CN113929787B

CN113929787B - Strategy for regulating and controlling biofilm to improve shikimic acid production performance

Info

Publication number: CN113929787B
Application number: CN202111255656.4A
Authority: CN
Inventors: 刘立明; 丁强; 陈修来; 高聪; 宋伟; 郭亮; 胡贵鹏; 刘佳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2023-08-25
Anticipated expiration: 2041-10-27
Also published as: CN113929787A

Abstract

The invention discloses a strategy for regulating and controlling biofilm to improve shikimic acid production performance, and belongs to the technical field of bioengineering. According to the invention, by means of molecular biology, a biological film self-assembly system is utilized to fix heterologous target proteins on a cell biological film so as to improve the contact area, improve the expression of the target proteins, effectively improve the utilization efficiency of substrate starch and improve the shikimic acid concentration in a fermentation system. The method has the advantages of simple design, few system elements, low strain growth load and no obvious influence on the growth condition and survival rate of the strain. The shikimic acid production strain constructed by the invention is used as a fermentation strain, starch is used as a substrate for fermentation production, and the shikimic acid yield and the production intensity are respectively improved to 47.5g/L and 0.66g/L/h, so that the method has good industrial application prospect.

Description

Strategy for regulating and controlling biofilm to improve shikimic acid production performance

Technical Field

The invention relates to a strategy for regulating and controlling biofilm to improve shikimic acid production performance, belonging to the technical field of bioengineering.

Background

Shikimic acid is an intermediate of antiviral and anticancer drugs, is slightly soluble in ethanol and diethyl ether, is almost insoluble in chloroform and benzene, and has sufficient raw material sources and can be regenerated. The shikimic acid production process is pollution-free, and the product can be synthesized by microorganism to realize circulation in the nature, so the shikimic acid is an ideal medical intermediate.

In native surface organelles, biofilms have been shown to act as extracellular enzyme self-assembly platforms to increase catalytic efficiency. For example, the rate of formation of trehalose is a limiting factor in the conversion of starch to maltose in large quantities using free amylase. To overcome this limitation, we performed fusion expression of the biofilm protein (CsgA) and amylase with Spy-catcher as a self-assembled system in E.coli to immobilize the β -amylase and starch. The current method for producing shikimic acid mainly comprises the following steps: (i) Knocking out the E.coli KPM1 SA1/pKPM-SA1pgiGenes, which allow the key enzymes in the shikimate synthesis pathway to obtain greater NADPH; (ii) Inactivation of phosphotransferase System genes ptsHIcrr, shikimate kinase I and IIaroKAndaroLpyruvate kinase IpykFTo inhibit downstream gene expression and enhance flux of upstream carbon metabolism; (iii) Overexpression of PTS and endogenous inositol transporter IolT1 and glucokinase to increase key precursors for shikimate production; (iv) Glucose-xylose co-substrates were used to isolate cell growth and biosynthesis of shikimate pathway chemicals. However, the reaction system has the problems of low content of final products and low substrate utilization efficiency.

The SpyTag/SpyCatcher system derived from CnaB2 domain has been widely used in the protein field, with the advantage that it spontaneously forms stable isopeptidic bonds under various conditions. CnaB2 isStreptcoccuspyogenesA domain isolated from fibronectin FbaB. CnaB2 can be split into two parts: spyTag (13 amino acid residues) and SpyCatcher (116 amino acid residues). Asp in SpyTag and Lys on Spycatcher can spontaneously react to generate covalent bonds under the conditions of various temperatures (4-37 ℃), pH (5-8) and buffers and the like. It was found that both SpyTag and SpyCatcher contain no cysteine residues, and that specific binding occurs whether SpyTag, spyCatcher is fused at the N-terminus, C-terminus, or in the middle of the protein.

Disclosure of Invention

In order to solve the above problems, the present invention improves the contact area by immobilizing a heterologous target protein on an extracellular biofilm, further improving the expression of the target protein and increasing the yield of the target product.

It is a first object of the present invention to provide a biofilm self-assembly control system consisting of fusion protein a and fusion protein B.

In one embodiment, the fusion protein a comprises an anchoring protein and SpyCatcher; the fusion protein B comprises a biological envelope protein and SpyTag.

In one embodiment, the anchoring protein comprises a green fluorescent protein or an amylase; the biofilm proteins include frizzled cilium fiber forming proteins.

