CN113462626B - Expression optimization of multi-subunit enzymes for monolignol synthesis of high value-added compounds - Google Patents

Abstract

The invention relates to a method for optimizing expression of multi-subunit enzyme and synthesizing high added value compound by using lignin monomer, which constructs RBS library of pE1K-GcoAB-M3 by Golden gate cloning method, screens RBS library of library G/C by researching growth rate, conversion rate and enzyme expression proportion of library, enzyme activity of crude enzyme liquid and the like, and has high strain growth rate and enzyme activity. The RBS library is then used in the construction of an enzymatic reaction pathway to catalyze the production of the monolignol compounds guaiacol (guaiacol) and pyrogallol (pyrogallol) to high value added compounds such as catechol, catechol-O-sulfate, and erythrosepolyl. The invention utilizes the constructed biocatalyst, realizes the high-efficiency utilization of aromatic compounds (guaiacol and 3-methoxy catechol) from lignin through biological conversion, and constructs the biosynthesis way of catechol-O-sulfate and erythrosepolyol for the first time.

Description

Expression optimization of multi-subunit enzymes for monolignol synthesis of high value-added compounds

Technical Field

The invention belongs to the technical field of microorganisms, and relates to expression optimization of multi-subunit enzyme for synthesizing high added value compounds from lignin monomers.

Background

In view of the gradual decrease of non-renewable resources such as petroleum, natural gas, etc., development and comprehensive utilization of renewable resources such as lignin are receiving attention. Lignin is a natural polymer compound, is widely present in higher plant cells, and is a biomass having a second natural content. Lignin is a natural aromatic polymer formed by connecting phenylpropane units through C-O-C and C-C bonds and performing enzymatic catalytic dehydrogenation polymerization and free radical polymerization. The prior belief is that lignin structural units mainly include: p-hydroxyphenyl propane (p-Hydroxyphenyl propane/H), guaiacyl propane (Guaiacyl/G) and syringyl propane (Syringyl/S). Lignin forms various aromatic compounds after thermochemical conversion and biological conversion, most of the compounds also have methyl groups, and intermediate metabolites such as catechol, protocatechuic acid, 3,4, 5-trihydroxybenzoic acid and the like are formed after microbial demethylation, so that the lignin can further enter central carbon metabolism and downstream high-added-value product generation, and therefore, the demethylation of lignin-derived monomer compounds is an important link for lignin utilization.

There have been many studies on the establishment of cell factories for the efficient use of lignin and the biorefinery of high added value compounds, such as in the fields of medicine, biofuel, food and chemical industry, etc., by modifying microorganisms. Genetic modification of microorganisms has been common, but in the genetic modification process, metabolic flow imbalance, growth retardation and low yield of target compounds are caused by modification of endogenous genes and insertion of exogenous genes. To address the imbalance of metabolic fluxes, speed strain optimization and improve the production of various high-value bio-based chemicals, multi-module metabolic engineering (multivariate modular metabolic engineering), module co-culture engineering (modular coculture engineering), and space-time and integrated genome technologies have been widely used. The above techniques focus on one or more metabolic pathways, focusing on the regulation of expression of multiple enzymes, while there is little focus on single-function multi-subunit enzymes.

The P450 protein is a multifunctional enzyme capable of catalyzing monomeric compounds derived from various lignin sources. O-demethylation consists of cytochrome P450 proteins (GcoA) of the CYP255A family and a three-domain constituent reductase (GcoB). The expression proportion and the enzyme activity in cells, the growth rate and the like of the multi-component O-demethylated enzyme double subunits GcoA and GcoB are researched, and a basis is provided for the utilization of aromatic compounds.

Catechol (Catchol, 1,2-benzenediol, hereinafter referred to as CAT) is also known as catechol, which is a white crystalline compound soluble in water, ethanol, diethyl ether, benzene, toluene, chloroform, and easily soluble in pyridine and alkaline aqueous solutions. Is an important chemical intermediate, and is mainly used for producing antioxidants, tanning agents, fragrances and the like. For example, in the production of pesticides, for the synthesis of diethofencarb, propoxur, furadan, etc.; in the aspect of medicine synthesis, the preparation method is used for preparing the cumeneepinephrine, berberine and the like. At present, the industrialized production of catechol mainly adopts a chemical synthesis method, phenol is used as a raw material, strong acid or hydrogen peroxide and the like are used as catalysts, but the method has the factors of harsh reaction conditions, low conversion rate, multiple byproducts, complex components, complex product separation process, serious environmental pollution and the like.

