CN112126610A

CN112126610A - Engineering bacterium for producing hydroxytyrosol

Info

Publication number: CN112126610A
Application number: CN201910554741.7A
Authority: CN
Inventors: 赵华; 张婷; 宋田青; 蒋发现
Original assignee: Fengyang Biological Research And Development Nanjing Co ltd
Current assignee: Fengyang Biological Research And Development Nanjing Co ltd
Priority date: 2019-06-25
Filing date: 2019-06-25
Publication date: 2020-12-25

Abstract

The invention discloses an engineering bacterium for producing hydroxytyrosol, which simultaneously expresses hydroxylase catalyzing tyrosol to generate hydroxytyrosol and NAD⁺Coenzyme for generating NADH is reduced, and a NAD coenzyme cyclic regeneration system is constructed. The engineering bacteria are used for producing the hydroxytyrosol by taking the tyrosol as a substrate, the process and the extraction method are simple, the product is single, the yield and the productivity are obviously improved, and the engineering bacteria have good industrial application prospect.

Description

Engineering bacterium for producing hydroxytyrosol

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an engineering bacterium for producing hydroxytyrosol.

Background

Hydroxytyrosol (HT), a chinese alias: 3, 4-dihydroxyphenethyl alcohol. The molecular formula is as follows: c₈H₁₀O₃Molecular weight: 154.1632.

hydroxytyrosol is a natural polyphenol compound with fat solubility, water solubility and biological activity, and is mainly in the form of esterified oleuropein which exists in each part of olive, and free hydroxytyrosol can be obtained after oleuropein is hydrolyzed. The research shows that: hydroxytyrosol is the strongest natural antioxidant discovered so far, has great benefits for human health, such as activities of cancer chemoprevention, atherosclerosis resistance, DNA oxidative damage inhibition, skin photodamage protection, anti-inflammation and the like, and is deeply valued by the biological and medical fields in recent years. Capsules, tablets and powder products of hydroxytyrosol have been made abroad one after another.

At present, hydroxytyrosol is mainly obtained by a plant extraction method and a chemical synthesis method. The chemical synthesis of hydroxytyrosol has the advantages of low degradation yield, complex reaction process, expensive catalyst, high toxicity of chemical reagents and serious environmental pollution, and is not suitable for industrial production. At present, hydroxytyrosol used in food additives and health care products in the market is mainly obtained by a plant extraction method, for example, the Chinese invention patent application CN20161083391.5 discloses an olive leaf hydroxytyrosol extraction method, and the Chinese invention patent application CN201710195462.7 discloses a method for extracting hydroxytyrosol from olive leaves, but the plant extraction is limited by plant resources and content, so that the price is high, and the large-scale application cannot be realized. Compared with the method for obtaining hydroxytyrosol by plant extraction and chemical synthesis, the method for synthesizing hydroxytyrosol by using a biological method has the advantages of stability, safety, no pollution, low cost and the like. Therefore, the biosynthesis of hydroxytyrosol has been widely focused by constructing a completely new biosynthesis pathway in engineered microorganisms using synthetic biology techniques.

The production of hydroxytyrosol by microbiological methods has been partially reported. Chinese patent application CN201510242626.8 discloses a monooxygenase gene cluster HpaBC from Escherichia coli over-expressed in Escherichia coli, which takes glucose as a substrate to synthesize hydroxytyrosol from the beginning, and the final yield of the hydroxytyrosol is 349.05mg/L and is only 0.017mol/mol due to the toxicity of the hydroxytyrosol to the gene.

Satoh Y, et al, 2012,14(6) proposed the Metabolic synthesis of hydroxytyrosol from tyrosine as a substrate under the combined action of tyrosine hydroxylase, L-dopa decarboxylase and tyramine oxidase (Engineering of L-tyrosine oxidation in Escherichia coli and microbial production of hydroxybutanols [ J ]. Metabolic Engineering, 2012). In this method, 1mM tyrosine (0.18g/L) produced only 0.19mM (0.029g/L) hydroxytyrosol, with a yield of 19%; 1mM L-DOPA (0.19g/L) produced only 0.74mM (0.11g/L) hydroxytyrosol, with a yield of 74%. And the key genes expressed in the escherichia coli host bacteria are mainly from mice and people, and the situation that heterologous host expression is incompatible exists, so that the utilization rate of the substrate is low, and industrial production is difficult to realize.

Chinese patent application CN107586794A discloses a method for producing tyrosol or hydroxytyrosol by escherichia coli heterometabolism. The scheme expresses Aminotransferase (amino transferase), ketoacid decarboxylase (ketoacid decarboxylase) and Alcohol dehydrogenase (Alcohol dehydrogenase) in a host efficiently to produce tyrosol, and then produces hydroxytyrosol under the action of 4-hydroxyphenylacetic acid hydroxylase. The scheme has the disadvantages that the yield of hydroxytyrosol can reach 1243 +/-165 mg/L (8mM) when 6g/L (33mM) tyrosine is taken as a substrate, the yield is only 0.24mol/mol, the scheme needs to add a plurality of expensive coenzyme pyridoxal phosphate (PLP) and reduced coenzyme I (NADH), the efficiency of adding hydroxyl on a benzene ring is obviously reduced, the yield of hydroxytyrosol is low, and the intermediate metabolite tyrosol is accumulated in a large quantity.

Based on the defects of the existing methods, the research of an engineering bacterium capable of industrially producing hydroxytyrosol is especially necessary.

Disclosure of Invention

The invention provides an engineering bacterium for producing hydroxytyrosol, which can produce high-yield and high-yield hydroxytyrosol and has a good industrial application prospect.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides an engineering bacterium for producing hydroxytyrosol, which expresses hydroxylase catalyzing tyrosol to generate hydroxytyrosol and NAD⁺Reducing the coenzyme to NADH.

In some embodiments of the invention, the hydroxylase that catalyzes the production of hydroxytyrosol from tyrosol is tyrosinase or 4-hydroxyphenylacetate 3-monooxygenase (HpaBC).

In some embodiments of the invention, the hydroxylase that catalyzes the production of hydroxytyrosol from tyrosol is tyrosinase.

In some embodiments of the invention, the tyrosinase is derived from stenotrophomonas maltophilia (smtyrosinase), Bacillus megaterium (bmystinase), Bacillus thuringiensis (bttyrosinase), Bacillus endophyticus (betapyronase), bifidobacterium Diplocarpon rosae (drtyrosinase), or ralstonia solanacearum (rstenylase).

In some embodiments of the invention, the amino acid sequence of the betanin ase is accession No. WP _063592733.1 on NCBI.

In some embodiments of the invention, the drtyrosinase has the amino acid sequence access No. PBP28426.1 on NCBI.

In some embodiments of the invention the amino acid sequence of smtyrosinase is ACCESSION NO. AAC16658.1 on NCBI and the nucleotide sequence encoding smtyrosinase is SEQ ID NO. 5.

In some embodiments of the invention, the amino acid sequence of the bmtyrosinase is ACC86108.1 at NCBI and the nucleotide sequence encoding bmtyrosinase is SEQ ID NO. 4.

In some embodiments of the invention, the amino acid sequence of the bttyrosinase is AAR88107.1 on NCBI and the nucleotide sequence encoding the bttyrosinase is SEQ ID NO 6.

In some embodiments of the invention, the amino acid sequence of rstyrrosinase is accession No. AFR68815.1 at NCBI and the nucleotide sequence encoding rstyrrosinase is SEQ ID NO 7.

In some embodiments of the invention, the hydroxylase that catalyzes the production of hydroxytyrosol from tyrosol is HpaBC, HpaBC and NAD⁺The coenzyme reduced to NADH constitutes a NAD coenzyme cyclic regeneration system.

In some embodiments of the invention, the HpaBC is derived from escherichia coli BL21(DE3) (ecHpaBC) or Pseudomonas aeruginosa BAR65782 (paHpaBC).

In some embodiments of the invention, the nucleotide sequence derived from echpaBC is accession No. NC-012892 REGION:4498782..4500874 at NCBI; the nucleotide sequence derived from paHpaBC is SEQ ID NO: 3.

in some embodiments of the invention, the hydroxylase catalyzing the production of hydroxytyrosol from tyrosol is derived from a natural or protein engineered microorganism, e.g. from a bacterium or a fungus.

In some embodiments of the invention, the NAD is⁺The coenzyme which is reduced to NADH is Formate Dehydrogenase (FDH), Alcohol Dehydrogenase (ADH), Glucose Dehydrogenase (GDH) or Phosphite Dehydrogenase (PDH).

In some embodiments of the invention, the NAD is⁺The coenzyme which is reduced to NADH is Formate Dehydrogenase (FDH) or Alcohol Dehydrogenase (ADH).

