Bacterial strain for producing sclareol as well as construction method and application of bacterial strain

Abstract

The invention belongs to the technical field of biosynthesis, and particularly relates to a strain for producing sclareol as well as a construction method and application of the strain. In order to solve the problem of microbial synthesis of sclareol, phosphoketolase in an NOG pathway is mutated and optimized, phosphoketolase NaXpk from nanoarchaea archaeon is subjected to site-directed mutagenesis, a sclareol synthesis pathway is constructed in an escherichia coli host, the influence of NaXpk before and after mutation on the yield of sclareol is compared, and the yield of sclareol is improved. The S472F mutant and the S472Y mutant are determined, the S472F mutant and the S472Y mutant are applied to a sclareol production path, the sclareol yield of the finally obtained production strains can reach 623.3 mg / L and 655.2 mg / L, and the effect is remarkable.

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: 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; Furthermore, the phosphoketokinase mutant is S472F, and its amino acid sequence is shown in SEQ ID NO.17; Furthermore, the phosphotransketolase mutant is S472Y, and its amino acid sequence is shown in SEQ ID NO.18.

[0011] The application also provides a gene encoding the phosphoketolase mutant. Further, the gene encoding the phosphoketolase mutant has a nucleotide sequence as shown in SEQ ID NO. 19 or SEQ ID NO. 20.

[0012] The second technical solution provided by the application is a recombinant vector or a recombinant strain comprising the gene encoding the phosphoketolase mutant. 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. 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. Still further, the host is Escherichia coli. Preferably, the Escherichia coli includes but is not limited to: E. coli BL21 (DE3).

[0013] 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.

[0014] 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. Still further, the application is achieved by expressing the phosphoketolase mutant of the first technical solution in a sclareol production strain. Still further, the sclareol production strain further comprises part or all of the pathways in the sclareol synthesis pathway (SC1 pathway) and / or part or all of the pathways in the MVA pathway and / or a phosphotransacetylase. Still further, the sclareol production strain takes Escherichia coli as the host, and on the basis of expressing the phosphoketolase mutant of the first technical solution, further gene editing is performed on any one or more of the following (1)-(4): (1) overexpression of a gene encoding geranylgeranyl diphosphate synthase PaGGPPSat least one gene selected from the group consisting of a gene encoding geranylgeranyl diphosphate synthase SsLPS , a gene encoding sclareol synthase SsScS ; (2) overexpressing at least one gene selected from the group consisting of a gene encoding acetyl-CoA acetyltransferase EcatoB , a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase EfmvaS , a gene encoding bifunctional acetyl-CoA thiolase EfmvaE , a gene encoding methylmalonyl-CoA kinase Samvk1 , a gene encoding phosphomevalonyl-CoA kinase Samvk2 , a gene encoding mevalonate pyrophosphate decarboxylase SamvaD , a gene encoding isopentenyl pyrophosphate Δ-isomerase Bsfni ; (3) overexpressing at least one gene selected from the group consisting of a gene encoding phosphotransferase CkPta ; (4) knocking out at least one gene selected from the group consisting of a gene encoding phosphogluconate dehydratase edd , and / or overexpressing at least one gene selected from the group consisting of a gene encoding 6-phosphogluconate dehydrogenase zwf ; Further, the gene encoding geranylgeranyl diphosphate synthase PaGGPPS has a nucleotide sequence as shown in SEQ ID NO. 1; Further, the gene encoding geranylgeranyl diphosphate synthase SsLPS has a nucleotide sequence as shown in SEQ ID NO. 2; Further, the gene encoding sclareol synthase SsScS has a nucleotide sequence as shown in SEQ ID NO. 3; Further, the gene encoding acetyl-CoA acetyltransferase EcatoB has a nucleotide sequence as shown in SEQ ID NO. 4; Further, the gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase EfmvaS has a nucleotide sequence as shown in SEQ ID NO. 5; Further, the gene encoding bifunctional acetyl-CoA thiolase EfmvaE has a nucleotide sequence as shown in SEQ ID NO. 6; Further, the gene encoding methylmalonyl-CoA kinase Samvk1 has a nucleotide sequence as shown in SEQ ID NO. 7; Further, the gene encoding phosphomevalonyl-CoA kinase Samvk2 has a nucleotide sequence as shown in SEQ ID NO. 8; Further, the gene encoding mevalonate pyrophosphate decarboxylaseSamvaD , the nucleotide sequence is shown as SEQ ID NO. 9; Further, the isopentenyl pyrophosphate Δ-isomerase encoding gene Bsfni , the nucleotide sequence is shown as SEQ ID NO. 10; Further, the phosphotransacetylase encoding gene CkPta , the nucleotide sequence is shown as SEQ ID NO. 12; Further, the edd gene, the NCBI accession number of the encoded protein is WP_322035618.1; Further, the zwf gene, the NCBI accession number of the encoded protein is WP_272457244.1.