In one embodiment, the amino acid sequence of the fusion protein A is shown as SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the fusion protein B is shown as SEQ ID NO. 3.

The second object of the invention is to provide a genetically engineered bacterium which expresses the above-mentioned biofilm self-assembly regulation system and shikimic acid synthesis pathway related genes.

In one embodiment, the shikimate synthesis pathway-related gene comprises a gene encoding DAHP (3-deoxy-D-arabino-hepulosonate-7-phosphate) synthasearoGGenes encoding 3-dehydroquinic acid synthasearoBAnd genes encoding transketolasetktA。

In one embodiment, the genearoGThe nucleotide sequence of (2) is shown as SEQ ID NO.4, and the genearoBThe nucleotide sequence of (2) is shown as SEQ ID NO.5, and the genetktAThe nucleotide sequence of (2) is shown as SEQ ID NO. 6.

In one embodiment, the biofilm self-assembly control system uses pSC101 as an expression vector.

In one embodiment, the genetically engineered bacterium hosts escherichia coli.

In one embodiment, the genetically engineered bacterium is a host of E.coli JM109 or E.coli MG 1655.

The third object of the invention is a method for producing shikimic acid by fermenting the genetically engineered bacteria.

In one embodiment of the invention, the conditions of shake flask fermentation are 35-38deg.C, 200-220 rpm, strain fermentation initial OD ₆₀₀ 0.04-0.1, illuminating for 45-50 h, fermenting for 70-75 h; or the fermentation condition of the fermentation tank is 35-38 ℃,480-530 rpm, the inoculum size is 5-10%, the liquid loading amount is 30-50%, the pH is 6.0-7.0, and the initial OD of the strain fermentation is achieved ₆₀₀ 0.04-0.3, ventilation of 1-2 vvm, illumination for 45-60 h, and fermentation of 70-100 h.

The invention also provides application of the biofilm self-assembly regulation system in preparation of target protein or improvement of target protein yield.

In one embodiment, the protein of interest comprises an enzymatic protein or a non-enzymatic protein.

The invention also provides application of the genetically engineered bacterium or the method for producing shikimic acid in preparing shikimic acid or shikimic acid derivatives.

The invention also provides application of the biofilm self-assembly regulation system or the genetically engineered bacterium or the method in the fields of biology, pharmacy, food or chemical industry.

The beneficial effects are that:

the invention provides a biofilm self-assembly regulation system, which can fix heterologous target proteins on extracellular biofilms to improve contact area, further improve target protein expression and increase target product yield. The method has the advantages of simple design, few system elements, low strain growth load and no obvious influence on the growth condition and survival rate of the strain.

The engineering strain is used for fermenting and producing shikimic acid in a fermentation tank by constructing the shikimic acid production engineering strain and introducing a biofilm regulation system, and the fermentation is 72-h, so that the shikimic acid yield in a reaction system is up to 47.5g/L, and the engineering strain has a good industrial application prospect.

Drawings

Fig. 1: regulating and controlling cell parameters of the biological envelope; a: congo red absorption peak, B: congo red binding amount, C: biofilm width, D: osmotic pressure, E: cell growth, F: cell viability, G: congo red staining and aliskiren blue congo red staining.

Fig. 2: a biofilm self-assembly system; a: biofilm self-assembly system, B: expression and extracellular secretion of biofilm self-assembly system, C: secretory expression of GFP fluorescent protein, D: congo red absorption peak.

Fig. 3: the production path and self-assembly regulation of shikimic acid; a: shikimic acid synthesis pathway, B: evaluation of the production performance of shikimic acid producing strain S1, C: color-changing ring, D: relative glucose concentration.

Fig. 4: variation of shikimic acid content in shake flasks and 3.6L fermenters; a: amylase concentration, B: shake flask culture shikimic acid concentration, C: the fermentation tank cultures shikimic acid concentration.

Detailed Description

Plasmids and strains referred to in the following examples:

plasmid construction was performed using classical molecular biology means.

The construction method of the plasmid PJ01-GAB-K-SBA comprises the following steps: the specific construction method is as follows: the PJ01-GAB-K-SBA is overexpressedaroG ^fbr 、tktAAndaroB ^opt a plasmid of the gene;aroG ^fbr namely DAHP (3-oxygen-D-arabino-hepulosonate-7-phosphate) synthetase mutant D146N with elimination of product feedback inhibition;aroB ^opt i.e.codon optimised with the first 15 amino acidsaroBAnd (3) a gene.

