final strain. At 213h, the sclareol yield of the control strain SGN10 was 7.6g / L, while that of SJQ04 reached 9.0g / L, indicating that the present invention significantly improves the growth efficiency of the strain in the late fermentation period by regulating the lifespan factor. At the same time, through the systematic optimization of the MVA pathway, the ethanol utilization pathway and the ERG9 weakening strategy, the strength of the endogenous and exogenous metabolic pathways is well adapted, which further promotes product synthesis and lays a good foundation for subsequent industrial production.

[0096] Example 6 Highly Efficient Synthesis of Sclareol by Fed-Batch Fermentation

[0097] The present invention provides a method for improving the synthesis efficiency of sclareol, which comprises regulating the cell activity of the biosynthesis process of a cell life-extending strain, coupling system metabolic engineering transformation, optimizing and adapting the endogenous pathway (MVA pathway, ethanol synthesis and ERG9 weakening) and the exogenous sclareol synthesis pathway, providing sufficient precursors for the engineered strain with extended life-span, reducing high-concentration ethanol stress, and further improving the biosynthesis efficiency.

[0098] In order to systematically compare the advantages of the methods and strategies proposed in the present invention, the starting strain SCX42 and the engineered strains SZL37 and SJQ04 constructed by the present invention were selected, and the auxotrophic genes URA3 and HIS3 were complemented, respectively, to obtain strains SCX42UH, SZL37UH and SJQ04UH, and the upper tank batch fermentation was carried out. The feeding conditions and process were similar to those in Example 5. Fermentation for 160 hours, the results are as follows Figure 6 As shown. Although the starting strain SCX42UH grew well, due to ethanol accumulation and decreased cell viability in the late fermentation period, the production of sclareol increased slowly, and only 12g / L was accumulated at the end of the fermentation. In contrast, the cell lifespan-regulated strain SZL37UH (tor1Δppg1Δ) was able to maintain higher cell viability and more efficient synthesis capacity in the later stage, and the final sclareol production increased to 15.9g / L, and the yield reached 0.061g / g glucose, which were 32.5% and 22% higher than the starting strain, respectively. On this basis, due to the enhanced ethanol utilization pathway, the ethanol accumulation of the engineered strain SJQ04UH constructed by the present invention was significantly reduced throughout the fermentation process, and the coupled lifespan regulation strategy maintained the continuous product synthesis capacity in the later stage, and the sclareol production was further increased to 18.9g / L, and the yield reached 0.071g / g glucose, which is the highest yield of sclareol synthesized by microorganisms reported so far, and has laid a good foundation for the large-scale industrial production of sclareol.

Claims

1. A method for improving the biosynthesis efficiency of sclareol, characterized in that: In the host strain, gene knockout is achieved by CRISPR / Cas9 technology, and / or DNA fragments are introduced to adjust the metabolic pathway of the strain to obtain the corresponding engineered strain; and then the obtained engineered strain is used for biosynthesis to obtain sclareol; The gene knockout is achieved by introducing donor DNA containing upstream and downstream homologous arms of the coding frame; The DNA fragment includes, from upstream to downstream, an upstream homology arm, a promoter, a gene, a terminator, and a downstream homology arm; The host strain is selected from any one of Saccharomyces cerevisiae.

2. The method for improving the biosynthesis efficiency of sclareol according to claim 1, characterized in that: Knock out at least one of the following lifespan regulator genes using CRISPR / Cas9 technology; The lifespan regulatory factor genes include Tor1, Sch9, Gpa2, Gpr1, Ras1, Ras2, Bcy1, Tpk1, Tpk3, Hxt1, Hxk2, Vhs1, Reg1, Mig1, and Ppg1.

3. The method for improving the biosynthesis efficiency of sclareol according to claim 1 or claim 2, characterized in that: By using CRISPR / Cas9 technology, at least one DNA fragment in (a) to (e) is introduced into the strain obtained by regulating the lifespan regulatory factor gene as the host strain, and the metabolic pathway of the strain is adjusted to obtain the corresponding engineered strain; and then the obtained engineered strain is used for biosynthesis to obtain sclareol; wherein at least one of (a) to (e): (a) Introducing the ERG8 gene into the host strain genome; (b) introducing the ERG19 gene into the host strain gene; (c) introducing the IDI1 gene into the host strain genome; (d) introducing the ERG12 gene into the host strain genome; (e) The EfmvaE and EfmvaS genes were simultaneously introduced into the host strain genome.

4. The method for improving the biosynthesis efficiency of sclareol according to claim 3, characterized in that: At least one DNA fragment in (a) to (c) is introduced into a host strain by CRISPR / Cas9 technology, and the metabolic pathway of the strain is adjusted to obtain a corresponding engineered strain; and then the obtained engineered strain is used for biosynthesis to obtain sclareol; wherein at least one of (a) to (c): (a) Introducing ACS1,2 genes into the host genome; (b) introducing the ALD6 gene into the host gene; (c) Introducing the ADH2 gene into the host gene.

5. The method for improving the biosynthesis efficiency of sclareol according to claim 4, characterized in that: The (a) engineering strain is obtained using a strong constitutive promoter P TEF1 , wherein (b) the engineered strain is obtained using a strong constitutive promoter P TEF1 The (c) engineering strain is obtained by using any of the following promoters, the promoter being a strong constitutive promoter P TDH3 , ethanol-inducible promoter P SSA1 , low glucose responsive promoter P HXT7 .

6. The method for improving the biosynthesis efficiency of sclareol according to claim 4, characterized in that: The host strain is any one of the Saccharomyces cerevisiae or a strain obtained by using at least one DNA fragment as described in claim 3 above.

7. The method for improving the biosynthesis efficiency of sclareol according to claim 1, characterized in that: At least one DNA fragment in (a) to (e) is introduced into a host strain by CRISPR / Cas9 technology, and the metabolic pathway of the strain is adjusted to obtain a corresponding engineered strain; and then the obtained engineered strain is used for biosynthesis to obtain sclareol; wherein at least one of (a) to (e): (a) Using high glucose responsive promoter P HXT1 Initiate ERG9 gene expression; (b) Using endogenous ERG9 promoter P ERG9 Initiate ERG9 gene expression; (c) The endogenous ERG9 promoter with the upstream activation sequence removed ERG9-ΔUAS Initiate ERG9 gene expression; (d) Using methionine-repressible promoter P MET3 Initiate ERG9 gene expression; (e) The ERG9 gene was fused with the protein degradation tag protein CLN2 for expression.

8. The method for improving the biosynthesis efficiency of sclareol according to claim 7, characterized in that: The host strain is any one of the Saccharomyces cerevisiae or a strain obtained by using at least one DNA fragment as described in claim 7.

9. An engineered bacterium constructed by the method according to claim 1.

10. An application of the engineered bacteria according to claim 9, characterized in that: The application of the engineered bacteria in the preparation of sclareol.

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