A strain for producing sclareol and a construction method and application thereof

Abstract

This invention belongs to the field of biosynthesis technology, specifically relating to a strain for producing perillaldehyde, its construction method, and its application. Addressing the microbial synthesis problem of perillaldehyde, this invention mutates and optimizes the phosphotransketase in the NOG pathway, using a strain derived from nanoarchaemonas (…). Nanoarchaeota archaeon Site-directed mutagenesis was performed on the phosphoketolase NaXpk, and a perillaldehyde synthesis pathway was constructed in E. coli. The effects of NaXpk mutation on perillaldehyde yield were compared to identify the S472F and S472Y mutants. These mutants were then applied to the perillaldehyde production pathway, and the resulting production strains achieved perillaldehyde yields of 623.3 mg / L and 655.2 mg / L, respectively, demonstrating significant effectiveness.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosynthesis, and particularly relates to a strain for producing sclareol and a construction method and application thereof. BACKGROUND

[0002] Sclareol is a diterpenoid compound with important biological activities, which exists widely in plants such as sclarea. It not only has pharmacological activities such as antibacterial, anti-inflammatory and antioxidant, but also is an important spice ingredient, which is widely used in the pharmaceutical, cosmetic and food industries. With the growing market demand, it is of great economic and social significance to develop an efficient and sustainable production method of sclareol.

[0003] At present, the production of sclareol mainly depends on plant extraction and chemical synthesis. However, the content of sclareol in plants such as sclarea is low, and the cost is high, which is difficult to meet the demand of large-scale industrial production. Chemical synthesis of sclareol usually requires harsh reaction conditions such as high temperature and high pressure, and a large amount of organic solvents and waste are produced in the chemical synthesis process, which pollutes the environment. In recent years, with the rapid development of synthetic biology and metabolic engineering, microbial synthesis of natural products has gradually become an efficient and sustainable production method. Compared with plant extraction and chemical synthesis, microbial fermentation has the advantages of short growth cycle, stable yield, and little environmental impact.

[0004] The mevalonate pathway (MVA) and the methylerythritol phosphate pathway (MEP) are the main pathways for synthesizing IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate), the precursors of sclareol. The methylerythritol phosphate pathway (MEP) mainly exists in prokaryotes (bacteria) and plant plastids, and the starting material is pyruvate and 3-phosphoglyceraldehyde, which generates MEP through a series of reactions, and finally synthesizes IPP and DMAPP. Similarly, the mevalonate pathway (MVA pathway) mainly exists in eukaryotes (animals, fungi, and some bacteria) and plant cytoplasm, and the starting material is acetyl-CoA, which generates MVA through a series of enzymatic reactions, and finally also synthesizes IPP and DMAPP.

[0005] In traditional glycolysis, carbon dioxide is released, resulting in carbon loss, and only two molecules of acetyl-CoA are generated from one molecule of glucose. The non-oxidative glycolysis pathway (NOG pathway) realizes the non-carbon loss conversion of one molecule of glucose to three molecules of acetyl-CoA through the reactions related to the pentose phosphate pathway, improves the supply of acetyl-CoA, and significantly enhances the flux of the MVA pathway, thereby improving the synthesis efficiency of sclareol.

[0006] IPP and DMAPP undergo a stepwise condensation under the catalysis of geraniol-geraniol pyrophosphate synthases (GGPPs) to generate geraniol-geraniol pyrophosphate (GGPP). Geraniol-geraniol pyrophosphate (GGPP) then undergoes cyclization and hydroxylation under the action of lysine-diol pyrophosphate synthase (SsLPS) to generate hemisinyl diterpene diphosphate (LDPP), which is then converted to the final product, perillyl alcohol, under the action of perillyl alcohol synthase (SsScs). This application aims to construct a perillyl alcohol synthesis pathway comprising GGPPs, SsLPS, and SsScs, referred to as the SC1 pathway.

[0007] Currently, the synthesis of perillaldehyde using microorganisms generally involves constructing a perillaldehyde expression pathway in a chassis strain or converting a substrate and key enzyme in vitro to produce perillaldehyde. For example, Chinese invention patent application CN 104031945 A, entitled "Method for Producing Perillaldehyde," discloses a method for producing perillaldehyde by contacting a specific polypeptide with perillaldehyde synthase activity with lysine pyrophosphate.

[0008] Although perillaldehyde has been synthesized by microorganisms, its biosynthetic pathway involves multiple enzymatic reactions, and the yield from Escherichia coli is low, hindering industrial production. Therefore, constructing a stable and efficient perillaldehyde synthesis pathway and production strains is crucial for realizing the industrial-scale microbial production of perillaldehyde. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention involves the mutation and optimization of the phosphotransketase (Xpk) in the NOG pathway. The enzyme derived from nanoarchaea (… Nanoarchaeota archaeon Site-directed mutagenesis was performed on the phosphate transketolase NaXpk, and the perillaldehyde synthesis pathway was constructed in E. coli host. The effects of NaXpk mutation on perillaldehyde yield were compared, thereby identifying the S472F mutant and the S472Y mutant, which were then applied to the perillaldehyde production pathway to achieve efficient perillaldehyde production.

