Saccharomyces cerevisiae genetically engineered bacteria with high yield of sclareol and construction and application thereof

Abstract

This invention discloses a high-yield *Saccharomyces cerevisiae* genetically engineered strain and its construction and application, belonging to the fields of microbiology, genetic engineering, and synthetic biology. Using *Saccharomyces cerevisiae* as the starting strain, this invention enhances the supply of gerany-gerany-pyrophosphate precursors by overexpressing the endogenous BTS1 gene, obtaining the chassis strain SC001; further, through screening using a combination of multi-species lysine-2-pyrophosphate synthase and gerany-2-perillyl alcohol synthase, *Saccharomyces cerevisiae* (Saccharomyces cerevisiae) was identified as a high-yield gerany-2-perillyl alcohol synthase. Mesosphaerum suaveolens Source: MsLPPS and QiYa ( Salvia hispanica The optimal catalytic combination is ShTPS; by integrating the above two enzymes into the SC001 genome, an engineered strain capable of efficiently synthesizing perillaldehyde was obtained. This invention solves the bottlenecks of low enzyme activity, poor synergy, and insufficient yield in existing perillaldehyde microbial synthesis.

Description

Technical Field

[0001] This invention relates to a genetically engineered Saccharomyces cerevisiae strain that produces high levels of perillaldehyde, and its construction and application, belonging to the fields of microbiology, genetic engineering and synthetic biology. Background Technology

[0002] Sclareol is a typical hemispherane-type bicyclic diterpenoid diterpenoid diterpenoid alcohol with the molecular formula C2. 20 H 36 O2, with a molecular weight of 308.5, is formed by the condensation of four isoprene units and was initially isolated from Salvia sclarea (sage) in southern Europe. Perillyl alcohol possesses both excellent aroma properties and biocompatibility, and is widely used not only as a fragrance base in cosmetics and high-end perfumes and as a food flavoring agent, but also as a core precursor for the industrial synthesis of ambergris. It is currently the only key chemical intermediate on the market that can be scalably replaced by natural ambergris, making it extremely valuable in the market.

[0003] Currently, the production of industrial-grade perillaldehyde relies heavily on traditional plant extraction processes, which involve cultivating perillaldehyde plants and extracting their active ingredients. This production model has significant drawbacks: yield is constrained by multiple factors such as plant variety, planting region, seasonal climate, and land resources; raw material supply is unstable; extraction and purification processes are complex; production costs are high; and overall capacity is low, making it difficult to meet the growing industrial market demand in the fragrance and fine chemical industries.

[0004] Compared to plant extraction, microbial synthesis technology has significant advantages such as short growth cycle, controllable production, environmental friendliness, lack of natural condition limitations, and large-scale fermentation production, making it the mainstream research direction for the green synthesis of terpenoid natural products. Saccharomyces cerevisiae, as a mature eukaryotic model microorganism, possesses a complete terpenoid precursor synthesis pathway, a clear protein folding modification system, a mature genetic modification system, and high safety, making it the optimal chassis strain for the synthesis of perillaldehyde.

[0005] The main synthetic pathway of perillyl alcohol is as follows: Glucose is converted to acetyl-CoA via glycolysis. Acetyl-CoA then generates isoprene pyrophosphate (IPP) via the mevalonate pathway or the erythrosine phosphate pathway, which in turn generates geranyl geranyl pyrophosphate (GGPP). Under the action of Class II diterpene synthase (lysine pyrophosphate diol ester synthase), the diphosphate group of GGPP undergoes ionization, leading to cyclization and the formation of a cyclized diterpene diphosphate intermediate. This intermediate is then hydroxylated to generate hemispheryl diterpene diphosphate (LDPP), which is finally converted to the final product, perillyl alcohol, under the action of Class I diterpene synthase (perillyl alcohol synthase). Current research has reconstructed the heterologous synthetic pathway by heterologously introducing the genes of key enzymes (lysine pyrophosphate diol ester synthase and perillyl alcohol synthase) into *Saccharomyces cerevisiae*, thus achieving the preliminary microbial synthesis of perillyl alcohol. However, existing technologies still face core bottlenecks: the catalytic activity of wild-type LPPS and TPS derived from sage is low, resulting in insufficient overall conversion efficiency of the two-step catalytic pathway. When only two wild-type enzymes are heterologously co-expressed, terpene synthesis precursors accumulate in large quantities, severely limiting product synthesis capacity. For example, in 2013, Yang Wei et al. expressed SsLPPS and SsTPS separately in Saccharomyces cerevisiae while simultaneously enhancing the precursor GGPP, and the yield of perillyl alcohol after 72 h of YPD shake-flask fermentation was only 2.05 mg / L; the control group in patent CN112574897B, which co-expressed wild-type dual enzymes in wild-type Saccharomyces cerevisiae, also yielded only 2.13 mg / L. Strains expressing only two key enzymes exhibit extremely low product concentrations and poor substrate conversion rates, failing to meet the economic requirements of large-scale industrial production. Summary of the Invention

