Recombinant bacterium for synthesizing hydroxytyrosol as well as construction method and application of recombinant bacterium

Abstract

The invention discloses a recombinant bacterium for synthesizing hydroxytyrosol as well as a construction method and application thereof, and relates to the technical field of microorganisms. One of the recombinant bacteria expresses aromatic aldehyde synthetase, alcohol dehydrogenase and 3-hydroxylase, and overexpresses PspF and / or CycA. According to the method, by means of replacing a promoter of a pspF gene and / or a cycA gene with a strong promoter and the like, genetic modification is carried out on a strain expressing aromatic aldehyde synthetase, alcohol dehydrogenase and 3-hydroxylase, so that the strain overexpresses the pspF gene and / or the cycA gene, and the yield of hydroxytyrosol can be remarkably increased.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to recombinant bacteria for synthesizing hydroxytyrosol, their construction methods, and applications. Background Technology

[0002] Hydroxytyrosol is a class of bisphenol compounds, mainly found in olives. It has excellent antioxidant activity and was the first natural phenolic compound approved by the European Food Inspection Agency as a food additive. It has anti-cancer, anti-inflammatory, antibacterial, anti-aging, antioxidant and anti-thrombotic effects and is widely used in video, medical, cosmetic, and agricultural fields.

[0003] Currently, the production of hydroxytyrosol mainly relies on natural extraction and chemical synthesis, but this still falls short of market demand. Biosynthesis is one of the most promising alternative technologies. Current biosynthesis methods for hydroxytyrosol include enzyme-catalyzed precursor synthesis (ZL201910882020.9, CN202211345112.1, ZL202210292883.2, ZL202210291600.2, CN202410135354.0, CN202410345500.2, CN202410547447.4, Nature Communications 11(2020)). The two main categories are (1515) and de novo fermentation synthesis (ZL202010582557.6, ZL202110176003.0, ZL202110546281.0, ZL202110292118.6, ZL202210930591.7, CN202210798069.8, ZL202210595170.3, CN202411808578.X, ZL202410510744.1, CN202410396605.0, CN202310840027.0). Among them, de novo fermentation synthesis has attracted much attention because it can utilize inexpensive carbon sources. Existing technologies have constructed recombinant Escherichia coli (ZL202010582557.6, ZL202110176003.0, ZL202210930591.7, CN202210798069.8, CN202411808578.X, ZL202410510744.1, CN202410396605.0, CN202310840027.0), Bacillus licheniformis (ZL202210595170.3), Saccharomyces cerevisiae (ZL202110546281.0), and Yersinia lipolytica (ZL202110292118.6) to produce hydroxytyrosol. However, the relevant invention patents and literature (Biochemical and Biophysical Research Communications 663 (2023) 16-24; ACS Synthetic Biology 11 (2022) 3706-3713; Journal of Agricultural Food Chemistry 71 (2023) 716681-16690) mainly focus on pathway enzymes, while less attention is paid to the modification of host cells. Summary of the Invention

[0004] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a recombinant bacterium capable of efficiently producing hydroxytyrosol.

[0005] The present invention also provides a method for constructing the above-mentioned recombinant bacteria.

[0006] The present invention also provides applications of the above-mentioned recombinant bacteria.

[0007] The present invention also provides a method for producing hydroxytyrosol.

[0008] According to a first aspect of the present invention, a recombinant bacterium expresses aromatic aldehyde synthase, alcohol dehydrogenase and 3-hydroxylase, and overexpresses PspF and / or CycA.

[0009] The recombinant bacteria according to embodiments of the present invention have at least the following beneficial effects: By pspF Genes and / or cycA Modifying hydroxytyrosol-producing strains by replacing the gene promoter with a strong promoter (such as promoter P37) to overexpress PspF and / or CycA can significantly increase the yield of hydroxytyrosol.

[0010] According to some embodiments of the present invention, the overexpression is achieved by any one of A1) to A5): A1) Introduce a vector containing the coding genes of PspF and / or CycA into the starting bacteria; A2) Increase the copy number of the genes encoding PspF and / or CycA on the chromosome; A3) Alter the promoter sequence of the gene encoding PspF and / or CycA on the chromosome; A4) The strong promoter is operatively linked to the coding gene of the PspF and / or CycA; A5) Change the nucleotide sequence encoding the PspF and / or CycA.

