A recombinant Amycolatopsis for fully synthesizing vanillin from glucose, its construction method and application

Abstract

The present invention provides an application of an O-methyltransferase gene OMT in improving the production of vanillin by fermentation of recombinant Amycolatopsis, wherein the OMT gene is derived from sea urchin, streptomyces or mycobacteria. The present invention also provides a recombinant Amycolatopsis, which is obtained by knocking out vdh, pcaGH, vanAB and pyk genes in Amycolatopsis HM-141, expressing DSD, ubiC, pobA, aroF, CAR and PPTase genes, and expressing the O-methyltransferase gene OMT. The present invention screens the source of the OMT gene, 以 It preferentially catalyzes the meta-methylation of protocatechuic acid or protocatechuic aldehyde rather than the para-methylation, and it was confirmed that the highest vanillin yield could be obtained when expressed in Amycolatopsis spp. without the production of the by-product isovanillin.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering. More specifically, the present invention relates to a recombinant Amycolatopsis that fully synthesizes vanillin from glucose, a construction method of the Amycolatopsis, and an application thereof in producing vanillin. Background Art

[0002] Vanillin (3-methoxy-4-hydroxybenzaldehyde), also known as vanillin, is widely used in food, medicine, cosmetics, agriculture and other fields. There are three types of vanillin on the market, including natural vanillin extracted from vanilla beans, vanillin produced by chemical synthesis, and vanillin produced by microbial transformation. Among them, in the microbial transformation technology for producing vanillin, eugenol, isoeugenol, ferulic acid and glucose are the main substrates for producing vanillin. However, eugenol and isoeugenol are highly toxic, and ferulic acid is expensive, while glucose is cheap, the raw material is sufficient, and it is safe and non-toxic, making it an ideal raw material for biosynthesis of vanillin.

[0003] Strains used to synthesize vanillin from glucose include Saccharomyces cerevisiae, Escherichia coli, Corynebacterium glutamicum, etc. Chinese invention patent CN 116751730A discloses a recombinant Escherichia coli for synthesizing vanillin. By knocking out aroE and 6 vanillin-degrading genes, heterologously expressing DSD, LiOMT and NiCAR genes, the vanillin yield is only 63 mg / L, which is low, and vanillin is highly toxic to Escherichia coli, which is not conducive to its accumulation. Chinese invention patent CN 117004544B synthesizes vanillin with protocatechuic acid as an intermediate by transforming the chassis cells of Corynebacterium glutamicum, introducing a vanillin synthesis module and a methyl cycle regeneration module, and synthesizing vanillin with protocatechuic acid as an intermediate. The yield is 765.85 mg / L, which shows that the yield is still relatively low.

[0004] In this field, the low yield of vanillin synthesized from glucose is the main reason why it is difficult to achieve industrial production. Summary of the invention

[0005] The purpose of the present invention is to overcome the existing technical solutions and provide a recombinant Amycolatopsis sp. that fully synthesizes vanillin from glucose through genetic modification.

[0006] The idea of ​​the invention is to start from the engineered strain of Amycolatopsis , knock out the vanillin dehydrogenase gene vdh, the protocatechuate 3,4-dioxygenase gene pcaGH, the vanillate demethylase gene vanAB and the pyruvate kinase gene pyk, express the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA, the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, the carboxylic acid reductase gene CAR and the phosphopantetheinyl transferase gene PPTase, and then screen a better O-methyltransferase gene OMT to increase the yield of vanillin and reduce the concentration of by-products.

[0007] To this end, the present invention provides an application of an O-methyltransferase gene OMT in improving the fermentation production of vanillin by recombinant Amycolatopsis sp., wherein the O-methyltransferase gene OMT is derived from sea urchin (Lytechinus pictus), chain algae (Klebsormidium nitens) or mycobacterium (Mycobacterium lentiflavum).

[0008] Among them, the recombinant Amycolatopsis knocks out the vanillin dehydrogenase gene vdh, the protocatechuate 3,4-dioxygenase gene pcaGH and the vanillic acid demethylase gene vanAB, and expresses the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA, the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, the carboxylic acid reductase gene CAR, and the phosphopantetheinyl transferase gene PPTase.

[0009] Preferably, the O-methyltransferase gene OMT is derived from Mycobacterium lentiflavum to obtain the highest vanillin production.

[0010] The present invention also provides application of promoter SP44 in improving the production of vanillin by fermentation of recombinant Amycolatopsis spp., and the sequence of promoter SP44 is shown in nucleotide sequence as SEQ ID NO:19.

[0011] Specifically, the recombinant Amycolatopsis knocks out the vanillin dehydrogenase gene vdh, the protocatechuate 3,4-dioxygenase gene pcaGH and the vanillic acid demethylase gene vanAB, expresses the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA, the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, the carboxylic acid reductase gene CAR, the phosphopantetheinyl transferase gene PPTase, and expresses the O-methyltransferase gene OMT derived from sea urchin (Lytechinus pictus), chain algae (Klebsormidium nitens) or mycobacterium (Mycobacterium lentiflavum).

[0012] The invention also provides the use of over-expressing O-methyltransferase gene OMT in improving the fermentation production of vanillin by recombinant Amycolatopsis sp., expressing two copies of O-methyltransferase gene OMT derived from Mycobacterium lentiflavum.

[0013] Among them, the recombinant Amycolatopsis knocks out the vanillin dehydrogenase gene vdh, the protocatechuate 3,4-dioxygenase gene pcaGH and the vanillic acid demethylase gene vanAB, and expresses the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA, the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, the carboxylic acid reductase gene CAR, and the phosphopantetheinyl transferase gene PPTase.