In some embodiments of the invention, the NAD is⁺The coenzyme which is reduced to NADH is Formate Dehydrogenase (FDH).

In some embodiments of the invention, the FDH is derived from Candida boidinii (CbFDH), Saccharomyces cerevisiae (ScFDH) or Mycobacterium Intracellulare (MiFDH).

In some embodiments of the invention, the amino acid sequence of CbFDH is accession No. AF004096 at NCBI and the nucleotide sequence encoding CbFDH is SEQ ID NO 9.

In some embodiments of the invention, the amino acid sequence of ScFDH is accession No. NM-001183808.1 at NCBI and the nucleotide sequence encoding ScFDH is SEQ ID NO. 8.

In some embodiments of the invention, the amino acid sequence of the MiFDH is accession No. WP-009957650 at NCBI and the nucleotide sequence encoding MiFDH is SEQ ID NO. 10.

In some embodiments of the invention, the NAD is⁺The coenzyme which is reduced to NADH is Alcohol Dehydrogenase (ADH)

In some embodiments of the invention, the ADH is derived from Lactobacillus brevis (LbADH).

In some embodiments of the invention, the amino acid sequence of the LbADH is accession No. WP _107696682 on NCBI, and the nucleotide sequence encoding the LbADH is SEQ ID NO. 11.

In a further aspect, the present invention provides an engineered bacterium for the production of hydroxytyrosol, which is obtained by transforming the plasmid pETDuet-1, which carries the hydroxylase gene catalyzing tyrosol to hydroxytyrosol and NAD, into the host Escherichia coli BL21(DE3)⁺Coenzyme gene for generating NADH by reduction.

In some embodiments of the invention, the genes on E.coli BL21(DE3) that break down phenolic compounds are knocked out.

In some embodiments of the invention, the genes that decompose phenolic compounds are hpaD and/or hpaE.

In some embodiments of the invention, the genes that decompose phenolic compounds are hpaD and hpaE.

In some embodiments of the invention, the hpaD has an amino acid sequence accession NO at the NCBI of: ACT 46009.1; the amino acid sequence access NO on the NCBI of the hpaE is: ACT 46010.1.

In some embodiments of the invention, the tyrosinase is derived from Stenotrophomonas maltophilia (smtyrosinase), Bacillus megaterium (bmystinase), Bacillus thuringiensis (bttyrosinase), Bacillus endophyticus (betapyranose), bifidobacterium diplocarpus roseus (drtyrosinase) or Ralstonia solanacearum (rstyrosinase).

In some embodiments of the invention, the NAD is⁺Reduction to NADHThe enzyme is Formate Dehydrogenase (FDH), Alcohol Dehydrogenase (ADH), Glucose Dehydrogenase (GDH), or Phosphite Dehydrogenase (PDH).

The term "OD 600" refers to the absorbance of a solution at a wavelength of 600 nm.

The term "yield" refers to the ratio of the yield of the target product (hydroxytyrosol) actually obtained by consuming a unit amount of substrate (tyrosol) to the theoretically calculated yield of the target product in the catalytic process. For example: consumption of 1mM tyrosol would theoretically yield 1mM hydroxytyrosol, and actual yield of 0.6mM hydroxytyrosol would yield 0.6mM/1mM, i.e. 0.6 mol/mol.

The invention has the beneficial effects that:

the invention provides an engineering bacterium for producing hydroxytyrosol, which simultaneously expresses hydroxylase catalyzing tyrosol to generate hydroxytyrosol and NAD⁺Coenzyme for generating NADH is reduced, and a NAD coenzyme cyclic regeneration system is constructed. Furthermore, the genes related to the decomposition of phenolic compounds on host Escherichia coli are knocked out. The engineering bacteria of the invention are used for producing hydroxytyrosol by taking tyrosol as a substrate, the process is simple, the product is single, the yield and the productivity are obviously improved, the industrial production is promoted, and the extraction method is simple and has good industrial application prospect.

Detailed Description

1. The invention relates to a strain and a plasmid

pETDuet-1 plasmid, Escherichia coli BL21(DE3), Escherichia coli DH 5. alpha. from Novagen.

2. Selection of enzymes

(1) Selection of tyrosol hydroxylase (hydroxylase catalyzing tyrosol to hydroxytyrosol)

Various sources of tyrosinase, as well as various sources of HpaBC enzyme (4-hydroxyphenylacetate 3hydroxylase), were selected herein. The selected tyrosinases are derived from Stenotrophomonas maltophilia (smtyrosinase), Bacillus megaterium (bmtyrosinase), Bacillus thuringiensis (bttyrosinase) and Ralstonia solanacearum (rstyrosinase), respectively; the HpaBC selected were derived from E.coli BL21(DE3) (echpaBC) and Pseudomonas aeruginosa BAR65782(paHpaBC), respectively.

(2) Reducible NAD⁺Selection of coenzymes

Need to consume NADH in the reduction process of tyrosol hydroxylase and enhance the expression of Escherichia coli NAD⁺The key enzyme of the synthetic pathway can improve the intracellular NADH level and is beneficial to the synthesis of the hydroxytyrosol. The genes selected were FDH and ADH. NCBIThe amino acid sequence of (A) is: AF004096, NM-001183808.1, WP-009957650, WP-107696682.

3. Construction of Co-expression System and culture of cells

The tyrosol hydroxylase and reducible NAD selected above⁺Coenzymes, from which one enzyme is selected, are co-expressed. Co-expression of the Dual Gene, pETDuet-1 loaded with tyrosol hydroxylase and reducible NAD, Using pETDuet-1 plasmid⁺The gene is transferred into Escherichia coli BL21(DE3) competent cells at the same time, and a positive transformant is obtained by screening Ampicillin (Ampicillin) -containing resistant plates, so that the recombinant Escherichia coli is obtained.

And (3) culturing the cells: according to the classical recombinant Escherichia coli culture and induced expression scheme, the recombinant Escherichia coli is transferred into TB culture medium (10 g/L peptone and 5g/L, NaCl 10g/L yeast powder) according to the volume ratio of 1%, when the OD600 of the cells reaches 0.6-0.8, isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.2mM is added, and the induced expression culture is carried out for 18h at 18 ℃. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 20min at 4 ℃.

4. Cell catalytic synthesis of hydroxytyrosol compounds

The cell catalytic system is as follows: the tyrosol concentration is 0.5-50g/L, the optional coenzyme substrate concentration is 0.5-50g/L, the pH is adjusted to be 4.0-8.0, the thallus OD600 is 10-600, and the conversion is carried out for 0.5-60h at the temperature of 15-40 ℃. And (3) measuring the yield of the hydroxytyrosol by liquid chromatography after the conversion is finished.

5. Detection analysis of samples

Quantitative analysis of hydroxytyrosol: the conversion solution is detected and analyzed by an Agilent 1260 high performance liquid chromatograph under the chromatographic conditions that: the mobile phase was 0.1% formic acid water and methanol, using an Agilent C18 column (Agilent Polaris C18-A4.6X 100mm,3.5 μm), with a flow rate of 0.8mL/min, a column temperature of 35 ℃, a sample size of 5 μ L, and a detection wavelength of 280 nm.

The present invention will be described in detail with reference to examples. It should be noted that the specific embodiments described herein are only for explaining the present invention and are not used to limit the present invention.

Example 1 plasmid construction

(1) Synthesis of primers

Primers were designed for PCR amplification.

Primer name	Primer 5 '-3'	Sequence numbering
			ecHpaBC-F	gatgaaaccagaagatttccgcg	SEQ ID NO:1
ecHpaBC-R	ttaaatcgcagcttccatttccagcacta	SEQ ID NO:2

(2) PCR amplification

Genomic DNA of a wild strain in the logarithmic growth phase was extracted according to the instruction manual of Genomic DNA Purification Kit of Takara, and PCR amplification was carried out using the primers shown in Table 1 and the genome extracted from the corresponding strain as a template. The amplification system is as follows: prime STARHS DNA Polymerase (2.5U/. mu.L) 0.5. mu.L, 10 XPrime STAR Buffer 10. mu.L, dNTP mix (2.5mM each) 4. mu.L, template DNA 1. mu. L, Up primer (20. mu.M) 1. mu. L, Down primer (20. mu.M) 1. mu. L, ddH₂O make up to 50. mu.L. The amplification procedure was: 15sec at 98 ℃; 98 ℃ for 10 sec; 55 ℃ for 30 sec; 30 cycles at 72 ℃ for 2 min; 72 ℃ for 10 min. The PCR product was subjected to gene sequencing by Biotech Ltd, Kingkunshire, Nanjing.

4 tyrosinase from different sources and 2 HpaBC enzymes from different sources are selected, wherein the echHpaBC from escherichia coli is not subjected to codon optimization, and in addition, 5 genes are optimized according to the codon preference of the escherichia coli by using JCAT online codon optimization software (http// www.jcat.de) in combination with an OPTIMIZER online codon optimization tool (http:// genes. uv. es/OPTIMIZER /), so as to design a tyrosinase gene and a paHpaBC gene.