[0010] To achieve the above objectives, the technical approach adopted by the present invention is as follows:

[0011] One of the technical solutions provided by the present invention is a phosphoketonease NaXpk mutant, which is obtained by mutating the wild-type phosphoketonease NaXpk shown in SEQ ID NO.16 to phenylalanine or tyrosine at position 472;

[0012] Furthermore, the phosphoketolase mutant is S472F, and its amino acid sequence is shown in SEQ ID NO.17;

[0013] Further, the phosphoketolase mutant is S472Y, and the amino acid sequence is shown as SEQ ID NO. 18.

[0014] The application further provides a coding gene of the above-mentioned phosphoketolase mutant.

[0015] Further, the coding gene of the phosphoketolase mutant has a nucleotide sequence shown as SEQ ID NO. 19 or SEQ ID NO. 20.

[0016] The second technical solution provided by the application is a recombinant vector or a recombinant strain comprising the coding gene of the above-mentioned phosphoketolase mutant.

[0017] Further, the expression plasmid used by the recombinant vector includes but is not limited to pACYDuet-1, pET-28a(+), pETDuet-1, pCDFDuet-1, pRSFDuet-1, pCOLADuet-1, pBAD / His A, pCold I, pMAL-c2x, pGEX-6P-1, pQE-80L, pTrcHis2B, pET21d, pUCmod, pSB1C3, pSEVA321, etc.

[0018] Further, the host used by the recombinant strain includes but is not limited to Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Aspergillus nidulans, Streptomyces, etc.

[0019] Still further, the host is Escherichia coli.

[0020] Preferably, the Escherichia coli includes but is not limited to: E. coli BL21 (DE3).

[0021] The third technical solution provided by the application is the application of the recombinant vector or the recombinant strain of the second technical solution, in particular, the application in the production of the phosphoketolase mutant of the first technical solution.

[0022] The fourth technical solution provided by the application is the application of the phosphoketolase mutant of the first technical solution or the recombinant vector or the recombinant strain of the second technical solution, in particular, the application in the production of sclareol.

[0023] Still further, the application is achieved by expressing the phosphoketolase mutant of the first technical solution in a sclareol production strain.

[0024] Further, the sylvestrol-producing strain further comprises part or all of the sylvestrol synthesis pathway (SC1 pathway), and / or part or all of the MVA pathway, and / or a phosphotransacetylase;

[0025] Further, the sylvestrol-producing strain, based on the phosphoketolase mutant of any one of the technical solutions, further comprises gene editing of any one or more of the following (1)-(4) in the host of Escherichia coli:

[0026] (1) overexpression of at least one gene of the following: PaGGPPS a geranylgeranyl diphosphate synthase coding gene SsLPS a sylvestrol heterologous sylvestrol synthase coding gene SsScS ;

[0027] (2) overexpression of at least one gene of the following: EcatoB an acetyl-CoA acetyltransferase coding gene EfmvaS a bifunctional acetyl-CoA thiolase coding gene EfmvaE a methylglutaconate kinase coding gene Samvk1 a phosphomevalonate kinase coding gene Samvk2 a mevalonate pyrophosphate decarboxylase coding gene SamvaD an isopentenyl pyrophosphate Δ-isomerase coding gene Bsfni ;

[0028] (3) overexpression of a phosphotransacetylase coding gene CkPta ;

[0029] (4) knockout of a phosphogluconate dehydratase coding gene edd , and / or overexpression of a 6-phosphogluconate dehydrogenase coding gene zwf ;

[0030] Further, the geranylgeranyl diphosphate synthase coding gene PaGGPPS has a nucleotide sequence as shown in SEQ ID NO. 1;

[0031] Further, the geranylgeranyl diphosphate synthase coding gene SsLPS has a nucleotide sequence as shown in SEQ ID NO. 2;

[0032] Further, the sylvestrol heterologous sylvestrol synthase coding gene SsScS has a nucleotide sequence as shown in SEQ ID NO. 3;

[0033] Further, the acetyl-CoA acetyltransferase coding gene EcatoB, the nucleotide sequence is shown as SEQ ID NO. 4;

[0034] Further, the 3-hydroxy-3-methylglutaryl coenzyme A synthetase encoding gene EfmvaS , the nucleotide sequence is shown as SEQ ID NO. 5;

[0035] Further, the bifunctional acetyl-CoA thiolase encoding gene EfmvaE , the nucleotide sequence is shown as SEQ ID NO. 6;

[0036] Further, the methylglutaconate kinase encoding gene Samvk1 , the nucleotide sequence is shown as SEQ ID NO. 7;

[0037] Further, the phosphomevalonate kinase encoding gene Samvk2 , the nucleotide sequence is shown as SEQ ID NO. 8;

[0038] Further, the mevalonate pyrophosphate decarboxylase encoding gene SamvaD , the nucleotide sequence is shown as SEQ ID NO. 9;

[0039] Further, the isopentenyl pyrophosphate Δ-isomerase encoding gene Bsfni , the nucleotide sequence is shown as SEQ ID NO. 10;

[0040] Further, the phosphotransacetylase encoding gene CkPta , the nucleotide sequence is shown as SEQ ID NO. 12;

[0041] Further, the edd gene, the NCBI accession number of the encoded protein is WP_322035618.1;

[0042] Further, the zwf gene, the NCBI accession number of the encoded protein is WP_272457244.1.