[0014] On the other hand, the present invention provides a recombinant Amycolatopsis, which is obtained by knocking out the vanillin dehydrogenase gene vdh, the protocatechuate 3,4-dioxygenase gene pcaGH, the vanillic acid demethylase gene vanAB and the pyruvate kinase gene pyk in Amycolatopsis HM-141, expressing the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA, the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, the carboxylic acid reductase gene CAR and the phosphopantetheinyl transferase gene PPTase, and expressing the O-methyltransferase gene OMT.

[0015] In particular, the dehydroshikimate dehydratase gene DSD is derived from Amycota, and its nucleotide sequence is shown in SEQ ID NO: 1; the chorismate pyruvate lyase gene ubiC is derived from Providencia rustigianii, and its nucleotide sequence is shown in SEQ ID NO: 2; the 4-hydroxybenzoate-3-monooxygenase gene pobA is derived from Amycota, and its nucleotide sequence is shown in SEQ ID NO: 3; the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF is derived from Amycota, and its nucleotide sequence is shown in SEQ ID NO: 4; the carboxylic acid reductase gene CAR is derived from the thermophilic fusilli, and its nucleotide sequence is shown in SEQ ID NO: 5; the phosphopantetheinyl transferase gene PPTase is derived from Amycota, and its nucleotide sequence is shown in SEQ ID NO: 6; the O-methyltransferase gene OMT is derived from Mycobacterium lentiflavum, and its nucleotide sequence is shown in SEQ ID NO: NO:16; the phosphoenolpyruvate synthase gene ppsA is derived from Amycolatopsis, and its nucleotide sequence is shown in SEQ ID NO:21.

[0016] In order to further increase the production of vanillin, the recombinant Amycolatopsis also expressed the phosphoenolpyruvate synthase gene ppsA and knocked out the pyruvate kinase gene pyk.

[0017] Particularly preferably, the recombinant Amycolatopsis expresses 2 copies of the O-methyltransferase gene OMT gene.

[0018] According to another preferred embodiment, the O-methyltransferase gene OMT is promoted by promoter SP44, and the nucleotide sequence of promoter SP44 is shown in SEQ ID NO:19.

[0019] The present invention also provides the use of the recombinant Amycolatopsis in producing vanillin by fermentation with glucose as a substrate.

[0020] Specifically, the recombinant Amycolatopsis was inoculated into 50 mL of seed culture medium M1, cultured at 30°C and 200 rpm for 48 h, the seed solution was inoculated into a 250 mL conical flask containing 50 mL of fermentation medium M1 at a mass ratio of 5%, and fermented at 37°C and 200 rpm for 96 h;

[0021] The M1 culture medium formula is: 30 g / L glucose, 10 g / L yeast extract powder, 4 g / L disodium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, and the rest is water, and the pH is adjusted to 7.2.

[0022] On the other hand, the present invention also provides a method for constructing the above-mentioned recombinant Amycolatopsis, which comprises the following steps:

[0023] (1) Knockout of the genes vdh, pcaGH, and vanAB in the vanillin degradation pathway and byproduct degradation pathway

[0024] Knockout plasmids pKG1132-vdh-2500, pKG1132-pcaGH-2500 and pKG1132-vanAB-2500 were constructed. pKG1132-vdh-2500 was transformed into Amycolatopsis pseudomycoticus HM-141, and the VAN-1 strain was obtained by single and double exchange screening and verification. pKG1132-pcaGH-2500 was transformed into VAN-1, and the VAN-2 strain was obtained by single and double exchange screening and verification. pKG1132-vanAB-2500 was transformed into VAN-2, and the VAN-3 strain was obtained by single and double exchange screening and verification.

[0025] (2) Integration of DSD, ubiC, pobA and aroF genes into the chromosome of Amycolatopsis spp.

[0026] The integration plasmids pKG1132-258-DSD-PrubiC and pKG1132-vdh-pobA-aroF were constructed. pKG1132-258-DSD-PrubiC was transformed into VAN-3, and the VAN-4 strain was obtained by single and double crossover screening. pKG1132-vdh-pobA-aroF was transformed into VAN-4, and the VAN-5 strain was obtained by single and double crossover screening.

[0027] (3) Integration of CAR and PPTase genes into the PcaGH locus of Amycolatopsis spp.

[0028] The integration plasmid pKG1132-pcaGH-TtCAR-PPTase was constructed and transformed into VAN-5, and the VAN-6 strain was obtained by single and double exchange screening.

[0029] (4) Integration of two copies of the SP44-M10MT gene into the vanAB locus of Amycolatopsis spp.

[0030] The plasmid pKG1132-vanAB-M1OMT×2 with two copies of SP44-M1OMT gene integrated into the vanAB locus was constructed. The pKG1132-vanAB-M1OMT×2 plasmid was transformed into VAN-6, and the VAN-10 strain was obtained by single and double crossover screening.

[0031] (5) Construction of Amycolatopsis pseudomycoticola VAN-11 with integrated ppsA gene

[0032] The integration plasmid pKG1132-pyk-ppsA was transformed into VAN-10, and the recombinant Amycolatopsis spp. with high vanillin production was obtained through single and double exchange screening.