[0015] 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. 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; Further, the engineered bacterium also knocks out the phosphogluconate dehydratase encoding gene edd , and overexpresses the 6-phosphogluconate dehydrogenase encoding gene zwf ; Still further, the sclareol synthesis pathway comprises at least one gene selected from the group consisting of geranylgeranyl pyrophosphate synthase encoding gene PaGGPPS , rishubang enediol pyrophosphate synthase encoding gene SsLPS , sclareol homologous sclareol synthase encoding gene SsScS ; Still further, the MVA pathway comprises at least one gene selected from the group consisting of acetyl-CoA acetyltransferase encoding gene EcatoB , 3-hydroxy-3-methylglutaryl-CoA synthase encoding gene EfmvaS , bifunctional acetyl-CoA thiolase encoding gene EfmvaE , methylpenthydroxy acid kinase encoding gene Samvk1 , phosphomevalonate kinase encoding gene Samvk2 , mevalonate pyrophosphate decarboxylase encoding gene SamvaD , isopentenyl pyrophosphate Δ-isomerase encoding gene Bsfni ; Still further, the phosphotransacetylase encoding gene is CkPta ; Preferably, the host is Escherichia coli BL21.

[0016] Beneficial effects 1. This invention utilizes site-directed mutagenesis to mutate wild-type phosphotransketolase, obtaining the S472F and S472Y mutants, which are then applied to the production of perillyl alcohol. Experimental verification shows that the phosphotransketolase mutation increases perillyl alcohol yield by 12.8% and 18.6%, respectively, indicating that the NaXpk mutants S472F or S472Y have significant effects on perillyl alcohol production.

[0017] 2. This invention uses *E. coli* as the chassis strain for perillyl alcohol production. Based on the endogenous MEP pathway in *E. coli*, the SC1 pathway gene is inserted to construct an *E. coli* strain that expresses perillyl alcohol production. Furthermore, the MVA and NOG pathways are introduced to enhance expression and increase yield. The *E. coli* chassis is also modified (knockout). edd Overexpression zwf Simultaneously, introducing the NaXpk mutants S472F or S472Y of this invention into the SC1 pathway yielded perillaldehyde-producing strains with yields of 623.3 mg / L and 655.2 mg / L, respectively, significantly increasing the perillaldehyde yield. Therefore, the engineered strains provided by this invention can efficiently synthesize natural perillaldehyde, offering advantages such as high yield, greater suitability for industrial production, and fulfillment of industrialization needs. Attached Figure Description

[0018] Figure 1 The pET21d-SC1 plasmid map; Figure 2 The pACYCDuet-MVA plasmid map; Figure 3 This is the pACYCDuet-MVA-NOG-1 plasmid map. Detailed Implementation

[0019] The present invention will now be described through specific embodiments. All technical means not specifically described herein are methods well-known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of the present invention.

[0020] In this invention, NaXpk represents a type of archaea derived from nano-archaea ( Nanoarchaeota archaeon The Xpk of ) indicates that it is derived from Leuconostoc membranaceus (LmXpk). Leuconostoc mesenteroides Xpk; Fxpk indicates it is derived from Bifidobacterium adolescentis (Bifidobacterium adolescentis). Bifidobacterium adolescentis) Xpk; BaXpk represents Xpk derived from animal bifidobacterium (‌ Bifidobacterium animalis ) Xpk.