[0011] According to some embodiments of the present invention, the PspF is a phage shock protein transcription activator.

[0012] According to some embodiments of the present invention, CycA is a D-serine / D-alanine / glycine transporter.

[0013] According to some embodiments of the present invention, the recombinant bacteria overexpress... purR PurR is a hypoxanthine DNA-binding transcriptional repressor. The overexpression... purR Achieve this through any one of methods 1) to 5): 1) Introduce a vector containing the encoding gene of PurR into the starting bacteria; 2) Increase the copy number of the gene encoding PurR on the chromosome; 3) Alter the promoter sequence of the gene encoding PurR on the chromosome; 4) The strong promoter is operatively linked to the coding gene of PurR; 5) Change the nucleotide sequence encoding the PurR.

[0014] According to some embodiments of the present invention, the vector described in A1) or 1) is an expression vector. The expression vector is capable of normal replication and expression in host cells.

[0015] According to some embodiments of the present invention, the vectors include, but are not limited to, pETDuet series expression vectors, pACYCDuet series expression vectors, pRSFDuet series expression vectors, pCOLADuet series expression vectors, or pCDFDuet series expression vectors.

[0016] According to some embodiments of the present invention, A3) or 3) includes replacing the original promoter of the coding gene for pspF and / or cycA on the chromosome with a strong promoter.

[0017] According to some embodiments of the present invention, the strong promoter includes at least one of the P37 promoter, P8 promoter, J23100 promoter, J23119 promoter, P21285 promoter, M1-93 promoter, and PL1118 promoter. The strong promoter can be recognized by the recombinant bacteria.

[0018] According to some embodiments of the present invention, the changes in A5) or 5) include codon optimization.

[0019] According to some embodiments of the present invention, the aromatic aldehyde synthase is at least one of AAS (Uniprot Q06086), ipdC (Uniprot O07043), ARO10 (Uniprot Q06408), KDC4 (Uniprot C4R7I0) or PDC (Uniprot C5MDS4).

[0020] According to some embodiments of the present invention, the alcohol dehydrogenase is at least one of yahK (Uniprot P75691), ADH6 (Uniprot Q04894), ahr (Uniprot A0A024L8S1) or adh (Uniprot A0A7M3GDV1).

[0021] According to some embodiments of the present invention, such as the 3-hydroxylase described in patent CN202411627473.4, all are applicable to this application. As described in CN202411627473.4, a heterozygote of PlHpaB (GenBank AAO17197.1) with PaHpaC (GenBank PKG21041.1), EhHpaC (GenBank PJG38871.1), PlHpaC (GenBank AAO17198.1), SeHpaC (GenBank GAR62209.1), CpHpaC (GenBank SDV46364.1), EcHpaC (GenBank ACT46002.1), or KpHpaC (GenBank CDO16164.1); preferably a heterozygote of PlHpaB or its mutant with KpHpaC; or a heterozygote of CpHpaB mutant (GenBank SDV46363.1) / KpHpaC. The PlHpaB mutant is a Ser 210 position with Gly substitution (S210G) or a Ser 210 position with Ala substitution (S210A); the CpHpaB mutant is a Ser 217 position with Trp substitution (S217W).

[0022] According to some embodiments of the present invention, the originating bacterium of the recombinant bacteria is Escherichia coli.

[0023] According to some embodiments of the present invention, the *Escherichia coli* is a common *Escherichia coli*, such as K12 derivatives (BW25113, MG1665, W3110, DH10B, BW2952, MDS42 and their derivatives), BL21 (DE3) and its derivatives, BREL606, W, DH1, etc., or it may be a tyrosine-producing *Escherichia coli*. Tyrosine-producing *Escherichia coli* is preferred.

[0024] According to some embodiments of the present invention, the *Escherichia coli* is selected from *Escherichia coli* K12 derivatives, BL21(DE3) and its derivatives, BREL606 strain, E.coli W strain or DH1 strain.