[0033] The invention knocks out vanillin dehydrogenase gene vdh, protocatechuate 3,4-dioxygenase gene pcaGH, vanillate demethylase gene vanAB and pyruvate kinase gene pyk in Amycolatopsis pseudomycoticus, and expresses dehydroshikimate dehydratase gene DSD, chorismate pyruvate lyase gene ubiC, 4-hydroxybenzoate-3-monooxygenase gene pobA, 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, carboxylic acid reductase gene CAR, phosphopantetheinyl transferase gene PPTase, O-methyltransferase gene OMT and phosphoenolpyruvate synthase gene ppsA, so as to obtain VAN-11 strain. The vanillin yield of VAN-11 is 5.09 g / L, which is the highest yield of vanillin fully synthesized from glucose by biological method in related research reported so far. Glucose is low in price and the vanillin yield is high, so as to provide technical support for industrial production of biosynthetic vanillin.

[0034] The invention obtains the M1OMT gene from Mycobacterium lentiflavum by screening the source of the OMT gene, and preferentially catalyzes the meta-methylation of protocatechuic acid or protocatechuic aldehyde rather than the para-methylation, and can obtain the highest vanillin yield when expressed in Amycolatopsis lentiflavum, without the generation of the byproduct isovanillin. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the pathway for the total synthesis of vanillin by Pseudomonas amycolata

[0036] Figure 2 The map of pKG1132-258-DSD-PrubiC plasmid

[0037] Figure 3 The map of pKG1132-vdh-pobA-aroF plasmid

[0038] Figure 4 The map of pKG1132-pcaGH-TtCAR-PPTase plasmid

[0039] Figure 5 Fermentation results for screening of O-methyltransferase gene OMT

[0040] Figure 6The map of pKG1132-vanAB-M10MT×2 plasmid

[0041] Figure 7 The map of pKG1132-pyk-ppsA plasmid DETAILED DESCRIPTION

[0042] The present invention will be better understood through the following examples.

[0043] In the present invention, unless otherwise specified, "%" used to explain concentration means mass percentage, and ":" used to explain ratio means mass ratio.

[0044] The strains and plasmids used in the examples are shown in Table 1, and the synthesized primer sequences are shown in Table 2.

[0045] Table 1 Bacterial species and plasmids used in the study

[0046]

[0047]

[0048] Table 2 Primers used in the present invention

[0049]

[0050]

[0051]

[0052] The present invention relates to the following culture medium:

[0053] The LB medium formula is: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L.

[0054] The formula of GYM medium is: glucose 4g / L, yeast extract 4g / L, malt extract 10g / L.

[0055] The formula of GYM solid culture medium is: glucose 4g / L, yeast extract 4g / L, malt extract 10g / L, calcium carbonate 2g / L, and agar powder 20g / L.

[0056] The formula of M1 culture medium is: 30g / L glucose, 10g / L yeast extract powder, 4g / L disodium hydrogen phosphate, 1g / L potassium dihydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, and the rest is water, and the pH is adjusted to 7.2.

[0057] The conjugation transfer experiment used in the following examples includes the following steps:

[0058] (1) Amycolatopsis sp. (or other engineered strains) was activated on GYM solid medium and cultured at 30°C for 3-4 days until colonies grew. The strain was then inoculated into 50 mL GYM liquid medium and cultured at 30°C and 200 rpm for 2 days.

[0059] (2) The constructed plasmid was heat-shocked into E. coli ET12567 (pUZ8002) strain, spread on LB solid plates containing 25 μg / mL chloramphenicol, 25 μg / mL kanamycin and 50 μg / mL apramycin resistance, and cultured at 37°C for 12 h until a single colony grew. The single colony was inoculated into 4 mL LB liquid medium containing resistance and cultured at 37°C and 200 rpm overnight. Then, it was inoculated into 20 mL LB at a 1% inoculum and cultured at 37°C and 200 rpm for 4-5 h until the OD 600 It is 0.4-0.6.

[0060] (3) Take 2 mL of Amycolatopsis sp. bacterial solution and 1 mL of E. coli ET12567 (pUZ8002) bacterial solution carrying the target plasmid, centrifuge at 5000 g for 1 min, wash twice with antibiotic-free LB, add 100 μL of antibiotic-free LB medium, mix Amycolatopsis sp. and E. coli ET12567 (pUZ8002) in a volume ratio of 7:1, take 30 μL of the mixed bacterial solution and spot it on antibiotic-free GYM solid medium at 30°C and culture upright for 14 h.

[0061] (4) The grown plaques were scraped off and spread on GYM solid culture medium containing 50 μg / mL apramycin and 25 μg / mL nalidixic acid solution, and cultured at 30°C for 4 days until single colonies grew, and then the single colonies were verified by PCR.

[0062] The fermentation experiment for producing vanillin used in the following examples includes:

[0063] The recombinant strains were inoculated into 50 mL of seed medium M1, cultured at 30°C and 200 rpm for 48 h, and the seed solution was inoculated into a 250 mL conical flask containing 50 mL of fermentation medium M1 at a 5% inoculum, and fermented at 37°C and 200 rpm for 96 h. After the fermentation stopped, the concentration of vanillin in the fermentation broth was measured.

[0064] The method for determining vanillin and isovanillin in the fermentation broth by HPLC is as follows: the fermentation broth is centrifuged, and the supernatant is taken for HPLC analysis. The conversion product is analyzed by Agilent HPLC1260, the chromatographic column is Elite Hypersil ODS2, 5μm, 4.6mm×250mm; the detection wavelength is 295nm; the column temperature is 30℃; the flow rate is 1mL / min; the injection volume is 10μL; the mobile phase is acetonitrile and 0.5% trifluoroacetic acid aqueous solution, 0-5min, 10% acetonitrile; 5-30min, 10-30% acetonitrile; 30-35min, 30%-10% acetonitrile.

