Expression optimization of multi-subunit enzyme for synthesizing high value-added compound by using lignin monomer

文档序号：62631 发布日期：2021-10-01 浏览：37次中文

阅读说明：本技术 多亚基酶的表达优化用于木质素单体合成高附加值化合物 (Expression optimization of multi-subunit enzyme for synthesizing high value-added compound by using lignin monomer ) 是由肖毅于 2021-06-24 设计创作，主要内容包括：本发明涉及一种多亚基酶的表达优化及利用木质素单体生物合成高附加值化合物的方法,通过Golden gate克隆的方法,构建了pE1K-GcoAB-M3的RBS文库,通过研究库的生长速率,转化速率和酶的表达比例,粗酶液的酶活等筛选到文库G/C的RBS文库,菌株生长速率高,酶活也高。之后,将该RBS文库用于酶促反应途径构建,催化木质素单体化合物愈创木酚(guaiacol)以及连苯三酚(pyrogallol)生产高附加值的化合物如邻苯二酚、儿茶酚-O-硫酸盐、红倍酚。本发明利用构建的生物催化剂,通过生物转化实现了木质素来源的芳香化合物(愈创木酚和3-甲氧基邻苯二酚)的高效利用,首次构建了儿茶酚-O-硫酸盐和红倍酚的生物合成途径。(The invention relates to a method for expression optimization of multi-subunit enzyme and biosynthesis of a high value-added compound by utilizing a lignin monomer, wherein an RBS library of pE 1K-GcobA-M3 is constructed by a Golden gate cloning method, the RBS library of the library G/C is screened by researching the growth rate, the conversion rate, the expression ratio of the enzyme, the enzyme activity of crude enzyme liquid and the like of the library, the growth rate of a strain is high, and the enzyme activity is also high. Thereafter, the RBS library was used for enzymatic reaction pathway construction, catalyzing monolignol compounds guaiacol (guaiacol) and pyrogallol (pyrogallol) to produce high value-added compounds such as catechol, catechol-O-sulfate, and erythrophenol. The invention utilizes the constructed biocatalyst to realize the high-efficiency utilization of aromatic compounds (guaiacol and 3-methoxy catechol) from lignin through biotransformation, and constructs the biosynthesis route of catechol-O-sulfate and rhodophenol for the first time.)

1. A method for synthesizing a high value-added compound by using a lignin monomer through expression optimization of multi-subunit enzyme is characterized in that the high value-added compound is synthesized by taking the lignin monomer compound as a starting raw material through a biocatalyst;

the catalyst is prepared by one of the following methods:

the method comprises the following steps: constructing an RBS library of a multi-subunit enzyme GcoAB containing GcoA and GcoB by a Golden gate method, converting the RBS library into E.coli, and screening to obtain a biocatalyst;

screening genes corresponding to the obtained biocatalyst and an aryl sulfotransferase mutant ASTB-OM2(Q191Y/Y218W/L225V) in the E.coli overexpression method I to obtain the biocatalyst;

coli by over-expressing laccase GoL3 in e.

2. The method for synthesizing high value-added compounds according to claim 1, wherein the catalyst obtained in the first method is E.

3. The method of claim 2, wherein the E.coli (GcoaB; RBS: G/C) is obtained by: constructing an RBS library of a multi-subunit enzyme GcoAB containing GcoA and GcoB by a Golden gate method, converting the RBS library into E.coli, and screening to obtain a biocatalyst E.coli (GcoAB; RBS: G/C); the genes GcoA and GcoB are derived from Amycolatopsis sp ATCC 39116, and the genes GcoA and GcoB are codon optimized.

4. The method of claim 2, wherein the sequence of the RBS core region of GcoA is AGGGG and the sequence of the RBS core region of GcoB is AGGCGGG.

5. The method for synthesizing high value-added compounds according to claim 1, wherein the catalyst obtained by the second method is E.

6. The method for synthesizing high value-added compounds according to claim 1, wherein the catalyst obtained by method three is E.coli (GoL 3).

7. The method for synthesizing high value-added compounds according to claim 1, wherein the monolignol compound comprises guaiacol or 3-methoxycatechol.

8. The method of synthesizing high value-added compounds according to claim 1, wherein the high value-added compounds comprise at least one of catechol, catechol-O-sulfate, and rhodophenol.

9. The method for synthesizing high value-added compounds according to claim 1, wherein the guaiacol is used as a starting material to synthesize catechol in the presence of a biocatalyst E.