[0043] The fifth technical solution of the present application provides a genetically engineered bacterium for producing sclareol, wherein the engineered bacterium expresses the phosphoketolase NaXpk mutant in the host.

[0044] Further, the engineered bacterium also expresses part or all of the pathway in the sclareol synthesis pathway, and / or part or all of the pathway in the MVA pathway, and / or phosphotransacetylase;

[0045] Further, the engineered bacterium also knocks out the phosphogluconate dehydratase encoding gene eddMeanwhile, the 6-phosphogluconate dehydrogenase encoding gene is overexpressed zwf ;

[0046] Further, the sylvestrol synthesis pathway comprises at least one of the following genes: PaGGPPS a lavandulyl diphosphate synthase encoding gene SsLPS a sylvestrol heterologous sylvestrol synthase encoding gene SsScS ;

[0047] Further, the MVA pathway comprises at least one of the following genes: EcatoB an acetyl-CoA acetyltransferase encoding gene EfmvaS a bifunctional acetyl-CoA thiolase encoding gene EfmvaE a mevalonate kinase encoding gene Samvk1 a phosphomevalonate kinase encoding gene Samvk2 a mevalonate pyrophosphate decarboxylase encoding gene SamvaD an isopentenyl pyrophosphate Delta-isomerase encoding gene Bsfni ;

[0048] Further, the phosphotransacetylase encoding gene is CkPta ;

[0049] Preferably, the host is Escherichia coli BL21.

[0050] Advantages

[0051] 1. The present application uses site-directed mutagenesis technology to mutate wild-type phosphoketolase, obtains S472F mutant and S472Y mutant, and applies the mutants to the production of sylvestrol. It is verified through experiments that the yield of sylvestrol is increased by 12.8% and 18.6% respectively after the mutation of phosphoketolase, which shows that the application effect of NaXpk mutant S472F or S472Y in the production of sylvestrol is remarkable.

[0052] 2. The present application uses Escherichia coli as the chassis strain for the production of sylvestrol, inserts SC1 pathway genes based on the endogenous MEP pathway of Escherichia coli, constructs Escherichia coli for expressing and generating sylvestrol, introduces MVA pathway and NOG pathway on this basis, enhances expression, improves yield, and performs chassis modification (knocking out edd , overexpression zwf). Meanwhile, the NaXpk mutant S472F or S472Y of the application is introduced into the SC1 pathway, and the obtained sclareol production strain has a yield of 623.3 mg / L, 655.2 mg / L, which significantly improves the yield of sclareol. It can be seen that the engineering strain provided by the application can efficiently synthesize natural sclareol, has the advantages of high yield, is more suitable for industrial production, and meets the needs of industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is a pET21d-SC1 plasmid map;

[0054] Figure 2 is a pACYCDuet-MVA plasmid map;

[0055] Figure 3 is a pACYCDuet-MVA-NOG-1 plasmid map. DETAILED DESCRIPTION

[0056] The application will be described in detail below through specific embodiments. The technical means not specifically described in the application are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the application, and the essence and scope of the application are only limited by the claims. For those skilled in the art, various changes or modifications to the material components and amounts in these embodiments without departing from the essence and scope of the application also fall within the protection scope of the application.

[0057] In the application, NaXpk represents Xpk derived from nanohaloarchaea ( Nanoarchaeota archaeon ), LmXpk represents Xpk derived from Leuconostoc mesenteroides ( Leuconostoc mesenteroides ), Fxpk represents Xpk derived from Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), and BaXpk represents Xpk derived from Bifidobacterium animalis ( Bifidobacterium animalis ).

[0058] 1. Some of the culture medium information used in the application:

[0059] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, agar 15-20 g / L.

[0060] M9Y medium: potassium dihydrogen phosphate (KH2PO4) 4.2 g / L, dipotassium hydrogen phosphate (K2HPO4) 12 g / L, ammonium sulfate ((NH4)2SO4) 2 g / L, citric acid monohydrate 1.96 g / L, glycerol 25 g / L, yeast extract 10 g / L.

[0061] 2. Nomenclature of amino acid and DNA nucleic acid sequences

[0062] (1) The accepted IUPAC nomenclature for amino acid residues is used in the three letter / single letter code format. The accepted IUPAC nomenclature is used for DNA nucleic acid sequences.