[0065] Example 1: Construction of Amycolatopsis chassis cells for the full synthesis of vanillin from glucose

[0066] 1. Knockout of genes vdh, pcaGH and vanAB in the vanillin decomposition pathway and byproduct decomposition pathway

[0067] Knocking out the vanillin dehydrogenase gene vdh (protein accession number WP_020422127.1) can block the degradation of vanillin into vanillic acid; knocking out the protocatechuic acid 3,4 dioxygenase gene pcaGH (protein accession numbers WP_020421708.1, WP_020421709.1) can block the degradation of the intermediate protocatechuic acid; knocking out the vanillic acid demethylase gene vanAB (protein accession numbers WP_020422153.1, WP_020422151.1) can prevent the conversion of vanillic acid into protocatechuic acid.

[0068] Construction of vdh, pcaGH and vanAB gene knockout plasmids:

[0069] With reference to the description of Chinese invention patent CN113717914B, the pKG1132-vdh-2500 plasmid was constructed.

[0070] Construction of pKG1132-pcaGH-2500 plasmid: Using Amy-F1 / Amy-R1 and Amy-F2 / Amy-R2 as primers and the genome of Amycota as a template, PCR was used to amplify the upstream homology arm and downstream homology arm of the pcaGH gene, each of which was about 2.5 kb. The two fragments obtained were ligated to the HindIII / EcoRI site of pKG1132 using an Assembly kit to obtain pKG1132-pcaGH-2500.

[0071] Construction of pKG1132-vanAB-2500 plasmid: Using Amy-F3 / Amy-R3 and Amy-F4 / Amy-R4 as primers and the genome of Amycota as a template, PCR was used to amplify the upstream homology arm and downstream homology arm of vanAB gene, each of which was about 2.5 kb. The two fragments obtained were connected to the HindIII / EcoRI site of pKG1132 using an Assembly kit to obtain pKG1132-vanAB-2500.

[0072] Construction of the vdh gene-knockout strain of Amycolatopsis pseudomycoticus VAN-1: Using the conjugation transfer experiment, pKG1132-vdh-2500 was transformed into Amycolatopsis pseudomycoticus HM-141, and the single colony grown on the GYM solid medium with apramycin resistance (final concentration of 50 μg / mL) was the homologous single exchange strain. The obtained single exchange strain was subcultured in the GYM medium without resistance, and the strain that grew on the GYM plate without resistance and did not grow on the apramycin resistance plate was the strain that underwent homologous double exchange. The double exchange strain was verified by PCR using the HM-141 genome as a control, and the verification primers were vdh-F1-test / vdh-R1-test (control band 4375bp, correct band 2964bp) and vdh-F2-test / vdh-R2-test (control band 4287bp, correct band 2876bp). The correct bands were sent for sequencing, and the ones with correct sequencing were the Amycolatopsis spp. VAN-1 with the vdh gene knocked out.

[0073] Construction of the pcaGH gene-knockout strain of Amycolatopsis spp. VAN-2: pKG1132-pcaGH-2500 was transformed into VAN-1, and the screening of single and double exchange was the same as that of VAN-1. The double exchange strain was verified by PCR using the VAN-1 genome as a control, and the verification primers were pcaGH-F1-test / pcaGH-R1-test (control band 4514bp, correct band 3157bp) and pcaGH-F2-test / pcaGH-R2-test (control band 4350bp, correct band 2993bp). The correct band was sent for sequencing, and the one with the correct sequencing was the pcaGH gene-knockout strain VAN-2.

[0074] Construction of vanAB gene knockout strain VAN-3 of Amycolatopsis pseudomycoticola: pKG1132-vanAB-2500 was transformed into VAN-2, and the screening of single and double exchange was the same as that of VAN-1. The double exchange strain was verified by PCR using the VAN-2 genome as a control, and the verification primers were vanAB-F1-test / vanAB-R1-test (control band 4951bp, correct band 2904bp) and vanAB-F2-test / vanAB-R2-test (control band 5454bp, correct band 3407bp). The correct band was sent for sequencing, and the one with the correct sequencing was the vanAB gene knockout strain VAN-3.

[0075] 2. Integration of DSD, ubiC, pobA and aroF genes into the chromosome of Amycolatopsis

[0076] It was verified that overexpression of the dehydroshikimate dehydratase gene DSD endogenous to Amycolatopsis, the chorismate pyruvate lyase gene PrubiC from Providencia rustigianii, the 4-hydroxybenzoate-3-monooxygenase gene pobA and the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF endogenous to Amycolatopsis was beneficial to the accumulation of the intermediate product protocatechuic acid.

[0077] The gene sequence of DSD is shown in SEQ ID NO:1, and the protein accession number is WP_020421538.1, the gene sequence of PrubiC is shown in SEQ ID NO:2, and the protein accession number is WP_006816067.1, the gene sequence of pobA is shown in SEQ ID NO:3, and the protein accession number is WP_020418261.1, and the gene sequence of aroF is shown in SEQ ID NO:4, and the protein accession number is WP_020418838.1.

[0078] Construction of plasmid pKG1132-258-DSD-PrubiC with DSD and ubiC genes integrated into the WP_020419258.1 protein site:

[0079] First, the pSET152-DSD plasmid was constructed: using Amy-F5 / Amy-R5 as primers and the genome of Amycota as a template, the DSD gene was amplified by PCR, and the DSD fragment was connected to the BamHI / NsiI site of the pSET152-permE plasmid using an Assembly kit to obtain pSET152-DSD.