The optimized gene sequence numbers are as follows:

name of Gene	Sequence numbering
		paHpaBC	SEQ ID NO:3
bmtyrosinase	SEQ ID NO:4
		smtyrosinase	SEQ ID NO:5
bttyrosinase	SEQ ID NO:6
		rstyrosinase	SEQ ID NO:7

The key genes, formate dehydrogenase and alcohol dehydrogenase genes, are preferably transformed based on a cofactor regeneration system, and the codon preference is optimized in E.coli using JCAT online codon optimization software (http:// www.jcat.de) in combination with the OPTIMIZER online codon optimization tool (http:// genes. urv. es/OPTIMIZER /), to design full-length FDH and ADH genes. The optimized gene sequence numbers are as follows:

name of Gene	Sequence numbering
		ScFDH	SEQ ID NO:8
CbFDH	SEQ ID NO:9
		MiFDH	SEQ ID NO:10
LbADH	SEQ ID NO:11

(3) Construction of pETDuet-1 monogene plasmid

The pETDuet-1 plasmid, HpaBC genes from 2 sources and tyrosinase products from 4 sources are placed in a water bath condition at 37 ℃ for enzyme digestion for 1 h. The enzyme cutting system is as follows: 10 Xcut buffer 5. mu.L, DNA 10. mu.L, restriction enzyme SacI and restriction enzyme SalI each 1. mu.L, sterile water 33. mu.L. Then respectively recovering enzyme digestion products, connecting the enzyme digestion products for 12h-16h in a water bath at the temperature of 16 ℃, and carrying out enzyme digestion connection on pETDuet and rstyrosine, bmtyrosinase, smtyrosinase, bttyrosinase, paHpaBC and echPaBC respectively, wherein the connecting body is as follows: 10 XDNA ligase buffer 2.5. mu.L, DNA fragment 8. mu.L, vector DNA 2. mu.L, T4DNA ligase 1. mu.L, sterile water 11.5. mu.L for a total of 25. mu.L. The following recombinant plasmids were obtained: pETDuet-rstyrrosinase, pETDuet-bmtyrosinase, pETDuet-smtyrosinase, pETDuet-bttyrosinase, pETDuet-echPaBC, and pETDuet-paHpaBC.

EXAMPLE 2 screening of tyrosol hydroxylase

A variety of recombinant engineered bacteria containing tyrosol hydroxylase genes were obtained from example 1 and expressed in Escherichia coli BL21(DE 3). The induction expression method comprises the following steps: transferring the recombinant Escherichia coli into TB fermentation medium at a volume ratio of 1%, adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with a final concentration of 0.2mM when the OD600 of the cells reaches 0.6-0.8, and performing induced expression culture at 18 ℃ for 12 h. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 20min at 20 ℃. The recombinant strains were compared for catalysis by the addition of the 10mM substrate tyrosol, as shown in Table 1.

Table 1: comparing the catalytic behavior of tyrosol hydroxylase from different sources

As can be seen from Table 1, the host strain containing the HpaBC gene of E.coli (pETDuet-echHpaBC) exhibited the best catalytic effect.

Example 3 Gene knockout

According to the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System [ J ]. Appl Environ Microbiol, 2016, 82 (12): 3693 the hpaD and hpaE were knocked out by the method described in Escherichia coli BL21(DE3) using the primers shown in Table 2. The plasmid for gene knockout is pCasRed, pCRISPR-gDNA (hpaED sgRNA) and a homology arm (hpaED donor) are introduced into Escherichia coli BL21(DE3), Cas9/sgRNA induces double strand breaks of a host at hpaD and hpaE gene sites, recombinase Red integrates the hpaED donor to the hpaD and hpaE genes, gene knockout is realized, and sequencing verification is carried out.

Table 2: PCR primer

A solution of pH 6.5 was prepared, 30mM tyrosol was added, OD600 was 20, and catalysis was carried out at 30 ℃ for 48 hours. The hydroxytyrosol was measured by liquid chromatography after the completion of the conversion, and the amounts of produced hydroxytyrosol before and after the removal of Escherichia coli HpaE and HpaD in the reaction system are shown in Table 3.

Table 3: hydroxytyrosol yield before and after knocking out HpaE and HpaD

As can be seen from Table 3, after the HpaE and HpaD are knocked out, the yield of hydroxytyrosol is improved by 43.7%, and the yield is improved by 55.2%.

Also, Table 3 shows that tyrosol is not completely consumed by the addition of 30mM tyrosol, mainly due to the catalytic process consuming the intracellular cofactor NADH to NAD⁺In the absence of NADH regeneration, the catalytic reaction is difficult to continue, i.e.higher concentrations of substrate are not consumed. Thus, if a catalytic reaction at a higher substrate concentration is to be achieved, it is necessary to provide the cofactor NADH required for the catalytic reaction continuously in the cell, which can be achieved by introducing a cofactor-regenerating enzyme, i.e.NAD⁺Reducing to NADH.

Example 4 reducible NAD⁺Coenzyme selection and high yield hydroxytyrosol production

The pETDuet-echPaBC plasmid and the recovered products of MiFDH, CbFDH, ScFDH and LbADH in the step (2) of example 1 were subjected to double digestion for 1h in a water bath at 37 ℃. The enzyme cutting system is as follows: 10 Xcut buffer 5. mu.L, DNA 10. mu.L, restriction enzymes Nde I and Xho I each 1. mu.L, sterile water 33. mu.L. Then respectively recovering enzyme digestion products, connecting for 12h-16h in water bath at 16 ℃, and carrying out enzyme digestion connection on pETDuet-echPaBC and MiFDH, CbFDH, ScFDH and LbADH respectively, wherein the connecting body is as follows: 10 XDNA ligase buffer 2.5. mu.L, DNA fragment 8. mu.L, vector DNA 2. mu.L, T4DNA ligase 1. mu.L, sterile water 11.5. mu.L for a total of 25. mu.L.

Subsequently, 25. mu.L of the ligation solution was transferred to 100. mu.L of DH 5. alpha. competent cells and ice-cooled for 30 min. Placing into preheated 42 deg.C water bath, standing for 90s for heat shock treatment, and immediately ice-cooling for 2 min. 1mL of LB medium containing no antibiotic was added and cultured at 37 ℃ for 1 hour to recover the cells. And finally, uniformly coating the cells containing the pETDuet-1 recombinant plasmid on an LB (Langmuir-Blodgett) plate containing ampicillin, selecting a single colony for culturing for 12h, then extracting the plasmid, verifying whether the plasmid is correct by double enzyme digestion, simultaneously carrying out gene sequencing to ensure the accuracy of the plasmid, and finally storing a correct transformant to obtain the following plasmids: recombinant plasmids pETDuet-echPaBC-MiFDH, pETDuet-echPaBC-CbFDH, pETDuet-echPaBC-ScFDH and pETDuet-echPaBC-LbADH containing tyrosol hydroxylation gene.

The 4 recombinant plasmids were expressed in Escherichia coli BL21(DE3) after HpaE and HpaD knockout. The induction expression method comprises the following steps: the recombinant Escherichia coli is transferred into TB culture medium according to the volume ratio of 1%, when the OD600 of the cells reaches 0.6-0.8, isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.2mM is added, and the cells are induced to express and cultured for 8h at the temperature of 30 ℃. After the induction expression was completed, the cells were collected by centrifugation at 30 ℃ and 8000rpm for 20 min. 120mM (16.5g/L) tyrosol and 120mM (7.56g/L) ammonium formate were added and catalyzed for 48 hours, and hydroxytyrosol was measured by liquid chromatography after the conversion was completed, comparing the catalysis of the above 4 engineering strains in shake flasks, as shown in Table 4.

Table 4: comparison of catalytic results under different coenzyme conditions

As can be seen from the table, the yield and the yield of hydroxytyrosol are improved after the coenzyme is added, particularly under the condition that the concentration of the hydroxytyrosol is kept at the same high concentration, the consumption of the tyrosol by adding the coenzymes ScFDH and MiFDH is higher, the produced hydroxytyrosol is also higher, particularly when the coenzyme MiFDH is added, 15.1g/L of hydroxytyrosol is produced, the yield reaches 0.99mol/mol and is far beyond the yield and the yield of the hydroxytyrosol reported in the prior art.