10. The method for synthesizing high value-added compounds according to claim 1, wherein the catechol-O-sulfate is synthesized from guaiacol (GcoAB-ASTB-OM2) as a starting material.

11. The method for synthesizing high value-added compounds according to claim 1, wherein when the biocatalyst is e.coli (GoL3), 3-methoxy catechol is used as a starting material to synthesize rhodol.

Technical Field

The invention belongs to the technical field of microorganisms, and relates to the expression optimization of multi-subunit enzyme for synthesizing a high-added-value compound by using a lignin monomer.

Background

In view of the gradual decrease of non-renewable resources such as petroleum, natural gas, etc., the development and comprehensive utilization of renewable resources such as lignin are receiving much attention. Lignin is a natural high molecular compound, widely present in higher plant cells, and is a second-order biomass in nature. The lignin is a natural aromatic polymer formed by connecting phenylpropane units through C-O-C and C-C bonds and carrying out catalytic dehydrogenation polymerization and free radical polymerization of enzyme. It is believed from a prior view that lignin building blocks mainly comprise: p-Hydroxyphenyl propane (p-Hydroxyphenyl propane/H), Guaiacyl propane (Guaiacryl/G) and Syringyl propane (Syringyl/S). Lignin forms various aromatic compounds after thermochemical conversion and biological conversion, most compounds also contain methyl, after microbial demethylation, intermediate metabolites such as catechol, protocatechuic acid, 3,4, 5-trihydroxybenzoic acid and the like are formed, and then the intermediate metabolites can further enter central carbon metabolism and downstream high-added-value product generation, so that demethylation of a monomer compound from lignin is an important link for lignin utilization.

Many studies have been made to establish cell factories for efficient utilization of lignin and biorefinery of high value-added compounds, such as in the fields of medicine, biofuel, food and chemical industry, by modifying microorganisms. Genetic modification of microorganisms is common, but in the process of genetic modification, due to modification of endogenous genes and insertion of exogenous genes, metabolic flow imbalance is caused, growth is delayed, and yield of target compounds is low. In order to solve the imbalance of metabolic flux, accelerate strain optimization and improve the production of various high-value bio-based chemicals, technologies such as multivariate modular metabolic engineering (multivariate metabolic engineering), modular co-culture engineering (modular co-culture engineering) and spatio-temporal and genome integration have been widely used. The above techniques, focusing on one or more metabolic pathways, focus on the regulation of expression of multiple enzymes, while there is little interest in single-function multi-subunit enzymes.

The P450 protein is a multifunctional enzyme and can catalyze monomer compounds from various lignin sources. O-demethylation, consisting of the cytochrome P450 protein (GcoA) of the CYP255A family and a three-domain constitutive reductase (GcoB). By optimizing the multicomponent O-demethylase double subunits GcoA and GcoB, the expression ratio and enzyme activity, growth rate and the like of the enzymes in cells are researched, and a foundation is provided for the utilization of aromatic compounds.

Catechol (hereinafter referred to as CAT) is a white crystalline compound which is soluble in water, ethanol, diethyl ether, benzene, toluene, and chloroform, and is easily soluble in pyridine and an alkaline aqueous solution. Is an important chemical intermediate, and is mainly used for producing antioxidants, tanning agents, spices and the like. For example, in the aspect of pesticide production, the compound is used for synthesizing diethofencarb, propoxur, carbofuran and the like; in the aspect of medical synthesis, the medicine is used for preparing the cumin epinephrine, the berberine and the like. At present, the industrial production of catechol is mainly a chemical synthesis method, phenol is taken as a raw material, strong acid or hydrogen peroxide is taken as a catalyst, but the method has the factors of harsh reaction conditions, low conversion rate, more byproducts, complex components, complex product separation process, serious environmental pollution and the like.

It has been reported in the literature (Production of carbonate from Benzoate by the Wild Strain metabolites Ba-0323and charaterization of Its salts 1,2-Dioxygenase, Bioscience, Biotechnology, and Biochemistry,2014, 65:9,1957 and 1964) that Ralstonia sp.Ba-0323 can convert sodium Benzoate to form 1.9mg/ml Catechol.

catechol-O-sulfate is an aryl sulfate, which is catechol having one of the two hydroxyl groups substituted with a sulfo group. It is a member of the aryl sulfates and phenols. The catechol-O-sulfate salt may improve cardiac muscle cell beating and Ca2+ signaling in response to sustained stimulation of β -adrenergic receptors by promoting cardioprotection in the following manner.