[0021] 1、Part of the culture medium information used in this application: LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, agar 15~20 g / L.

[0022] 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.

[0023] 2、Naming of amino acids and DNA nucleic acid sequences (1) The recognized IUPAC naming of amino acid residues is used in the form of three-letter / one-letter code. The DNA nucleic acid sequence adopts the recognized IUPAC naming.

[0024] (2) Identification (naming) principle of NaXpk mutant 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:

[0025] In the present application, the wild type NaXpk is derived from nanobacterium (‌ Nanoarchaeota archaeon ); the amino acid sequence is shown in SEQ ID NO. 16: MVKIKELQIMADRLRVDSIIATTAAGSGHPTSCMSCAEIMSTLFFNTITKDDDFILSKGHAVPILWSVYAEAGIISQAKLKTLRKISSNLEGHPTPNMPYIQVATGSLGQGLSAGLGMALAKTKGKTFVLLGDSESTEGSVWEAANTAAYYKTKNLIAIIDVNRLGQSQETMHGHKIRVYKKKFKAFGWKAVSINGHSIKQILWALKLARKSKKPFAIIAKTYKGKGVSFLENKEGWHGKALSKDEAKLAIEEINPQKIKLKSQIKKPKVNYKRSNFKLNNYELKEEVATRDAFGKALVNAGKTNNKIITIDGEVRNSTKTEEFFKKFPKRSFESFIAEQNMVGMALGFSTQGFTPVVATFGTFFTRAFDFIRMANYSKANIKFVGSHVGVHIGEDGPSQMGLEDISMFLSVPNSTILYPSDAPSTEYLTKEMLNLKGISYLRTTRGTTPVIYSEKEKFPVGKFKVVKKSKSDKVLIIAAGITLHESLKAYEILQKKKINVRIIDLYSIRPLDSKNLIKNAKECKNKVIVVEDHYPYGISAVITEILGKVTSLNIKETPRSGDPDKLLKKYQIDSSTIIKTVEKLK In the present application, the mutant S472F has an amino acid sequence as shown in SEQ ID NO. 17: MVKIKELQIMADRLRVDSIIATTAAGSGHPTSCMSCAEIMSTLFFNTITKDDDFILSKGHAVPILWSVYAEAGIISQAKLKTLRKISSNLEGHPTPNMPYIQVATGSLGQGLSAGLGMALAKTKGKTFVLLGDSESTEGSVWEAANTAAYYKTKNLIAIIDVNRLGQSQETMHGHKIRVYKKKFKAFGWKAVSINGHSIKQILWALKLARKSKKPFAIIAKTYKGKGVSFLENKEGWHGKALSKDEAKLAIEEINPQKIKLKSQIKKPKVNYKRSNFKLNNYELKEEVATRDAFGKALVNAGKTNNKIITIDGEVRNSTKTEEFFKKFPKRSFESFIAEQNMVGMALGFSTQGFTPVVATFGTFFTRAFDFIRMANYSKANIKFVGSHVGVHIGEDGPSQMGLEDISMFLSVPNSTILYPSDAPSTEYLTKEMLNLKGISYLRTTRGTTPVIYSEKEKFPVGKFKVVKKSKFDKVLIIAAGITLHESLKAYEILQKKKINVRIIDLYSIRPLDSKNLIKNAKECKNKVIVVEDHYPYGISAVITEILGKVTSLNIKETPRSGDPDKLLKKYQIDSSTIIKTVEKLK In the present application, the mutant S472Y, the amino acid sequence is shown as SEQ ID NO. 18: MVKIKELQIMADRLRVDSIIATTAAGSGHPTSCMSCAEIMSTLFFNTITKDDDFILSKGHAVPILWSVYAEAGIISQAKLKTLRKISSNLEGHPTPNMPYIQVATGSLGQGLSAGLGMALAKTKGKTFVLLGDSESTEGSVWEAANTAAYYKTKNLIAIIDVNRLGQSQETMHGHKIRVYKKKFKAFGWKAVSINGHSIKQILWALKLARKSKKPFAIIAKTYKGKGVSFLENKEGWHGKALSKDEAKLAIEEINPQKIKLKSQIKKPKVNYKRSNFKLNNYELKEEVATRDAFGKALVNAGKTNNKIITIDGEVRNSTKTEEFFKKFPKRSFESFIAEQNMVGMALGFSTQGFTPVVATFGTFFTRAFDFIRMANYSKANIKFVGSHVGVHIGEDGPSQMGLEDISMFLSVPNSTILYPSDAPSTEYLTKEMLNLKGISYLRTTRGTTPVIYSEKEKFPVGKFKVVKKSKYDKVLIIAAGITLHESLKAYEILQKKKINVRIIDLYSIRPLDSKNLIKNAKECKNKVIVVEDHYPYGISAVITEILGKVTSLNIKETPRSGDPDKLLKKYQIDSSTIIKTVEKLK 3. The partial primer information involved in the embodiments of the application is shown in Table 1.