Sequence information

SEQ ID NO:1ecHpaBC-F

atgaaaccagaagatttccgcg

SEQ ID NO:2ecHpaBC-R

ttaaatcgcagcttccatttccagcatca

Nucleotide sequence of SEQ ID NO 3 coding paHpaBC

atgaaaccggaagacttccgtgcttctgctacccgtccgttcaccggtgaagaatacctggcttctctgcgtgacgaccgtgaaatctacatctacggtgaccgtgttaaagacgttacctctcacccggctttccgtaacgctgctgcttctatggctcgtctgtacgacgctctgcacgacccgcagtctaaagaaaaactgtgctgggaaaccgacaccggtaacggtggttacacccacaaattcttccgttacgctcgttctgctgacgaactgcgtcagcagcgtgacgctatcgctgaatggtctcgtctgacctacggttggatgggtcgtaccccggactacaaagctgctttcggttctgctctgggtgctaacccgggtttctacggtcgtttcgaagacaacgctaaaacctggtacaaacgtatccaggaagcttgcctgtacctgaaccacgctatcgttaacccgccgatcgaccgtgacaaaccggttgaccaggttaaagacgttttcatctctgttgacgaagaagttgacggtggtatcgttgtttctggtgctaaagttgttgctaccaactctgctctgacccactacaacttcgttggtcagggttctgctcagctgctgggtgacaacaccgacttcgctctgatgttcatcgctccgatgaacaccccgggtatgaaactgatctgccgtccgtcttacgaactggttgctggtatcgctggttctccgttcgactacccgctgtcttctcgtttcgacgaaaacgacgctatcctggttatggacaaagttttcatcccgtgggaaaacgttctgatctaccgtgacttcgaacgttgcaaacagtggttcccgcagggtggtttcggtcgtctgttcccgatgcagggttgcacccgtctggctgttaaactggacttcatcaccggtgctctgtacaaagctctgcagtgcaccggttctctggaattccgtggtgttcaggctcaggttggtgaagttgttgcttggcgtaacctgttctggtctctgaccgacgctatgtacggtaacgcttctgaatggcacggtggtgctttcctgccgtctgctgaagctctgcaggcttaccgtgttctggctccgcaggcttacccggaaatcaaaaaaaccatcgaacaggttgttgcttctggtctgatctacctgccgtctggtgttcgtgacctgcacaacccgcagctggacaaatacctgtctacctactgccgtggttctggtggtatgggtcaccgtgaacgtatcaaaatcctgaaactgctgtgggacgctatcggttctgaattcggtggtcgtcacgaactgtacgaaatcaactacgctggttctcaggacgaaatccgtatgcaggctctgcgtcaggctatcggttctggtgctatgaaaggtatgctgggtatggttgaacagtgcatgggtgactacgacgaaaacggttggaccgttccgcacctgcacaacccggacgacatcaacgttctggaccgtatccgtcagtaacgcagcaggaggttaagatgtctcagctggaaccgcgtcagcaggctttccgtaacgctatggctcacctgtctgctgctgttaacgttatcacctctaacggtccggctggtcgttgcggtatcaccgctaccgctgtttgctctgttaccgactctccgccgaccctgatgctgtgcatcaaccgtaactctgaaatgaacaccgttttcaaagctaacggtcgtctgtgcgttaacgttctgtctggtgaacacgaagaagttgctcgtcacttcgctggtatgaccgaagttccgatggaacgtcgtttcgctctgcacgactggcgtgaaggtctggctggtctgccggttctgcacggtgctctggctaacctgcagggtcgtatcgctgaagttcaggaaatcggtacccactctgttctgctgctggaactggaagacatccaggttctggaacagggtgacggtctggtttacttctctcgttctttccaccgtctgcagtgcccgcgtcgtgctgcttaa

Nucleotide sequence of SEQ ID NO. 4 encoding bmtyrosinase

atggaatctaacaaataccgtgttcgtaaaaacgttctgcacctgaccgacaccgaaaaacgtgacttcgttcgtaccgttctgatcctgaaagaaaaaggtatctacgaccgttacatcgcttggcacggtgctgctggtaaattccacaccccgccgggttctgaccgtaacgctgctcacatgtcttctgctttcctgccgtggcaccgtgaatacctgctgcgtttcgaacgtgacctgcagtctatcaacccggaagttaccctgccgtactgggaatgggaaaccgacgctcagatgcaggacccgtctcagtctcagatctggtctgctgacttcatgggtggtaacggtaacccgatcaaagacttcatcgttgacaccggtccgttcgctgctggtcgttggaccaccatcgacgaacagggtaacccgtctggtggtctgaaacgtaacttcggtgctaccaaagaagctccgaccctgccgacccgtgacgacgttctgaacgctctgaaaatcacccagtacgacaccccgccgtgggacatgacctctcagaactctttccgtaaccagctggaaggtttcatcaacggtccgcagctgcacaaccgtgttcaccgttgggttggtggtcagatgggtgttgttccgaccgctccgaacgacccggttttcttcctgcaccacgctaacgttgaccgtatctgggctgtttggcagatcatccaccgtaaccagaactaccagccgatgaaaaacggtccgttcggtcagaacttccgtgacccgatgtacccgtggaacaccaccccggaagacgttatgaaccaccgtaaactgggttacgtttacgacatcgaactgcgtaaatctaaacgttcttcttaa

SEQ ID NO 5 nucleotide sequence coding for smtyrosinase

atggaccgtggtgttaacgttgctaaacagatgaaattcaccaacgctccggacttctctggtgctctgaacgttgaataccgtaccgaactggcttctgctggtaacctgtctgctcgtgtttcttactcttaccagtctgaagtttggccgaccaccgacctgtctccggttatccgtcaggacggttacggtctggttaacgctggtgttatctggaaactggacgacgcttggaccttctctctgcagggtaccaacctggctgacaaagaataccgtaccaccggttacaacatcccggctgttggtaccctgatcggtttctacggtccgccgcgtcagtacacctctgcttctgttaccatctctcgtaaccgtctgcacgacaccgttctgcgtctgatctgcaccggtaaagttactatggtttgcgcttgcatgcgtgctatcatgccgcaggctcgtgactctcacccggttgctcgttggtctgcttctcgtgcttaa

Nucleotide sequence of SEQ ID NO 6 coding for bttyrosinase

atgggtatccgtaaaaaccaggcttgcctgaccgacgaagaaaaagctgctttcgttgacgctatccaggaactgaaacgtaacggtgaataccagccgtacgttgacgttcaccgtaaacacttcttccacccgatccaccagtctgctatgttcctgccgtggcaccgtgaattcctgcacaaattcgaaatcgaactgcagaaagttgaccgtaacgttaccatcccgtactgggactggaccgttgacaactctatcacctcttctatctggcgtggtaacttcatgggtgctttcaccggtctgaaccgtcagctgggtgctaacccgttcctgccgacccgtacccaggttaaagaagctatcgacaccaccccgtacgacaccgctccgtggcgtcaggttacctctggtttccgttctgctctggaagaactgcacaacggtccgcacaactgggttggtggtgttatggctggtgctggttctccggaagacccggttttctggctgcaccactctaacatcaaccgtctgtgggctatctggcagcgtgaacacctgaacgaaccgtacctgccgacctctggtaccaccggtgctgacgaactgggtctggacgacccgatgcacgaattccgtgaaggtgaaaaaaacaccctgaccccgaaagacgttctggaccacacctctctgggttaccagtacgacaactactctctggacccggttgactgctaa

Nucleotide sequence of SEQ ID NO 7 encoding rstyrrosinase

ttgttcgtcgtaccgttctgaaagctatcgctggtacctctgttgctaccgttttcgctggtaaactgaccggtctgtctgctgttgctgctgacgctgctccgctgcgtgttcgtcgtaacctgcacggtatgaaaatggacgacccggacctgtctgcttaccgtgaattcgttggtatcatgaaaggtaaagaccagacccaggctctgtcttggctgggtttcgctaaccagcacggtaccctgaacggtggttacaaatactgcccgcacggtgactggtacttcctgccgtggcaccgtggtttcgttctgatgtacgaacgtgctgttgctgctctgaccggttacaaaaccttcgctatgccgtactggaactggaccgaagaccgtctgctgccggaagctttcaccgctaaaacctacaacggtaaaaccaacccgctgtacgttccgaaccgtaacgaactgaccggtccgtacgctctgaccgacgctatcgttggtcagaaagaagttatggacaaaatctacgctgaaaccaacttcgaagttttcggtacctctcgttctgttgaccgttctgttcgtccgccgctggttcagaactctctggacccgaaatgggttccgatgggtggtggtaaccagggtatcctggaacgtaccccgcacaacaccgttcacaacaacatcggtgctttcatgccgaccgctgcttctccgcgtgacccggttttcatgatgcaccacggtaacatcgaccgtgtttgggctacctggaacgctctgggtcgtaaaaactctaccgacccgctgtggctgggtatgaaattcccgaacaactacatcgacccgcagggtcgttactacacccagggtgtttctgacctgctgtctaccgaagctctgggttaccgttacgacgttatgccgcgtgctgacaacaaagttgtttctaacgctcgtgctgaacacctgctggctctgttcaaaaccggtgactctgttaaactggctgaccacatccgtctgcgttctgttctgaaaggtgaacacccggttgctaccgctgttgaaccgctgaactctgctgttcagttcgaagctggtaccgttaccggtgctctgggtgctgacgttggtaccggttctaccaccgaagttgttgctctgatcaaaaacatccgtatcccgtacaacgttatctctatccgtgttttcgttaacctgccgaacgctaacctggacgttccggaaaccgacccgcacttcgttacctctctgtctttcctgacccacgctgctggtcacgaccaccacgctctgccgtctactatggttaacctgaccgacaccctgaaagctctgaacatccgtgacgacaacttctctatcaacctggttgctgttccgcagccgggtgttgctgttgaatcttctggtggtgttaccccggaatctatcgaagttgctgttatctaa