As reported in literature (Production of Catechol from Benzoate by the Wild Strain Ralstonia Species Ba-0323and Characterization of Its Catechol 1,2-Dioxygenase,Bioscience,Biotechnology,and Biochemistry,2014,65:9,1957-1964), ralstonia sp.Ba-0323 can convert sodium benzoate to 1.9mg/ml catechol.

Catechol-O-sulfate is an aryl sulfate, which is catechol with one of the two hydroxyl groups replaced by a sulfo group. It is a member of the group of aryl sulphates and phenols. catechol-O-sulfate may improve cardiac protection by improving the beating of cardiomyocytes and the response of ca2+ signals to sustained stimulation of the β -adrenergic receptor in the following manner.

The biosynthesis of Guan Ercha phenol-O-sulfate has been recently reported, and only ASTB-OM2 (arylsulfonyltransferase B) has been reported to enzymatically catalyze catechol to catechol-O-sulfate (Loop engineering of aryl sulfotransferase B for improving catalytic performance in regioselective sulfation,Catal.Sci.Technol.,2020,10,2369).

Red bisphenol (Purpurogallin) is a natural phenol extracted from nuts and oak bark, has strong xanthine oxidase (Xanthine Oxidase, XO) inhibitory activity, and has an IC of 0.2 μm. The red bisphenol has antioxidant, anticancer and antiinflammatory properties. Xanthine oxidase is a terminal enzyme of human purine catabolism, catalyzing the oxidation reaction of hypoxanthine and xanthine. These reactions produce uric acid and reactive oxygen species such as superoxide anions and hydrogen peroxide. It is well known that gout is caused by excessive accumulation of uric acid: this excessive accumulation is thought to be related to eating habits and thus, although gout is a classical disease, it is currently recognized as a lifestyle-related disease, and more patients with such diseases have recently grown. Cardiovascular disease is another lifestyle-related disease, well known to be associated with oxidative stress caused by superoxide anions, hydrogen peroxide, nitric oxide and its metabolites (hydroxyl radicals, peroxynitrite, etc.). Thus, inhibition of the excessive work of xanthine oxidase is important for the prevention of such lifestyle-related diseases. Erythropolis inhibit the production of proinflammatory cytokines by inhibiting the mRNA and protein expression of IL-1 beta and TNF-alpha in LPS stimulated BV2 microglia. Erythropolins exert anti-inflammatory effects by inhibiting LPS-stimulated BV2 microglial phosphatidylinositol 3-kinase/Akt and mitogen activated protein kinase signaling pathways. Erythropolins inhibit esophageal squamous cell carcinoma by directly targeting mitogen-activated protein kinase 1/2 (MEK 1/2) signaling pathways. Therefore, the synthesis of erythrosepolyol is of great importance. The synthesis of the reported red-fold phenols is focused on chemical methods.

Francesco Ferlin et al report that (Heterogeneous Manganese-Catalyzed Oxidase C-H/C-OCyclization to Access Pharmaceutically Active Compounds,ChemCatChem 2020,12,449–454), produced 0.94mmol of rhodol with H ₂O₂ and O ₂ and 1mmol of pyrogallol as reactants and Mn ₈KO₁₆ as catalyst.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for synthesizing high added value compounds by using lignin monomers through expression optimization of multi-subunit enzyme in order to research the expression optimization of the multi-subunit enzyme.

The invention constructs RBS library of pE1K-GcoAB-M3 by Golden gate cloning, screens RBS library of library G/C by researching growth rate, conversion rate and enzyme expression proportion of library, enzyme activity of crude enzyme liquid and the like, and has high strain growth rate and high enzyme activity. The RBS library is then used in the construction of an enzymatic reaction pathway to catalyze the production of the monolignol compounds guaiacol (guaiacol) and pyrogallol (pyrogallol) to high value added compounds such as catechol, catechol-O-sulfate, and erythrosepolyl.

The invention aims at realizing the following technical scheme:

the invention provides a method for synthesizing a high-added-value compound by using a monolignol through expression optimization of a multi-subunit enzyme, which takes the monolignol compound as a starting material and synthesizes the high-added-value compound through a biocatalyst;

the catalyst is prepared by one of the following methods:

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

The second method is to obtain the biocatalyst by screening the gene GcoAB (RBS: G/C) and the aryl sulfotransferase mutant ASTB-OM2 (Q191Y/Y218W/L225V) corresponding to the biocatalyst obtained in the first E.coli over-expression method;

And thirdly, obtaining the biocatalyst by over-expressing laccase GoL in E.coli.