[0026] Table 1 Primer Table

[0027] The application will be further explained and described with specific examples below.

[0028] Example 1 Plasmid Construction 1. Plasmid construction of sclareol synthase pathway (SC1) 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.

[0029] 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.

[0030] 2. MVA pathway plasmid construction 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.

[0031] 3. Construction of plasmids via the MVA-NOG-1 pathway The gene encoding phosphate transketolase was determined according to the sequence shown in SEQ ID NO.11-12. LmXpk (SEQ ID NO.11), gene encoding phosphorylated acetyltransferase CkPta (SEQ ID NO.12) was artificially synthesized to obtain the full-length DNA sequence.

[0032] Using pACYCDuet-1 as the expression vector, a recombinant expression plasmid was constructed using the In-Fusion strategy, and PCR amplification primers containing homologous arms were designed for cloning (see Table 1). LmXpk, CkPta Homologous recombination with the linearized pACYCDuet-MVA vector yielded recombinant plasmids containing both the MVA and NOG pathways. Details are as follows: (1) Using plasmid pACYCDuet-MVA as a template, F-pMVA and R-pMVA were used as upstream and downstream primers for PCR amplification to obtain linearized pACYCDuet-MVA vector fragment; (2) Synthetic LmXpk Using F-LmXpk and R-LmXpk as templates, PCR amplification was performed with F-LmXpk and R-LmXpk as upstream and downstream primers, respectively, to obtain... LmXpk Fragment; (3) Synthetic CkPta Using F-CkPta and R-CkPta as templates, PCR amplification was performed with F-CkPta and R-CkPta as upstream and downstream primers, respectively, to obtain... CkPta Fragment; (4) Following the kit instructions, place the linearized pACYCDuet-MVA vector fragment obtained above, LmXpk Fragments and CkPta The fragment underwent homologous recombination reaction at 50°C for 1 hour, followed by transformation. E. coli DH5α competent cells were plated on CMR-resistant plates and cultured overnight at 37°C. Colony PCR was performed using VF-NOG and VR-NOG primers as upstream and downstream verification primers to screen positive clones. Positive clones were picked and placed in 5 mL of LB medium containing CMR for sequencing verification. The correct recombinant plasmid pACYCDuet-MVA-NOG-1 (CMR resistant) was extracted. The plasmid map is shown below. Figure 3 As shown.

[0033] 4. Construction of plasmids via the MVA-NOG-2 pathway Using the exact same method as step 3, only... LmXpk Replace with NaXpk (SEQ ID NO.13) NaXpkThe amplification primers of F-NaXpk and R-NaXpk are used to replace F-NOG-2 and R-NOG-2, and the rest remains unchanged, to obtain the recombinant plasmid pACYCDuet-MVA-NOG-2 (CMR resistance).

[0034] 5. MVA-NOG-3 pathway plasmid construction The amplification primers of F-BaXpk and R-BaXpk are used to replace F-NOG-3 and R-NOG-3, and the rest remains unchanged, to obtain the recombinant plasmid pACYCDuet-MVA-NOG-3 (CMR resistance). LmXpk BaXpk (SEQ ID NO. 14), BaXpk The amplification primers of F-BaXpk and R-BaXpk are used to replace F-NOG-3 and R-NOG-3, and the rest remains unchanged, to obtain the recombinant plasmid pACYCDuet-MVA-NOG-3 (CMR resistance).