[0006] Technical issues The technical problem to be solved by the present invention is that when existing recombinant Saccharomyces cerevisiae produces perillyl ester synthase (LPPS) and perillyl ester synthase (TPS) using lysine pyrophosphate diol ester synthase (LPPS), the expression efficiency of the two enzyme combination is low, the synergistic catalytic ability is weak, the product concentration is extremely low, and the substrate conversion rate is poor.

[0007] Technical solution This invention uses *Saccharomyces cerevisiae*, a yeast known for its high safety and genetic modifiability, as the chassis host. It focuses on screening for efficient expression combinations of LPPS and TPS. Through heterologous expression adaptability screening, expression intensity optimization, and comparison of synergistic activities of lysine pyrophosphate diol ester synthase (LPPS) and perillyl alcohol synthase (TPS) from multiple species, an optimal LPPS-TPS dual-enzyme expression combination was obtained, exhibiting efficient and stable intracellular expression and outstanding synergistic catalytic activity within *Saccharomyces cerevisiae*. This addresses the core issues of low expression efficiency, weak synergistic catalytic ability, and low product yield in existing perillyl alcohol synthesis technologies, providing a simple, efficient, and stable technical solution for the industrial-scale green production of perillyl alcohol.

[0008] This invention provides a genetically engineered strain for the efficient synthesis of perillaldehyde, using *Saccharomyces cerevisiae* as the starting strain, overexpressing the endogenous BTS1 (geranyl-geranyl diphosphate synthase) gene of *Saccharomyces cerevisiae*, and derived from *Heliotropium indicum* (a type of plant). Mesosphere sweet-smelling LPPS genes from Chia ( Spanish sage The TPS gene.

[0009] The amino acid sequence of the protein encoded by the endogenous BTS1 gene of *Saccharomyces cerevisiae* is shown in SEQ ID NO. 1. The sequence of the endogenous BTS1 gene of *Saccharomyces cerevisiae* is shown in SEQ ID NO. 4.

[0010] The amino acid sequence of the protein encoded by the LPPS gene derived from *Syzygium aromaticum* is shown in SEQ ID NO.2. The sequence of the LPPS gene derived from *Syzygium aromaticum* is shown in SEQ ID NO.5.

[0011] The source is Qiya ( Spanish sage The amino acid sequence of the protein encoded by the TPS gene of [a specific organism] is shown in SEQ ID NO. 3. [The protein is derived from] [a specific organism]. Spanish sage The TPS gene sequence is shown in SEQ ID NO.6.

[0012] In some embodiments of the present invention, the overexpression is performed by integrating the BTS1, LPPS, and TPS genes into the Saccharomyces cerevisiae genome.

[0013] In some embodiments of the present invention, the starting strain is Saccharomyces cerevisiae IMX581.

[0014] This invention provides a method for constructing the genetically engineered bacteria, the specific construction steps of which are as follows: 1) Construction of a chassis strain SC001 overexpressing BTS1: Using Saccharomyces cerevisiae IMX581 as the starting strain, the endogenous BTS1 gene of Saccharomyces cerevisiae was overexpressed through gene integration to enhance the synthesis and accumulation of intracellular GGPP precursors in the strain, thereby obtaining a chassis strain SC001 with sufficient substrate supply; the gene integration site can be a commonly used integration site of Saccharomyces cerevisiae. 2) The ingredients are derived from mountain fragrance ( Sweet-smelling mesosphere LPPS gene and Chia ( Sage Spanish The TPS gene was integrated into the genome of strain SC001 to obtain a genetically engineered bacterium that synthesizes perillaldehyde efficiently, thus constructing a perillaldehyde-specific core synthetic pathway with sufficient substrate, high catalytic efficiency, and strong stability.

[0015] The present invention provides a method for producing perillaldehyde using the genetically engineered bacteria, which involves inoculating the genetically engineered bacteria into YPD medium and synthesizing perillaldehyde using glucose as a carbon source.

[0016] Beneficial effects This invention enhances the strain's own GGPP synthesis ability by overexpressing the endogenous BTS1 gene of Saccharomyces cerevisiae, thereby solving the problem of insufficient intracellular precursor substrate in wild-type strains and obtaining a stable chassis strain SC001.

