CN113462626A - Expression optimization of multi-subunit enzyme for synthesizing high value-added compound by using lignin monomer - Google Patents

Abstract

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.

Description

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

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:

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;

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

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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

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 cttttaagaa ggagatatac atatgcgtac 1500

ctacctgaat accgaaaaac atctgattac cctgcaagct gaatctgaag aacgtttcct 1560

ggccgaactg cgtgctggta actacaccgc cgaatcaccg ctggttgtga aaaacccgta 1620

tattatcaat ccgctggcgg ccgttatttg ctttaatacg gatgaagaaa ccacggccga 1680

aattaccgtc aaaggcaaag caatcgaagg tgacctgtct cataccttcg cagctgcgaa 1740

agaacacgtt ctgccggtct atggcctgta cgatgactat gtgaacacgg tcgtgatcaa 1800

actgagtaat ggtaaaacca gcgaagtgaa aattgaagtg gaagaactga acgttaataa 1860

agccctgtac tgccgcacca cgccggaata cttcggcaaa gatttcatgc tgatctcaac 1920

cacgaccccg ctgatcgaat cggctcgtac ggcaggcttt gattacgcag gtgacctgcg 1980

ttggtgtatt accaacctgc agtcatggga tatcaaaaaa ctggaaaatg gtcgcctgct 2040

gtatacgtcg catcgtaccg tgtataaacc gtattacaac gtgggcgtta tggaaatgga 2100

tttctgtggt aaaatctaca aagaataccg tctgccgggc ggttggcatc acgacgcggt 2160

tgaagtggaa aacggcaata ttctggccgc aagtgataac gactttaatg attccgtgga 2220

agacttcgtt gtcgaaattg aacgcgccac cggcgcagtt atcaaaagtt gggatctgca 2280

gaaaattctg ccgcgcggcc agggtaaagc tggtgattgg aaccatcacg actggtttca 2340

taacaatgcg gtgtggtacg ataaaccgac gaatagcatc accatgtctg gccgccacat 2400

ggacgctgtt attaacttcg attatgacag cggtgcgctg aattggatcc tgggcgatcc 2460

ggaaggttgg tctgaagaat ggcagaaata ctttttcaaa aacgtgacca aaggcgattt 2520

tgactggcag tatgaacaac atgctgcgcg tattctgccg aatggcgatg tttttctgtt 2580

cgacaacggc acgtatcgca gtaaaaatga agctacccgt gtggatccgg aacagaattt 2640

ttcccgcggt gttatttacc gtatcgatac cgacaaaatg gaaatcgaac aagtgtggca 2700

atatggcaaa gaacgcggtg ccgaattcta cagcccgtat atctgcaacg tcgattatta 2760

cggcgaaggt cattacatgg tgcactctgg cggtattgcc acgtatcgtg gcaaacacac 2820

cgatggcctg ggtgcaatgc tgctgaacaa atacaaagac gaacatatcc acctgacgct 2880

ggaatcaatc accgtcgaag tgcagaacga tcaactgaaa tacgaactga aagtgcaggg 2940

cggtaattat taccgcgcac gtcgcgtttc gccgtatgat gaaaaaacca acctggtcct 3000

gggcaaaggt gaactgctgg gcggttttgg tgttacgccg gaatttatga aagtcaattt 3060

caaagatgcg gaaaccgaac tgagcgaaaa acataacctg aatgtcatcc tggaagaaga 3120

ccgtctggct attcgcgcgt catttcgtga aggctcgcag gttttcctgg aactgaaggg 3180

tgcggaacaa agtaaatttt ataacattcc gacggaagtg cacgatgtta ccgccgcatg 3240

tgtctccttc gaagaacaga acgataatga ctttcaattc tatgtgagcc gtgaaggcct 3300

gtctggtgaa ttcggcatct acctgaacat tgatagcaaa cgctacgata cgcatctgtc 3360