[0035] 6. MVA-NOG-4 pathway plasmid construction The amplification primers of F-Fxpk and R-Fxpk are used to replace F-NOG-4 and R-NOG-4, and the rest remains unchanged, to obtain the recombinant plasmid pACYCDuet-MVA-NOG-4 (CMR resistance). LmXpk Fxpk (SEQ ID NO. 15), Fxpk The amplification primers of F-BaXpk and R-BaXpk are used to replace F-NOG-3 and R-NOG-3, and the rest remains unchanged, to obtain the recombinant plasmid pACYCDuet-MVA-NOG-3 (CMR resistance).

[0036] 7. Construction of NaXpk mutant (1) The mutant primers (F-S472F, R-S472F) are designed to perform S472F site-directed mutation on the 472th amino acid of NaXpk using pACYCDuet-MVA-NOG-2 as the template.

[0037] The PCR reaction system is as follows:

[0038] The PCR reaction program is as follows: Pre-denaturation: 98°C, 3 min; denaturation: 98°C, 10 sec; annealing: 55°C-65°C, 5 sec; extension: 68°C, 5 sec / kb; final extension: 68°C, 10 min; storage: 4°C.

[0039] After the PCR is completed, the PCR product is digested with restriction endonuclease Dpn I for 2 h to remove the template; after agarose gel electrophoresis, the target band is recovered, eluted with ddH2O during recovery, and then recombined to transform E. coli DH5α competent cells, and coated on Cmr resistance plates for overnight culture at 37°C; VF-MUT and VR-MUT are used as upstream and downstream verification primers to perform colony PCR screening of positive clones, and the positive clones are picked into 5 mL Cmr-containing LB medium, sequenced, and the correct plasmid pACYCDuet-MVA-NOG (S472F)-2 is extracted.​​

[0040] (2) Using the same method as step (1), the S472Y site mutation was performed on the 472th amino acid of NaXpk, only the mutation primer was replaced by F-S472Y, R-S472Y, and the rest remained unchanged, finally obtaining the plasmid pACYCDuet-MVA-NOG (S472Y)-2.

[0041] Example 2 Knockout edd Overexpression zwf of chassis modification The N20 sequence was 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) was used to PCR amplify the plasmid pEcgRNA to obtain the linearized plasmid pEcgRNA, and then the resulting PCR product was circularized to construct the plasmid pEcgRNA-edd.

[0042] Using the primer pair F / R-D1, F / R-zwf, F / R-D3, the target homologous regions D1, D2, and D3 were amplified, respectively, and the above amplification products were assembled by overlap extension PCR to obtain Donor-edd-zwf.

[0043] pEcgRNA-edd and Donor-edd-zwf were transformed into E. coli BL21 (DE3) cells carrying pEcCas9 plasmid at the same time; after colony PCR screening of positive clones with VF-zwf and VR-zwf as upstream and downstream verification primers, DNA sequencing was performed to confirm that edd knockout zwf overexpression.

[0044] For positive clones, L-rhamnose was added to the culture medium to eliminate the pEcgRNA-edd plasmid, and sucrose was added to eliminate the pEgCas9 plasmid, thereby obtaining the empty chassis strain SCR01 which knocked out edd and overexpressed zwf in E. coli BL21 (DE3) based on the chassis strain.

[0045] Example 3 Construction of Recombinant Strains According to Table 2, different plasmids were transformed into different chassis strains to obtain recombinant strains, as follows: 1. Construction of Comparative Example G00 Strain (Containing SC1 Pathway): The recombinant plasmid pET21d-SC1 was transformed into E. coliBL21 (DE3) chassis cells were coated on LB plates containing Apr (100 μg / ml) resistance, incubated at 37°C overnight, and single colonies were picked into 50 μL of LB medium containing Apr (100 μg / ml) resistance and incubated at 37°C to the logarithmic growth phase to obtain recombinant strain G00, which was stored in a -80°C refrigerator.