(1) Based on commercial Plasmid pTargetF (Addgene Plasmid # 62226), a T7Te terminator sequence was inserted after rrnB T1 terminator in a full Plasmid PCR manner to reduce leakage expression; in the same way, the sgRNA expression cassette is removed to obtain a plasmid PJ01 only containing the Pj23119 constitutive promoter and terminator;

(2) The genome of the escherichia coli MG1655 is used as a template to be amplified respectively to obtain the RBS containing B0034aroB ^opt 、 aroG ^fbr 、tktAFragments, willaroB ^opt The fragment adopts multiple fragments and one-step homologous weightGroup mode, inserted into the expression cassette of PJ01 to obtain PJ01-B plasmid, and the same procedure is followedaroG ^fbr 、tktAThe two fragments are inserted into an expression frame of PJ01 by adopting a multi-fragment one-step homologous recombination mode to obtain PJ01-GA plasmid;

(3) To be used forStreptococcus bovisNRIC 1535 as template, then amplifying to obtain amylase gene containing B0034RBS after codon optimization, inserting the fragment into PJ01 plasmid to obtain PJ01-SBA plasmid, utilizing enzyme cutting siteBglII and IISpeI double restriction plasmid PJ01-SBA;

(4) Adopting the mode of linking with tail enzymeBamHI+BglII+XbaI) Respectively assembling PJ01-GA, PJ01-B and PJ01-SBA plasmids to finally obtain plasmids PJ01-GAB-K-SBA; and assembling the PJ01-GA and the PJ01-B plasmids to finally obtain the plasmid PJ01-GAB-K.

Plasmid pETac-CsgA-SpyTag: the plasmid pETac (info: doi/10.1002/bit.26580) is used as a template, ecoRI and BamHI are used as cleavage sites, and a fusion fragment (the nucleotide sequence of which is shown as SEQ ID NO. 7) of the CsgA-SpyTag is connected through homologous recombination to form the plasmid pETac-CsgA-SpyTag.

Plasmid pETac-GFP-SpyCatcher: the plasmid pETac (info: doi/10.1002/bit.26580) is used as a template, ecoRI and BamHI are used as cleavage sites, and fusion fragments of GFP-SpyCatcher (the nucleotide sequence of which is shown as SEQ ID NO. 8) are connected through homologous recombination to form the plasmid pETac-GFP-SpyCatcher.

Plasmid pETac-SBA-SpyCatcher: the plasmid pETac (info: doi/10.1002/bit.26580) is used as a template, ecoRI and BamHI are used as cleavage sites, and fusion fragments (the nucleotide sequence of which is shown as SEQ ID NO. 9) of the SBA-SpyCatcher are connected through homologous recombination to form the plasmid pETac-SBA-SpyCatcher.

Plasmid pETac-CsgA-SpyTag/SBA-SpyCatcher: the plasmid pETac-SBA-SpyCatcher and the constructed pETac-CsgA-SpyTag are subjected to homologous recombination through single enzyme digestion of the AvRII, and a single plasmid system pETac-CsgA-SpyTag/SBA-SpyCatcher is assembled.

The following examples relate to media:

seed culture medium: LB medium, the ingredients comprise peptone 10 g/L, yeast powder 5g/L and sodium chloride 10 g/L.

Fermentation medium: the composition comprises standard fermentation medium (NBS medium, 1L) containing K ₂ HPO ₄ (7.5 g), ferric ammonium (III) citric acid (0.3 g), citric acid monohydrate (2.1 g), L-phenylalanine (0.7 g), L-tyrosine (0.7 g), L-tryptophan (0.35 g), concentrated H ₂ SO ₄ (1.2, mL). Concentrated ammonia is added before high pressure to adjust the pH of the fermentation medium to 7.0. The supplement is added immediately before fermentation is started, and is glucose, mgSO ₄ (0.24 g), parahydroxybenzoic acid (0.010 g), potassium paraaminobenzoate (0.010 g), 2, 3-dihydroxybenzoic acid (0.010 g), and trace minerals (NH) ₄ ) ₆ (Mo ₇ O ₂₄ )·4H ₂ O (0.0037 g)、ZnSO ₄ ·7H ₂ O (0.0029 g)、H ₃ BO ₃ (0.0247 g)、CuSO ₄ ·5H ₂ O (0.0025 g)、MnCl ₂ ·4H ₂ O (0.0158, g), methyl-alpha-d-glucopyranoside final concentration is 1 mM. For glucose and MgSO respectively ₄ And autoclaving the methyl-alpha-d-glucopyranoside solution, sterilizing the aromatic vitamin and trace mineral solution with a 0.22 μm membrane. Before addition of the fermentation medium, the pH was adjusted to 7 by KOH and sterilization was carried out by means of a 0.22 μm membrane. Defoamer (Sigma 204) was added as needed.