Nucleotide sequence of SEQ ID NO 8 encoding ScFDH

atgtctaaaggtaaagttctgctggttctgtacgaaggtggtaaacacgctgaagaacaggaaaaactgctgggttgcatcgaaaacgaactgggtatccgtaacttcatcgaagaacagggttacgaactggttaccaccatcgacaaagacccggaaccgacctctaccgttgaccgtgaactgaaagacgctgaaatcgttatcaccaccccgttcttcccggcttacatctctcgtaaccgtatcgctgaagctccgaacctgaaactgtgcgttaccgctggtgttggttctgaccacgttgacctggaagctgctaacgaacgtaaaatcaccgttaccgaagttaccggttctaacgttgtttctgttgctgaacacgttatggctaccatcctggttctgatccgtaactacaacggtggtcaccagcaggctatcaacggtgaatgggacatcgctggtgttgctaaaaacgaatacgacctggaagacaaaatcatctctaccgttggtgctggtcgtatcggttaccgtgttctggaacgtctggttgctttcaacccgaaaaaactgctgtactacgactaccaggaactgccggctgaagctatcaaccgtctgaacgaagcttctaaactgttcaacggtcgtggtgacatcgttcagcgtgttgaaaaactggaagacatggttgctcagtctgacgttgttaccatcaactgcccgctgcacaaagactctcgtggtctgttcaacaaaaaactgatctctcacatgaaagacggtgcttacctggttaacaccgctcgtggtgctatctgcgttgctgaagacgttgctgaagctgttaaatctggtaaactggctggttacggtggtgacgtttgggacaaacagccggctccgaaagaccacccgtggcgtaccatggacaacaaagaccacgttggtaacgctatgaccgttcacatctctggtacctctctggacgctcagaaacgttacgctcagggtgttaaaaacatcctgaactcttacttctctaaaaaattcgactaccgtccgcaggacatcatcgttcagaacggttcttacgctacccgtgcttacggtcagaaaaaataa

Nucleotide sequence of SEQ ID NO 9 encoding CbFDH

atgaaaatcgttctggttctgtacgacgctggtaaacacgctgctgacgaagaaaaactgtacggttgcaccgaaaacaaactgggtatcgctaactggctgaaagaccagggtcacgaactgatcaccacctctgacaaagaaggtgaaacctctgaactggacaaacacatcccggacgctgacatcatcatcaccaccccgttccacccggcttacatcaccaaagaacgtctggacaaagctaaaaacctgaaactggttgttgttgctggtgttggttctgaccacatcgacctggactacatcaaccagaccggtaaaaaaatctctgttctggaagttaccggttctaacgttgtttctgttgctgaacacgttgttatgaccatgctggttctggttcgtaacttcgttccggctcacgaacagatcatcaaccacgactgggaagttgctgctatcgctaaagacgcttacgacatcgaaggtaaaaccatcgctaccatcggtgctggtcgtatcggttaccgtgttctggaacgtctgctgccgttcaacccgaaagaactgctgtactacgactaccaggctctgccgaaagaagctgaagaaaaagttggtgctcgtcgtgttgaaaacatcgaagaactggttgctcaggctgacatcgttaccgttaacgctccgctgcacgctggtaccaaaggtctgatcaacaaagaactgctgtctaaattcaaaaaaggtgcttggctggttaacaccgctcgtggtgctatctgcgttgctgaagacgttgctgctgctctggaatctggtcagctgcgtggttacggtggtgacgtttggttcccgcagccggctccgaaagaccacccgtggcgtgacatgcgtaacaaatacggtgctggtaacgctatgaccccgcactactctggtaccaccctggacgctcagacccgttacgctgaaggtaccaaaaacatcctggaatctttcttcaccggtaaattcgactaccgtccgcaggacatcatcctgctgaacggtgaatacgttaccaaagcttacggtaaacacgacaaaaaataa

SEQ ID NO 10 nucleotide sequence encoding MiFDH

Tactgctaaatggcttacgtcgctcgctcgcttagctcgctcgctcgctggctcgctcgctcgctggctcgctggctcgctggctggctcgctggctggctggctcgctggctcgctggctggctcgctggctggctggctcgctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctcgctggctggctggctggctggctggctggctggctggctggctggctggctggctggctcgctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctcgctggctggctggctggctggctggctcgctggctggctggctggctggctggctggctggctggctggctcgctggctggctggctggctggctggctggctggctcgctggctggctggctggctcgctggctggctggctggctggctggctggctggctggctcgctggctcgctggctggctggctggctggctggctggctggctggctggctggctcgctggctggctggctggctggctggctggctggctggctcgctggctggctcgctggctcgctggctcgctggctggctggctggctggctggctggctggctggctggctggctggctggctggctcgctcgctggctggctggctggcgcgcgcgctcgctggcgcgcgctggctggcgcgctggctcgctggctggctcgcgcgcgctggctggctggctggctggctcgctggctggctggctggctggctcgctggctggctggctggctggctggcgctggctggctggcgctggctggctggctcgctcgctcgctggctggctggctggctggctggctggctggctcgctggctggctggctggctggctggctggcgctcgctcgctggctggctggctggctggctggcgcgcgcgcgcgcgcgcgcgcgctcgcgcgcgctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctcgctcgcgctggctggctggctggctggctggctggctggctggctggcgcgcgcgctggctggctggctggcgcgctcgctggctggctggctcgcgcgcgcgctggctggcgcgcgcgcgcgcgcgctggctggcgcgcgcgcgctggcgcgcgcgcgcgcgcgcgcgcgcgctggctggctggctggctggctggctggctggctcgctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctggctg

atgcagatcaaaaccgctttctctgacggtacctctgaacagctgaaaatcatcgacgctaccctggacgaaccgaaagctaacgaagttctgatcaaaatggttgctaccggtgtttgccacaccgacaccgctggtcgtgacggtctgaccaccccgctgccggtttctctgggtcacgaaggtgctggtatcgttgaacaggttggttcttctgttatggacctgcagccgggtgaccacgttgttctgtctttcgcttactgcggtcgttgccagcactgcctgaccggtcacccgggtatgtgcgaacacttcaacgaactgaacttcgctggtcacaacttcgacggttctcaccgtatccacaccctggacggtcagccgatctctaccttcttcggtcagtcttctttctctacccacaccgttgttgaccagcacaacgttatcaaagttgacccgaccgttgacctgcgtctgctgggtccgctgggttgcggtatccagaccggtgctggtaccatcctgaactacctgcagccgaaattcggtgaatctctggttatcttcggtaccggtgctgttggtctgtctgctatcatggctgctaaaatcaccggtgctaaccacatcatcgctatcgacatcttcgacaaccgtctggctctggctaaagaactgggtgctaccgaagttatcaactctcaccaggctgacgttgaagctaccctgaaagaaatcctgccgaccggtgctgactacgctatcgacaccaccggtgtttctccggttgttatgaacgctctgcacgctctgaaaccgggtggtgaatgcgctgttgttggtatgggtcgtggtctgaccttcaacctgatggacgacctgatggctgaaggtaaaaaaatctctggtgttatcgaaggtgacgctatcccgcagctgttcatcccgaaactggttgactactacaaacagggtcgtttcccgttcgacaaactgatcaaattctacgacttcgaccagatcaacgacggtttcgctgcttctaaagacggttctgttctgaaaccggttatcaccttctaa

SEQ ID NO:12HpaE-homo-F

tagtgaacggcaggtatatgtgatgggttaaaaaggatccgcccaaacaatattgcatacatgc

SEQ ID NO:13HpaE-FRT-R

ggttccttacgcgggcagaagttcctattctctagaaagtataggaacttcttttttcatttcgctgtttcctcactc

SEQ ID NO:14FRT-HpaD-F

aaacagcgaaatgaaaaaagaagttcctatactttctagagaataggaacttctgcccgcgtaaggaacc

SEQ ID NO:15HpaD-sgRNA-R

cttaagcatatggcgatcagggtgtgggtg

SEQ ID NO:16HpaD-sgRNA-F

accctgatcgccatatgcttaagctggatccttgacag

Sequence listing

<110> Pterocarya stenoptera biological research & development (Nanjing) Co., Ltd

<120> an engineering bacterium for producing hydroxytyrosol

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gatgaaacca gaagatttcc gcg 23