[0025] The method for constructing the recombinant bacteria described in the first aspect embodiment or for increasing hydroxytyrosol production according to a second aspect embodiment of the present invention includes the following steps: Genetic engineering techniques were used to overexpress PspF and / or CycA in the starting bacteria; The starting bacteria express aromatic aldehyde synthase, alcohol dehydrogenase and 3-hydroxylase.

[0026] According to some embodiments of the present invention, the genetic engineering methods include, but are not limited to, gene editing technology.

[0027] According to some embodiments of the present invention, the method includes: using gene editing technology to modify the starting bacteria... pspF and / or cycA The original promoter is replaced with a strong promoter.

[0028] According to some embodiments of the present invention, the strong promoter includes at least one of the P37 promoter, P8 promoter, J23100 promoter, J23119 promoter, P21285 promoter, M1-93 promoter, and PL1118 promoter. The strong promoter can be recognized by the recombinant bacteria.

[0029] According to some embodiments of the present invention, the gene editing technology includes, but is not limited to, gene replacement. For example, gene replacement can be performed using the method described in the reference (Applied and Environmental Microbiology 2015, 81(7):2506-2514).

[0030] According to some embodiments of the present invention, the gene replacement method includes, but is not limited to, the CRISPR / Cas method.

[0031] Application of the recombinant bacteria according to the first aspect embodiment of the third aspect of the present invention in any one of B1) to B3): B1) Production of hydroxytyrosol or its derivatives; B2) Preparation of products for the production of hydroxytyrosol or its derivatives; B3) Increase the yield of hydroxytyrosol or its derivatives.

[0032] According to some embodiments of the present invention, the product includes at least one of reagents and kits.

[0033] A method for producing hydroxytyrosol according to a fourth aspect of the present invention includes: culturing the recombinant bacteria described in the first aspect of the present invention.

[0034] According to some embodiments of the present invention, the initial OD of the culture 600 The range is 0.01-0.2. For example, it can be 0.01, 0.05, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2.

[0035] According to some embodiments of the present invention, the culture temperature is 30℃-37℃. For example, it can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃ or 37℃.

[0036] According to some embodiments of the present invention, the culture time is 48 h-96 h. For example, it can be 48 h, 52 h, 56 h, 60 h, 64 h, 68 h, 72 h, 76 h, 80 h, 84 h, 88 h, 92 h or 96 h.

[0037] According to some embodiments of the present invention, the rotation speed of the culture is 150 rpm to 300 rpm. For example, it can be 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm, or 300 rpm.

[0038] According to some embodiments of the present invention, the method further includes the step of separating and purifying hydroxytyrosol from the fermentation broth.

[0039] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the biosynthetic pathway of hydroxytyrosol. Detailed Implementation

[0041] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0042] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0043] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0044] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0045] Using CRISPRa activation screening technology (Advanced Biotechnology 2024, 2:15), all endogenous stress protein (123) and transport protein (396) genes of Escherichia coli were activated and screened. Some target genes that could improve the robustness of the strain were identified, and the following promoter substitutions were performed to enhance expression.

[0046] Promoter replacement was performed using CRISPR gene editing technology (Applied Environmental Microbiology, 2015, 81: 2506-2514), and the plasmids used (pCas*, pTargetB) were the modified plasmids used in this experiment (Fronters Microbiology 2018, 9:1623).

[0047] Example 1 purR Replacement of gene promoters This embodiment uses the high-tyrosine-producing bacterium TYR-14B1 (Front Microbiol. 2021;12: 710405), which was previously constructed in the laboratory, as the research object for chassis cell modification. purR The natural promoter of the gene (Uniprot P0ACP7) was replaced with promoter P37 to construct TYR-14B1 (P purR strain ::P37). The specific experimental methods are as follows: (1) Construction of the shooting segment: Using the genome of *E. coli* BW25113 as a template, the upstream homologous arm of *purR* was amplified from the genome using primer pair *purR up-F / R*, and the downstream homologous arm of *purR* was amplified from the genome using primer pair *purR down-F / R*. Using plasmid pZBK (Biotechnology Biofuels 2019, 12:94) as a template, the P37-UTR fragment A was amplified using primer pair *purR37-F / R*. Then, using the upstream homologous arm of *purR*, P37-UTR fragment A, and the downstream homologous arm of *purR* as templates, the targeted fragment *purR up-P37-UTR-purR down* was obtained using overlap PCR with primers *purR up-F* and *purR down-R*.