The following examples relate to detection methods:

the method for detecting the parameters of the biological envelope comprises the following steps: the measurement was performed using a fluorescence microscope (Nikon microscope, 80 i) and a transmission electron microscope, and the ambient temperature was controlled at 30 degrees.

And (3) preparing a fermentation sample: taking a fermentation liquor sample, centrifuging at 12000 rpm for 5 min, taking supernatant, diluting, filtering by a 0.22 mu m water-based membrane, and using the filtrate for liquid chromatography.

Determination of shikimic acid content: dai Angao A liquid chromatograph (equipped with UV-visible detector) using a Berle Aminex HPX-87H (300×7.8mm,9 μm) column with mobile phase of 0.005M H ₂ SO ₄ Filtering the mobile phase with 0.22 μm filter membrane, ultrasonically degassing at flow rate of 0.6 mL/min and column temperature of 35deg.C, and detecting with ultravioletDetection was performed at length 210 nm.

Determination of amylase enzyme activity: after culturing 12. 12 h, the mixture was centrifuged at 4℃for 15 min, and 200. Mu.L of the enzyme solution was collected. The enzyme activity of alpha-amylase was quantified by the 3, 5-dinitrosalicylic acid method using 200. Mu.L reaction solution, pH=5.5, containing 0.5. 0.5 wt% soluble starch in 20 mM sodium acetate buffer and 50. Mu.L enzyme solution. The pH of the reaction mixture was controlled with 5M NaOH solution. The enzymolysis reaction was terminated by adding a solution composed of 0.4M NaOH, 22 mM 3, 5-dinitrosalicylic acid and 1.1. 1.1M potassium sodium tartrate tetrahydrate and incubating for 5 min. Enzyme activity was then detected using DNS method.

Color-changing ring: the amylase can hydrolyze when meeting starch, iodine solution is added, the iodine solution reacts with the starch to form a color-changing ring, the larger the color-changing ring is, the higher the concentration of the amylase is, and the smaller the color-changing ring is, the lower the concentration of the amylase is. The principle is that the central cavity of the starch helix can just hold iodine molecules, and blue-black complex is formed by Van der Waals force.

Measurement of enzyme Activity index: starch and iodine solution are hydrolyzed by amylase to form a blue transparent ring, and the value of the enzyme activity index is obtained by dividing the diameter of the transparent ring by the diameter of a colony. Enzyme activity index as another way to characterize enzyme activity.

Congo red staining: cells were inoculated in YESCA or M63 minimal medium for 12 h, temperature 30℃and 180 rpm. Subsequently, 10. Mu.L of the culture was spotted on YESCA-CR or M63-CR plates with the addition of 25. Mu.g/mL CR and 5. Mu.g/mL Brilliant Blue G250, incubated at 30℃for 48 h. The plate was then imaged and biofilm-producing bacteria formed red colonies, while biofilm-producing cells formed white colonies.

EXAMPLE 1 evaluation of biofilm

Will encode a frizzled ciliated fiber-forming proteincsgA、csgB、csgC、csgD、csgEThe gene was added with B0034RBS by PCR at the ATG position in front of the gene. The PCR products were recovered and ligated with the vector pSC101 plasmid by cleavage to obtain recombinant plasmids pETac-CsgA, pETac-CsgB, pETac-CsgC, pETac-CsgD, pETac-CsgE, respectively.

The obtained recombinant plasmid pETac-CsgA, pETac-CsgB and pETacThe CsgC, pETac-CsgD and pETac-CsgE are respectively introduced into competent cellsE. coliIn JM109, a series of recombinant strains carrying the frizzled fiber gene were obtained.

The above strains were evaluated for biofilm parameters in LB medium using fluorescence microscopy and transmission electron scanning electron microscopy, and were tested by Congo red staining experiments.

As shown in figure 1 of the drawings,csgA、csgB、csgC、csgD、csgEthe genes all successfully induce the formation of the biological film of the escherichia coli MG 1655.