Preferably, the catalyst prepared by the first method is E.coli (GcoAB; RBS: G/C).

Preferably, the catalyst prepared by the second method is E.coli (GcoAB-ASTB-OM 2).

Preferably, the catalyst prepared by the third method is E.coli (GoL).

Preferably, the E.coli (GcoAB; RBS: G/C) is obtained by: RBS library containing GcoA and GcoB of the multi-subunit enzyme GcoAB was constructed by Golden gate method, gcoA and GcoB were first transformed into BL21 to form pE1k-GcoAB-M3 (RBS: N/N), and then the RBS library of GcoAB was obtained by sequencing. Wherein, the biocatalyst E.coli (GcoAB; RBS: G/C) is obtained by screening. Screening the obtained GcoAB; the gene sequence of RBS G/C is SEQ ID NO.1.

Genes GcoA and GcoB were derived from Amycolatopsis sp ATCC 39116 and genes GcoA and GcoB were codon optimized. The best biocatalyst E.coli (GcoAB; RBS: G/C) is selected by comparing the protein expression condition of GcoAB RBS library, the exponential phase growth rate, the guaiacol conversion rate and the crude enzyme liquid enzyme activity (figures 1 and 2), the catalyst grows fast, the protein GcoA and GcoB expression amounts are proper, the conversion rate is fast, and the crude enzyme liquid enzyme activity is high.

Preferably, the ASTB-OM2 (Q191Y/Y218W/L225V) is a mutant of an arylsulfonyl transferase derived from Desulfitobacterium hafniens. ASTB-OM2 (Q191Y/Y218W/L225V) refers to the mutation of amino acid at position 191 of ASTB-OM2 from Glutamine (Q) to Tyrosine (Y), the mutation of amino acid Tyrosine (Y) at position 218 to trytophan (W), and the mutation of Leucine (L) at position 225 to Valine (V) as compared with wild-type ASTB. The gene sequence of pB1c-ASTB-OM2 is SEQ ID NO.2.

Preferably, the laccase GoL is derived from Gramella forsetii KT0803. The gene sequence of pB1c-GoL3 SEQ ID NO.3.

Preferably, gcoA has an RBS core sequence of AGGGGG and a RBS core sequence of AGGCGG.

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

The biocatalyst is prepared by adopting an electrotransformation method, 2 mu L of plasmids are taken and mixed with 100 mu L of electrotransformation competent BL21, the mixture is transferred to an electric shock cup, 1700V is pulsed, 900 mu L of LB culture medium is immediately added and transferred to an EP tube, after 1h of culture at 37 ℃ at 180rpm/min, 200 mu L of bacterial liquid is taken and coated on an LB solid plate with corresponding resistance, the culture is carried out overnight at 37 ℃, and monoclonal is selected and carried out in an LB test tube with corresponding antibiotics, and the culture is carried out at 37 ℃ at 220rpm/min, thus obtaining the corresponding biocatalyst.

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

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

Preferably, when the biocatalyst is E.coli (GcoAB; RBS: G/C), catechol is synthesized starting material from guaiacol.

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

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

Preferably, the biocatalyst is E.coli (GoL) and E.coli (GcoAB; RBS: G/C) in combination, and 3-methoxy catechol is used as a starting material to synthesize the erythropolin.

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, and the expression of the bi-components GcoA and GcoB is optimized, so that the RBS combination with high conversion rate, high growth rate and good enzyme activity is obtained, the 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, realizes the high-efficiency utilization of aromatic compounds (guaiacol and 3-methoxy catechol) from lignin through biological conversion, and constructs the biosynthesis way of catechol-O-sulfate and erythrosepolyol for the first time.

Drawings

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

FIG. 1 is a schematic representation of GcoAB ribosomal library (RBS library), growth rate, and protein fraction; a is a ribosome library (RBS library) pE1k-GcoAB-M3 schematic representation of GcoAB; b is GcoA and GcoB, which catalyze guaiacol to form catechol schematic; c is a SDS-PAGE map of the ribosomal library (RBS library) of GcoAB, protein expression of GcoA and GcoB; d is the molar ratio of GcoA to GcoB protein expression in the GcoAB ribosomal library (RBS library); e is GcoAB, the left side refers to the case where no guaiacol is added, and the right side refers to the case where guaiacol is added;