Little has been reported about the biosynthesis of catechol-O-sulfate, and only ASTB-OM2 (aryl sulfotransferase B) catalyzes catechol to produce catechol-O-sulfate (Loop engineering of aryl sulfotransferase B for improving catalytic performance in a metabolic substrate, Catal. Sci. technol.,2020,10, 2369).

Hongbaolin (Purpurogenin) is a natural phenol extracted from nuts and oak bark, and has strong Xanthine Oxidase (XO) inhibitory activity, and IC of 0.2 μ M. The rhodophenol has antioxidant, anticancer and anti-inflammatory properties. Xanthine oxidase is the terminal enzyme of human purine catabolism, catalyzing the oxidation of hypoxanthine and xanthine. These reactions produce uric acid and active oxygen such as superoxide anion and hydrogen peroxide. It is well known that the onset of gout is due to an excessive accumulation of blood uric acid: this excessive accumulation is considered to be associated with eating habits, and therefore, although gout is a classic disease, it is now recognized as a lifestyle-related disease, and more patients are recently suffering from the disease. Cardiovascular disease is another lifestyle-related disease that is well known to be associated with oxidative stress caused by superoxide anions, hydrogen peroxide, nitric oxide and its metabolites (hydroxyl radicals, peroxynitrite, etc.). Therefore, inhibition of xanthine oxidase overworking is important for prevention of such lifestyle-related diseases. Repazol inhibits the production of proinflammatory cytokines by inhibiting the mRNA and protein expression of IL-1 β and TNF- α in LPS stimulated BV2 microglia. Erythrophenol exerts an anti-inflammatory effect by inhibiting LPS stimulated BV2 microglia phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways. Honegyphenol inhibits esophageal squamous cell carcinoma by directly targeting the mitogen-activated protein kinase 1/2(MEK1/2) signaling pathway. Therefore, the synthesis of the rhodophenol is of great significance. The reported synthesis of rhodophenol has focused on chemical methods.

France sco Ferlin et al report (heterologous Man-catalyst oxide C-H/C-catalysis to Access pharmaceutical Active Compounds, ChemCat Chem 2020,12, 449-454) as H₂O₂And O₂And 1mmol pyrogallol as a reaction reagent, Mn₈KO₁₆As a catalyst, 0.94mmol of red-ployphenol can be produced.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for synthesizing a high value-added compound by using expression optimization of multi-subunit enzyme for researching expression optimization of the multi-subunit enzyme.

In the invention, an RBS library of pE 1K-GcobA-M3 is constructed by a Golden gate cloning method, the RBS library of the library G/C is screened by researching the growth rate, the conversion rate, the expression ratio of enzyme, the enzyme activity of crude enzyme solution and the like of the library, the growth rate of a strain is high, and the enzyme activity is also high. Thereafter, the RBS library was used for enzymatic reaction pathway construction, catalyzing monolignol compounds guaiacol (guaiacol) and pyrogallol (pyrogallol) to produce high value-added compounds such as catechol, catechol-O-sulfate, and erythrophenol.

The purpose of the invention is realized by the following technical scheme:

the invention provides a method for synthesizing a high value-added compound by using a lignin monomer through expression optimization of a multi-subunit enzyme, wherein the high value-added compound is synthesized by using the lignin monomer compound as an initial raw material through a biocatalyst;

the catalyst is prepared by one of the following methods:

secondly, screening a gene GcobA (RBS: G/C) corresponding to the obtained biocatalyst and an aryl sulfotransferase mutant ASTB-OM2(Q191Y/Y218W/L225V) in the E.coli overexpression method I to obtain the biocatalyst;

coli by over-expressing laccase GoL3 in e.

Preferably, the catalyst prepared by Process one is E.coli (GcobA; RBS: G/C).

Preferably, the catalyst prepared by process two is E.coli (GcoaB-ASTB-OM 2).

Preferably, the catalyst produced by process three is e.coli (GoL 3).

Preferably, the method for obtaining the E.coli (GcoaB; RBS: G/C) is as follows: an RBS library of the multi-subunit enzyme GcoAB comprising GcoA and GcoB is constructed by the method of Golden gate, the GcoA and the GcoB are firstly transformed into BL21 to form pE1k-GcoAB-M3(RBS: N/N), and then the RBS library of the GcoAB is obtained by sequencing. Wherein, the biocatalyst E.coli (GcoaB; RBS: G/C) is obtained by screening. Screening the obtained GcobA; the gene sequence of RBS G/C is SEQ ID NO. 1.