[0046] 2. Construction of G01 strain (containing MVA+SC1 pathway): The recombinant plasmids pACYCDuet-MVA and pET21d-SC1 were co-transformed into E. coli BL21 (DE3) chassis cells were 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 recombinant strain G01, which was stored in a -80°C refrigerator.

[0047] 3. Construction of G02 strain (containing MVA-NOG-1+SC1 pathway) The recombinant plasmids pACYCDuet-MVA-NOG-1 and pET21d-SC1 were co-transformed into E. coli BL21 (DE3) chassis cells were 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 recombinant strain G02, which was stored in a -80°C refrigerator.

[0048] 4. Construction of G03 strain (containing MVA-NOG-1+SC1 pathway) 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 recombinant strain G03, which was stored in a -80°C refrigerator.

[0049] 5. Construction of G04 strain (containing MVA-NOG-2+SC1 pathway) 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, incubated at 37°C to the logarithmic growth phase, to obtain the recombinant strain G04, and stored in a -80°C refrigerator.

[0050] 6. Construction of G05-G07 strains 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, incubated at 37°C to the logarithmic growth phase, to obtain the recombinant strain G05, and stored in a -80°C refrigerator.

[0051] 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.

[0052] 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.

[0053] Similarly, the recombinant plasmids pACYCDuet-MVA-NOG (S472F)-2 and pET21d-SC1 were co-transformed into SCR01 chassis cells to obtain the recombinant strain G08.

[0054] Similarly, the recombinant plasmids pACYCDuet-MVA-NOG (S472Y)-2 and pET21d-SC1 were co-transformed into SCR01 chassis cells to obtain the recombinant strain G09.

[0055] Table 2. Recombinant strain information table

[0056] Example 4. Production experiment verification The G00-G09 strains constructed above were taken for fermentation verification: Frozen strain G00 was streaked on solid LB medium containing Apr (100 μg / mL) resistance and incubated at 37°C overnight; single colony was picked and inoculated in 25 mL LB medium containing Cmr and Apr resistance and incubated at 37°C overnight to prepare fermentation seed liquid; the seed liquid was inoculated in 25 mL M9Y medium at 1% inoculation amount, and incubated at 250 rpm, 37°C until OD600~0.6-0.8, then 0.1 mM IPTG and 10% (v / v) dodecane were added, and the expression was induced at 22°C for 72 h, then the fermentation liquid was collected and centrifuged at 12,000 rpm, 4°C to collect the dodecane layer, which was stored at -20°C for standby. The dodecane layer was diluted 10 times with anhydrous ethanol, and the supernatant after filtration through a 0.22 μm filter membrane was used for HPLC analysis to determine the output of sclareol in the reaction sample. The results are shown in Table 3.

[0057] Frozen strains G01-G09 were streaked on solid LB medium containing Cmr (50 μg / mL) and Apr (100 μg / mL) resistance and incubated at 37°C overnight; single colony was picked and inoculated in 25 mL LB medium containing Cmr and Apr resistance and incubated at 37°C overnight to prepare fermentation seed liquid; the seed liquid was inoculated in 25 mL M9Y medium at 1% inoculation amount, and incubated at 250 rpm, 37°C until OD600~0.6-0.8, then 0.1 mM IPTG and 10% dodecane were added, and the expression was induced at 22°C for 72 h, then the fermentation liquid was collected and centrifuged at 12,000 rpm, 4°C to collect the dodecane layer, which was stored at -20°C for standby. The dodecane layer was diluted 10 times with anhydrous ethanol, and the supernatant after filtration through a 0.22 μm filter membrane was used for HPLC analysis to determine the output of sclareol in the reaction sample. The results are shown in Table 3.

[0058] Sclareol HPLC detection conditions: The mobile phase was water:acetonitrile=3:7, the flow rate was 1 mL / min, the chromatographic column was C18 (4.6×250 mm), the column temperature was 40°C, the detection wavelength was 195 nm (ultraviolet detector), and the collection time was 45 min.