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

Guaiacol conversion rate for a ribosomal library (RBS library) where a is GcoAB; b is GcoAB enzyme activity of ribosome library (RBS library) crude enzyme solution; c is a catalyst E.coli (GcoAB; RBS: G/C) for converting guaiacol to catechol;

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

fig. 4 is a graph of catechol-O-sulfate synthesis results and mass spectrum: a is a schematic diagram of E.coli (GcoAB-ASTB-OM 2) catalyzing guaiacol to generate catechol-O-sulfate; b is E.coli (GcoAB-ASTB-OM 2) catalyzing guaiacol to generate catechol-O-sulfate; c product and catechol-O-sulfate standard mass spectra;

FIG. 5 is a graph with biocatalyst screening and optimal pH optimization for conversion of pyrogallol to rhodol; wherein a is whether 3 biocatalysts have activity of producing the red bisphenol or not; b is the conversion rate of the biocatalyst E.coli (GoL) to the formation of the red-sesquiphenol by the catalysis of the pyrogallol;

FIG. 6 is a diagram of the synthesis of erythrosepolyol, a result chart and a mass spectrum; wherein a is a schematic diagram of the generation of the erythrosephenol, and the biocatalysts E.coli (GcoAB; RBS: G/C) and E.coli (GoL 3) catalyze 3-methoxy catechol to generate the erythrosephenol; b is a biological catalyst E.coli (GcoAB; RBS: G/C) for catalyzing 3-methoxy catechol to generate pyrogallol; c is production of red bisphenol; d, sample and red bisphenol standard quality spectrograms;

FIG. 7 is a general diagram showing the use of a biocatalyst for the production of catechol, catechol-O-sulfate and erythrosepolyol. a is GcoA and GcoB, which catalyze guaiacol to form catechol schematic; b is E.coli (GcoAB-ASTB-OM 2) catalyzing guaiacol to generate catechol-O-sulfate; and C is a schematic diagram of the formation of the erythrosephenol, and the biocatalysts E.coli (GcoAB; RBS: G/C) and E.coli (GoL) catalyze 3-methoxy catechol to form the erythrosephenol.

Detailed Description

The invention will now be described in detail with reference to the drawings and specific examples. The following examples, which are presented to provide those of ordinary skill in the art with a detailed description of the invention and to provide a further understanding of the invention, are presented in terms of implementation and operation. It should be noted that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made on the premise of the inventive concept are all within the protection scope of the present invention.

The method of the following embodiment includes:

step one, a ribosome library (RBS library) of the multi-subunit enzyme GcoAB is constructed, and an E.coli (GcoAB; RBS: G/C) biocatalyst with high conversion efficiency, high growth rate and high enzyme activity is obtained, and catechol is produced by taking guaiacol as a substrate;

Step two, modifying escherichia coli to obtain a high-efficiency biocatalyst E.coli (GcoAB-ASTB-OM 2), taking guaiacol as a starting material, adding p-nitrophenyl sulfate as a sulfo donor, and successfully synthesizing catechol-O-sulfate;

Step three, screening from various laccase, reforming a biocatalyst E.coli (GoL) obtained for the first time by escherichia coli and combining with an E.coli (GcoAB; RBS: G/C) catalyst, and successfully synthesizing the erythropolin by taking 3-methoxy catechol as a raw material;

the obtaining method

In the first step, the construction of the biocatalyst E.coli (GcoAB; RBS: G/C) constructs a ribosome library (RBS library) of GcoA and GcoB by a Golden gate cloning method, and the optimal RBS combination is obtained by screening and is used for catalyzing guaiacol to produce catechol;

in the second step, the construction of the biocatalyst E.coli (GcoAB-ASTB-OM 2) is realized by over-expressing GcoAB (RBS: G/C) and an arylsulfonyl transferase mutant ASTB-OM2 (Q191Y/Y218W/L225V) which is derived from Desulfitobacterium hafniens in the E.coli and is used for catalyzing guaiacol to synthesize catechol-O-sulfate;

In the third step, the biocatalysts E.coli (GoL 3), E.coli (Spr-CotA), E.coli (Bsu-CotA) and the like are realized by over-expressing laccase GoL3 derived from Gramella forsetii KT0803, laccase Spr-CotA derived from Streptomyces PRISTINAESPIRALIS ATCC25486 and laccase Bsu-CotA derived from Bacillus subtilis in E.coli. Then, a biocatalyst with laccase activity is obtained by screening, and GcoAB (RBS: G/C) is further introduced for the synthesis of the erythropolis.