The genes GcoA and GcoB are derived from Amycolatopsis sp ATCC 39116, and the genes GcoA and GcoB are codon optimized. The screening is to screen the best biocatalyst E.coli (GcobA; RBS: G/C) by comparing the protein expression, exponential phase growth rate, converted guaiacol rate and crude enzyme liquid enzyme activity of the RBS library of GcobA, wherein the catalyst has the advantages of fast growth, proper expression amount of the proteins GcobA and GcobB, fast conversion rate and high crude enzyme liquid enzyme activity.

Preferably, the ASTB-OM2(Q191Y/Y218W/L225V) is a mutant of an aryl sulfotransferase derived from Desutobacterium hafniens. ASTB-OM2(Q191Y/Y218W/L225V) refers to that the 191 th amino acid of ASTB-OM2 is mutated from Glutamine (Q) to Tyrosine (Y), the 218 th amino acid Tyrosine (Y) is mutated to tryptophan (W), and the 225 th Leucine (L) is mutated to Valine (V) compared with wild type ASTB. pB1c-ASTB-OM2 gene sequence SEQ ID NO. 2.

Preferably, the laccase GoL3 is derived from Gramella forsetii KT 0803. The gene sequence of pB1c-GoL3 is SEQ ID NO. 3.

Preferably, the sequence of the RBS core region of GcoA is AGGGGG, and the sequence of the RBS core region of GcoB is AGGCGG.

Preferably, the method for obtaining e.coli (GoL 3): plasmid pB1c-GoL3 was synthesized and subsequently achieved in e.coli BL21 by chemical transformation.

The biocatalyst is prepared by adopting an electrotransfer method, 2 mu L of plasmid is taken and mixed with 100 mu L of electrotransfer competence BL21, the mixture is transferred to an electric shock cup, pulse is carried out at 1700V, 900 mu L of LB culture medium is immediately added, the mixture is transferred to an EP tube, after the mixture is cultured for 1h at 37 ℃ and 180rpm/min, 200 mu L of bacterial liquid is taken and coated on an LB solid plate containing corresponding resistance, overnight culture is carried out at 37 ℃, the bacterial liquid is picked and monoclone is selected in an LB test tube containing corresponding antibiotic, and culture is carried out at 37 ℃ and 220rpm/min, thus obtaining the corresponding biocatalyst.

Preferably, the monolignol compound comprises guaiacol or 3-methoxy catechol.

Preferably, the high value-added compound comprises at least one of catechol, catechol-O-sulfate, and rhodol.

Preferably, when the biocatalyst is E.coli (GcobA; RBS: G/C), guaiacol is used as a starting material to synthesize catechol.

Preferably, when the biocatalyst is E.coli (GcobA-ASTB-OM 2), the catechol-O-sulfate is synthesized by using guaiacol as a starting material.

Preferably, when the biocatalyst is E.coli (GoL3), 3-methoxy catechol is used as a starting material to synthesize the rhodol.

Preferably, when the biocatalyst is E.coli (GoL3) and E.coli (GcoaB; RBS: G/C), 3-methoxy catechol is used as the starting material to synthesize the Hongbao phenol.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, a GcoAB library is constructed by a molecular cloning means, the expression of two components of GcoA and GcoB is optimized, an RBS combination with high conversion rate, high growth rate and good enzyme activity is obtained, an optimal biocatalyst is obtained, and the demethylation reaction of guaiacol and 3-methoxy catechol can be rapidly catalyzed.

(2) The invention utilizes the constructed biocatalyst to realize the high-efficiency utilization of aromatic compounds (guaiacol and 3-methoxy catechol) from lignin through biotransformation, and constructs the biosynthesis route of catechol-O-sulfate and rhodophenol for the first time.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic representation of the ribosome library (RBS library) of GcobA, growth rate and protein ratio; wherein a is a ribosome library of GcobA (RBS library) pE1 k-GcobA-M3 schematic diagram; b is a schematic diagram of catalyzing guaiacol to form catechol by GcoA and GcoB; c is SDS-PAGE pattern of protein expression of GcoA and GcoB in the ribosome library of GcoAB (RBS library); d is the molar ratio of GcoA and GcoB protein expression in the ribosome library of GcoAB (RBS library); e is thermodynamic diagram of the growth rate of the ribosome library of GcoAB (RBS library) in exponential phase, the left side refers to the case where no guaiacol is added, the right side to the case where guaiacol is added;