tgtgaaactg gagctctctg catggagcca tccgcagttc gaaaagtaag gatccaaact 3420

cgagtaagga tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt 3480

cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg 3540

ggtgggcctt tctgcgttta tacctaggct acagccgata gtctggaaca gcgcacttac 3600

gggttgctgc gcaacccaag tgctaccggc gcggcagcgt gacccgtgtc ggcggctcca 3660

acggctcgcc atcgtccaga aaacacggct catcgggcat cggcaggcgc tgctgcccgc 3720

gccgttccca ttcctccgtt tcggtcaagg ctggcaggtc tggttccatg cccggaatgc 3780

cgggctggct gggcggctcc tcgccggggc cggtcggtag ttgctgctcg cccggataca 3840

gggtcgggat gcggcgcagg tcgccatgcc ccaacagcga ttcgtcctgg tcgtcgtgat 3900

caaccaccac ggcggcactg aacaccgaca ggcgcaactg gtcgcggggc tggccccacg 3960

ccacgcggtc attgaccacg taggccaaca cggtgccggg gccgttgagc ttcacgacgg 4020

agatccagcg ctcggccacc aagtccttga ctgcgtattg gaccgtccgc aaagaacgtc 4080

cgatgagctt ggaaagtgtc ttctggctga ccaccacggc gttctggtgg cccatctgcg 4140

ccacgaggtg atgcagcagc attgccgccg tgggtttcct cgcaataagc ccggcccacg 4200

cctcatgcgc tttgcgttcc gtttgcaccc agtgaccggg cttgttcttg gcttgaatgc 4260

cgatttctct ggactgcgtg gccatgctta tctccatgcg gtaggggtgc cgcacggttg 4320

cggcaccatg cgcaatcagc tgcaactttt cggcagcgcg acaacaatta tgcgttgcgt 4380

aaaagtggca gtcaattaca gattttcttt aacctacgca atgagctatt gcggggggtg 4440

ccgcaatgag ctgttgcgta cccccctttt ttaagttgtt gatttttaag tctttcgcat 4500

ttcgccctat atctagttct ttggtgccca aagaagggca cccctgcggg gttcccccac 4560

gccttcggcg cggctccccc tccggcaaaa agtggcccct ccggggcttg ttgatcgact 4620

gcgcggcctt cggccttgcc caaggtggcg ctgccccctt ggaacccccg cactcgccgc 4680

cgtgaggctc ggggggcagg cgggcgggct tcgcccttcg actgccccca ctcgcatagg 4740

cttgggtcgt tccaggcgcg tcaaggccaa gccgctgcgc ggtcgctgcg cgagccttga 4800

cccgccttcc acttggtgtc caaccggcaa gcgaagcgcg caggccgcag gccggaggca 4860

ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa 4920

tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag cgagctcgat 4980

atcaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct 5040

gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac 5100

cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat 5160

attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa 5220

catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc 5280

ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga 5340

aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac 5400

cagctcaccg tctttcattg ccatacgaaa ttccggatga gcattcatca ggcgggcaag 5460

aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc 5520

cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc 5580

aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt 5640

ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag 5700

tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt 5760

tcgccagata tc 5772

<210> 3

<211> 5439

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

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 cttttaagaa ggagatatac atatggatac 1500

