A method for improving the biosynthesis efficiency of sclareol

Abstract

The present invention belongs to the field of application of microbial genetic engineering and metabolic engineering, and specifically relates to a method for improving the biosynthesis efficiency of sclareol. Specifically, in a host strain, gene knockout is achieved through the CRISPR / Cas9 technology, and / or a DNA fragment is introduced to regulate the metabolic pathway of the strain to obtain a corresponding engineered strain; then, the obtained engineered strain is used for biosynthesis to obtain sclareol; the gene knockout is achieved by introducing a donor DNA containing homologous arms upstream and downstream of the coding frame; the DNA fragment sequentially includes, from upstream to downstream: an upstream homologous arm, a promoter, a gene, a terminator, and a downstream homologous arm; the host strain is selected from any one of Saccharomyces cerevisiae. The present invention associates cell lifespan with product synthesis and couples metabolic balance at the same time, thereby improving the synthesis efficiency of the target product and laying a foundation for industrial fed-batch fermentation.

Description

Technical Field

[0001] The present 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 labdane-type diterpenoid tertiary alcohol. As a natural plant fragrance, it is widely used in the fields of essence and fragrance, medicine, cosmetics, health products, etc. In particular, as a substitute for the precious fragrance ambergris, sclareol can be used to synthesize sclareolide and ambrein, and has high economic value and market application prospects (Acta Biochimica et Biophysica Sinica, 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 hinder the industrial production and application of sclareol. Therefore, there is an urgent need to develop a new sclareol preparation process that can achieve an efficient, clean, and renewable production process.

[0003] Microbial cell factories provide an economical and environmentally friendly method for producing high-value chemicals from renewable and inexpensive raw materials, including biofuels, fine chemicals, and pharmaceutical chemicals, etc. In recent years, with the development of functional genomics, metabolic engineering, and synthetic biology, yeast has become an ideal cell factory at present and is widely used in the biosynthesis process of target products (Front. Bioeng. Biotechnol., 2020, 8: 594347). Therefore, yeast cell factories are expected to become a new technical route for sclareol synthesis. For example, in Saccharomyces cerevisiae, through systematic optimization of the mevalonate pathway, the sclareol yield was increased to 403 mg / L (Metab. eng. 2015, 27, 65-75). Further, through the modification 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 systematic pathway engineering modification and combined with the optimization of two-phase fermentation conditions, batch-fed fermentation was carried out in a 5 L fermenter, and the sclareol yield reached 12.9 g / L (Green Chem., 2024, 26, 5202-5210). The applicant's team previously integrated metabolic module switching, modular pathway engineering, and regulation target modification in Saccharomyces cerevisiae, and the sclareol yield was increased to 11.4 g / L (Metab. Eng., 2023, 75, 19-28; Patent Application: 202111539688.7).

[0004] Although conventional metabolic engineering approaches, such as pathway engineering and enzyme engineering, can significantly enhance the ability of microbial cell factories to synthesize sclareol, existing technologies have overlooked a key issue in industrial production processes: cell viability and sustained production during prolonged fermentation. Cell viability is typically determined by cell lifespan, including both replicative and chronological lifespans (Science, 2010, 328, 321–326). Therefore, by regulating cell lifespan, sclareol-producing cells can maintain high activity and efficient production during prolonged fed-batch fermentation. Coupled with pathway engineering optimization, this approach is expected to further improve sclareol synthesis efficiency and promote its industrial application. Summary of the Invention

[0005] The object of the present invention is to improve the synthesis efficiency of the target product sclareol, and further propose a method for improving the biosynthesis efficiency of sclareol.

[0006] In order to achieve the above purpose, the present invention adopts the technical solution of

[0007] A method for improving the biosynthesis efficiency of sclareol, characterized by: using CRISPR / Cas9 technology to achieve gene knockout and / or introduce DNA fragments in a host strain to regulate the strain's metabolic pathway to obtain a corresponding engineered strain; and then using the obtained engineered strain to biosynthesize 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 regulatory gene 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 knockout the target gene. First, donor DNA is obtained by fusion PCR, including a 500bp upstream homologous arm and a 500bp downstream homologous arm. Second, a gRNA plasmid targeting the target gene is constructed (for the specific method, refer to the inventor's previous authorized patent ZL 202010428088.2). Then, 500 ng each of the gRNA plasmid and donor DNA are transformed into the Salvinolone-synthesizing yeast strain SCX42 (Metab. Eng., 2023, 75, 19-28). The obtained transformants are used to extract genomic DNA for PCR verification, and the strains with correct verification are used for Salvinolone fermentation.