[0080] The PrubiC gene was sent to GenScript for codon optimization (as shown in SEQ ID NO: 2) and synthesized into the BamHI / NsiI site of the pSET152-permE plasmid to obtain the plasmid pSET152-PrubiC.

[0081] Construction of pKG1132-258-2500 plasmid: Using Amy-F6 / Amy-R6 and Amy-F7 / Amy-R7 as primers and the genome of Pseudomonas aeruginosa as template, PCR amplified the upstream homology arm and downstream homology arm of WP_020419258.1 protein of about 2.5 kb each, and ligated the two fragments into the HindIII / EcoRI site of pKG1132 using the Assembly kit to obtain pKG1132-258-2500.

[0082] Construction of pKG1132-258-DSD-PrubiC plasmid: Using Amy-F8 / Amy-R8 as primers and pSET152-DSD as template, the DSD fragment was amplified, using Amy-F9 / Amy-R9 as primers and pSET152-PrubiC as template, the PrubiC fragment was amplified, and the DSD and PrubiC fragments were ligated to the speI site of the pKG1132-258-2500 plasmid to obtain pKG1132-258-DSD-PrubiC.

[0083] Construction of plasmid pKG1132-vdh-pobA-aroF with pobA and aroF genes integrated into the vdh locus:

[0084] First, construct the pSET152-pobA plasmid: use Amy-F10 / Amy-R10 as primers and the genome of Amycota as a template to PCR amplify the pobA gene, and connect the pobA fragment to the BamHI / NsiI site of the pSET152-permE plasmid using an Assembly kit to obtain pSET152-pobA. Construct the pSET152-aroF plasmid according to the description of Chinese invention patent CN117417952B.

[0085] Construction of pKG1132-vdh-pobA-aroF plasmid: Using Amy-F11 / Amy-R11 as primers and pSET152-pobA as template, the pobA fragment was amplified, using Amy-F12 / Amy-R12 as primers and pSET152-aroF as template, the aroF fragment was amplified, and the pobA and aroF fragments were ligated to the speI site of the pKG1132-vdh-2500 plasmid to obtain pKG1132-vdh-pobA-aroF.

[0086] Construction of Amycolatopsis pseudomycoticola VAN-4 strain: pKG1132-258-DSD-PrubiC was transformed into VAN-3, and the screening of single and double exchange was the same as that of VAN-1. The double exchange strain was verified by PCR using the VAN-3 genome as a control, and the verification primers were 258-F1-test / DSD-R1-test (no band in the control, correct band 3357bp) and ubiC-F2-test / 258-R2-test (no band in the control, correct band 3194bp). The correct band was sent for sequencing, and the strain with the correct sequencing was strain VAN-4, in which the vanillin dehydrogenase gene vdh, protocatechuate 3,4-dioxygenase gene pcaGH and vanillate demethylase gene vanAB were knocked out, and the dehydroshikimate dehydratase gene DSD and chorismate pyruvate lyase gene ubiC were expressed.

[0087] Construction of Amycolatopsis pseudomycoticola VAN-5 strain: pKG1132-vdh-pobA-aroF was transformed into VAN-4. The screening of single and double exchange was the same as that of VAN-1. The double exchange strain was verified by PCR using the VAN-4 genome as a control. The verification primers were vdh-F1-test / pobA-R1-test (no band in the control, correct band was 3264bp) and aroF-F2-test / vdh-R2-test (no band in the control, correct band was 3275bp). The correct bands were sent for sequencing, and the strain with the correct sequencing was strain VAN-5. This engineered strain had the vanillin dehydrogenase gene vdh, protocatechuate 3,4-dioxygenase gene pcaGH and vanillate demethylase gene vanAB knocked out, and expressed the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA and the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF.

[0088] Example 2: Construction of Amycolatopsis spp. for the complete synthesis of vanillin from glucose

[0089] 1. Integration of CAR and PPTase genes into the PcaGH locus of Amycolatopsis

[0090] The carboxylic acid reductase gene TtCAR from Thermothelomyces thermophilus and the endogenous phosphopantetheinyl transferase gene PPTase from Amycolatopsis thermophilus were expressed in Amycolatopsis thermophilus, wherein the gene sequence of TtCAR is shown in SEQ ID NO:5, the gene sequence of PPTase is shown in SEQ ID NO:6, and the protein accession number is WP_020416839.1.

[0091] Construction of plasmid pKG1132-pcaGH-TtCAR-PPTase with TtCAR and PPTase genes integrated into the pcaGH site:

[0092] The TtCAR gene was sent to GenScript for codon optimization (as shown in SEQ ID NO: 5) and synthesized into the BamHI / NsiI site of the pSET152-permE plasmid to obtain pSET152-TtCAR.

[0093] Construction of pSET152-PPTase plasmid: Using Amy-F13 / Amy-R13 as primers and the genome of Amycota as a template, the PPTase gene was amplified by PCR, and the PPTase fragment was connected to the BamHI / NsiI site of the pSET152-permE plasmid using an Assembly kit to obtain pSET152-PPTase.

[0094] Construction of pKG1132-pcaGH-TtCAR-PPTase plasmid: Using Amy-F14 / Amy-R14 as primers and pSET152-TtCAR as template, the TtCAR fragment was amplified, using Amy-F15 / Amy-R15 as primers and pSET152-PPTase as template, the PPTase fragment was amplified, and the TtCAR and PPTase fragments were connected to the speI site of the pKG1132-pcaGH-2500 plasmid to obtain pKG1132-pcaGH-TtCAR-PPTase.