gagcacccgc catccggaaa ccggcaacaa aatgaacaaa gattttattg cggatctgga 1560

tattcagctg accgcgagcc cgagtcagac caacattttt ccggataaaa gcaccaacac 1620

ctatagctat aaagcgagca ttattaaagg cagcgaagat aacctgcaga acattgaagg 1680

cagctatctg ggcccggtgc tgcgcgtgaa aaaaggcgat aaagtgcgcg tgcgctatga 1740

aaatcagatt ccggcggaaa gcattgtgca ttggcatggc ctgcatgtga gccatgaaaa 1800

cgatggccat ccggcgcatg tgattggcga aggcgatacc tattactatg aatttgaagt 1860

gatgaaccgc gcgggcacct attggtttca tccgcatccg catcgccata ccggcgaaca 1920

agtgtatcaa ggcctggcgg gcctgtttat tgtgagcgat aaagaagagg aaaaactgaa 1980

cctgccgcaa ggcgaatatg atattccggt ggtgattcaa gatcgcacct ttgatgataa 2040

caaacagctg cagtatctgg gcgatggtca gatggatcgc atgcaaggct ttctgggcga 2100

acagattctg attaacggca aaattgataa caccctggaa ctgggcgcga acggcaaata 2160

tcgcctgcgc ctgctgaacg gcagcaacag ccgcgcgtat aaactggcgt gggatcatgg 2220

cgaagcgatt accgtgctgg gcgtggatgg cggcctgctg aaagcgccga aacgcatgcc 2280

gtatctgatg ctgggcccgg cgcagcgcgt ggatatttgg ctggatctga gtcagcaagt 2340

ggaaaacagc cgcattaaac tggtgcatct gccgattagc ctggatatga tgggcggtgg 2400

tatgatgaac ggcggcatga tgggcaacag taacagcaac catctgccgt atgatacgca 2460

gtttgatatt atggaaatta acgtgggcgc gagcgcggaa aacgatgcgc agctgccggg 2520

cgaactgagc agcctgaaca ccctggcggc gaccgatgcg attaacaaaa acaacccgcg 2580

cacctttacc tttgcgatgg gcggtatgat ggaatggacc attaacggcc atacctataa 2640

cggcaccgaa gtggcggaag aggaaaccgt gaaactggat accaccgaaa tttggcgcat 2700

taacaacggc agtcagttta gcagcgatcc ggatgacgat agcggcatga tgggtggcgg 2760

catgcatggc aacggcggca tgatgggcgg ccaaggcggc atgggcaaca tgatgcagat 2820

gccgcatccg gtgcatattc atcagctgca gtttaacatt ctgaaccgca acgcggataa 2880

agtggatgat aaactgtggg aagcgaccaa agatggcttt attaacgaag gccgccaaga 2940

tagcgtgtat ctgctgccgg gcatgcagat ggatctgatt atgcgctttg aagattttaa 3000

aggcctgttt ctgtatcatt gccataacct ggaacatgaa gatatgggca tgatgcgcaa 3060

ctttaaaatt gtgtaaggat ccaaactcga gtaaggatct ccaggcatca aataaaacga 3120

aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 3180

tactagagtc acactggctc accttcgggt gggcctttct gcgtttatac ctaggctaca 3240

gccgatagtc tggaacagcg cacttacggg ttgctgcgca acccaagtgc taccggcgcg 3300

gcagcgtgac ccgtgtcggc ggctccaacg gctcgccatc gtccagaaaa cacggctcat 3360

cgggcatcgg caggcgctgc tgcccgcgcc gttcccattc ctccgtttcg gtcaaggctg 3420

gcaggtctgg ttccatgccc ggaatgccgg gctggctggg cggctcctcg ccggggccgg 3480

tcggtagttg ctgctcgccc ggatacaggg tcgggatgcg gcgcaggtcg ccatgcccca 3540