FIG. 2 shows the conversion rate and catechol synthesis of the ribosome library of GcobA (RBS library);

wherein a is the guaiacol conversion rate of the ribosome library of GcoAB (RBS library); b is the enzyme activity of the crude enzyme liquid of ribosome library (RBS library) of GcobA; c is catalyst E.coli (GcoaB; RBS: G/C) to convert guaiacol into catechol;

FIG. 3 shows the production of catechol by the multiple addition of catalyst E.coli (GcoaB; RBS: G/C) and guaiacol;

FIG. 4 is a chart of the catechol-O-sulfate synthesis results and mass spectra: a is a schematic diagram of E.coli (GcobA-ASTB-OM 2) catalyzing guaiacol to generate catechol-O-sulfate; coli (GcoAB-ASTB-OM2) catalyzes guaiacol to generate catechol-O-sulfate; mass spectrograms of the product and catechol-O-sulfate;

FIG. 5 shows the biocatalyst screening and optimum pH optimization with the conversion of pyrogallol to erythrophenol; wherein a is the activity of comparing 3 biocatalysts for production of red betaxol; coli (GoL3) catalyzes the conversion rate of pyrogallol to erythrophenol;

FIG. 6 is a schematic diagram of synthesis of rhodophenol, a result diagram and a mass spectrum diagram; wherein a is a schematic diagram of generation of the rhodophenol, and the biocatalysts E.coli (GcobA; RBS: G/C) and E.coli (GoL3) catalyze 3-methoxy catechol to generate the rhodophenol; coli (GcobA; RBS: G/C) catalyzes 3-methoxy catechol to generate pyrogallol; c is the production of the red betal; d, mass spectrograms of the sample and the red double phenol standard substance;

FIG. 7 is a general diagram showing the use of biocatalysts for the production of catechol, catechol-O-sulfate and Hongbiol. a is a schematic diagram of GcoA and GcoB catalyzing guaiacol to form catechol; coli (GcoAB-ASTB-OM2) catalyzes guaiacol to generate catechol-O-sulfate; c is a schematic diagram of the formation of the rhodophenol, and the biocatalysts E.coli (GcobA; RBS: G/C) and E.coli (GoL3) catalyze 3-methoxy catechol to form the rhodophenol.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments. The following examples, which are set forth to provide a detailed description of the invention and a detailed description of the operation, will help those skilled in the art to further understand the present invention. It should be noted that the scope of the present invention is not limited to the following embodiments, and that several modifications and improvements made on the premise of the idea of the present invention belong to the scope of the present invention.

The method of the following example includes:

step one, constructing a ribosome library (RBS library) of a multi-subunit enzyme GcobA, obtaining an E.coli (GcobA; RBS: G/C) biocatalyst with high conversion efficiency, high growth rate and high enzyme activity, and producing catechol by using guaiacol as a substrate;

coli (GcoAB-ASTB-OM2) as an efficient biocatalyst, and catechol-O-sulfate is successfully synthesized by taking guaiacol as a starting material and adding p-nitrophenyl sulfate as a sulfo donor;

step three, screening various laccase, modifying a biocatalyst E.coli (GoL3) obtained by escherichia coli for the first time, combining the E.coli (GcoaB; RBS: G/C) catalyst, and successfully synthesizing the rhodophenol by using 3-methoxy catechol as a raw material;

the obtaining method

Constructing a ribosome library (RBS library) of GcoA and GcoB by a Golden gate cloning method, and screening to obtain an optimal RBS combination for catalyzing guaiacol to produce catechol;

in the second step, the biocatalyst E.coli (GcobA-ASTB-OM 2) is constructed by over-expressing GcobA (RBS: G/C) and an aryl sulfotransferase mutant ASTB-OM2(Q191Y/Y218W/L225V) derived from DesF bacterium hafniens in E.coli, and is used for catalyzing guaiacol to synthesize catechol-O-sulfate;

in step three, the biocatalysts E.coli (GoL3), E.coli (Spr-CotA) and E.coli (Bsu-CotA) etc. were achieved by overexpressing in E.coli the laccase GoL3 derived from Gramela forsetii KT0803, the laccase Spr-CotA derived from Streptomyces pristinaespiralis ATCC25486, and the laccase Bsu-CotA derived from Bacillus subtilis. Then, through screening, the biocatalyst with laccase activity is obtained, and is further introduced into GcoaB (RBS: G/C) for the synthesis of the rhodophenol.