[0063] (2) Identification (nomenclature) principle of NaXpk mutants

[0064] The "original amino acid residue + residue position + replaced amino acid residue" is used to represent the mutated amino acid in the NaXpk mutant. For example, S472F, which means that the amino acid at position 472 is replaced by phenylalanine (F) from the wild-type NaXpk serine (S), and the position number corresponds to the amino acid sequence number of the wild-type NaXpk in SEQ ID NO. 16. The information is as follows:

[0065]

[0066] In the present application, the wild-type NaXpk is from a nanogemus ( Nanoarchaeota archaeon ); the amino acid sequence is shown in SEQ ID NO. 16:

[0067] MVKIKELQIMADRLRVDSIIATTAAGSGHPTSCMSCAEIMSTLFFNTITKDDDFILSKGHAVPILWSVYAEAGIISQAKLKTLRKISSNLEGHPTPNMPYIQVATGSLGQGLSAGLGMALAKTKGKTFVLLGDSESTEGSVWEAANTAAYYKTKNLIAIIDVNRLGQSQETMHGHKIRVYKKKFKAFGWKAVSINGHSIKQILWALKLARKSKKPFAIIAKTYKGKGVSFLENKEGWHGKALSKDEAKLAIEEINPQKIKLKSQIKKPKVNYKRSNFKLNNYELKEEVATRDAFGKALVNAGKTNNKIITIDGEVRNSTKTEEFFKKFPKRSFESFIAEQNMVGMALGFSTQGFTPVVATFGTFFTRAFDFIRMANYSKANIKFVGSHVGVHIGEDGPSQMGLEDISMFLSVPNSTILYPSDAPSTEYLTKEMLNLKGISYLRTTRGTTPVIYSEKEKFPVGKFKVVKKSKSDKVLIIAAGITLHESLKAYEILQKKKINVRIIDLYSIRPLDSKNLIKNAKECKNKVIVVEDHYPYGISAVITEILGKVTSLNIKETPRSGDPDKLLKKYQIDSSTIIKTVEKLK

[0068] In the present application, the mutant S472F has an amino acid sequence as shown in SEQ ID NO. 17:

[0069] MVKIKELQIMADRLRVDSIIATTAAGSGHPTSCMSCAEIMSTLFFNTITKDDDFILSKGHAVPILWSVYAEAGIISQAKLKTLRKISSNLEGHPTPNMPYIQVATGSLGQGLSAGLGMALAKTKGKTFVLLGDSESTEGSVWEAANTAAYYKTKNLIAIIDVNRLGQSQETMHGHKIRVYKKKFKAFGWKAVSINGHSIKQILWALKLARKSKKPFAIIAKTYKGKGVSFLENKEGWHGKALSKDEAKLAIEEINPQKIKLKSQIKKPKVNYKRSNFKLNNYELKEEVATRDAFGKALVNAGKTNNKIITIDGEVRNSTKTEEFFKKFPKRSFESFIAEQNMVGMALGFSTQGFTPVVATFGTFFTRAFDFIRMANYSKANIKFVGSHVGVHIGEDGPSQMGLEDISMFLSVPNSTILYPSDAPSTEYLTKEMLNLKGISYLRTTRGTTPVIYSEKEKFPVGKFKVVKKSKFDKVLIIAAGITLHESLKAYEILQKKKINVRIIDLYSIRPLDSKNLIKNAKECKNKVIVVEDHYPYGISAVITEILGKVTSLNIKETPRSGDPDKLLKKYQIDSSTIIKTVEKLK

[0070] In the present application, the mutant S472Y, the amino acid sequence is shown as SEQ ID NO. 18:

[0071] MVKIKELQIMADRLRVDSIIATTAAGSGHPTSCMSCAEIMSTLFFNTITKDDDFILSKGHAVPILWSVYAEAGIISQAKLKTLRKISSNLEGHPTPNMPYIQVATGSLGQGLSAGLGMALAKTKGKTFVLLGDSESTEGSVWEAANTAAYYKTKNLIAIIDVNRLGQSQETMHGHKIRVYKKKFKAFGWKAVSINGHSIKQILWALKLARKSKKPFAIIAKTYKGKGVSFLENKEGWHGKALSKDEAKLAIEEINPQKIKLKSQIKKPKVNYKRSNFKLNNYELKEEVATRDAFGKALVNAGKTNNKIITIDGEVRNSTKTEEFFKKFPKRSFESFIAEQNMVGMALGFSTQGFTPVVATFGTFFTRAFDFIRMANYSKANIKFVGSHVGVHIGEDGPSQMGLEDISMFLSVPNSTILYPSDAPSTEYLTKEMLNLKGISYLRTTRGTTPVIYSEKEKFPVGKFKVVKKSKYDKVLIIAAGITLHESLKAYEILQKKKINVRIIDLYSIRPLDSKNLIKNAKECKNKVIVVEDHYPYGISAVITEILGKVTSLNIKETPRSGDPDKLLKKYQIDSSTIIKTVEKLK

[0072] 3. The primer information involved in the embodiments of the application is shown in Table 1.

[0073] Table 1 Primer Table

[0074]

[0075] The application will be further explained and described by specific examples.

[0076] Example 1 Plasmid Construction

[0077] 1. Plasmid construction of sclareol synthase pathway (SC1)

[0078] IPP and DMAPP undergo a stepwise condensation under the catalysis of geraniol-geraniol pyrophosphate synthases (GGPPs) to generate geraniol-geraniol pyrophosphate (GGPP). Geraniol-geraniol pyrophosphate (GGPP) then undergoes cyclization and hydroxylation under the action of lysine-diol pyrophosphate synthase (SsLPS) to generate hemisinyl diterpene diphosphate (LDPP), which is then converted to the final product—geraniol—under the action of perillyl alcohol synthase (SsScs). This example will construct a perillyl alcohol synthesis pathway including GGPPs, SsLPS, and SsScs, referred to as the SC1 pathway.