acagcgattc gtcctggtcg tcgtgatcaa ccaccacggc ggcactgaac accgacaggc 3600

gcaactggtc gcggggctgg ccccacgcca cgcggtcatt gaccacgtag gccaacacgg 3660

tgccggggcc gttgagcttc acgacggaga tccagcgctc ggccaccaag tccttgactg 3720

cgtattggac cgtccgcaaa gaacgtccga tgagcttgga aagtgtcttc tggctgacca 3780

ccacggcgtt ctggtggccc atctgcgcca cgaggtgatg cagcagcatt gccgccgtgg 3840

gtttcctcgc aataagcccg gcccacgcct catgcgcttt gcgttccgtt tgcacccagt 3900

gaccgggctt gttcttggct tgaatgccga tttctctgga ctgcgtggcc atgcttatct 3960

ccatgcggta ggggtgccgc acggttgcgg caccatgcgc aatcagctgc aacttttcgg 4020

cagcgcgaca acaattatgc gttgcgtaaa agtggcagtc aattacagat tttctttaac 4080

ctacgcaatg agctattgcg gggggtgccg caatgagctg ttgcgtaccc ccctttttta 4140

agttgttgat ttttaagtct ttcgcatttc gccctatatc tagttctttg gtgcccaaag 4200

aagggcaccc ctgcggggtt cccccacgcc ttcggcgcgg ctccccctcc ggcaaaaagt 4260

ggcccctccg gggcttgttg atcgactgcg cggccttcgg ccttgcccaa ggtggcgctg 4320

cccccttgga acccccgcac tcgccgccgt gaggctcggg gggcaggcgg gcgggcttcg 4380

cccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca aggccaagcc 4440

gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa ccggcaagcg 4500

aagcgcgcag gccgcaggcc ggaggcacta gtgcttggat tctcaccaat aaaaaacgcc 4560

cggcggcaac cgagcgttct gaacaaatcc agatggagtt ctgaggtcat tactggatct 4620

atcaacagga gtccaagcga gctcgatatc aaattacgcc ccgccctgcc actcatcgca 4680

gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg 4740

aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggt 4800

gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact 4860

cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc 4920

caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc 4980

gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta 5040

acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca tacgaaattc 5100

cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt 5160

atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt 5220

acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc 5280

aacggtggta tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct 5340

cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc 5400

tcttacgtgc cgatcaacgt ctcattttcg ccagatatc 5439

<210> 4

<211> 4848

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

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 cttttaagaa ggagatatac atatggacag 1500