As shown in FIG. 1A, D, overexpressioncsgAThe congo red absorption peak and osmotic pressure of the recombinant strain of the gene were 43.47% and 20.46% lower, respectively, than the control strain, and the congo red binding capacity and biofilm diameter increased to 9.21 ng and 6.7 nm, respectively (fig. 1B-C).

Further, overexpression will occurcsgAThe recombinant strain of the gene was inoculated in LB medium, cultured at 37℃for 12 h, and the cell growth (OD ₆₀₀ ) And survival (CFU/mL), the results are shown in FIGS. 1E and 1F to be over-expressedcsgAThe formation of the gene-induced biofilm has no effect on the growth of the strain.

In addition, biofilms were characterized and identified by congo red staining, congo red-alisxin blue staining and transmission electron microscopy (fig. 1G). The above results show that,csgAgenes are effective targets for increasing congo red adsorption.

Example 2 biofilm self-assembly Regulation System

A biofilm self-assembly system for expressing GFP protein on the cell surface was successfully constructed in E.coli MG1655 by co-electrotransformation of the plasmid pETac-CsgA-SpyTag and the plasmid pETac-GFP-SpyCatcher (FIG. 2A).

The results show that: (i) Transmission electron microscopy showed the expression and extracellular secretion of the biofilm self-assembly system (fig. 2B); (ii) The fluorescent microscope observation shows that the surface of the escherichia coli has the secretion expression of GFP fluorescent protein (FIG. 2C); (iii) After biofilm self-assembly, the absorbance of congo red by e.coli decreased to 0.54 (fig. 2D).

Based on these results, it was demonstrated that the biofilm-based SpyTag/SpyCatcher protein self-assembly system can improve contact area by immobilizing heterologous proteins on extracellular biofilms.

EXAMPLE 3 shake flask of shikimic acid and detection of shikimic acid content in fermenter

The shikimic acid producing strain was shake-cultured in NBS medium 72 h to identify the content.

As shown in FIG. 3, a shikimic acid producing strain S1 is constructed, and the strain S1 overexpresses the shikimic acid synthesis pathwayaroB、aroGAndtktAgenes and amylase genes (fig. 3A). Then, the starch utilization by the engineering strain S1 was analyzed: (1) The amylase color-changing experiment shows that the color-changing circle of the engineering strain S1 is 68.35 percent larger than that of the wild type (figure 3B); (2) The enzyme activity index, amylase concentration and glucose concentration of the engineering strain S1 are respectively 0.7, 3.5U and 45 g/L (FIG. 3B, D); (3) The shikimic acid concentration and the production intensity of the engineering strain S1 were 10.24 g/L and 0.14 g/L/h, respectively (FIG. 3B).

The constructed vectors PJ01-GAB-K and pETac-CsgA-SpyTag/SBA-SpyCatcher for expressing shikimic acid synthesis path related genes are introduced into competent cells of a production strain MG1655 to obtain shikimic acid production strains S8 and S9 carrying PJ01-GAB-K and pETac-CsgA-SpyTag/SBA-SpyCatcher plasmids, and the shikimic acid production strains S8 and S9 are respectively shake-flask cultured under dark conditions or light conditions. As shown in FIG. 4A, the amylase and glucose concentrations were increased by 38.26% and 22.56% respectively in the fermentation culture under light conditions compared to the fermentation culture under dark conditions.

The shikimic acid production strains S8 and S9 were shake-cultured, and as shown in FIG. 4B, the shikimic acid concentration and production intensity of the strain S9 reached 14.65 g/L and 0.2 g/L/h under the illumination condition, which was 46.5% higher than that of the strain S8. The reaction system was expanded to a 3.6L fermenter and the shikimic acid yield and production intensity of E.coli S9 under light conditions were increased to 47.5g/L and 0.66g/L/h, respectively (FIG. 4C).