[0079] The plasmid pET21d-SC1 (General Biotechnology (Anhui) Co., Ltd.) was constructed. Specifically, the gene encoding geranium pyrophosphate synthase was constructed according to the sequences shown in SEQ ID NO. 1-3. PaGGPPS (SEQ ID NO.1), gene encoding lysine diol pyrophosphate synthase SsLPS (SEQ ID NO.2), gene encoding heterologous perilla alcohol synthase. SsScS (SEQ ID NO.3) was synthesized, and the synthesized PaGGPPS , SsLPS SsScS was integrated into the pET21d plasmid to construct the plasmid pET21d-SC1 (Apr resistance). The plasmid map is shown below. Figure 1 As shown.

[0080] 2. MVA pathway plasmid construction

[0081] The plasmid pACYCDuet-MVA (General Biotechnology (Anhui) Co., Ltd.) was constructed. Specifically, the gene encoding acetyl-CoA acetyltransferase was constructed according to the sequence shown in SEQ ID NO.4-10. EcatoB (SEQ ID NO.4), gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase EfmvaS (SEQ ID NO.5), gene encoding bifunctional acetyl-CoA thiolase EfmvaE (SEQ ID NO.6), mevalonate kinase encoding gene Samvk1 (SEQ ID NO.7), gene encoding mevalonate kinase (MGT). Samvk2 (SEQ ID NO.8), gene encoding mevalonate pyrophosphate decarboxylase SamvaD (SEQ ID NO. 9), gene encoding isopentenyl pyrophosphate Δ-isomerase Bsfni (SEQ ID NO.10) was synthesized, and the synthesized... EcatoB, EfmvaS, EfmvaE, Samvk1, Samvk2, SamvaD, Bsfni Integrating into the pACYCDuet-1 plasmid, the plasmid pACYCDuet-MVA (CMR resistance) was constructed. The plasmid map is shown below.Figure 2 as shown.

[0082] 3. MVA-NOG-1 pathway plasmid construction

[0083] According to the sequences shown in SEQ ID NO. 11-12, the phosphoketolase encoding gene LmXpk (SEQ ID NO. 11), the phosphotransacetylase encoding gene CkPta (SEQ ID NO. 12) were artificially synthesized to obtain the full-length DNA sequence.

[0084] Using pACYCDuet-1 as an expression vector, the recombinant expression plasmid was constructed by In-Fusion strategy, and the PCR amplification primers containing homologous arms (see Table 1) were used to clone LmXpk, CkPta and linearized pACYCDuet-MVA vector, and the recombinant plasmid containing MVA pathway and NOG pathway was obtained after homologous recombination. Specifically as follows:

[0085] (1) Using plasmid pACYCDuet-MVA as a template, F-pMVA and R-pMVA as upstream and downstream primers, PCR amplification was performed to obtain linearized pACYCDuet-MVA vector fragment;

[0086] (2) Using synthesized LmXpk as a template, F-LmXpk and R-LmXpk as upstream and downstream primers, PCR amplification was performed to obtain LmXpk fragment;

[0087] (3) Using synthesized CkPta as a template, F-CkPta and R-CkPta as upstream and downstream primers, PCR amplification was performed to obtain CkPta fragment;

[0088] (4) According to the kit instructions, the linearized pACYCDuet-MVA vector fragment, LmXpk fragment and CkPta fragment obtained above were subjected to homologous recombination reaction, and after 1 h of reaction at 50°C, the DH5α competent cells were transformed, and the Cmr-resistant plate was incubated at 37°C overnight; VF-NOG and VR-NOG were used as upstream and downstream verification primers for colony PCR screening of positive clones, and the positive clones were picked into 5 mL of LB medium containing Cmr, and the correct recombinant plasmid pACYCDuet-MVA-NOG-1 (CMR resistance) was extracted and verified by sequencing, and the plasmid map is shown in E. coli . Figure 3

[0089] 4. MVA-NOG-2 pathway plasmid construction

[0090] ​The same method as step 3 is used, only LmXpk is replaced by NaXpk (SEQ ID NO. 13), NaXpk The amplification primer of F-NaXpk, R-NaXpk is replaced, and the rest remains unchanged, to obtain the recombinant plasmid pACYCDuet-MVA-NOG-2 (CMR resistance).

[0091] 5. MVA-NOG-3 pathway plasmid construction

[0092] The same method as step 3 is used, only LmXpk is replaced by BaXpk (SEQ ID NO. 14), BaXpk The amplification primer of F-BaXpk, R-BaXpk is replaced, and the rest remains unchanged, to obtain the recombinant plasmid pACYCDuet-MVA-NOG-3 (CMR resistance).