<210> 2

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ttaaatcgca gcttccattt ccagcacta 29

<210> 3

<211> 2093

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaaaccgg aagacttccg tgcttctgct acccgtccgt tcaccggtga agaatacctg 60

gcttctctgc gtgacgaccg tgaaatctac atctacggtg accgtgttaa agacgttacc 120

tctcacccgg ctttccgtaa cgctgctgct tctatggctc gtctgtacga cgctctgcac 180

gacccgcagt ctaaagaaaa actgtgctgg gaaaccgaca ccggtaacgg tggttacacc 240

cacaaattct tccgttacgc tcgttctgct gacgaactgc gtcagcagcg tgacgctatc 300

gctgaatggt ctcgtctgac ctacggttgg atgggtcgta ccccggacta caaagctgct 360

ttcggttctg ctctgggtgc taacccgggt ttctacggtc gtttcgaaga caacgctaaa 420

acctggtaca aacgtatcca ggaagcttgc ctgtacctga accacgctat cgttaacccg 480

ccgatcgacc gtgacaaacc ggttgaccag gttaaagacg ttttcatctc tgttgacgaa 540

gaagttgacg gtggtatcgt tgtttctggt gctaaagttg ttgctaccaa ctctgctctg 600

acccactaca acttcgttgg tcagggttct gctcagctgc tgggtgacaa caccgacttc 660

gctctgatgt tcatcgctcc gatgaacacc ccgggtatga aactgatctg ccgtccgtct 720

tacgaactgg ttgctggtat cgctggttct ccgttcgact acccgctgtc ttctcgtttc 780

gacgaaaacg acgctatcct ggttatggac aaagttttca tcccgtggga aaacgttctg 840

atctaccgtg acttcgaacg ttgcaaacag tggttcccgc agggtggttt cggtcgtctg 900

ttcccgatgc agggttgcac ccgtctggct gttaaactgg acttcatcac cggtgctctg 960

tacaaagctc tgcagtgcac cggttctctg gaattccgtg gtgttcaggc tcaggttggt 1020

gaagttgttg cttggcgtaa cctgttctgg tctctgaccg acgctatgta cggtaacgct 1080

tctgaatggc acggtggtgc tttcctgccg tctgctgaag ctctgcaggc ttaccgtgtt 1140

ctggctccgc aggcttaccc ggaaatcaaa aaaaccatcg aacaggttgt tgcttctggt 1200

ctgatctacc tgccgtctgg tgttcgtgac ctgcacaacc cgcagctgga caaatacctg 1260

tctacctact gccgtggttc tggtggtatg ggtcaccgtg aacgtatcaa aatcctgaaa 1320

ctgctgtggg acgctatcgg ttctgaattc ggtggtcgtc acgaactgta cgaaatcaac 1380

tacgctggtt ctcaggacga aatccgtatg caggctctgc gtcaggctat cggttctggt 1440

gctatgaaag gtatgctggg tatggttgaa cagtgcatgg gtgactacga cgaaaacggt 1500

tggaccgttc cgcacctgca caacccggac gacatcaacg ttctggaccg tatccgtcag 1560

taacgcagca ggaggttaag atgtctcagc tggaaccgcg tcagcaggct ttccgtaacg 1620

ctatggctca cctgtctgct gctgttaacg ttatcacctc taacggtccg gctggtcgtt 1680

gcggtatcac cgctaccgct gtttgctctg ttaccgactc tccgccgacc ctgatgctgt 1740

gcatcaaccg taactctgaa atgaacaccg ttttcaaagc taacggtcgt ctgtgcgtta 1800

acgttctgtc tggtgaacac gaagaagttg ctcgtcactt cgctggtatg accgaagttc 1860

cgatggaacg tcgtttcgct ctgcacgact ggcgtgaagg tctggctggt ctgccggttc 1920

tgcacggtgc tctggctaac ctgcagggtc gtatcgctga agttcaggaa atcggtaccc 1980

actctgttct gctgctggaa ctggaagaca tccaggttct ggaacagggt gacggtctgg 2040

tttacttctc tcgttctttc caccgtctgc agtgcccgcg tcgtgctgct taa 2093

<210> 4

<211> 897

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggaatcta acaaataccg tgttcgtaaa aacgttctgc acctgaccga caccgaaaaa 60

cgtgacttcg ttcgtaccgt tctgatcctg aaagaaaaag gtatctacga ccgttacatc 120

gcttggcacg gtgctgctgg taaattccac accccgccgg gttctgaccg taacgctgct 180

cacatgtctt ctgctttcct gccgtggcac cgtgaatacc tgctgcgttt cgaacgtgac 240

ctgcagtcta tcaacccgga agttaccctg ccgtactggg aatgggaaac cgacgctcag 300

atgcaggacc cgtctcagtc tcagatctgg tctgctgact tcatgggtgg taacggtaac 360

ccgatcaaag acttcatcgt tgacaccggt ccgttcgctg ctggtcgttg gaccaccatc 420

gacgaacagg gtaacccgtc tggtggtctg aaacgtaact tcggtgctac caaagaagct 480

ccgaccctgc cgacccgtga cgacgttctg aacgctctga aaatcaccca gtacgacacc 540

ccgccgtggg acatgacctc tcagaactct ttccgtaacc agctggaagg tttcatcaac 600

ggtccgcagc tgcacaaccg tgttcaccgt tgggttggtg gtcagatggg tgttgttccg 660

accgctccga acgacccggt tttcttcctg caccacgcta acgttgaccg tatctgggct 720

gtttggcaga tcatccaccg taaccagaac taccagccga tgaaaaacgg tccgttcggt 780

cagaacttcc gtgacccgat gtacccgtgg aacaccaccc cggaagacgt tatgaaccac 840

cgtaaactgg gttacgttta cgacatcgaa ctgcgtaaat ctaaacgttc ttcttaa 897

<210> 5

<211> 510

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggaccgtg gtgttaacgt tgctaaacag atgaaattca ccaacgctcc ggacttctct 60

ggtgctctga acgttgaata ccgtaccgaa ctggcttctg ctggtaacct gtctgctcgt 120

gtttcttact cttaccagtc tgaagtttgg ccgaccaccg acctgtctcc ggttatccgt 180

caggacggtt acggtctggt taacgctggt gttatctgga aactggacga cgcttggacc 240

ttctctctgc agggtaccaa cctggctgac aaagaatacc gtaccaccgg ttacaacatc 300

ccggctgttg gtaccctgat cggtttctac ggtccgccgc gtcagtacac ctctgcttct 360

gttaccatct ctcgtaaccg tctgcacgac accgttctgc gtctgatctg caccggtaaa 420

gttactatgg tttgcgcttg catgcgtgct atcatgccgc aggctcgtga ctctcacccg 480

gttgctcgtt ggtctgcttc tcgtgcttaa 510

<210> 6

<211> 744

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgggtatcc gtaaaaacca ggcttgcctg accgacgaag aaaaagctgc tttcgttgac 60

gctatccagg aactgaaacg taacggtgaa taccagccgt acgttgacgt tcaccgtaaa 120

cacttcttcc acccgatcca ccagtctgct atgttcctgc cgtggcaccg tgaattcctg 180

cacaaattcg aaatcgaact gcagaaagtt gaccgtaacg ttaccatccc gtactgggac 240

tggaccgttg acaactctat cacctcttct atctggcgtg gtaacttcat gggtgctttc 300

accggtctga accgtcagct gggtgctaac ccgttcctgc cgacccgtac ccaggttaaa 360

gaagctatcg acaccacccc gtacgacacc gctccgtggc gtcaggttac ctctggtttc 420

cgttctgctc tggaagaact gcacaacggt ccgcacaact gggttggtgg tgttatggct 480

ggtgctggtt ctccggaaga cccggttttc tggctgcacc actctaacat caaccgtctg 540

tgggctatct ggcagcgtga acacctgaac gaaccgtacc tgccgacctc tggtaccacc 600

ggtgctgacg aactgggtct ggacgacccg atgcacgaat tccgtgaagg tgaaaaaaac 660

accctgaccc cgaaagacgt tctggaccac acctctctgg gttaccagta cgacaactac 720

tctctggacc cggttgactg ctaa 744

<210> 7

<211> 1487

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttgttcgtcg taccgttctg aaagctatcg ctggtacctc tgttgctacc gttttcgctg 60