TABLE 1 Strain information

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

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165 170

<210> 4

<211> 1053

<212> DNA

<213> artificial sequence

<400> 4

atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60

gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120

aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180

tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240

gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300

acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360

gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420

gcaggtgagt ttctcaatat gatcacccca caatatctcg ctgacctgat gagctggggc 480

gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540

tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600

aatgccgccg gtgcgccgca ctgcttcctg tccgtaacga aatgggggca ttcggcgatt 660

gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720

tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780

caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840

gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900

gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960

ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020

ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053

<210> 5

<211> 1089

<212> DNA

<213> artificial sequence

<400> 5

atggagcgta ttgtcgttac tctcggggaa cgtagttacc caattaccat cgcatctggt 60

ttgtttaatg aaccagcttc attcttaccg ctgaaatcgg gcgagcaggt catgttggtc 120

accaacgaaa ccctggctcc tctgtatctc gataaggtcc gcggcgtact tgaacaggcg 180

ggtgttaacg tcgatagcgt tatcctccct gacggcgagc agtataaaag cctggctgta 240

ctcgataccg tctttacggc gttgttacaa aaaccgcatg gtcgcgatac tacgctggtg 300

gcgcttggcg gcggcgtagt gggcgatctg accggcttcg cggcggcgag ttatcagcgc 360

ggtgtccgtt tcattcaagt cccgacgacg ttactgtcgc aggtcgattc ctccgttggc 420

ggcaaaactg cggtcaacca tcccctcggt aaaaacatga ttggcgcgtt ctaccaacct 480

gcttcagtgg tggtggatct cgactgtctg aaaacgcttc ccccgcgtga gttagcgtcg 540

gggctggcag aagtcatcaa atacggcatt attcttgacg gtgcgttttt taactggctg 600

gaagagaatc tggatgcgtt gttgcgtctg gacggtccgg caatggcgta ctgtattcgc 660

cgttgttgtg aactgaaggc agaagttgtc gccgccgacg agcgcgaaac cgggttacgt 720

gctttactga atctgggaca cacctttggt catgccattg aagctgaaat ggggtatggc 780

aattggttac atggtgaagc ggtcgctgcg ggtatggtga tggcggcgcg gacgtcggaa 840

cgtctcgggc agtttagttc tgccgaaacg cagcgtatta taaccctgct caagcgggct 900

gggttaccgg tcaatgggcc gcgcgaaatg tccgcgcagg cgtatttacc gcatatgctg 960

cgtgacaaga aagtccttgc gggagagatg cgcttaattc ttccgttggc aattggtaag 1020

agtgaagttc gcagcggcgt ttcgcacgag cttgttctta acgccattgc cgattgtcaa 1080

tcagcgtaa 1089

<210> 6

<211> 1992

<212> DNA

<213> artificial sequence

<400> 6

atgtcctcac gtaaagagct tgccaatgct attcgtgcgc tgagcatgga cgcagtacag 60

aaagccaaat ccggtcaccc gggtgcccct atgggtatgg ctgacattgc cgaagtcctg 120

tggcgtgatt tcctgaaaca caacccgcag aatccgtcct gggctgaccg tgaccgcttc 180

gtgctgtcca acggccacgg ctccatgctg atctacagcc tgctgcacct caccggttac 240

gatctgccga tggaagaact gaaaaacttc cgtcagctgc actctaaaac tccgggtcac 300

ccggaagtgg gttacaccgc tggtgtggaa accaccaccg gtccgctggg tcagggtatt 360

gccaacgcag tcggtatggc gattgcagaa aaaacgctgg cggcgcagtt taaccgtccg 420

ggccacgaca ttgtcgacca ctacacctac gccttcatgg gcgacggctg catgatggaa 480