[0095] Construction of the Amycolatopsis pseudomycota strain VAN-6: pKG1132-pcaGH-TtCAR-PPTase was transformed into VAN-5. The screening of single and double exchange was the same as that of VAN-1. The double exchange strain was verified by PCR using the VAN-5 genome as a control. The verification primers were pcaGH-F1-test / CAR-R1-test (no band in the control, the correct band was 3116bp) and PPTase-F2-test / pcaGH-R2-test (no band in the control, the correct band was 3038bp). The correct bands were sent for sequencing, and the strain with correct sequencing was strain VAN-6. In this engineered strain, the vanillin dehydrogenase gene vdh, the protocatechuate 3,4-dioxygenase gene pcaGH and the vanillate demethylase gene vanAB were knocked out, and the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA, the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, the carboxylic acid reductase gene CAR and the phosphopantetheinyl transferase gene PPTase were expressed.

[0096] 2. Screening of the source of O-methyltransferase gene OMT

[0097] According to the records in Table 3, OMT gene sequences from Arabidopsis thaliana, Homo sapiens, Fragaria ananassa, Rattus norvegicus, Mus musculus, Vanilla planifolia, Lytechinus pictus, Klebsormidium nitens, Medicago sativa and Mycobacterium lentiflavum were obtained from NCBI and sent to GenScript for synthesis after codon optimization (such as SEQ ID No.7-16), and the synthesized gene fragments were respectively inserted into the BamHI / NsiI sites of the plasmid pSET152-permE plasmid to obtain pSET152-AtOMT, pSET152-HsOMT, pSET152-FaOMT, pSET152-RnOMT, pSET152-MmOMT, pSET152-VpOMT, pSET152-LpOMT, pSET152-KnOMT, pSET152-MsOMT, and pSET152-MlOMT plasmids. The above 10 plasmids were transformed into strain VAN-6 to obtain VAN-7At, VAN-7Hs, VAN-7Fa, VAN-7Rn, VAN-7Mm, VAN-7Vp, VAN-7Lp, VAN-7Kn, VAN-7Ms, and VAN-7Ml strains, and the above 10 strains were used for fermentation production of vanillin, that is, on the basis of knocking out the vanillin dehydrogenase gene vdh, the protocatechuate 3,4-dioxygenase gene pcaGH and the vanillate demethylase gene vanAB, expressing the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA, the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, the carboxylic acid reductase gene CAR and the phosphopantetheinyl transferase gene PPTase, the O-methyltransferase gene OMT from different sources was expressed.

[0098] By detecting the content of vanillin in the fermentation broth, the recombinant strain with high vanillin production can be screened out. The fermentation results are as follows: Figure 5 shown.

[0099] The experimental results showed that by comparing the yields of vanillin and byproduct isovanillin of each recombinant strain, the VAN-7M1 strain had the highest vanillin yield of 2.39 g / L, and no byproduct isovanillin. Although the vanillin yields of the VAN-7Lp and VAN-7Kn strains were lower, they also did not have byproduct isovanillin, so they have development potential.

[0100] From this courseware, the M10MT gene from Mycobacterium lentiflavum enables the recombinant bacteria to preferentially catalyze the meta-methylation of protocatechuic acid or protocatechuic aldehyde rather than the para-methylation, and can obtain the highest vanillin yield when expressed in Amycolatopsis . Therefore, the M10MT gene from Mycobacterium lentiflavum was selected for subsequent research.

[0101] Table 3 O-methyltransferase genes and their sources

[0102]

[0103]

[0104] 3. Screening of promoter and copy number of O-methyltransferase gene OMT

[0105] (1) Screening of promoters of O-methyltransferase gene OMT

[0106] In order to further improve the vanillin production of VAN-7M1 strain, the promoter of the key gene OMT was screened.

[0107] The three promoters KasOp, SP44, and P21 were synthesized by GenScript (as shown in SEQ ID No. 18-20), and the pSET152-KasOp, pSET152-SP44, and pSET152-P21 plasmids were obtained respectively. The M10MT gene sequence was amplified using pSET152-M10MT as a template and Amy-F16 / Amy-R16 as primers, and then connected to the KpnI-NsiI sites of the pSET152-KasOp, pSET152-SP44, and pSET152-P21 plasmids, respectively, to obtain pSET152-KasOp-M10MT, pSET152-SP44-M10MT, and pSET152-P21-M10MT plasmids. The above three plasmids were transformed into strain VAN-6 to obtain VAN-8-KasOp, VAN-8-SP44, and VAN-8-P21 strains, that is, on the basis of knocking out the vanillin dehydrogenase gene vdh, protocatechuate 3,4-dioxygenase gene pcaGH and vanillate demethylase gene vanAB, expressing the dehydroshikimate dehydratase gene DSD, chorismate pyruvate lyase gene ubiC, 4-hydroxybenzoate-3-monooxygenase gene pobA, 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, carboxylic acid reductase gene CAR and phosphopantetheinyl transferase gene PPTase, and expressing the OMT gene with promoter KasOp, SP44 or P21, respectively.

[0108] Fermentation test was carried out and the content of vanillin in the fermentation broth was detected, as shown in Table 4.