gtaaactgac cggtctgtct gctgttgctg ctgacgctgc tccgctgcgt gttcgtcgta 120

acctgcacgg tatgaaaatg gacgacccgg acctgtctgc ttaccgtgaa ttcgttggta 180

tcatgaaagg taaagaccag acccaggctc tgtcttggct gggtttcgct aaccagcacg 240

gtaccctgaa cggtggttac aaatactgcc cgcacggtga ctggtacttc ctgccgtggc 300

accgtggttt cgttctgatg tacgaacgtg ctgttgctgc tctgaccggt tacaaaacct 360

tcgctatgcc gtactggaac tggaccgaag accgtctgct gccggaagct ttcaccgcta 420

aaacctacaa cggtaaaacc aacccgctgt acgttccgaa ccgtaacgaa ctgaccggtc 480

cgtacgctct gaccgacgct atcgttggtc agaaagaagt tatggacaaa atctacgctg 540

aaaccaactt cgaagttttc ggtacctctc gttctgttga ccgttctgtt cgtccgccgc 600

tggttcagaa ctctctggac ccgaaatggg ttccgatggg tggtggtaac cagggtatcc 660

tggaacgtac cccgcacaac accgttcaca acaacatcgg tgctttcatg ccgaccgctg 720

cttctccgcg tgacccggtt ttcatgatgc accacggtaa catcgaccgt gtttgggcta 780

cctggaacgc tctgggtcgt aaaaactcta ccgacccgct gtggctgggt atgaaattcc 840

cgaacaacta catcgacccg cagggtcgtt actacaccca gggtgtttct gacctgctgt 900

ctaccgaagc tctgggttac cgttacgacg ttatgccgcg tgctgacaac aaagttgttt 960

ctaacgctcg tgctgaacac ctgctggctc tgttcaaaac cggtgactct gttaaactgg 1020

ctgaccacat ccgtctgcgt tctgttctga aaggtgaaca cccggttgct accgctgttg 1080

aaccgctgaa ctctgctgtt cagttcgaag ctggtaccgt taccggtgct ctgggtgctg 1140

acgttggtac cggttctacc accgaagttg ttgctctgat caaaaacatc cgtatcccgt 1200

acaacgttat ctctatccgt gttttcgtta acctgccgaa cgctaacctg gacgttccgg 1260

aaaccgaccc gcacttcgtt acctctctgt ctttcctgac ccacgctgct ggtcacgacc 1320

accacgctct gccgtctact atggttaacc tgaccgacac cctgaaagct ctgaacatcc 1380

gtgacgacaa cttctctatc aacctggttg ctgttccgca gccgggtgtt gctgttgaat 1440

cttctggtgg tgttaccccg gaatctatcg aagttgctgt tatctaa 1487

<210> 8

<211> 1131

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgtctaaag gtaaagttct gctggttctg tacgaaggtg gtaaacacgc tgaagaacag 60

gaaaaactgc tgggttgcat cgaaaacgaa ctgggtatcc gtaacttcat cgaagaacag 120

ggttacgaac tggttaccac catcgacaaa gacccggaac cgacctctac cgttgaccgt 180

gaactgaaag acgctgaaat cgttatcacc accccgttct tcccggctta catctctcgt 240

aaccgtatcg ctgaagctcc gaacctgaaa ctgtgcgtta ccgctggtgt tggttctgac 300

cacgttgacc tggaagctgc taacgaacgt aaaatcaccg ttaccgaagt taccggttct 360

aacgttgttt ctgttgctga acacgttatg gctaccatcc tggttctgat ccgtaactac 420

aacggtggtc accagcaggc tatcaacggt gaatgggaca tcgctggtgt tgctaaaaac 480

gaatacgacc tggaagacaa aatcatctct accgttggtg ctggtcgtat cggttaccgt 540

gttctggaac gtctggttgc tttcaacccg aaaaaactgc tgtactacga ctaccaggaa 600

ctgccggctg aagctatcaa ccgtctgaac gaagcttcta aactgttcaa cggtcgtggt 660

gacatcgttc agcgtgttga aaaactggaa gacatggttg ctcagtctga cgttgttacc 720

atcaactgcc cgctgcacaa agactctcgt ggtctgttca acaaaaaact gatctctcac 780

atgaaagacg gtgcttacct ggttaacacc gctcgtggtg ctatctgcgt tgctgaagac 840

gttgctgaag ctgttaaatc tggtaaactg gctggttacg gtggtgacgt ttgggacaaa 900

cagccggctc cgaaagacca cccgtggcgt accatggaca acaaagacca cgttggtaac 960

gctatgaccg ttcacatctc tggtacctct ctggacgctc agaaacgtta cgctcagggt 1020

gttaaaaaca tcctgaactc ttacttctct aaaaaattcg actaccgtcc gcaggacatc 1080

atcgttcaga acggttctta cgctacccgt gcttacggtc agaaaaaata a 1131

<210> 9

<211> 1095

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgaaaatcg ttctggttct gtacgacgct ggtaaacacg ctgctgacga agaaaaactg 60

tacggttgca ccgaaaacaa actgggtatc gctaactggc tgaaagacca gggtcacgaa 120

ctgatcacca cctctgacaa agaaggtgaa acctctgaac tggacaaaca catcccggac 180

gctgacatca tcatcaccac cccgttccac ccggcttaca tcaccaaaga acgtctggac 240

aaagctaaaa acctgaaact ggttgttgtt gctggtgttg gttctgacca catcgacctg 300

gactacatca accagaccgg taaaaaaatc tctgttctgg aagttaccgg ttctaacgtt 360

gtttctgttg ctgaacacgt tgttatgacc atgctggttc tggttcgtaa cttcgttccg 420

gctcacgaac agatcatcaa ccacgactgg gaagttgctg ctatcgctaa agacgcttac 480

gacatcgaag gtaaaaccat cgctaccatc ggtgctggtc gtatcggtta ccgtgttctg 540

gaacgtctgc tgccgttcaa cccgaaagaa ctgctgtact acgactacca ggctctgccg 600

aaagaagctg aagaaaaagt tggtgctcgt cgtgttgaaa acatcgaaga actggttgct 660

caggctgaca tcgttaccgt taacgctccg ctgcacgctg gtaccaaagg tctgatcaac 720

aaagaactgc tgtctaaatt caaaaaaggt gcttggctgg ttaacaccgc tcgtggtgct 780

atctgcgttg ctgaagacgt tgctgctgct ctggaatctg gtcagctgcg tggttacggt 840

ggtgacgttt ggttcccgca gccggctccg aaagaccacc cgtggcgtga catgcgtaac 900

aaatacggtg ctggtaacgc tatgaccccg cactactctg gtaccaccct ggacgctcag 960

acccgttacg ctgaaggtac caaaaacatc ctggaatctt tcttcaccgg taaattcgac 1020

taccgtccgc aggacatcat cctgctgaac ggtgaatacg ttaccaaagc ttacggtaaa 1080

cacgacaaaa aataa 1095

<210> 10

<211> 1155

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggctaaat gcgttatggt tctgtacccg gacccggttg acggttaccc gccggcttac 60

gctcgtgact ctatcccgac catccacggt tacccggacg gttctaccgt tccgaccccg 120

tctaccatcg acttcacccc gggtgaactg ctgggttgcg tttctggtgc tctgggtctg 180

cgtaaattct tcgaagacgc tggtcacgaa ctggttgtta cctctgacaa agacggtccg 240

gactctgaat tcgaacgtgc tctgccggac gctgaaatcg ttatctctca gccgttctgg 300

ccggcttacc tgaccaaaga acgtatcgct aaagctccga aactgaaact ggctctgacc 360

gctggtatcg gttctgacca cgttgacctg gacgctgcta aagaacgtgg tatcaccgtt 420

gctgaagtta cctactctaa ctctatctct gttgctgaac acgctgttat gcagatcctg 480

gctctggttc gtaacttcgt tccgtctcac cgttgggctg ttgaaggtgg ttggaacatc 540

gctgactgcg ttgaacgtgc ttacgacctg gaaggtatgg acgttggtgt tatcgctgct 600

ggtcgtatcg gtcgtgctgt tctgcgtcgt ctggctccgt tcgacgttaa cctgcactac 660

accgacaccc gtcgtctggc tccggaagtt gaaaaagaac tgaacgttac cttccacccg 720

accgttcagg aactggttcg tgctgttgac gttgtttcta tccactctcc gctgtacgct 780

gacacccgtg ctatgttcga cgaaaaactg atctctacca tgcgtcgtgg ttcttacatc 840

gttaacaccg ctcgtgctga agaaaccgtt ccggaagcta tcgctgacgc tctgcgttct 900

ggtcagctgg gtggttacgc tggtgacgtt tggtacccgc agccgccgcc ggttgctcac 960

ccgtggcgta ccatgccgaa caacgctatg accccgcacg tttctggtac caccctgtct 1020

gctcaggctc gttacgctgc tggtacccgt gaaatcctgg aatcttggtt cgctggtacc 1080

ccgatccgtc cggaatacct gatcgttgaa ggtggtcgtc tggctggtac cggtgctctg 1140

tcttaccaga aatga 1155

<210> 11

<211> 1089

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgcagatca aaaccgcttt ctctgacggt acctctgaac agctgaaaat catcgacgct 60