Example 1

(1) E.acquisition of the coll (GcoAB; RBS: G/C) biocatalyst and production of catechol (construction schematic diagrams are shown in FIG. 1, FIG. 2).

A Golden gate clone method is adopted to construct GcoA and GcoB ribosome library (RBS library), the ribosome library is transformed and introduced into BL21, and E.coli (GcoAB; RBS: G/C) biocatalyst is obtained by screening. The specific method comprises the following steps:

From the company, codon optimized GcoA and GcoB were synthesized, ligated into 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 pE1k-GcoAB-M3 (RBS: N/N), namely RBS library of GcoAB for short, are obtained through sequencing, all RBS conversion efficiency, growth rate and enzyme activity are compared, the optimal E.coli (pE 1k-GcoAB-M3; RBS: G/C) biocatalyst is obtained, the protein expression condition of GcoAB RBS library, the exponential phase growth rate, the guaiacol conversion rate and the enzyme activity of crude enzyme solution are compared, and the optimal biocatalyst E.coli (GcoAB; RBS: G/C) is selected, wherein the catalyst grows fast, the protein GcoA and GcoB are suitable in expression quantity, the conversion rate is fast, and the enzyme activity of crude enzyme solution is high. The catalyst can be used for producing catechol by adding the substrate guaiacol once or multiple times.

(2) Bioconversion of catechol synthesis (synthetic scheme is shown in FIG. 2 and FIG. 3)

Inoculating the selected biocatalyst E.coli (pE 1k-GcoAB-M3, RBS: G/C) into 3mL LB for activation, transferring the activated biocatalyst into 100mL LB according to the proportion of 1:100 after 12-16h at 37 ℃, adding IPTG inducer with the final concentration of 0.2mM and cofactor of 100 mg/L5 aminolevulinic acid and 200mg/L ammonium ferric (III) when OD600 reaches 0.6, centrifuging at 8000rpm/min for 3min after 4h induction at 37 ℃, collecting thalli, re-suspending in M9Y culture medium until OD600 = 40.0, adding guaiacol with the final concentration of 10mM,10G/L glucose, and converting for 1h at 37 ℃, thus obtaining catechol with the final concentration of 8.41mM, wherein the conversion rate is 84.1%. Or the centrifugally collected bacteria were resuspended to od=10.0, and 18.0mM catechol was produced by adding the bacteria and 3mM guaiacol multiple times, with a conversion rate of 62.96%.

Example 2

(1) Construction of biocatalysts for the Synthesis of catechol-O-sulfate

Plasmid pB1C-ASTB-OM2 was synthesized from Jin Weizhi, and BL21 was transformed together with pE1k-GcoAB-M3 (RBS: G/C) obtained by the screening in example 1 to obtain biocatalyst E.coli (GcoAB-ASTB-OM 2).

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

Inoculating a biocatalyst E.coli (GcoAB-ASTB-OM 2) into 3mL LB for activation, transferring the activated biocatalyst into 100mL LB according to the proportion of 1:100 after 12-16 hours at 37 ℃, adding an IPTG inducer with the final concentration of 0.2mM and a cofactor of 100 mg/L5-aminolevulinic acid (GcoA) and 200mg/L ammonium ferric citrate (III) when the OD600 reaches 0.6, centrifuging for 3 minutes at 8000rpm after induction for 4 hours at 25 ℃, collecting thalli, re-suspending in an M9Y medium until the OD=40.0, adding guaiacol 10mM,10mM p-nitrophenyl sulfate, 10g/L glucose and converting for 24 hours at 25 ℃, and obtaining catechol-O-sulfate with the final concentration of 2.21mM, wherein the conversion rate is 22.1%.

Example 3

(1) Construction of biocatalyst for the Synthesis of Red bisphenol (synthetic scheme is shown in FIG. 5)