[0059] Table 3 Sclareol production results

[0060] From the sclareol production shown in Table 3: (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 substantial increase in sclareol production to 302.1 mg / L; on this basis, the G02 strain obtained by further expressing the NOG pathway has a 7% increase in production.

[0061] (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 a sclareol production of 399.2 mg / L, 314.1 mg / L and 305.4 mg / L respectively. It is illustrated that the Xpk from different sources also has a great influence on the sclareol production, and the NaXpk has the best effect.

[0062] (3) On the basis of (1), the strain SCR01 obtained by knocking out edd and overexpressing zwf in the BL21 host is used as the host, and the production strain G03 obtained by introducing the plasmids (pACYCDuet-MVA-NOG-1, pET21d-SC1) containing the SC1 pathway, MVA pathway and NOG pathway. edd Compared with the G02 strain without knocking out zwf and overexpressing edd in the host, the sclareol production is increased from 323.4 mg / L to 419.8 mg / L, which illustrates that knocking out zwf and overexpressing has a significant effect on the sclareol production.

[0063] (4) According to the results of (2) and (3), the strain SCR01 obtained by knocking out edd and overexpressing zwf is used as the host, and the production strain G04 is obtained by introducing the pACYCDuet-MVA-NOG-2 containing the NaXpk and the pET21d-SC1. Compared with the G03, the sclareol production of the G04 is further increased by 31.6%, and the sclareol production reaches 552.6 mg / L.

[0064] (5) On the basis of the strain G04, the wild-type NaXpk is further mutated into S472F or S472Y to obtain the strains G08 and G09, and the sclareol production reaches 623.3 mg / L and 655.2 mg / L respectively, which is increased by 12.8% and 18.6% respectively compared with the G04, and it is illustrated that the mutation of the NaXpk into S472F or S472Y has a significant effect.

[0065] Although the present application has been disclosed as above with preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various changes, modifications, replacements and variations in form and details to these 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. The phosphoketolase mutant of claim 1, wherein, The mutant is S472F, and the amino acid sequence is shown in SEQ ID NO. 17; or the mutant is S472Y, and the amino acid sequence is shown in SEQ ID NO.

18.

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

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

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

6. Use of the phosphoketolase mutant of claim 1, or the recombinant vector or recombinant strain of claim 4 in the production of sclareol.

7. Use according to claim 6, wherein The use is achieved by expressing the phosphoketolase mutant of claim 1 in a sclareol-producing strain.

8. A genetically engineered bacterium, characterized by, The engineered bacterium is obtained by expressing the phosphoketolase mutant of claim 1 in a host.

9. The engineered bacterium of claim 8, wherein, The engineered bacterium has Escherichia coli as the host, and is further genetically edited for any one or more of the following (1)-(4) based on the expression of the phosphoketolase mutant of claim 1: (1) overexpression of at least one gene of a geranylgeranyl pyrophosphate synthase encoding gene PaGGPPS , a labdane-type enediyne pyrophosphate synthase encoding gene SsLPS , a clivisate heterologous clivisate synthase encoding gene SsScS , a clivisate heterologous clivisate synthase encoding gene (2) overexpression of at least one gene encoding an acetyl-CoA acetyltransferase EcatoB , 3-hydroxy-3-methylglutaryl-CoA synthase EfmvaS , bifunctional acetyl-CoA thiolase EfmvaE , methylglutaconyl-CoA kinase Samvk1 , phosphomevalonate kinase Samvk2 , mevalonate pyrophosphate decarboxylase SamvaD , isopentenyl pyrophosphate Δ-isomerase Bsfni ​ (3) overexpression of a phosphoacetyltransferase-encoding gene CkPta ; (4) knocking out a phosphogluconate dehydratase-encoding gene edd and / or overexpressing a 6-phosphogluconate dehydrogenase-encoding gene zwf .

10. The engineered bacterium of claim 9, 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 of which 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, NCBI Accession No. WP_322035618 encoding the protein; The zwf Gene, NCBI Accession No. WP_272457244.1 encoding the protein.

11. Use of the engineered bacterium of any one of claims 8-10 in the production of sclareol.

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