[0017] This invention utilizes the SC001 chassis to screen for multi-species dual-enzyme combinations. It involves pairwise heterologous expression of LPPS and TPS genes from various species, verifying the gene expression levels and synergistic catalytic activity for the synthesis of perillaldehyde within SC001 cells for each combination. After multiple rounds of comparative screening, unexpectedly, enzyme combinations with close sequence relationships and high homology showed significantly poorer target product synthesis capabilities. Most highly homologous pairings resulted in strains that produced almost no perillaldehyde. Ultimately, the enzymes were determined to originate from *Ligustrum lucidum* (a type of wild mushroom). Sweet-smelling mesosphere LPPS genes from Chia ( Sage Spanish The TPS derived from this enzyme is the optimal and efficient expression combination adapted to the SC001 chassis. Both enzymes can be expressed efficiently and stably within the SC001 strain, and can efficiently synergistically catalyze the core synthesis reaction of perillaldehyde using sufficient GGPP precursors, significantly improving the synthesis efficiency and yield of perillaldehyde. Compared to other dual-enzyme combinations and wild-type chassis expression systems, the modified system of the above LPPS-TPS dual-enzyme combination exhibits high expression efficiency and significantly improved product synthesis, making it the optimal modified system for the efficient synthesis of perillaldehyde by *Saccharomyces cerevisiae*.

[0018] This invention effectively overcomes the shortcomings of existing perillaldehyde biosynthesis technologies, such as low enzyme catalytic efficiency, low yield, and poor stability. It lays a solid technical foundation for the green, simple, and efficient synthesis and production of perillaldehyde by brewing yeast fermentation, and has extremely high industrial application value. Attached Figure Description

[0019] Figure 1 Metabolic pathway diagram of perillyl alcohol in Saccharomyces cerevisiae.

[0020] Figure 2 Gas chromatogram of GGOH standard.

[0021] Figure 3 Gas chromatogram of perillyl alcohol standard.

[0022] Figure 4 Comparison of yield between SC001 and control strain GGOH in Example 1.

[0023] Figure 5Gas chromatogram of fermentation broth sample of engineered bacteria SC017.

[0024] Figure 6 Homology trees of LPPS from different sources.

[0025] Figure 7 Homology trees of TPS from different sources.

[0026] Figure 8 Comparison of perillyl alcohol yields in different LPPS-TPS combinations in Example 2. Detailed Implementation

[0027] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention. The pROS10 plasmid involved in the following embodiments is a laboratory-preserved plasmid and is a universal tool plasmid for the modification of Saccharomyces cerevisiae. The pROS10 plasmid is described in the following literature: Mans et al FEMS Yeast Res. 2015 Mar;15(2). pii: fov004. doi: 10.1093 / femsyr / fov004. Epub 2015 Mar 4. The Saccharomyces cerevisiae involved in the following embodiments IMX581 This strain, *Saccharomyces cerevisiae*, is a commonly used CRISPR / Cas9 editing chassis strain in the fields of synthetic biology and metabolic engineering, and is intended for laboratory storage. IMX581 genotype: MATa ura3-52 can1Δ::cas9- natNT2 TRP1 LEU2 HIS3 This refers to the genome containing the Cas9 protein expression cassette from the CRISPR-Cas9 system, along with the Ura trophic deficiency selection marker, as described in the following literature: CRISPR / Cas9: a molecular Swissarmy knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. The culture medium used in the following examples is as follows: Seed culture medium (YPD) (1L): 10 g yeast extract, 20 g tryptone, 20 g glucose.

[0028] Screening medium (SD-U) (1 L): YNB 6.7g, DO Supplement-Ura 1.29g, glucose 0.5g / L.

[0029] The strain information involved in the following examples is shown in Table 1 below: Table 1: Information on the main plasmids and strains involved in this invention

[0030] Table 2: Primers used in Implementation Case 1

[0031] The detection method of perillaldehyde involved in the following examples is as follows: (1) Sample preparation: Take 700 μL of fermentation broth into a 2 mL centrifuge tube, add an equal volume of n-hexane and an appropriate amount of quartz sand, vortex and shake for 30 min; centrifuge at 12000 rpm for 10 min, filter the supernatant through a 0.22 μm organic filter membrane, and test.