acggagcttc aaccggcgtc tcctcgcggg cggcgcggcc gcggcggcga caggcgtgac 1560

atcgttgtcg atcacttccg cctccaacgc ggcgccggcc ccggcgaagg gcgcgccccg 1620

caccgcgcag gccggcggtc aggtgcgcca cctcaagatg tacgccgaga aacgggccga 1680

cggatcgatg ggctacggcc tcgagaaggg caaggccacc gtccccgggc cgctgatcga 1740

actcgtcgag ggcgacacgc tgcacatcga gttcgagaac ctgatggacg tgccggtcag 1800

cctgcatccg cacggcgtcg actacgacat ctccaacgac ggcacgaaga tgagccgcag 1860

ccatgtcgag ccgggcgcca cccgcaccta cacctggcgc acccacgcac ccggccgccg 1920

cgcggacggc acctggcggc cgggcagcgc gggttactgg cactaccacg accatgtcgt 1980

cggcacggat cacggcaccg gcggcatccg caagggcctc tacggtccga tggtggtgcg 2040

caggaaggac gacatccttc ccgacaagca gttcaccatc gtcttcaacg acatgacgat 2100

caacaaccgg cccgcggccg acccgcccaa cttccttgcc acggtggggg accgggtcga 2160

gatcatcatg atcacgcacg gcgagtacta ccacacgttc catatgcacg gtcaccgctg 2220

ggcggacaac cggaccggcc tgctgtcggg tcccgaggac gtcagccggg tgatcgacaa 2280

caagatcacc ggcccggcgg actccttcgg cttccaggtg atcgcgggcg aacacgtggg 2340

cccgggcgcg tggatgtacc actgccacgt ccagagccac tcggacatgg gcatggccgg 2400

gctgttcctc gtcgccaagg aggacggcac gatccccggg tacgagccgc accatcccac 2460

gtccgaggag ggccacgacc actaaggatc caaactcgag taaggatctc caggcatcaa 2520

ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg 2580

aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatacc 2640

taggctacag ccgatagtct ggaacagcgc acttacgggt tgctgcgcaa cccaagtgct 2700

accggcgcgg cagcgtgacc cgtgtcggcg gctccaacgg ctcgccatcg tccagaaaac 2760

acggctcatc gggcatcggc aggcgctgct gcccgcgccg ttcccattcc tccgtttcgg 2820

tcaaggctgg caggtctggt tccatgcccg gaatgccggg ctggctgggc ggctcctcgc 2880

cggggccggt cggtagttgc tgctcgcccg gatacagggt cgggatgcgg cgcaggtcgc 2940

catgccccaa cagcgattcg tcctggtcgt cgtgatcaac caccacggcg gcactgaaca 3000

ccgacaggcg caactggtcg cggggctggc cccacgccac gcggtcattg accacgtagg 3060

ccaacacggt gccggggccg ttgagcttca cgacggagat ccagcgctcg gccaccaagt 3120

ccttgactgc gtattggacc gtccgcaaag aacgtccgat gagcttggaa agtgtcttct 3180

ggctgaccac cacggcgttc tggtggccca tctgcgccac gaggtgatgc agcagcattg 3240

ccgccgtggg tttcctcgca ataagcccgg cccacgcctc atgcgctttg cgttccgttt 3300

gcacccagtg accgggcttg ttcttggctt gaatgccgat ttctctggac tgcgtggcca 3360

tgcttatctc catgcggtag gggtgccgca cggttgcggc accatgcgca atcagctgca 3420

acttttcggc agcgcgacaa caattatgcg ttgcgtaaaa gtggcagtca attacagatt 3480

ttctttaacc tacgcaatga gctattgcgg ggggtgccgc aatgagctgt tgcgtacccc 3540

ccttttttaa gttgttgatt tttaagtctt tcgcatttcg ccctatatct agttctttgg 3600

tgcccaaaga agggcacccc tgcggggttc ccccacgcct tcggcgcggc tccccctccg 3660

gcaaaaagtg gcccctccgg ggcttgttga tcgactgcgc ggccttcggc cttgcccaag 3720

gtggcgctgc ccccttggaa cccccgcact cgccgccgtg aggctcgggg ggcaggcggg 3780

cgggcttcgc ccttcgactg cccccactcg cataggcttg ggtcgttcca ggcgcgtcaa 3840

ggccaagccg ctgcgcggtc gctgcgcgag ccttgacccg ccttccactt ggtgtccaac 3900

cggcaagcga agcgcgcagg ccgcaggccg gaggcactag tgcttggatt ctcaccaata 3960

aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt 4020

actggatcta tcaacaggag tccaagcgag ctcgatatca aattacgccc cgccctgcca 4080

ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacaaac 4140

ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt 4200

gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact 4260

ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg 4320

gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg 4380

ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa 4440

aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat 4500

acgaaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa 4560

cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg 4620

gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg 4680

ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc 4740

tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa 4800

gttggaacct cttacgtgcc gatcaacgtc tcattttcgc cagatatc 4848

<210> 5

<211> 5397

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

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 cttttaagaa ggagatatac atatgacact 1500