ggcatctccc acgaagtttg ctctctggcg ggtacgctga agctgggtaa actgattgca 540

ttctacgatg acaacggtat ttctatcgat ggtcacgttg aaggctggtt caccgacgac 600

accgcaatgc gtttcgaagc ttacggctgg cacgttattc gcgacatcga cggtcatgac 660

gcggcatcta tcaaacgcgc agtagaagaa gcgcgcgcag tgactgacaa accttccctg 720

ctgatgtgca aaaccatcat cggtttcggt tccccgaaca aagccggtac ccacgactcc 780

cacggtgcgc cgctgggcga cgctgaaatt gccctgaccc gcgaacaact gggctggaaa 840

tatgcgccgt tcgaaatccc gtctgaaatc tatgctcagt gggatgcgaa agaagcaggc 900

caggcgaaag aatccgcatg gaacgagaaa ttcgctgctt acgcgaaagc ttatccgcag 960

gaagccgctg aatttacccg ccgtatgaaa ggcgaaatgc cgtctgactt cgacgctaaa 1020

gcgaaagagt tcatcgctaa actgcaggct aatccggcga aaatcgccag ccgtaaagcg 1080

tctcagaatg ctatcgaagc gttcggtccg ctgttgccgg aattcctcgg cggttctgct 1140

gacctggcgc cgtctaacct gaccctgtgg tctggttcta aagcaatcaa cgaagatgct 1200

gcgggtaact acatccacta cggtgttcgc gagttcggta tgaccgcgat tgctaacggt 1260

atctccctgc acggtggctt cctgccgtac acctccacct tcctgatgtt cgtggaatac 1320

gcacgtaacg ccgtacgtat ggctgcgctg atgaaacagc gtcaggtgat ggtttacacc 1380

cacgactcca tcggtctggg cgaagacggc ccgactcacc agccggttga gcaggtcgct 1440

tctctgcgcg taaccccgaa catgtctaca tggcgtccgt gtgaccaggt tgaatccgcg 1500

gtcgcgtgga aatacggtgt tgagcgtcag gacggcccga ccgcactgat cctctcccgt 1560

cagaacctgg cgcagcagga acgaactgaa gagcaactgg caaacatcgc gcgcggtggt 1620

tatgtgctga aagactgcgc cggtcagccg gaactgattt tcatcgctac cggttcagaa 1680

gttgaactgg ctgttgctgc ctacgaaaaa ctgactgccg aaggcgtgaa agcgcgcgtg 1740

gtgtccatgc cgtctaccga cgcatttgac aagcaggatg ctgcttaccg tgaatccgta 1800

ctgccgaaag cggttactgc acgcgttgct gtagaagcgg gtattgctga ctactggtac 1860

aagtatgttg gcctgaacgg tgctatcgtc ggtatgacca ccttcggtga atctgctccg 1920

gcagagctgc tgtttgaaga gttcggcttc actgttgata acgttgttgc gaaagcaaaa 1980

gaactgctgt aa 1992

<210> 7

<211> 522

<212> DNA

<213> artificial sequence

<400> 7

atgaaacttt taaaagtagc agcaattgca gcaatcgtat tctccggtag cgctctggca 60

ggtgttgttc ctcagtacgg cggcggcggt aaccacggtg gtggcggtaa taatagcggc 120

ccaaattctg agctgaacat ttaccagtac ggtggcggta actctgcact tgctctgcaa 180

actgatgccc gtaactctga cttgactatt acccagcatg gcggcggtaa tggtgcagat 240

gttggtcagg gctcagatga cagctcaatc gatctgaccc aacgtggctt cggtaacagc 300

gctactcttg atcagtggaa cggcaaaaat tctgaaatga cggttaaaca gttcggtggt 360

ggcaacggtg ctgcagttga ccagactgca tctaactcct ccgtcaacgt gactcaggtt 420

ggctttggta acaacgcgac cgctcatcag tacggcggcg gcggcagcgg cggcggcggc 480

agcgcgcaca tcgttatggt cgatgcatat aaacccacca aa 522

<210> 8

<211> 993

<212> DNA

<213> artificial sequence

<400> 8

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggtc 720

gacgggagtg gtggcagcgg agatagtgct acccatatta aattctcaaa acgtgatgag 780

gacggccgtg agttagctgg tgcaactatg gagttgcgtg attcatctgg taaaactatt 840

agtacatgga tttcagatgg acatgtgaag gatttctacc tgtatccagg aaaatataca 900

tttgtcgaaa ccgcagcacc agacggttat gaggtagcaa ctgctattac ctttacagtt 960

aatgagcaag gtcaggttac tgtaaatggc taa 993

<210> 9

<211> 2502

<212> DNA

<213> artificial sequence

<400> 9

atgacctttc agaacaaagt gaacctgaaa aaaaaaatga aaaaaagcct gggcagcctg 60

ctgattctga ccgcgattgg cgcgggcggc ctggtgcaag tgaaagtggt gaacgcggac 120

gaacaagtta gcatgaaaga tggcaccatt ctgcatgcgt ggtgctggag ctttaacacc 180

attaaagata acatgcaagc gattaaagat gcgggctaca ctagcgtgca gacgagcccg 240