[0032] (2) Gas chromatography conditions: Agilent HP-5 column (30 m × 0.32 mm, 0.25 μm), injector temperature: 250℃; detector temperature: 230℃; column temperature program: 150℃ for 1 min → 20℃ / min to 220℃ for 5.5 min → 10℃ / min to 300℃ for 15.5 min; carrier gas: nitrogen, flow rate 2 mL / min; injection volume 1 μL. The target substance was identified by comparing its retention time with that of the standard, and the substance was quantitatively analyzed based on its peak area. Example 1: Construction of the perillaldehyde synthesis chassis strain SC001 The specific steps are as follows: (1) Using the Saccharomyces cerevisiae genome as a template, five DNA fragments (site homologous arms, promoter, expression cassette and terminator) were amplified by primers X-1 UF and X-1 UR (GAL7p), GAL7p-F and GAL7p-R, GAL7p-BTS1-UF and TDH2t-BTS1-R, TDH2t-F and TDH2t-R, X-1-DF (TDH2t) and X-1 DR, respectively. Then, the above five fragments were connected by overlap PCR to be used as the Donor DNA for gene editing. (2) Using the original pROS10 plasmid as a template, the pROS10-X-1 plasmid was obtained by PCR amplification with primer X-1-SgRNA. The obtained Donor DNA and pROS10-X-1 plasmid were co-transformed into the competent strain of Saccharomyces cerevisiae IMX581 by chemical transformation. Screening was performed on SD-U medium, and the obtained single colonies were identified by PCR using primers X-1-JD-F and X-1-JD-R. Based on the PCR results and sequencing comparison analysis, strain SC001, which overexpresses the BTS1 gene on chromosome X-1 of Saccharomyces cerevisiae, was obtained.

[0033] Single clones of strain SC001 were picked from the plate and inoculated into 2 mL of YPD medium for overnight culture. After 12-16 h, the seed culture was converted to OD2000. 600 An initial inoculum of 0.2 g was inoculated into 20 mL of YPD shake flask fermentation medium; the flask was then incubated at 30°C and 220 rpm for 3 days. Figure 4 As shown, the shake-flask fermentation results indicated that, compared to the control strain IMX581, the BTS1 overexpressing strain SC001 accumulated 65 mg / L of GGOH in the fermentation broth after 3 days of shake-flask fermentation. This result confirms that strain SC001 synthesizes a large amount of GGPP precursors intracellularly. However, due to the lack of an efficient downstream catalytic pathway, a large amount of unused GGPP is hydrolyzed and converted into GGOH, accumulating extensively intracellularly, resulting in a waste of precursor substrate resources. This also indicates that this chassis strain possesses sufficient GGPP substrate supply potential.

[0034] Example 2: Expression screening using a combination of LPPS and TPS Because the yield of perillaldehyde varies when LPPS and TPS genes are expressed in combination from different sources, this invention uses *Saccharomyces cerevisiae* IMX581 as the starting strain and screens for the optimal expression combination of LPPS and TPS from different sources, including: *Sesamum indicum* (…). Winged sesame ), Shanxiang ( Sweet-smelling mesosphere ), Hairy throat coleopterus ( Plectranthus barbatus Teak ( Large tectona ), *Platycladus orientalis* leaf-derived herb ( Salvia yangii Vitex negundo () Vitex lamb-chaste ), Japanese cedar ( Cryptomeria japonica 7 types of LPPS; derived from date palms ( Phoenix dactylifera ), Salvia miltiorrhiza ( Salvia miltiorrhiza ), Qiya ( Spanish sage Three types of TPS were obtained. Under the drive of the GAL1 / 10 bidirectional promoter, 21 sets of dual enzyme expression cassettes were constructed and integrated into the HO site of SC001 to obtain strains SC002–SC022 (Table 5).

[0035] Single clones of all obtained strains were inoculated into 2 mL of YPD medium and cultured overnight on a shaker at 30°C and 220 rpm. Then, OD200 was used as the inoculum. 600 =0.2 g was inoculated into YPD medium for fermentation. Samples were taken after 72 hours for product analysis. Finally, shake-flask fermentation was compared, and the homology between different LPPS and TPS was analyzed (e.g., ...). Figure 6 , Figure 7 ) and perillyl alcohol production (e.g. Figure 8It was found that there were significant differences in the yield of LPPS and TPS among different combinations, and the protein sequence homology of the two enzymes LPPS and TPS was significantly negatively correlated with the fermentation yield of perillaldehyde: enzyme combinations with close sequence phylogenetic relationships and high homology had significantly poorer synthesis ability of the target product, and most high homology pairings produced almost no perillaldehyde. After screening all 21 groups of two enzyme combinations through fermentation, it was unexpectedly found that the product derived from *Heliotropium indicum* (a type of plant) produced the best yield. Sweet-smelling mesosphere MsLPPS and Chia ( Spanish sage When expressing the ShTPS combination from the source, the yield of perillaldehyde reached 55 mg / L, achieving the best synthesis effect; it is also the best combination of perillaldehyde synthases known to be screened in related studies to date. Table 3: LPPS from different sources involved in Example 2