[0093] 6. MVA-NOG-4 pathway plasmid construction

[0094] The same method as step 3 is used, only LmXpk is replaced by Fxpk (SEQ ID NO. 15), Fxpk The amplification primer of F-Fxpk, R-Fxpk is replaced, and the rest remains unchanged, to obtain the recombinant plasmid pACYCDuet-MVA-NOG-4 (CMR resistance).

[0095] 7. Construction of NaXpk mutant

[0096] (1) The template is pACYCDuet-MVA-NOG-2, and the mutant primer (F-S472F, R-S472F) is designed to perform S472F site-directed mutation on the 472th amino acid of NaXpk.

[0097] The PCR reaction system is as follows:

[0098]

[0099] The PCR reaction program is as follows:

[0100] Pre-denaturation: 98℃, 3min; denaturation: 98℃, 10sec; annealing: 55℃-65℃, 5sec; extension: 68℃, 5sec / kb; final extension: 68℃, 10min; storage: 4℃.

[0101] After the PCR is completed, the PCR product is digested with restriction endonuclease Dpn I for 2h to remove the template; after agarose gel electrophoresis, the target band is recovered, and ddH2O is used for elution during recovery, and then recombination transformationE. coli DH5α competent cells, coated Cmr resistance plate at 37°C overnight culture; VF-MUT, VR-MUT as the upstream and downstream validation primer for colony PCR screening positive clones, picking positive clones in 5 mL containing Cmr LB medium, sequencing, extracting the correct plasmid pACYCDuet-MVA-NOG (S472F)-2.

[0102] (2) The same method as step (1) is used to perform S472Y site-directed mutation on the 472th amino acid of NaXpk, only the mutation primer is replaced by F-S472Y, R-S472Y, and the rest remains unchanged, and finally the plasmid pACYCDuet-MVA-NOG (S472Y)-2 is obtained.

[0103] Example 2 Knockout edd Overexpression zwf of the chassis bacteria

[0104] The N20 sequence is designed using the online CRISPR gRNA design tool (https: / / chopchop.cbu.uib.no / ), and the primer pair F / R-edd (N20 design on the primer) is used to PCR amplify the plasmid pEcgRNA to obtain the linearized plasmid pEcgRNA, and then the resulting PCR product is circularized to construct the plasmid pEcgRNA-edd.

[0105] Using the primer pair F / R-D1, F / R-zwf, F / R-D3, the target homologous regions D1, D2, and D3 are amplified respectively, and the above amplification products are assembled by overlap extension PCR to obtain Donor-edd-zwf.

[0106] pEcgRNA-edd and Donor-edd-zwf are simultaneously transformed into E. coli BL21 (DE3) cells carrying pEcCas9 plasmid; VF-zwf, VR-zwf are used as upstream and downstream verification primers for colony PCR screening of positive clones, and DNA sequencing is performed to confirm that edd knockout zwf overexpression.

[0107] For positive clones, L-rhamnose is added to the culture medium to eliminate the pEcgRNA-edd plasmid, and sucrose is added to eliminate the pEgCas9 plasmid, thereby obtaining the empty chassis strain SCR01 which is knocked out edd and overexpressed zwf in E. coli BL21 (DE3) based on

[0108] Example 3 Recombinant strain construction

[0109] Different plasmids were transformed into different chassis according to Table 2 to obtain recombinant strains, as follows:

[0110] 1. Construction of strain G00 (containing SC1 pathway):

[0111] The recombinant plasmid pET21d-SC1 was transformed into E. coli BL21 (DE3) chassis cells, spread on LB plates containing Apr (100 μg / ml) resistance, and incubated at 37°C overnight. Single colonies were picked and inoculated in 50 μL of LB medium containing Apr (100 μg / ml) resistance and incubated at 37°C until the logarithmic growth phase. The recombinant strain G00 was obtained and stored in a -80°C freezer.

[0112] 2. Construction of strain G01 (containing MVA+SC1 pathway):

[0113] The recombinant plasmids pACYCDuet-MVA and pET21d-SC1 were co-transformed into E. coli BL21 (DE3) chassis cells, spread on LB plates containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance, and incubated at 37°C overnight. Single colonies were picked and inoculated in 50 μL of LB medium containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance and incubated at 37°C until the logarithmic growth phase. The recombinant strain G01 was obtained and stored in a -80°C freezer.

[0114] 3. Construction of strain G02 (containing MVA-NOG-1+SC1 pathway)

[0115] The recombinant plasmids pACYCDuet-MVA-NOG-1 and pET21d-SC1 were co-transformed into E. coli BL21 (DE3) chassis cells, spread on LB plates containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance, and incubated at 37°C overnight. Single colonies were picked and inoculated in 50 μL of LB medium containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance and incubated at 37°C until the logarithmic growth phase. The recombinant strain G02 was obtained and stored in a -80°C freezer.

[0116] 4. Construction of strain G03 (containing MVA-NOG-1+SC1 pathway)

[0117] The recombinant plasmids pACYCDuet-MVA-NOG-1 and pET21d-SC1 were co-transformed into SCR01 chassis cells, coated on LB plates containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance, incubated at 37°C overnight, and single colonies were picked into 50 μL of LB medium containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance, and incubated at 37°C to the logarithmic growth phase to obtain the recombinant strain G03, which was stored in a -80°C refrigerator.