Example 1

(1) Coli (GcoaB; RBS: G/C) biocatalyst acquisition and catechol production (construction schematic diagram is shown in figure 1 and figure 2).

A ribosome library (RBS library) of GcoA and GcoB is constructed by adopting a Golden gate cloning method, transformed and introduced into BL21, and screened to obtain the E.coli (GcoAB; RBS: G/C) biocatalyst. The specific method comprises the following steps:

codon optimized GcoA and GcoB were synthesized from a company, ligated to pE1k-RFP by the Golden gate method, and transformed into BL21 to form pE1k-GcoAB-M3(RBS: N/N), wherein the pE1k-RFP gene sequence is SEQ ID NO.6, and N represents degenerate bases A, T, C, G. All RBS combinations of pE1 k-GcobA-M3 (RBS: N/N), namely RBS library of GcobA for short, are obtained through sequencing, the conversion efficiency, the growth rate and the enzyme activity of all RBS are compared, the optimal E.coli (pE1 k-GcobA-M3; RBS: G/C) biocatalyst is obtained, and the optimal biocatalyst E.coli (GcobA; RBS: G/C) is screened through comparing the protein expression condition, the exponential phase growth rate, the converted guaiacol rate and the crude enzyme liquid enzyme activity (figure 1 and figure 2) of the RBS library of the GcobA, wherein the optimal biocatalyst E.coli (GcobA; RBS: G/C) is obtained through comparison, the catalysts grow fast, the protein GcobA and the protein GcobB expression quantity are proper, the conversion rate is fast, and the crude enzyme liquid enzyme activity is high. The catalyst can produce catechol by adding the substrate guaiacol once or repeatedly.

(2) Biotransformation synthesis of catechol (the synthesis schematic diagram is shown in figure 2 and figure 3)

Inoculating the screened biocatalyst E.coli (pE1k-GcoaB-M3, RBS: G/C) into 3mL LB for activation, transferring the activated biocatalyst into 100mL LB according to the ratio of 1:100 after 12-16h at 37 ℃, adding 0.2mM IPTG inducer and cofactor 100 mg/L5 aminolevulinic acid and 200mg/L ammonium iron citrate (III) when OD600 reaches 0.6, inducing for 4h at 37 ℃, centrifuging at 8000rpm/min for 3min, collecting thalli, resuspending in M9Y culture medium to OD600 ═ 40.0, adding guaiacol 10mM, glucose 10G/L at final concentration, and converting for 1h at 37 ℃ to obtain 8.41mM catechol with the conversion rate of 84.1%. Or the centrifugally collected bacterial cells were resuspended at an OD of 10.0, and 18.0mM catechol was produced by adding the bacterial cells and 3mM guaiacol several times, with a conversion rate of 62.96%.

Example 2

(1) Method for synthesizing catechol-O-sulfate by constructing biocatalyst

BL21 was co-transformed with plasmid pB1C-ASTB-OM2 synthesized from Jinzhi and pE1 k-GcobA-M3 (RBS: G/C) obtained by screening in example 1 to obtain biocatalyst E.coli (GcobA-ASTB-OM 2).

(2) Biotransformation of catechol-O-sulfate (the synthetic scheme is shown in FIG. 4)

Inoculating a biocatalyst E.coli (GcoaB-ASTB-OM2) into 3mL LB for activation, after 12-16h at 37 ℃, transferring the activated biocatalyst into 100mL LB according to a ratio of 1:100, adding a final concentration of 0.2mM IPTG inducer, a cofactor of 100 mg/L5-aminolevulinic acid (GcoA) and 200mg/L ammonium iron citrate (III) when OD600 reaches 0.6, inducing at 25 ℃ for 4h, centrifuging at 8000rpm/min for 3min, collecting thalli, resuspending in an M9Y culture medium until OD is 40.0, adding a final concentration of guaiacol of 10mM, 10mM p-nitrophenyl sulfate, 10g/L glucose, and converting at 25 ℃ for 24h to obtain 2.21mM catechol-O-sulfate with a conversion rate of 22.1%.