[0036] Table 4: TPS from different sources involved in Example 2

[0037] Table 5: Strains involved in Example 2

[0038] SEQ ID NO.1 Geraniylgeraniyl diphosphate synthase MEAKIDELINNDPVWSSQNESLISKPYNHILLKPGKNFRLNLIVQINRVMNLPKDQLAIVSQIVELLHNSSLLIDDIEDNAPLRRGQTTSHLIFGVPSTINTANYMYFRAMQLVSQLTTKEPLYHNLITIFNEELINLHRGQGLDIYWRDFLPEIIPTQEMYLNMVMN KTGGLFRLTLRLMEALSPSSHGHSLVPFINLLGIIYQIRDDYLNLKDFQMSSEKGFAEDITEGKLSFPIVHALNFTKTKGQTEQHNEILRILLLRTSDKDIKLKLIQILEFDTNSLAYTKNFINQLVNMIKNDNENKYLPDLASHSDTATNLHDELLYIIDHLSEL*.

[0039] SEQ ID NO.2 LPPS *。

[0040] SEQ ID NO.3 TPS MSLALVTPFSGSRVGSLRNTFPRHGFPVTGNKSSLTFRCSLTTIDLMQNIKEKINDSLETAIVHIPSNLCVIDTLQRLGPDRYFQSEIDTILESTYMLWQQKHKIMFSNVTTHAMAFRLLRVKGYEISSEELAPYDNQDGVSQQTVDVAMVIELYRAAHERIDEEERCLVNILGWTTTFLNHQLHSKSIPDKKLHKLVEFYMNNYHGIPIRLGVRRNLDLYDMSHYQALRLKTRFSNICNEDFVALAMQDFTICQAQYHKELQQLQRWYADCRLDTLKFGRQVVFISYFLASLVIDDCATSAHARLAFTKTTVLATLIDDFFDYGGSRKECYNILELVNEWKEKASAEYESMEVEIIFTALYNTVNELAVMASVAQGRSTKEFIVGMWVEVVSSFKVELDAWSNGSQQSLDEYMSTSWLSVSGKIVVLMALLFIGVKLSDEMLMSEECTDLSRHVSIIIRLINDVCSFEKERKENKGNNCMSILLADGRGITEEEAIAEIKEIVENHRRKLMQIVYTKGTILPRRCRDIFVETCKAASYVYTSSDEFTTPQKLKEDMKSFLGL*.

[0041] SEQ ID NO.4 Gene encoding geranylgeranyl diphosphate synthase

[0042] SEQ ID NO.5 Gene encoding LPPS

[0043] SEQ ID NO.6 Gene encoding TPS

[0044] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered bacterium for synthesizing perillaldehyde, characterized in that, Using *Saccharomyces cerevisiae* as the starting strain, the endogenous BTS1 gene of *Saccharomyces cerevisiae* was overexpressed, and the strain was derived from *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Mesosphaerum suaveolens LPPS genes from Chia ( Salvia hispanica The TPS gene; The amino acid sequence of the protein encoded by the endogenous BTS1 gene of the *Saccharomyces cerevisiae* is shown in SEQ ID NO.

1. The source is mountain fragrance ( Mesosphaerum suaveolens The amino acid sequence of the protein encoded by the LPPS gene is shown in SEQ ID NO.

2. The source is Qiya ( Salvia hispanica The amino acid sequence of the protein encoded by the TPS gene is shown in SEQ ID NO.

3. The overexpression method involves integrating the BTS1, LPPS, and TPS genes into the Saccharomyces cerevisiae genome. The starting strain is Saccharomyces cerevisiae IMX581.

2. The method for constructing the genetically engineered bacteria for synthesizing perillaldehyde according to claim 1, characterized in that, Includes the following steps: 1) Construction of chassis strain SC001 overexpressing the endogenous BTS1 gene of *Saccharomyces cerevisiae*: Using *Saccharomyces cerevisiae* IMX581 as the starting strain, the endogenous BTS1 gene of *Saccharomyces cerevisiae* was overexpressed through gene integration. 2) The ingredients derived from mountain fragrance ( Mesosphaerum suaveolens The LPPS gene of ) and derived from Chia ( Salvia hispanica The TPS gene of the strain was integrated into the genome of the chassis strain SC001 to obtain a genetically engineered strain for the synthesis of perillaldehyde.

3. The application of the genetically engineered bacteria for synthesizing perillaldehyde as described in claim 1 in the production of perillaldehyde.

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