[0109] The results showed that compared with VAN-7M1 without promoter expression, the expression of SP44 promoter had the strongest effect on increasing the vanillin production of recombinant bacteria, with the vanillin production reaching 3.27 g / L, while the expression of promoters KasOp or P21 reduced the vanillin production of recombinant bacteria to varying degrees. Therefore, the OMT gene driven by the SP44 promoter was selected for subsequent research.

[0110] Table 4 Fermentation results of screening O-methyltransferase gene OMT promoter

[0111]

[0112] (2) Screening of the copy number of the O-methyltransferase gene OMT

[0113] Construction of pSET152-2×-M1OMT plasmid overexpressing 2 copies of OMT gene:

[0114] The pSET152-SP44-MlOMT plasmid was digested with XbaI-SpeI, the SP44-MlOMT fragment was recovered, and the fragment was ligated to the SpeI site of the pSET152-SP44-MlOMT plasmid to obtain the pSET152-2×-MlOMT plasmid.

[0115] Construction of pSET152-3×M1OMT plasmid overexpressing 3 copies of OMT gene:

[0116] The SP44-M10MT fragment was digested and ligated into the SpeI site of the pSET152-2×-M10MT plasmid to obtain the pSET152-3×M10MT plasmid.

[0117] The pSET152-2×MlOMT and pSET152-3×MlOMT plasmids were transformed into the strain VAN-6, respectively, to obtain VAN-9-MlOMT×2 and VAN-9-MlOMT×3 strains, that is, the vanillin dehydrogenase gene vdh, protocatechuate 3,4-dioxygenase gene pcaGH and vanillate demethylase gene vanAB were knocked out, and the dehydroshikimate dehydratase gene DSD, chorismate pyruvate lyase gene ubiC, 4-hydroxybenzoate-3-monooxygenase gene pobA, 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, carboxylic acid reductase gene CAR and phosphopantetheinyl transferase gene PPTase were expressed, and two or three copies of the MlOMT gene were overexpressed on the basis of using promoter SP44 to drive the O-methyltransferase gene OMT.

[0118] Fermentation test was carried out to detect the content of vanillin in the fermentation broth, as shown in Table 5.

[0119] The results showed that the recombinant bacteria containing 2 copies of the OMT gene had the best effect in producing vanillin, with a vanillin yield of 4.29 g / L. Further increasing the copy number of the OMT gene did not further increase the vanillin yield of the recombinant bacteria. Therefore, the 2 copies of the OMT gene were selected for subsequent research.

[0120] Table 5 Fermentation results for screening the copy number of O-methyltransferase gene OMT

[0121]

[0122] 4. Integrate 2 copies of the SP44-M10MT gene into the vanAB locus of Amycolatopsis

[0123] Construction of plasmid pKG1132-vanAB-MlOMT×2 with 2 copies of SP44-MlOMT gene integrated into the vanAB site:

[0124] The pSET152-2×-MlOMT plasmid was digested with XbaI-SpeI, and the 2×-MlOMT fragment was recovered and ligated to the SpeI site of the pKG1132-vanAB-2500 plasmid to obtain the pKG1132-vanAB-MlOMT×2 plasmid.

[0125] Construction of the Amycolatopsis pseudomycoticola VAN-10 strain: the pKG1132-vanAB-MlOMT×2 plasmid was transformed into VAN-6. The single and double exchange screening methods were the same as those of VAN-1. The double exchange strain was verified by PCR using the VAN-6 genome as a control. The verification primers were vanAB-F1-test / OMT-R1-test (no band in the control, the correct band was 3037bp) and OMT-F2-test / vanAB-R2-test (no band in the control, the correct band was 3537bp). The correct bands were sent for sequencing, and the strain with correct sequencing was strain VAN-10, which was an engineering strain in which the vanillin dehydrogenase gene vdh, protocatechuate 3,4-dioxygenase gene pcaGH and vanillate demethylase gene vanAB were knocked out, and the dehydroshikimate dehydratase gene DSD, chorismate pyruvate lyase gene ubiC, 4-hydroxybenzoate-3-monooxygenase gene pobA, 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, carboxylic acid reductase gene CAR and phosphopantetheinyl transferase gene PPTase were expressed, and the O-methyltransferase gene OMT was driven by promoter SP44, and two copies of the MlOMT gene were integrated into the vanAB site and driven by promoter SP44.

[0126] Fermentation experiments were conducted to detect the content of vanillin in the fermentation broth. The results showed that the vanillin product of the VAN-10 strain was 4.31 g / L.

[0127] It can be seen from this that inserting two copies of the M10MT gene into the vanAB locus can slightly increase the vanillin production of the engineered strain, but the increase is not significant.

[0128] Example 3: Construction of Amycolatopsis spp. VAN-11 with integrated ppsA gene

[0129] In order to further increase the yield of vanillin and increase the precursor phosphoenolpyruvate, the phosphoenolpyruvate synthase gene ppsA gene was integrated into the pyruvate kinase gene pyk site. The pKG1132-pyk-ppsA plasmid was constructed with reference to the description of Chinese invention patent CN117417952B.

[0130] Construction of Amycolatopsis pseudomycoticola VAN-11 strain: pKG1132-pyk-ppsA was transformed into VAN-10, and the screening of single and double exchange was the same as that of VAN-1. The double exchange strain was verified by PCR using the VAN-10 genome as a control, and the verification primers were pyk-F1-test / ppsA-R1-test (no band in the control, correct band 3253bp) and ppsA-F2-test / pyk-R2-test (no band in the control, correct band 3302bp). The correct band was sent for sequencing, and the strain with the correct sequencing was strain VAN-11.