[0118] 5. Construction of G04 strain (containing MVA-NOG-2+SC1 pathway)

[0119] The recombinant plasmids pACYCDuet-MVA-NOG-2 and pET21d-SC1 were co-transformed into SCR01 chassis cells, coated on LB plates containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance, incubated at 37°C overnight, and single colonies were picked into 50 μL of LB medium containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance, and incubated at 37°C to the logarithmic growth phase to obtain the recombinant strain G04, which was stored in a -80°C refrigerator.

[0120] 6. Construction of G05-G07 strains

[0121] The recombinant plasmids pACYCDuet-MVA-NOG-2 and pET21d-SC1 were co-transformed into E. coli BL21 (DE3) chassis cells, coated on LB plates containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance, incubated at 37°C overnight, and single colonies were picked into 50 μL of LB medium containing Cmr (50 μg / mL) and Apr (100 μg / ml) resistance, and incubated at 37°C to the logarithmic growth phase to obtain the recombinant strain G05, which was stored in a -80°C refrigerator.

[0122] Similarly, the recombinant plasmids pACYCDuet-MVA-NOG-3 and pET21d-SC1 were co-transformed into E. coli BL21 (DE3) chassis cells to obtain the recombinant strain G06.

[0123] Similarly, the recombinant plasmids pACYCDuet-MVA-NOG-4 and pET21d-SC1 were co-transformed into E. coli BL21 (DE3) chassis cells to obtain the recombinant strain G07.

[0124] Similarly, the recombinant plasmids pACYCDuet-MVA-NOG (S472F)-2 and pET21d-SC1 were co-transformed into the SCR01 chassis cells to obtain the recombinant strain G08.

[0125] Similarly, the recombinant plasmids pACYCDuet-MVA-NOG (S472Y)-2 and pET21d-SC1 were co-transformed into the SCR01 chassis cells to obtain the recombinant strain G09.

[0126] Table 2. Recombinant strain information table

[0127]

[0128] Example 4. Production experiment verification

[0129] The G00-G09 strains constructed above were taken for fermentation verification:

[0130] The frozen strain G00 was streaked on solid LB medium containing Apr (100 μg / mL) resistance and cultured at 37°C overnight; a single colony was picked and inoculated in 25 mL LB medium containing Cmr and Apr resistance and cultured at 37°C overnight to prepare a fermentation seed solution; the seed solution was inoculated in 25 mL M9Y medium at an inoculation amount of 1%, and cultured at 250 rpm and 37°C until OD600was about 0.6-0.8, then 0.1 mM IPTG and 10% (v / v) dodecane were added, and expression was induced at 22°C for 72 h, then the fermentation solution was collected and centrifuged at 12,000 rpm and 4°C to collect the dodecane layer, which was stored at -20°C for standby use. The dodecane layer was diluted 10 times with anhydrous ethanol, and the supernatant filtered through a 0.22 μm filter membrane was used for HPLC analysis to determine the production of vetiverol in the reaction sample. The results are shown in Table 3.

[0131] The frozen strains G01-G09 were respectively streaked on solid LB medium containing Cmr (50 μg / mL) and Apr (100 μg / mL) resistance and cultured at 37°C overnight; a single colony was picked and inoculated in 25 mL LB medium containing Cmr and Apr resistance and cultured at 37°C overnight to prepare a fermentation seed solution; the seed solution was inoculated in 25 mL M9Y medium at an inoculation amount of 1%, and cultured at 250 rpm and 37°C until OD600was about 0.6-0.8, then 0.1 mM IPTG and 10% dodecane were added, and expression was induced at 22°C for 72 h, then the fermentation solution was collected and centrifuged at 12,000 rpm and 4°C to collect the dodecane layer, which was stored at -20°C for standby use. The dodecane layer was diluted 10 times with anhydrous ethanol, and the supernatant filtered through a 0.22 μm filter membrane was used for HPLC analysis to determine the production of vetiverol in the reaction sample. The results are shown in Table 3.

[0132] HPLC detection conditions of sclareol:

[0133] Mobile phase: water: acetonitrile = 3:7, flow rate 1 mL / min, column: C18 (4.6 x 250 mm), column temperature 40℃, detection wavelength 195 nm (UV detector), collection time 45 min.

[0134] Table 3 Sclareol production results

[0135]

[0136] From the results of the production of sclareol shown in Table 3:

[0137] (1) The G00 strain containing only the SC1 pathway has very low sclareol production (3.5 mg / L); the G01 strain obtained by further introducing the MVA pathway has a significantly improved sclareol production of 302.1 mg / L; on this basis, the G02 strain obtained by further expressing the NOG pathway has a 7% increase in production.

[0138] (2) On the basis of (1), the strains G05, G06 and G07 obtained by replacing LmXpk in the G02 strain with NaXpk, BaXpk and FXpk, respectively, have sclareol production of 399.2 mg / L, 314.1 mg / L and 305.4 mg / L, respectively. This shows that the Xpk from different sources also has a very important effect on the production of sclareol, among which NaXpk is the best.