[0014] Then, the obtained strains are fermented and cultured. The fermentation medium is a basal medium containing 20 g / L glucose, and they are cultured at 30 °C and 220 rpm for 96 h. The Salvinolone yield is determined by gas chromatography.

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

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

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

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

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

[0020] (e) Simultaneously introduce the EfmvaE and EfmvaS genes into the host strain genome.

[0021] Furthermore, at least one DNA fragment of (a) to (c) is introduced into the host strain by the CRISPR / Cas9 technology to obtain corresponding engineered strains by regulating the strain metabolic pathway; then, the obtained engineered strains are used for biosynthesis to obtain Salvinolone; where at least one of (a) to (c):

[0022] (a) Introduce the ACS1,2 gene into the host genome;

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

[0024] (c) Introduce 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 locus 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 , (b) obtaining an engineered strain using a strong constitutive promoter P TEF1 , the (c) engineering strain is obtained using any of the following promoters, the promoter is 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 from (a) to (e) is introduced into a host strain using CRISPR / Cas9 technology to modulate the metabolic pathway of the strain to obtain a corresponding engineered strain; and the obtained engineered strain is then 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 P was removed from the upstream activation sequence. 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] Application of the engineered bacterium as described above, application of the engineered bacterium in the preparation of sclareol.

[0039] The beneficial effects that can be achieved by this application include:

[0040] The method for regulating cell lifespan and modifying metabolic pathways to improve the biosynthesis efficiency of target products in the present invention first associates cell lifespan with product synthesis. On this basis, by coordinating the intensity of metabolic pathways, adapting the endogenous metabolic pathway, and the exogenous sclareol synthesis pathway, product synthesis is maximized, promoting the industrial production of products represented by sclareol. Description of the Drawings

[0041] Figure 1 Mechanism diagram related to 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 intensity of the MVA pathway based on lifespan-regulated strains to further increase production

[0044] Figure 4 Enhancing ethanol utilization and coupling with MVA pathway optimization to synergistically improve sclareol production

[0045] Figure 5 Weakening the ERG9 strategy to optimize and promote batch fed-batch fermentation of products

[0046] Figure 6 Efficient synthesis of sclareol by batch fed-batch fermentation Detailed Embodiments

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

[0048] The embodiments of the present invention are based on the modification of the sclareol synthesis strain SCX42, which was reported in the paper Metab. Eng., 2023, 75, 19 - 28, but does not specifically limit the present invention. The method for improving the sclareol synthesis efficiency provided by the present invention is applicable to any Saccharomyces cerevisiae strain.

[0049] Example 1 Regulating Cell Lifespan to Improve Product Synthesis Efficiency

[0050] To explore the effect of regulating cell lifespan on sclareol production, genes related to the nutrient sensing signal pathway and the mitophagy 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 by the engineered strains is as follows:

[0051] (1) Medium

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

[0053] Fermentation medium (basic component medium): 20 g / L glucose, 2.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 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 when in use.

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

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

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

[0057] Reagent 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 procedure and conditions

[0059] The engineered strains were streaked and activated on YPD plates, and single colonies were picked and cultured in 3 / 15 mL YPD liquid medium at 30 °C with shaking at 220 rpm for 16 h; inoculation, the seed liquid was washed twice with the fermentation medium, and inoculated into the fermentation medium at an initial OD 600 = 0.1, the liquid loading was 20 mL / 100 mL conical flask, and fermentation was carried out at 30 °C with shaking at 220 rpm for 72 - 96 h. Samples were taken at fixed points or at the end point for analysis of biomass (expressed as the absorbance value at 600 nm) and sclareol production

[0060] (3) Sclareol synthesis

[0061] The results showed that compared with the starting strain SCX42 (0.67 g / L), the deletion of factors related to the nutrient sensing pathway and negative regulators of mitophagy had different degrees of effects on the production of sclareol (Table 3). Therefore, the above results indicate that regulating cell lifespan can indeed improve the synthesis efficiency of target products, which can be used as a new metabolic engineering modification strategy for efficient biosynthesis processes and subsequent industrial scale-up.

[0062] Table 3 Effects of knockout of factors related to regulating cell lifespan on sclareol production

[0063]

[0064] Example 2: Strategy Superposition Synergistically Improves the Yield of Salviol

[0065] The above results showed that the knockout of the mitophagy negative regulator Ppg1, the nutrient sensing factor Tor1, and Ras2 could improve the yield of salviol. Therefore, in this study, these three factors were further combined and superimposed in pairs according to the above records to verify whether they could coordinately improve the yield of salviol. The results showed that the combined deletion of tor1Δppg1Δ and ras2Δppg1Δ could indeed further increase the yield, up to 1.3 g / L and 1.2 g / L respectively( Figure 2 ).