Example 3

(1) Construction of biocatalyst for Synthesis of Red Diphenols (schematic for the Synthesis is shown in FIG. 5)

Plasmids pB1c-GoL3, pB1c-Spr-CotA, pB1c-Bsu-CotA and transformation of BL21 were synthesized to obtain biocatalysts E.coli (GoL3), E.coli (Spr-CotA) and E.coli (Bsu-CotA). The gene sequence of pB1c-Spr-CotA is SEQ ID NO. 4. The gene sequence of pB1c-Bsu-CotA is SEQ ID NO. 5. Biocatalysts E.coli (GoL3), E.coli (Spr-CotA) and E.coli (Bsu-CotA) were inoculated into 3ml of LB separately for activation at 37 deg.C12-16h later, the activated biocatalyst was transferred to 100mL LB5 at a ratio of 2:100, and when OD600 reached 0.5, 1mM IPTG inducer and 0.25mM CuCl were added to the final concentration₂After 4 hours of induction at 25 ℃ and 100rpm/min, the rotation speed was adjusted to 0, induction was carried out for 20 hours, the cells were collected, resuspended in M9Y medium until the OD became 40.0, pyrogallol was added at a final concentration of 2.5mM, and the cells were transformed at 37 ℃ for 0.5 hours, and only e.coli (GoL3) had the activity of catalyzing the production of pyrogallol into rhodotriphenol, with a transformation rate of 10.6%.

(2) Biotransformation to synthesize rhodophenol (the synthesis scheme is shown in FIGS. 6 and 7)

Inoculating a biocatalyst E.coli (GoL3) into 3mL LB for activation, transferring the activated biocatalyst into 100mL LB5 according to a ratio of 2:100 after 12-16h at 37 ℃, adding a final concentration of 1mM IPTG inducer and 0.25mM Cucl2 when OD600 reaches 0.5, inducing for 4h at 25 ℃ at 100rpm/min, regulating the rotation speed to 0, inducing for 20h, centrifuging for 3min at 8000rpm/min, collecting thalli, and resuspending in an M9Y culture medium until OD is 40.0.

Inoculating the screened biocatalyst E.coli (GcoaB, RBS: G/C) into 3ml LB to activate, after 12-16h at 37 ℃, the activated biocatalyst was transferred to 100mL LB at a ratio of 1:100, and when OD600 reached 0.6, adding 5-aminolevulinic acid (GcoA) and ammonium iron citrate (III) with a final concentration of 0.2mM IPTG inducer and cofactors of 100mg/L and 200mg/L, inducing for 4h at 37 ℃, the cells were centrifuged at 8000rpm/min for 3min, collected, resuspended in M9Y medium until OD is 40.0 and 10g/L glucose, 5mM 3-methoxycatechol was added to the final concentration, and the cells were transformed at 37 ℃ for 1.5h, after which the centrifuged cells were added with the above-mentioned biocatalyst E.coli (GoL3) whose volume OD is 40.0, the transformation rate was 100%, and 0.35mM rhodophenol was produced at the highest yield of 14%.

The invention has many applications, and the above description is only a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made without departing from the principles of the invention and these modifications are to be considered within the scope of the invention.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

<110> Shanghai university of transportation

<120> expression optimization of multi-subunit enzyme for synthesizing high value-added compound by using lignin monomer

<130> KAG47745

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5805

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gacgtcgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa 60

gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat 120

gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg 180

aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg 240

gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc 300

ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc 360

agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac 420

aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat 480

gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac 540

cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag 600

catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc 660

tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg 720

atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg 780

ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc 840

gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga 900

tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat 960

tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg 1020

gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc 1080

aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 1140

gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt 1200

gatctggttt gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca 1260

gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct 1320

caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa 1380

atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg tggaattgtg 1440

agcggataac aatttcagaa ttcaaaagat ctacaaacgt cttattaagg nggattttca 1500

tgaccaccac cgaacgccct gacctggcat ggctggatga agtgaccatg acccagctgg 1560

aacgtaatcc gtacgaagtt tacgaacgcc tgcgtgccga agcccctctg gcattcgttc 1620

ctgtcctggg aagctatgtt gctagtacgg ccgaagtgtg tcgcgaagtt gcaaccagcc 1680

ctgattttga ggcagttatt acccctgccg gtggtcgtac ctttggtcat cctgcaatta 1740

ttggtgttaa tggggatatt catgcagatt tacgtagtat ggttgagccg gcactgcagc 1800

cggctgaagt agatcgttgg attgatgatc tggttcgtcc gattgcccgt cgttatctgg 1860

aacgttttga gaatgatggt catgcagagc tggttgcgca gtattgtgaa cctgtttcag 1920

ttcgtagtct gggtgatctg ctgggtctgc aggaagttga cagcgataaa ctgcgtgaat 1980

ggtttgcaaa actgaatcgt agttttacta atgcagcagt tgatgagaat ggtgaatttg 2040

caaatcctga ggggtttgct gaaggtgatc aggcaaaagc agaaattcgt gcagtggttg 2100

atccgctgat tgacaaatgg attgaacacc cggatgatag cgcaatcagt cattggctgc 2160

acgatggaat gccgccgggt caaacacgtg atcgcgaata tatttatccg acgatttatg 2220

tttatctgct gggcgccatg caggaacctg gtcatggaat ggcaagcacc ctggttggtc 2280

tgtttagccg tcctgagcag ctggaagaag ttgtggatga cccgaccctg attccgcgtg 2340

ctattgctga aggactgcgt tggaccagtc cgatttggag tgcaaccgca cgtatttcta 2400

ccaaacctgt taccattgca ggtgttgatc tgccggcagg tacccctgtt atgctgagtt 2460

acggtagtgc aaatcatgat accggtaaat acgaagcacc gagccagtac gacctgcatc 2520

gccctcctct gcctcacctg gcatttggtg caggtaatca cgcatgtgcc ggtatttatt 2580

ttgcgaatca tgttatgcgc attgccctgg aagaactgtt cgaagccatt cctaatctgg 2640

aacgtgatac ccgcgaaggt gttgaatttt gggggtgggg ttttcgtggg ccgacatctc 2700

tgcatgttac ctgggaagtt taacatcatc tataaaataa ttaattaatt aaggnggtat 2760

ttttatgacc tttgccgttt ccgttggggg ccgtcgcgtt gattgtgaac cgggtcagac 2820

cctgctggag gcatttctgc gtgggggtgt gtggatgccg aacagttgta accaggggac 2880

ctgtggcacc tgtaaactgc aggttctgtc aggtgaggtt gatcatggtg gggcaccgga 2940

agatacactg tctgccgaag aacgtgcctc tgggctggca ctggcttgtc aagcacgccc 3000

tctggcagat accgaagttc gttctaccgc agatgcgggt cgtgttacac atcctctgcg 3060

tgacctgacc gcaacagtgc tggaagttgc tgatattgct cgtgataccc gtcgtgtcct 3120

gctgggactg gcagaacctc tggcatttga agcaggtcaa tatgttgaac tggttgttcc 3180

gggtagcggt gcccgtcgtc aatattctct ggcgaatacc gcggatgaag ataaagttct 3240

ggaactgcat gtgcgtcgtg taccgggggg tgttgcaaca gatggttggc tgtttgatgg 3300

tctggcggca ggtgatcgtg ttgaagcaac cggcccactg ggagattttc atctgcctcc 3360

gcctgatgaa gatgatggtg gtccgatggt tctgattggt ggtggtaccg gtctggcacc 3420

tctggttggt attgcacgta ccgcactggc acgtcatccg agtcgtgaag ttctgctgta 3480

tcatggggtt cgtggtgcag cagatttata tgatctgggg cgttttgcag aaattgctga 3540

agaacatccg ggttttcgtt ttgttccggt tctgagcgat gaaccggacc ctgcttatcg 3600

tggtggtttt ccgacagacg catttgtgga ggatgttcct agtggtcgtg ggtggagcgg 3660

ttggctgtgt ggtcctcctg caatggttga agcaggggtg aaagcattta aacgccgtcg 3720

tatgagtccg cgtcgtattc atcgtgaaaa atttacccca gcaagttaag gatccaaact 3780

cgagtaagga tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt 3840

cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg 3900

ggtgggcctt tctgcgttta tacctagggc gttcggctgc ggcgagcggt atcagctcac 3960

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 4020

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 4080

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 4140

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 4200

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4260

ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4320

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4380

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 4440

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 4500

ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 4560

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 4620

gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 4680

tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgact 4740

agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc 4800

cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcg agctctcgaa 4860

ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 4920

tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 4980

tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 5040

ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 5100

tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 5160

agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg 5220

gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 5280

gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg 5340

gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag 5400

tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 5460

agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc 5520

ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag 5580

ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa 5640

cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca 5700

gatcatgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg 5760

cagggcttcc caaccttacc agagggcgcc ccagctggca attcc 5805

<210> 2

<211> 5772

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2