tgaaaaattt gtggatgctc tcccaatccc agatacacta aagccagtac agcaatcaaa 1560

agaaaaaaca tactacgaag tcaccatgga ggaatgcact catcagctcc atcgcgatct 1620

ccctccaacc cgcctgtggg gctacaacgg cttatttccg ggaccgacca ttgaggttaa 1680

aagaaatgaa aacgtatatg taaaatggat gaataacctt ccttccacgc atttccttcc 1740

gattgatcac accattcatc acagtgacag ccagcatgaa gagcccgagg taaagactgt 1800

tgttcattta cacggcggcg tcacgccaga tgatagtgac gggtatccgg aggcttggtt 1860

ttccaaagac tttgaacaaa caggacctta tttcaaaaga gaggtttatc attatccaaa 1920

ccagcagcgc ggggctatat tgtggtatca cgatcacgcc atggcgctca ccaggctaaa 1980

tgtctatgcc ggacttgtcg gtgcatatat cattcatgac ccaaaggaaa aacgcttaaa 2040

actgccttca gacgaatacg atgtgccgct tcttatcaca gaccgcacga tcaatgagga 2100

tggttctttg ttttatccga gcgcaccgga aaacccttct ccgtcactgc ctaatccttc 2160

aatcgttccg gctttttgcg gagaaaccat actcgtcaac gggaaggtat ggccatactt 2220

ggaagtcgag ccaaggaaat accgattccg tgtcatcaac gcctccaata caagaaccta 2280

taacctgtca ctcgataatg gcggagattt tattcagatt ggttcagatg gagggctcct 2340

gccgcgatct gttaaactga attctttcag ccttgcgcct gctgaacgtt acgatatcat 2400

cattgacttc acagcatatg aaggagaatc gatcattttg gcaaacagcg cgggctgcgg 2460

cggtgacgtc aatcctgaaa cagatgcgaa tatcatgcaa ttcagagtca caaaaccatt 2520

ggcacaaaaa gacgaaagca gaaagccgaa gtacctcgcc tcataccctt cggtacagca 2580

tgaaagaata caaaacatca gaacgttaaa actggcaggc acccaggacg aatacggcag 2640

acccgtcctt ctgcttaata acaaacgctg gcacgatccc gtcacagaaa caccaaaagt 2700

cggcacaact gaaatatggt ccattatcaa cccgacacgc ggaacacatc cgatccacct 2760

gcatctagtc tccttccgtg tattagaccg gcggccgttt gatatcgccc gttatcaaga 2820

aagcggggaa ttgtcctata ccggtccggc tgtcccgccg ccgccaagtg aaaagggctg 2880

gaaagacacc attcaagcgc atgcaggtga agtcctgaga atcgcggcga cattcggtcc 2940

gtacagcgga cgatacgtat ggcattgcca tattctagag catgaagact atgacatgat 3000

gagaccgatg gatataactg atccccataa ataaggatcc aaactcgagt aaggatctcc 3060

aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt 3120

ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc 3180

gtttatacct aggctacagc cgatagtctg gaacagcgca cttacgggtt gctgcgcaac 3240

ccaagtgcta ccggcgcggc agcgtgaccc gtgtcggcgg ctccaacggc tcgccatcgt 3300

ccagaaaaca cggctcatcg ggcatcggca ggcgctgctg cccgcgccgt tcccattcct 3360

ccgtttcggt caaggctggc aggtctggtt ccatgcccgg aatgccgggc tggctgggcg 3420

gctcctcgcc ggggccggtc ggtagttgct gctcgcccgg atacagggtc gggatgcggc 3480

gcaggtcgcc atgccccaac agcgattcgt cctggtcgtc gtgatcaacc accacggcgg 3540

cactgaacac cgacaggcgc aactggtcgc ggggctggcc ccacgccacg cggtcattga 3600

ccacgtaggc caacacggtg ccggggccgt tgagcttcac gacggagatc cagcgctcgg 3660

ccaccaagtc cttgactgcg tattggaccg tccgcaaaga acgtccgatg agcttggaaa 3720

gtgtcttctg gctgaccacc acggcgttct ggtggcccat ctgcgccacg aggtgatgca 3780

gcagcattgc cgccgtgggt ttcctcgcaa taagcccggc ccacgcctca tgcgctttgc 3840

gttccgtttg cacccagtga ccgggcttgt tcttggcttg aatgccgatt tctctggact 3900

gcgtggccat gcttatctcc atgcggtagg ggtgccgcac ggttgcggca ccatgcgcaa 3960

tcagctgcaa cttttcggca gcgcgacaac aattatgcgt tgcgtaaaag tggcagtcaa 4020

ttacagattt tctttaacct acgcaatgag ctattgcggg gggtgccgca atgagctgtt 4080

gcgtaccccc cttttttaag ttgttgattt ttaagtcttt cgcatttcgc cctatatcta 4140

gttctttggt gcccaaagaa gggcacccct gcggggttcc cccacgcctt cggcgcggct 4200

ccccctccgg caaaaagtgg cccctccggg gcttgttgat cgactgcgcg gccttcggcc 4260

ttgcccaagg tggcgctgcc cccttggaac ccccgcactc gccgccgtga ggctcggggg 4320

gcaggcgggc gggcttcgcc cttcgactgc ccccactcgc ataggcttgg gtcgttccag 4380

gcgcgtcaag gccaagccgc tgcgcggtcg ctgcgcgagc cttgacccgc cttccacttg 4440

gtgtccaacc ggcaagcgaa gcgcgcaggc cgcaggccgg aggcactagt gcttggattc 4500

tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct 4560

gaggtcatta ctggatctat caacaggagt ccaagcgagc tcgatatcaa attacgcccc 4620

gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc 4680

atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt 4740

ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa 4800

atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa 4860

ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg 4920

tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg 4980

ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt 5040

cattgccata cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc 5100

cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg 5160

aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg 5220

atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc 5280

cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt 5340

atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc agatatc 5397

<210> 6

<211> 4206

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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 cttttaagaa ggagatatac atatggcgag 1500

tagcgaagac gttatcaaag agttcatgcg tttcaaagtt cgtatggaag gttccgttaa 1560

cggtcacgag ttcgaaatcg aaggtgaagg tgaaggtcgt ccgtacgaag gtacccagac 1620

cgctaaactg aaagttacca aaggtggtcc gctgccgttc gcttgggaca tcctgtcccc 1680

gcagttccag tacggttcca aagcttacgt taaacacccg gctgacatcc cggactacct 1740

gaaactgtcc ttcccggaag gtttcaaatg ggaacgtgtt atgaacttcg aagacggtgg 1800

tgttgttacc gttacccagg actcctccct gcaagacggt gagttcatct acaaagttaa 1860

actgcgtggt accaacttcc cgtccgacgg tccggttatg cagaaaaaaa ccatgggttg 1920

ggaagcttcc accgaacgta tgtacccgga agacggtgct ctgaaaggtg aaatcaaaat 1980

gcgtctgaaa ctgaaagacg gtggtcacta cgacgctgaa gttaaaacca cctacatggc 2040

taaaaaaccg gttcagctgc cgggtgctta caaaaccgac atcaaactgg acatcacctc 2100

ccacaacgaa gactacacca tcgttgaaca gtacgaacgt gctgaaggtc gtcactccac 2160

cggtgcttaa ggatccaaac tcgagtaagg atctccaggc atcaaataaa acgaaaggct 2220

cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag 2280

agtcacactg gctcaccttc gggtgggcct ttctgcgttt atacctaggg cgttcggctg 2340

cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 2400

aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 2460

gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 2520

tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 2580

agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 2640

ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 2700

taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 2760

gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 2820

gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 2880

ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 2940

ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 3000

gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 3060

caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 3120

taagggattt tggtcatgac tagtgcttgg attctcacca ataaaaaacg cccggcggca 3180

accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag 3240

gagtccaagc gagctctcga accccagagt cccgctcaga agaactcgtc aagaaggcga 3300

tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca 3360

gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag 3420

cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc 3480

atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg 3540

cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga 3600

tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc 3660

gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca 3720

gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc 3780

acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg 3840

caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc 3900

agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg 3960

aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc 4020

tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat 4080

cctcatcctg tctcttgatc agatcatgat cccctgcgcc atcagatcct tggcggcaag 4140

aaagccatcc agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc 4200

aattcc 4206

Claims (11)

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.

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