[0131] Fermentation tests were conducted to detect the content of vanillin in the fermentation broth. The vanillin product of the VAN-11 strain was 5.09 g / L, and the product did not contain isovanillin.

[0132] In summary, the present invention knocks out the vanillin dehydrogenase gene vdh, the protocatechuate 3,4-dioxygenase gene pcaGH, the vanillic acid demethylase gene vanAB and the pyruvate kinase gene pyk in Amycolatopsis, and expresses the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA, the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, the carboxylic acid reductase gene CAR, the phosphopantetheinyl transferase gene PPTase, the O-methyltransferase gene OMT and the phosphoenolpyruvate synthase gene ppsA, to obtain an engineered strain VAN-11 for vanillin, and the strain is capable of fermenting vanillin with glucose as a substrate, with a yield of 5.09 g / L, and no isovanillin is contained in the product.

[0133] The present invention screens the source of the OMT gene and confirms that the M1OMT gene derived from Mycobacterium lentiflavum preferentially catalyzes the meta-methylation of protocatechuic acid or protocatechuic aldehyde rather than the para-methylation, and can obtain the highest vanillin yield when expressed in Amycolatopsis lentiflavum without the production of the byproduct isovanillin.

Claims

1. Application of O-methyltransferase gene OMT in improving the fermentation production of vanillin by recombinant Amycolatopsis, wherein the recombinant Amycolatopsis is depleted of vanillin dehydrogenase gene vdh, protocatechuate 3,4-dioxygenase gene pcaGH and vanillic acid demethylase gene vanAB, and expresses dehydroshikimate dehydratase gene DSD, chorismate pyruvate lyase gene ubiC, 4-hydroxybenzoate-3-monooxygenase gene pobA, 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, carboxylic acid reductase gene CAR, phosphopantetheinyl transferase gene PPTase, and the O-methyltransferase gene OMT is derived from Mycobacterium ( Mycobacterium lentiflavum ), the nucleotide sequence of the O-methyltransferase gene OMT is shown in SEQ ID NO:

16.

2. Application of overexpression of O-methyltransferase gene OMT in improving the fermentation production of vanillin by recombinant Amycolatopsis, wherein the recombinant Amycolatopsis is knocked out of vanillin dehydrogenase gene vdh, protocatechuate 3,4-dioxygenase gene pcaGH and vanillate demethylase gene vanAB, and expresses dehydroshikimate dehydratase gene DSD, chorismate pyruvate lyase gene ubiC, 4-hydroxybenzoate-3-monooxygenase gene pobA, 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, carboxylic acid reductase gene CAR, phosphopantetheinyl transferase gene PPTase and two copies of Mycobacterium ( Mycobacterium lentiflavum ) has an O-methyltransferase gene OMT, the nucleotide sequence of which is shown in SEQ ID NO:

16.

3. A recombinant Amycolatopsis, wherein the recombinant Amycolatopsis is obtained by knocking out the vanillin dehydrogenase gene vdh, the protocatechuate 3,4-dioxygenase gene pcaGH and the vanillic acid demethylase gene vanAB in Amycolatopsis HM-141, expressing the dehydroshikimate dehydratase gene DSD, the chorismate pyruvate lyase gene ubiC, the 4-hydroxybenzoate-3-monooxygenase gene pobA, the 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene aroF, the carboxylic acid reductase gene CAR and the phosphopantetheinyl transferase gene PPTase, and expressing the O-methyltransferase gene OMT, wherein the O-methyltransferase gene OMT is derived from Mycobacterium ( Mycobacterium lentiflavum ), the nucleotide sequence of the O-methyltransferase gene OMT is shown in SEQ ID NO:

16.

4. The recombinant Amycolatopsis according to claim 3, characterized in that The dehydroshikimate dehydratase gene DSD is derived from Amycolatopsis spp., and its nucleotide sequence is shown in SEQ ID NO: 1; the chorismate pyruvate lyase gene ubiC is derived from Providencia ragenbergii ( Providencia rustigianii ), whose nucleotide sequence is shown in SEQ ID NO:2; the 4-hydroxybenzoate-3-monooxygenase gene pobA is derived from Amycolatopsis, and its nucleotide sequence is shown in SEQ ID NO:3; the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthase gene aroF is derived from Amycolatopsis, and its nucleotide sequence is shown in SEQ ID NO:4; the carboxylic acid reductase gene CAR is derived from Thermospora thermophila, and its nucleotide sequence is shown in SEQ ID NO:5; the phosphopantetheinyl transferase gene PPTase is derived from Amycolatopsis, and its nucleotide sequence is shown in SEQ ID NO:

6.

5. The recombinant Amycolatopsis according to claim 3, characterized in that The recombinant Amycolatopsis also expresses a phosphoenolpyruvate synthase gene ppsA and knocks out a pyruvate kinase gene pyk. The phosphoenolpyruvate synthase gene ppsA is derived from Amycolatopsis, and its nucleotide sequence is shown in SEQ ID NO:

21.

6. The recombinant Amycolatopsis according to claim 3, characterized in that The recombinant Amycolatopsis expresses two copies of the O-methyltransferase gene OMT.

7. The recombinant Amycolatopsis according to claim 3, characterized in that The O-methyltransferase gene OMT is activated under the action of promoter SP44, and the nucleotide sequence of promoter SP44 is shown in SEQ ID NO:

19.

8. Use of the recombinant Amycolatopsis according to any one of claims 5 to 7 in fermenting vanillin using glucose as a substrate.

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