[0048] (2) Construction of pTargetB-purR plasmid: Using pTargetB as a template, the N20 targeting plasmid pTargetB-purR-N20 was obtained by reverse PCR amplification using primer pair purR20-F / R.

[0049] (3) Containing pCas* plasmid E. coli Preparation and transformation of TYR-14B1 electrocompetent states: TYR-14B1 cells were prepared as competent cells, and the pCas* plasmid was transformed into the competent cells. The cells were then plated on Kansat resistant plates and incubated upside down at 30°C for 24 h. Successfully transformed single colonies were then picked and prepared as electroporation competent cells. 100 ng of the targeting plasmid pTargetB-purR-N20 and 1000 ng of the targeting fragment purRup-P37-UTR-purRdown were added, and electroporation was performed at 1.8 kV. Immediately after electroporation, 800 μL of LB medium was added, and the cells were incubated at 30°C and 200 rpm for 1.5 h. Then, 10 μL of arabinose was added for induction, followed by another 1.5 h of incubation. The cells were then plated on Kansat and Spe resistant plates.

[0050] (4) Transformation verification and removal of pTargetB-purR plasmid: Colony PCR was performed on transformed single colonies using the primer pair purR-F / R. The correctly validated strains were inoculated into Kan monoclonal antibody LB tubes, and IPTG was added to a final concentration of 2.5 mM. The cultures were then incubated overnight at 30°C. Afterward, the overnight cultures were streaked, and single colonies were spotted onto Kan and Spe plates, respectively. If a single colony survived on the Kan plate but did not grow on the Spe plate, it confirmed that the pTargetB-purR plasmid removal was successful.

[0051] (5) Removal of plasmid pCas*: Single colonies with successfully removed pTargetB-purR plasmid were picked and inoculated into antibiotic-free LB tubes and incubated at 42°C for 14-18 hours. The bacterial culture was then streaked onto antibiotic-free plates and incubated overnight at 37°C. Single colonies were then spotted onto both antibiotic-free and Kan plates. If a single colony survived on the antibiotic-free plate but did not grow on the Kan plate, it confirmed successful pCas removal and successful construction of TYR-14B1(P purR ::P37) strain.

[0052] The sequences of primers used in the above experimental methods are shown in Table 1.

[0053] Table 1

[0054] Quantitative PCR analysis of gene expression levels before and after promoter substitution revealed that... purRAfter the natural promoter of the gene is replaced with promoter P37, TYR-14B1 (P purR ::P37) strain purR The gene expression level was 5.5 times that of the TYR-14B1 strain.

[0055] Example 2 pspF Replacement of gene promoters Using TYR-14B1 (P) obtained in Example 1 purR ::P37) is the starting bacterium, and will pspF The natural promoter of the gene (UniprotP37344) was replaced with promoter P37 to construct TYR-14B1 (P purR ::P37, P pspF strain ::P37). The specific experimental methods are as follows: (1) Construction of the shooting segment: Using the *E. coli* BW25113 genome as a template, the upstream homologous arm of *pspF* was amplified from the genome using primer pair *pspFup-F / R*, and the downstream homologous arm of *pspF* was amplified from the genome using primer pair *pspFdown-F / R*. Using plasmid pZBK as a template, the P37-UTR fragment B was amplified using primer pair *pspF37-F / R*. Subsequently, using the upstream homologous arm of *pspF*, P37-UTR fragment B, and the downstream homologous arm of *pspF* as templates, the targeted fragment *pspFup-P37-UTR-pspFdown* was obtained using overlap PCR with primers *pspFup-F* and *pspFdown-R*.

[0056] (2) Construction of pTargetB-pspF plasmid: Using pTargetB as a template, the N20 targeting plasmid pTargetB-pspF-N20 was obtained by reverse PCR amplification using primer pair pspF20-F / R.