accctggacg aaccgaaagc taacgaagtt ctgatcaaaa tggttgctac cggtgtttgc 120

cacaccgaca ccgctggtcg tgacggtctg accaccccgc tgccggtttc tctgggtcac 180

gaaggtgctg gtatcgttga acaggttggt tcttctgtta tggacctgca gccgggtgac 240

cacgttgttc tgtctttcgc ttactgcggt cgttgccagc actgcctgac cggtcacccg 300

ggtatgtgcg aacacttcaa cgaactgaac ttcgctggtc acaacttcga cggttctcac 360

cgtatccaca ccctggacgg tcagccgatc tctaccttct tcggtcagtc ttctttctct 420

acccacaccg ttgttgacca gcacaacgtt atcaaagttg acccgaccgt tgacctgcgt 480

ctgctgggtc cgctgggttg cggtatccag accggtgctg gtaccatcct gaactacctg 540

cagccgaaat tcggtgaatc tctggttatc ttcggtaccg gtgctgttgg tctgtctgct 600

atcatggctg ctaaaatcac cggtgctaac cacatcatcg ctatcgacat cttcgacaac 660

cgtctggctc tggctaaaga actgggtgct accgaagtta tcaactctca ccaggctgac 720

gttgaagcta ccctgaaaga aatcctgccg accggtgctg actacgctat cgacaccacc 780

ggtgtttctc cggttgttat gaacgctctg cacgctctga aaccgggtgg tgaatgcgct 840

gttgttggta tgggtcgtgg tctgaccttc aacctgatgg acgacctgat ggctgaaggt 900

aaaaaaatct ctggtgttat cgaaggtgac gctatcccgc agctgttcat cccgaaactg 960

gttgactact acaaacaggg tcgtttcccg ttcgacaaac tgatcaaatt ctacgacttc 1020

gaccagatca acgacggttt cgctgcttct aaagacggtt ctgttctgaa accggttatc 1080

accttctaa 1089

<210> 12

<211> 64

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tagtgaacgg caggtatatg tgatgggtta aaaaggatcc gcccaaacaa tattgcatac 60

atgc 64

<210> 13

<211> 78

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggttccttac gcgggcagaa gttcctattc tctagaaagt ataggaactt cttttttcat 60

ttcgctgttt cctcactc 78

<210> 14

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaacagcgaa atgaaaaaag aagttcctat actttctaga gaataggaac ttctgcccgc 60

gtaaggaacc 70

<210> 15

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cttaagcata tggcgatcag ggtgtgggtg 30

<210> 16

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

accctgatcg ccatatgctt aagctggatc cttgacag 38

Claims

1. An engineering bacterium for producing hydroxytyrosol, which is characterized in that the engineering bacterium expresses hydroxylase catalyzing tyrosol to generate hydroxytyrosol and expresses NAD⁺Reducing the coenzyme to NADH.

2. The engineered bacterium of claim 1, wherein said hydroxylase catalyzing tyrosol to hydroxytyrosol is tyrosinase or 4-hydroxyphenylacetate 3-monooxygenase.

3. The engineered bacterium of claim 2, wherein the hydroxylase catalyzing tyrosol to hydroxytyrosol is tyrosinase.

4. The engineered bacterium of claim 3, wherein said tyrosinase is derived from stenotrophomonas maltophilia, Bacillus megaterium, Bacillus thuringiensis, Bacillus endophyticus, Sphaerotheca rosea, or Ralstonia solanacearum.

5. The engineered bacterium of claim 2, wherein said hydroxylase catalyzing tyrosol to hydroxytyrosol is 4-hydroxyphenylacetate 3-monooxygenase.

6. The engineered bacterium of claim 5, wherein said 4-hydroxyphenylacetate 3-monooxygenase is derived from Escherichia coli or Pseudomonas aeruginosa.

7. The engineered bacterium of claim 6, wherein said 4-hydroxyphenylacetic acid 3-monooxygenase is derived from E.

8. The engineered bacterium of claim 1, wherein said NAD is added to said bacteria⁺The coenzyme which is reduced to generate NADH is formate dehydrogenase, alcohol dehydrogenase, glucose dehydrogenase or phosphite dehydrogenase.

9. The engineered bacterium of claim 8, wherein said NAD is added to said bacteria⁺The coenzyme which is reduced to generate NADH is formate dehydrogenase or alcohol dehydrogenase.

10. The engineered bacterium of claim 9, wherein said NAD is added to said culture medium⁺The coenzyme which is reduced to NADH is formate dehydrogenase.

11. The engineered bacterium of claim 10, wherein said formate dehydrogenase is derived from Candida boidinii, Saccharomyces cerevisiae, or Mycobacterium intracellulare.

12. The engineered bacterium of claim 11, wherein said formate dehydrogenase is derived from M.

13. The engineered bacterium of claim 12, wherein the nucleotide sequence encoding formate dehydrogenase derived from mycobacterium intracellulare is SEQ ID NO: 10.

14. the engineered bacterium of claim 9, wherein said alcohol dehydrogenase is derived from lactobacillus brevis.

15. The engineered bacterium of claim 14, wherein the nucleotide sequence encoding an alcohol dehydrogenase derived from lactobacillus brevis is SEQ ID NO: 11.

16. an engineered bacterium for producing hydroxytyrosol, which is obtained by transforming a pETDuet-1 plasmid into a host Escherichia coli BL21(DE3), wherein the pETDuet-1 plasmid is loaded with a hydroxylase gene catalyzing tyrosol to generate hydroxytyrosol and NAD⁺Coenzyme gene for generating NADH by reduction.

17. The engineered bacterium of claim 16, wherein a gene for decomposing phenolic compounds in escherichia coli BL21(DE3) is knocked out.

18. The engineered bacterium of claim 17, wherein the genes that decompose phenolic compounds are hpaD and/or hpaE.

19. The engineered bacterium of claim 18, wherein the genes that decompose phenolic compounds are HpaD and HpaE.

20. The engineered bacterium of any one of claims 16 to 19, wherein the hydroxylase catalyzing tyrosol to hydroxytyrosol is tyrosinase or 4-hydroxyphenylacetate 3-monooxygenase.

21. The engineered bacterium of claim 20, wherein the hydroxylase that catalyzes the production of hydroxytyrosol from tyrosol is tyrosinase.

22. The engineered bacterium of claim 21, wherein said tyrosinase is derived from stenotrophomonas maltophilia, bacillus megaterium, bacillus thuringiensis, bacillus endophyticus, diplocarpus roseus, or ralstonia solanacearum.

23. The engineered bacterium of claim 20, wherein said hydroxylase catalyzing tyrosol to hydroxytyrosol is 4-hydroxyphenylacetate 3-monooxygenase.

24. The engineered bacterium of claim 23, wherein said 4-hydroxyphenylacetate 3-monooxygenase is derived from escherichia coli or pseudomonas aeruginosa.

25. The engineered bacterium of claim 24, wherein said 4-hydroxyphenylacetic acid 3-monooxygenase is derived from e.

26. The engineered bacterium of claim 20, wherein said NAD is added⁺The coenzyme which is reduced to generate NADH is formate dehydrogenase, alcohol dehydrogenase, glucose dehydrogenase or phosphite dehydrogenase.

27. The engineered bacterium of claim 26, wherein said NAD is added⁺The coenzyme which is reduced to generate NADH is formate dehydrogenase or alcohol dehydrogenase.

28. The engineered bacterium of claim 27, wherein said NAD is added to said culture medium⁺The coenzyme which is reduced to NADH is formate dehydrogenase.

29. The engineered bacterium of claim 28, wherein said formate dehydrogenase is derived from candida boidinii, saccharomyces cerevisiae, or mycobacterium intracellulare.

30. The engineered bacterium of claim 29, wherein said formate dehydrogenase is derived from mycobacterium intracellulare.

31. The engineered bacterium of claim 30, wherein the nucleotide sequence encoding formate dehydrogenase derived from mycobacterium intracellulare is SEQ ID NO: 10.

32. the engineered bacterium of claim 27, wherein said alcohol dehydrogenase is derived from lactobacillus brevis.

33. The engineered bacterium of claim 32, wherein the nucleotide sequence encoding an alcohol dehydrogenase derived from lactobacillus brevis is SEQ ID NO: 11.