[0066] Example 3: Optimization of the Mevalonate Pathway Promotes the Synthesis of Salviol

[0067] In this invention, the starting strain SCX42 for the synthesis of salviol only enhanced 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 regulation of cell lifespan in the above examples significantly improved the product synthesis efficiency, which might lead to insufficient precursors for the synthesis of salviol. To maximize the product synthesis, the MVA pathway was further optimized to make its expression intensity coordinated with the lifespan optimization. Using the engineering strain SZL37 (tor1Δppg1Δ) with the highest yield in Example 1 as the starting strain, IDI1, ERG8, ERG19, ERG12, EfmvaE, and EfmvaS were expressed separately or in combination. The sequence information is shown in Tables 4 and 5. The genes shown in Table 4 were integrated at neutral sites respectively. The gRNA sequences of the integration sites and the site sequence information can be found in the previous published article by the applicant's research group, FEMS Microbiol. Lett., 2022, 369, 1-5.

[0068] The strains with correct verification were fermented and detected according to the culture conditions in Example 1. The results are as Figure 3 shown. The individual overexpression all improved the yield of salviol to varying degrees. Compared with the starting strain, it increased by 4% - 11.2%. Similarly, the overexpression of the genes EfmvaE - EfmvaS also had a slight promoting effect on the yield of salviol. The above results indicate that the regulation of cell lifespan improved the synthesis efficiency of salviol, 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 the MVA Pathway Optimization Expression Cassette

[0070] Serial Number Gene NCBI No. Promoter Terminator Site 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 Sequences of partial genes, promoters and terminators

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] Example 4 Enhancement of ethanol utilization coupled with optimization of the MVA pathway to synergistically improve the production of sclareol

[0078] Ethanol is the main by-product of Saccharomyces cerevisiae fermentation, which will affect the carbon source utilization efficiency. Moreover, a relatively high ethanol accumulation may exert stress on cell survival and affect the function of lifespan regulatory factors. Therefore, in order to improve the ethanol utilization ability of the strain, the ethanol utilization pathway was further enhanced on the basis of the above SZL37 strain (tor1Δppg1Δ), including alcohol dehydrogenase (ADH2), aldehyde dehydrogenase (ALD6), and acetyl-CoA synthetase (ACS1,2). The gene sequence information is shown in Tables 6 and 7. The gRNA sequences of the integration sites and the site sequence information can be found in the previous article published by the applicant's research group, FEMS Microbiol. Lett., 2022, 369, 1-5.

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

[0080]

[0081] Table 7 Promoter and terminator sequences required for optimizing the ethanol utilization pathway

[0082]

[0083]

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

[0085] On this basis, by strengthening the ethanol utilization pathway, the supply of acetyl-CoA was increased, which 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. The experimental results Figure 4 are shown. When strain SGN01 was used as the host, the gene EfmvaE-EfmvaS was overexpressed, and strain SGN10 was obtained, which could significantly increase the production of sclareol to 1.7 g / L, while the expression of the remaining genes had no obvious effect.

[0086] Example 5 Weakening the ERG9 Strategy to Optimize the Fed-Batch Fermentation of the Product

[0087] Using strain SCX42 as the starting strain, the high-glucose concentration-responsive promoter P HXT1 was used to weaken the gene ERG9, which is crucial for the production of sclareol (Metab. Eng., 2023, 75, 19-28). However, considering the subsequent fed-batch fermentation process, due to the continuous addition of glucose, its weakening effect may be unstable. Moreover, if glucose is controlled at a low level for a long time, it is not conducive to cell growth. Therefore, different ERG9 weakening strategies were optimized to make it more suitable for the fed-batch fermentation process.

[0088] Based on the best strain SGN10 in Example 4 of this invention, its original P HXT1 -ERG9 was respectively replaced with the endogenous promoter P ERG9 , the endogenous promoter P ERG9-ΔUAS that removes the upstream activation sequence, the promoter P MET3 inhibited by methionine, and ERG9-CLN2 ([[]] Figure 5 ) fused with the degradation tag.

[0089] (1) Strain construction

[0090] Using SGN10 as the starting strain, first, the promoter P ERG9 of the endogenous ERG9 gene was complemented, and G418 was used as the resistance screening (kanMX gene). An integrated DNA fragment was constructed, including the P ERG9 fragment (upstream homologous arm), the fragment kanMX, the fragment P HXT1 , and the fragment ERG9 (downstream homologous arm). The above DNA fragments were amplified by PCR in turn, and then the above fragments were assembled by fusion PCR to obtain the complete integrated DNA fragment P ERG9 -kanMX-PHXT1 -ERG9, 500 ng was transformed into SGN10 strain and cultured and screened on YPD+G418 plate at 30 °C to obtain the correct transformant SGN10-kan.