[0057] (3) Containing pCas* plasmid E. coli Preparation and transformation of TYR-14B1 electrocompetent states: The starting strain TYR-14B1 (P purR::P37) cells were prepared as competent cells. The pCas* plasmid was transformed into the competent cells, plated on Kansat resistant plates, and incubated upside down at 30°C for 24 h. Successfully transformed single colonies were then picked and prepared as electroporation competent cells. 100 ng of the targeting plasmid pTargetB-pspF-N20 and 1000 ng of the targeting fragment pspFup-P37-UTR-pspFdown were added, and electroporation was performed at 1.8 kV. Immediately after electroporation, 800 μL of LB medium was added, and the cells were incubated at 30°C and 200 rpm for 1.5 h. Then, 10 μL of arabinose was added for induction, followed by another 1.5 h of incubation, and finally plated on Kansat and Spe dual-antibody plates.

[0058] (4) Transformation verification and removal of pTargetB-pspF plasmid: Colony PCR verification of the transformed single colonies was performed using primer pair pspF verification-F / R. The verified strains were inoculated into Kan monoclonal antibody LB tubes, and IPTG was added to a final concentration of 2.5 mM. The cultures were then incubated overnight at 30°C. Afterward, the overnight cultures were streaked, and single colonies were picked and spotted onto Kan plates and Spe plates, respectively. If the single colonies survived on the Kan plate but did not grow on the Spe plate, it proved that the removal of pTargetB-pspF plasmid was successful.

[0059] (5) Removal of plasmid pCas*: Single colonies with successfully removed pTargetB-pspF plasmid were picked and inoculated into antibiotic-free LB tubes and incubated at 42°C for 14-18 hours. The bacterial culture was then streaked onto antibiotic-free plates and incubated overnight at 37°C. Single colonies were then spotted onto both antibiotic-free and Kan plates. If a single colony survived on the antibiotic-free plate but did not grow on the Kan plate, it confirmed successful pCas removal and successful construction of TYR-14B1(P purR ::P37, P pspF ::P37) strain.

[0060] The sequences of primers used in the above experimental methods are shown in Table 2.

[0061] Table 2

[0062] Quantitative PCR analysis of gene expression levels before and after promoter substitution revealed that... pspF After the natural promoter of the gene is replaced with promoter P37, TYR-14B1(P purR ::P37, P pspF ::P37) strain pspF Gene expression level was TYR-14B1 (P purR ::P37) strain 1.9 times.

[0063] Example 3cycA Replacement of gene promoters Using TYR-14B1 (P) obtained in Example 1 purR ::P37) is the starting bacterium, and will cycA The natural promoter of the gene (UniprotP0AAE0) was replaced with promoter P37 to construct TYR-14B1 (P purR ::P37, P cycA strain ::P37). The specific experimental methods are as follows: (1) Construction of the shooting segment: Using the *E. coli* BW25113 genome as a template, the upstream homologous arm of cycA was amplified from the genome using primer pair cycA up-F / R, and the downstream homologous arm of cycA was amplified from the genome using primer pair cycA down-F / R. Using plasmid pZBK as a template, the P37-UTR fragment C was amplified using primer pair P37UTR-F / R. Subsequently, using the upstream homologous arm of cycA, P37-UTR fragment C, and the downstream homologous arm of cycA as templates, the targeted fragment cycA up-F and cycA down-R were obtained using overlap PCR.

[0064] (2) Construction of pTargetB-cycA plasmid: Using pTargetB as a template, the N20 targeting plasmid pTargetB-cycA-N20 was obtained by reverse PCR amplification using primer pair cycA20-F / R.

[0065] (3) Containing pCas* plasmid E. coli Preparation and transformation of TYR-14B1 electrocompetent states: The starting strain TYR-14B1 (P purR ::P37) cells were prepared as competent cells. The pCas* plasmid was transformed into the competent cells, plated on Kansas resistant plates, and incubated upside down at 30°C for 24 h. Successfully transformed single colonies were then picked and prepared as electroporation competent cells. 100 ng of the targeting plasmid pTargetB-cycA-N20 and 1000 ng of the targeting fragment cycA_up-P37-UTR-cycA_down were added, and electroporation was performed at 1.8 kV. Immediately after electroporation, 800 μL of LB medium was added, and the cells were incubated at 30°C and 200 rpm for 1.5 h. Then, 10 μL of arabinose was added for induction, followed by another 1.5 h of incubation, and finally plated on Kansas and Spesas resistant plates.