Plasmids pB1c-GoL3, pB1c-Spr-CotA, pB1c-Bsu-CotA were synthesized and BL21 was transformed to obtain biocatalysts E.coli (GoL), E.coli (Spr-CotA) and E.coli (Bsu-CotA). The pB1c-Spr-CotA gene sequence is SEQ ID NO.4. The gene sequence of pB1c-Bsu-CotA is SEQ ID NO.5. Biocatalysts E.coli (GoL), E.coli (Spr-CotA) and E.coli (Bsu-CotA) are respectively inoculated into 3ml LB for activation, after the temperature is 37 ℃ and the time is 12-16 hours, the activated biocatalysts are transferred into 100ml LB5 according to the proportion of 2:100, when the OD600 reaches 0.5, 1mM IPTG inducer and 0.25mM CuCl ₂ are added, after the induction is carried out for 4 hours at the temperature of 100rpm/min at the temperature of 25 ℃, the rotating speed is regulated to 0, the induction is carried out for 20 hours, bacterial cells are collected, the bacterial cells are resuspended in an M9Y culture medium until the OD=40.0, the final concentration of pyrogallol is added for 2.5mM, the pyrogallol is converted for 0.5 hours at the temperature of 37 ℃, and only E.coli (GoL 3) has the activity of catalyzing the pyrogallol to generate the red-bisphenol, and the conversion rate is 10.6%.

(2) Bioconversion of Red bisphenol (synthetic schematic diagrams are shown in FIGS. 6 and 7)

Inoculating a biocatalyst E.coli (GoL) into 3ml LB for activation, after 12-16 hours at 37 ℃, transferring the activated biocatalyst into 100ml LB5 according to a ratio of 2:100, when the OD600 reaches 0.5, adding a final concentration of 1mM IPTG inducer and 0.25mM Cucl2, after induction for 4 hours at 25 ℃, adjusting the rotating speed to 0, and after induction for 20 hours, centrifugation for 3 minutes at 800 rpm, collecting thalli, and re-suspending in M9Y culture medium until the OD=40.0.

Inoculating the selected biocatalyst E.coli (GcobB, RBS: G/C) into 3mL LB for activation, after the temperature is 37 ℃, transferring the activated biocatalyst into 100mL LB according to the proportion of 1:100 after 12-16 hours, adding an IPTG inducer with the final concentration of 0.2mM and a cofactor of 100 mg/L5-aminolevulinic acid (GcoA) and 200mg/L ammonium ferric citrate (III) when the OD600 reaches 0.6, centrifuging for 3 minutes at 37 ℃ after the induction of 4 hours at 8000rpm, collecting thalli, re-suspending in an M9Y culture medium until the OD=40.0, 10G/L glucose, adding 5mM 3-methoxy catechol with the final concentration, converting for 1.5 hours at 37 ℃, adding the obtained biocatalyst E.coli (GoL 3) with the final concentration of OD=40.0, centrifuging until the conversion rate is 100%, and the maximum yield is 14 percent.

There are many ways in which the invention may be practiced, and what has been described above is merely 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 modifications may be made without departing from the principles of the invention, and such modifications are intended to be within the scope of the invention.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Sequence listing

<110> Shanghai university of transportation

<120> Expression optimization of Multi-subunit enzyme for monolignol Synthesis of high value-added Compounds

<130> KAG47745

<160> 6

<170> SIPOSequenceListing 1.0

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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

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 (1)

1. A method for optimizing the expression of a multi-subunit enzyme for synthesizing a high-added-value compound by using a monolignol compound as a starting material, and synthesizing the high-added-value compound by using a biocatalyst;

the catalyst is prepared by one of the following methods:

The method comprises the following steps: constructing RBS library containing GcoA and GcoB multi-subunit enzyme GcoAB 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 second method is that the biocatalyst E.coli-GcoAB-ASTB-OM2 is obtained by screening the gene corresponding to the biocatalyst and the aryl sulfotransferase mutant ASTB-OM2 in the first method of over-expression in E.coli;

The method III, obtaining a biocatalyst E.coli-GoL3 by over-expressing laccase GoL3 in E.coli;

Screening the obtained GcoAB in the first method; the gene sequence of the RBS G/C is SEQ ID NO.1;

In the second method, the sequence of the plasmid pB1c-ASTB-OM2 gene used for over-expressing the aryl sulfotransferase mutant ASTB-OM2 is shown as SEQ ID NO. 2;

in the third method, the sequence of the plasmid pB1c-GoL3 gene used for over-expressing laccase GoL3 is shown as SEQ ID NO. 3;

The biocatalyst is E.coli-GcoAB; in the process of RBS G/C, guaiacol is taken as a starting material, and a synthesized high-added-value compound is catechol;

when the biocatalyst is E.coli-GcoAB-ASTB-OM 2, guaiacol is used as a starting material, and the synthesized high-added-value compound is catechol-O-sulfate;

The biocatalyst is E.coli-GoL and E.coli-GcoAB; when RBS/C is combined, 3-methoxy catechol is used as a starting material, and the synthesized high added value compound is red bisphenol.