[0091] On this basis, while recovering the kanMX resistance, the corresponding donor DNA fragments obtained using different promoters were integrated into the genome. Specifically, 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 (2917 bp). All donor DNA fragments were amplified and assembled using fusion PCR technology. 500 ng of the gRNA plasmid targeting kanMX and each of the above donor DNA fragments were separately transformed into SGN10-kan and cultured and screened on SD+His plate at 30 °C. After PCR identification of the obtained transformants, they were named SJQ01, SJQ02, SJQ03, and SJQ04 in sequence. For fed-batch fermentation verification, the auxotrophic genes URA3 and HIS3 were in situ complemented into the above transformants (i.e., SGN10-kan, SJQ01, SJQ02, SJQ03, SJQ04) to obtain strains SGN10UH, SJQ01UH, SJQ02UH, SJQ03UH, and SJQ04UH respectively.

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

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

[0094] (3) Analysis of fermentation results

[0095] The fermentation results are as Figure 5As shown, there was no significant difference in the growth of the 5 strains, and the final OD600 remained around 30 - 35. The strain SJQ04 was slightly higher than other strains in the later stage of fermentation. The yields of sclareol were measured at 93h, 137h, 161h, and 213h. There was no obvious difference in the early stage of fermentation. In the later stage of fermentation, especially 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 7.6 g / L, while that of SJQ04 reached 9.0 g / L. This indicates that by regulating the lifespan factor, the growth efficiency of the strain in the later stage of fermentation was significantly improved. At the same time, through the systematic optimization of the MVA pathway, ethanol utilization pathway, and ERG9 weakening strategy, the intensity of the endogenous and exogenous metabolic pathways was well adapted, further promoting product synthesis, laying a good foundation for subsequent industrial production.

[0096] Example 6 Batch Fed-Batch Fermentation for High-Efficiency Synthesis of Sclareol

[0097] The present invention provides a method for improving the synthesis efficiency of sclareol. By regulating cell lifespan, the cell activity during the biosynthesis process of the strain is prolonged. At the same time, coupled with systematic metabolic engineering modification, the endogenous pathways (MVA pathway, ethanol synthesis, and ERG9 weakening) and the exogenous sclareol synthesis pathway are optimized and adapted, providing sufficient precursors for the engineered strain with extended lifespan, and reducing the high-concentration ethanol stress, further improving the biosynthesis efficiency.

[0098] 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 in the present invention were selected, and the auxotrophic genes URA3 and HIS3 were supplemented respectively to obtain the strains SCX42UH, SZL37UH, and SJQ04UH, and fed-batch fermentation in a bioreactor was carried out. The feeding conditions and process were similar to those in Example 5. After 160h of fermentation, the results were as Figure 6 shown. Although the starting strain SCX42UH grew well, due to ethanol accumulation and the decline of cell viability in the later stage of fermentation, the growth rate of sclareol yield was slow, and only 12 g / L was accumulated at the end of fermentation. In contrast, the strain SZL37UH (tor1Δppg1Δ) with regulated cell lifespan could maintain higher cell viability and more efficient synthesis ability in the later stage, and the final sclareol yield was increased to 15.9 g / L, and the yield reached 0.061 g / g glucose, which was increased by 32.5% and 22% respectively compared with the starting strain. On this basis, due to the strengthening of the ethanol utilization pathway, the ethanol accumulation amount during the whole fermentation process of the engineered strain SJQ04UH constructed in the present invention was significantly reduced. At the same time, the coupled lifespan regulation strategy maintained the continuous product synthesis ability in the later stage, and the sclareol yield was further increased to 18.9 g / L, and the yield reached 0.071 g / g glucose, which is the highest reported yield of microbial synthesis of sclareol at present, laying 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, Tor1 and Ppg1 genes were knocked out by CRISPR / Cas9 technology, and then introduced ACS1,2 Gene was obtained from strain SGN01, and the gene was overexpressed in strain SGN01 EfmvaE-EfmvaS Strain SGN10 was obtained and SGN10 was used as the starting strain with endogenous ERG9 Promoter P ERG9 start up ERG9 Gene expression and ERG9 Gene and protein degradation tag protein CLN2 The corresponding engineered strain is obtained by fusion expression; and the obtained engineered strain is then used to biosynthesize sclareol; The host strain is selected from any one of Saccharomyces cerevisiae.

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

3. A use of the engineered bacteria according to claim 2, characterized in that: Application of the engineered bacteria in the preparation of sclareol.

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