[0066] (4) Transformation verification and removal of pTargetB-cycA plasmid: Colony PCR was performed on transformed single colonies using primer pair cycA-F / R. The correctly validated strains were inoculated into Kan monoclonal antibody LB tubes, and IPTG was added to a final concentration of 2.5 mM. The cultures were then incubated overnight at 30°C. Afterward, the overnight cultures were streaked, and single colonies were spotted onto Kan and Spe plates respectively. If a single colony survived on the Kan plate but did not grow on the Spe plate, it confirmed that the pTargetB-cycA plasmid removal was successful.

[0067] (5) Removal of plasmid pCas*: Single colonies with successfully removed pTargetB-cycA plasmid were picked and inoculated into antibiotic-free LB tubes and incubated at 42°C for 14-18 hours. The bacterial culture was then streaked onto antibiotic-free plates and incubated overnight at 37°C. Single colonies were then spotted onto both antibiotic-free and Kan plates. If a single colony survived on the antibiotic-free plate but did not grow on the Kan plate, it confirmed successful pCas removal and successful construction of TYR-14B1 (P purR ::P37, P cycA ::P37) strain.

[0068] The sequences of primers used in the above experimental methods are shown in Table 3.

[0069] Table 3

[0070] Quantitative PCR analysis of gene expression levels before and after promoter substitution revealed that... cycA After the natural promoter of the gene is replaced with promoter P37, TYR-14B1(P purR ::P37, P cycA ::P37) strain cycA Gene expression level was TYR-14B1 (P purR 12.6 times that of strain ::P37).

[0071] Detection example The hydroxytyrosol pathway plasmids pZBK-SpyTag-aro10-linker-yahK (Advanced Biotechnology (2024) 2:15) and pZEA-KpHpaB-(GGGS)3-PaHpaC (CN202411627473.4) were co-transferred into the above promoter-replaced tyrosine-producing bacteria (TYR-14B1 (P purR ::P37) strain, TYR-14B1 (P purR ::P37,P pspF ::P37) strain, TYR-14B1 (P purR ::P37, P cycARecombinant Escherichia coli producing hydroxytyrosol was obtained from strain P37. A schematic diagram of the biosynthetic pathway of hydroxytyrosol is shown below. Figure 1 As shown.

[0072] Fresh single colonies of recombinant *E. coli* were inoculated into 5 mL LB broth tubes containing the corresponding antibiotics as primary seed culture and incubated overnight at 37°C and 200 rpm for 12-16 h. Then, the primary seed culture was inoculated at a 1:100 ratio into small shake flasks containing 20 mL of the corresponding antibiotic-containing LB broth as secondary seed culture and incubated overnight at 37°C and 200 rpm for 12-16 h. After incubation, the secondary seed culture was inoculated at an initial inoculum size of OD0.05. 600 The culture medium was inoculated at a concentration of 0.1 mg / L into 50 mL of M9YGT medium containing the corresponding antibiotic, and incubated at 200 rpm and 30°C for 72 h. The OD value of the fermentation broth was measured after fermentation. 600 1 mL of bacterial culture was centrifuged at 12000 rpm for 1 min, filtered through a membrane, and then placed into a liquid chromatography bottle for HPLC analysis to determine the yield of hydroxytyrosol.