[0139] (3) On the basis of (1), the production strain G03 obtained by replacing the BL21 chassis strain with the strain SCR01 in which edd is knocked out and zwf is overexpressed, and simultaneously introducing plasmids containing the SC1 pathway, MVA pathway and NOG pathway (pACYCDuet-MVA-NOG-1, pET21d-SC1), has a sclareol production of 419.8 mg / L, compared with 323.4 mg / L of G02 which does not knock out edd and does not overexpress zwf This shows that knocking out edd and overexpressing zwf has a significant effect on the production of sclareol.

[0140] (4) Based on the results of (2) and (3), the strain G04 obtained by knocking out edd and overexpressing zwfThe expressed strain SCR01 is used as a host, pACYCDuet-MVA-NOG-2 containing NaXpk and pET21d-SC1 are introduced to obtain a production strain G04, and the yield of sclareol of the strain G04 reaches 552.6 mg / L, which is further increased by 31.6% than that of G03.

[0141] (5) On the basis of the strain G04, the wild-type NaXpk is further mutated into S472F or S472Y to obtain strains G08 and G09, and the yield of sclareol of the strains G08 and G09 reaches 623.3 mg / L and 655.2 mg / L respectively, which is increased by 12.8% and 18.6% respectively than that of G04, and it is indicated that the effect of mutating NaXpk into S472F or S472Y is remarkable.

[0142] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various forms and details of changes, modifications, replacements and variations to the embodiments without departing from the spirit and principles of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A phosphoketolase mutant, characterized in that, The mutant is obtained by mutating the serine at position 472 to phenylalanine or tyrosine based on the wild-type phosphoketolase NaXpk shown in SEQ ID NO.

16.

2. A gene encoding the phosphoketolase mutant of claim 1.

3. A recombinant vector or a recombinant strain comprising the gene of claim 2.

4. Use of the recombinant vector or the recombinant strain of claim 3 in the preparation of the phosphoketolase mutant of claim 1.

5. A genetically engineered bacterium, characterized by, The engineered bacteria take Escherichia coli as the host, and express the phosphoketolase mutant of claim 1, and further edit the following (1)-(4) genes: (1) overexpression of a geranylgeranyl pyrophosphate synthase encoding gene PaGGPPS , a labdane enediyol pyrophosphate synthase encoding gene SsLPS , a clivisate heterologous clivisyl alcohol synthase encoding gene SsScS ; (2) overexpression of acetyl-CoA acetyltransferase-encoding genes EcatoB , 3-hydroxy-3-methylglutaryl-CoA synthase-encoding genes EfmvaS , bifunctional acetyl-CoA thiolase-encoding genes EfmvaE , methylglutaconyl-CoA kinase-encoding genes Samvk1 , phosphomevalonate kinase-encoding genes Samvk2 , mevalonate pyrophosphate decarboxylase-encoding genes SamvaD , isopentenyl pyrophosphate Δ-isomerase-encoding genes Bsfni ; (3) overexpression of a phosphoacetyltransferase-encoding gene CkPta ; (4) Knocking out phosphogluconate dehydratase-encoding gene edd , overexpressing 6-phosphogluconate dehydrogenase-encoding gene zwf .

6. The engineered bacterium of claim 5, wherein, The gene encoding the geranylgeranyl pyrophosphate synthase PaGGPPS The nucleotide sequence is shown as SEQ ID NO.

1. The said Laidancheng alkene diol pyrophosphorylase coding gene SsLPS The nucleotide sequence is shown as SEQ ID NO.

2. The said clausena heterologous clausenase coding gene SsScS The nucleotide sequence is shown as SEQ ID NO.

3. The acetyl-CoA acetyltransferase-encoding gene EcatoB , the nucleotide sequence of which is shown as SEQ ID NO. 4; The 3-hydroxy-3-methylglutaryl coenzyme A synthetase encoding gene EfmvaS , the nucleotide sequence of which is shown as SEQ ID NO. 5; The bifunctional acetyl-CoA thiolase encoding gene EfmvaE , the nucleotide sequence of which is shown as SEQ ID NO. 6; The methylglutaconate kinase-encoding gene Samvk1 , the nucleotide sequence of which is shown as SEQ ID NO. 7; The phosphomevalonate kinase-encoding gene Samvk2 , the nucleotide sequence of which is shown as SEQ ID NO. 8; The mevalonate pyrophosphate decarboxylase encoding gene SamvaD , the nucleotide sequence is shown as SEQ ID NO. 9; The isopentenyl pyrophosphate delta-isomerase-encoding gene Bsfni , the nucleotide sequence of which is shown as SEQ ID NO. 10; The phosphoacetyltransferase-encoding gene CkPta , the nucleotide sequence of which is shown as SEQ ID NO. 12; The edd Gene, encoding protein with NCBI Accession No. WP_322035618.1; The zwf Gene, NCBI Accession No. WP_272457244.1 encoding the protein.

7. Use of the engineered bacteria of claim 5 or 6 in the production of sclareol.

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