[0073] M9YGT medium: Weigh appropriate amounts of yeast extract, Na2HPO4·12H2O, KH2PO4, NaCl, and NH4Cl, add 900 mL of distilled water and dissolve thoroughly. Adjust the pH to 7.0-7.2, and bring the volume to 1000 mL (final concentration: yeast extract 2.5 g / L, Na2HPO4·12H2O 17.48 g / L, KH2PO4 3 g / L, NaCl 0.5 g / L, NH4Cl 1 g / L). Sterilize at 121℃ for 20 min. Before use, add MgSO4 to a final concentration of 0.06 g / mL, CaCl2 to a final concentration of 0.011 g / mL, glucose to a final concentration of 20 g / L, and trace element stock solution to a final concentration of 10 mL / L. Filter for sterilization. The trace element mother liquor consisted of: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2.2 g / L, MnSO4·4H2O 0.58 g / L, CuSO4·5H2O 1 g / L, and (NH4)6Mo7O 24 ·4H2O 0.1g / L, Na2B4O7·10H2O 0.2 g / L, 35% HCl 10 mL / L.

[0074] The HPLC detection method was as follows: the chromatographic column was an Inertsil ODS-SP C18 reversed-phase column; the column temperature was 30℃; the mobile phase was 0.2% TFA aqueous solution and liquid-grade methanol; the flow rate was 0.5 mL / min; gradient elution was performed from 0 to 20 min, with the methanol concentration increasing uniformly from 14 (V / V)% to 45 (V / V)% over time. After 20 min, the methanol concentration immediately decreased from 45 (V / V)% to 14 (V / V)%, and the equilibration was completed after 10 min.

[0075] Table 4

[0076] As shown in Table 4. By replacement pspF Gene, cycA The gene's promoter, P37, is a strong promoter that can significantly increase the production of hydroxytyrosol.

[0077] In addition to those described in this embodiment, any expression vector and tyrosine-producing strain can be used for all expression vectors and tyrosine-producing strains.

[0078] In addition to the methods mentioned in this embodiment, hydroxytyrosol can also be produced using a whole-cell catalytic method.

[0079] In addition to the above embodiments, the hydroxytyrosol synthesis pathway enzyme gene can also be integrated into the Escherichia coli chromosome to construct the corresponding recombinant Escherichia coli.

[0080] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A recombinant bacterium, characterized in that, The recombinant bacteria express aromatic aldehyde synthase, alcohol dehydrogenase and 3-hydroxylase, and overexpress PspF and / or CycA.

2. The recombinant bacteria according to claim 1, characterized in that, The overexpression is achieved through any of the methods from A1) to A5): A1) Introduce a plasmid containing the coding genes of PspF and / or CycA into the starting bacteria; A2) Increase the copy number of the genes encoding PspF and / or CycA on the chromosome; A3) Alter the promoter sequence of the gene encoding PspF and / or CycA on the chromosome; A4) The strong promoter is operatively linked to the coding gene of the PspF and / or CycA; A5) Change the nucleotide sequence encoding the PspF and / or CycA.

3. The recombinant bacteria according to claim 2, characterized in that, A3) This includes replacing the original promoter of the gene encoding PspF and / or CycA on the chromosome with a strong promoter; Preferably, the strong promoter includes at least one of the following: P37 promoter, P8 promoter, J23100 promoter, J23119 promoter, P21285 promoter, M1-93 promoter, and PL1118 promoter.

4. The recombinant bacteria according to claim 2, characterized in that, The changes mentioned in A5 include codon optimization.

5. A method for constructing the recombinant bacteria according to any one of claims 1 to 4, or a method for increasing hydroxytyrosol production, characterized in that, Includes the following steps: Using genetic engineering techniques to overexpress the starting bacteria pspF and / or cycA ; The starting bacteria express aromatic aldehyde synthase, alcohol dehydrogenase and 3-hydroxylase.

6. The method according to claim 5, characterized in that, The method includes: using gene editing technology to modify the starting bacteria. pspF and / or cycA The original promoter is replaced with a strong promoter.

7. The method according to claim 6, characterized in that, The gene editing technology includes gene replacement.

8. The use of the recombinant bacteria according to any one of claims 1 to 4 in any one of B1) to B3): B1) Production of hydroxytyrosol or its derivatives; B2) Preparation of products for the production of hydroxytyrosol or its derivatives; B3) Increase the yield of hydroxytyrosol or its derivatives.

9. The application according to claim 8, characterized in that, The product includes at least one of reagents and reagent kits.

10. A method for producing hydroxytyrosol, characterized in that, include: Cultivate the recombinant bacteria according to any one of claims 1 to 4.

Images