attaacaccg tggtggcggg cgaaggcggc aacaaaagcc tgaaaaactg gtattatcag 300

tatcagccga ccatttataa aattggcaac tatcagctgg gcaccgaaga agaatttaaa 360

gaaatgaacc gcgtggcgga tcagtatggc attaaaatta ttgtggatgc ggtgctgaac 420

cataccacga gcgattataa tcagattagc caagaaatta aaaacattcc gaactggacc 480

catggcaaca ccctgattag cgattggcat aaccgctatg atgtgacgca gaacgcgctg 540

ctgaccctgt atgattggaa cacgcagaac gaatatgtgc agcagtatct gctgaactat 600

ctgaaacaag cggtggcgga tggggcggat ggattccgct atgatgcggc gaaacatatt 660

gaactgccgg gcgaatatgg cagcaacttt tggaacgtga ttctgaacaa cggcagcgaa 720

tttcagtatg gcgaaattct gcaagatgat gtgagcaacg atgcgggcta tggcaaactg 780

atgagcatta ccgcgagcaa ctatggtcag aaaattcgca gcgcgctgaa agatcgccat 840

attagcgcgg gcaacctgat gaactatcaa gtgagcggcg tggatgcggc gaacctggtg 900

acctgggtgg aaagccatga taactatgcg aacgatgatc aagaaagcac ctggatgaac 960

gatagcgata ttcgcctggg ctgggcgatg attaccgcgc gcgcgaaagg caccccgctg 1020

ttttttagcc gcccggtggg cggcggcaac ggcacccgct ttccgggtca gagtcagatt 1080

ggcgatgcgg gcagcaacct gtataaagat gcgaccgtga ccgcggtgaa caaatttcat 1140

aacgcgatgg tgggcgaaag cgaatatctg cgcaatccgg gcggcgacga gcaagtagcg 1200

atgattgaac gcggcacaaa aggcgcagta atcgtgaacc tggtggatgg cgataagcag 1260

attaacagcg aaaccaacct ggcggatggc acctataccg ataaagtgag cggccgtcag 1320

tttaacgtga gcaacggccg cattaccggc agcgtgccga gccgcagcgc ggtggtgctg 1380

tatgatgatc aagcgagcca agcggcgcaa gtgagcgtgg atggctataa agaaggcgat 1440

aacagcatta gcaaagcgac cgaagtgacc ctgaaagcga aaaacgcgga tagcgcgacc 1500

tataaactgg gcaacggcca agaagtggcg tataaagatg gcgataaagt aacggtgggc 1560

gaaggcctgg aagcgggaca gagcaccacc ctgacgctga ccgcgaccgg tgcggacggt 1620

cagagtacca ccaaaacata tacctttacc atgaaagatc cgagtgcgga aaccaacatt 1680

tattttcaga acccggataa ctggagcgat gtgtatgcgt atatgtatag cgcgaaagat 1740

aacaaactgc tgggcgcgtg gcctgggacc aagatgacca aggaagcgag cggccgctat 1800

agcattaccg tgccggcgag ctatgcggaa gaaggcgtga aagtgatttt taccaacaac 1860

caaggcagtc agtatccgca gaacgaaggc tttgatttta aagcggaagg cctgtatagc 1920

aaagcgggcc tgatgccgga tgtgccggcg ggcaaaaccc gcgtgacctt tgataacccg 1980

ggcggctggg acagcgcgaa cgcgtatctg tattatggca acccggtgca gtatccgctg 2040

ggcgtgtggc cgggcactca aatgaccaaa gatgatgcgg gcaactttta tctggatctg 2100

ccggaagaat atgcggatgt gaacgcgaaa attattttta atcagccggg cacgagcaat 2160

cagtttccgt atagcgaagg ctttaacctg gtgaaaagcg gcaactataa caaagatggc 2220

ctgaaagtcg acgggagtgg tggcagcgga gatagtgcta cccatattaa attctcaaaa 2280

cgtgatgagg acggccgtga gttagctggt gcaactatgg agttgcgtga ttcatctggt 2340

aaaactatta gtacatggat ttcagatgga catgtgaagg atttctacct gtatccagga 2400

aaatatacat ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc 2460

tttacagtta atgagcaagg tcaggttact gtaaatggct aa 2502

Claims

1. A genetically engineered bacterium is characterized by expressing a biofilm self-assembly regulation system and shikimic acid synthesis pathway related genes; the shikimic acid synthesis pathway related genes comprise genes encoding DAHP synthetasearoGGenes encoding 3-dehydroquinic acid synthasearoBAnd genes encoding transketolasetktA；

The biofilm self-assembly regulation system consists of a fusion protein A with an amino acid sequence shown as SEQ ID NO.2 and a fusion protein B with an amino acid sequence shown as SEQ ID NO. 3;

the escherichia coli is taken as a host,

genearoGThe nucleotide sequence of (2) is shown as SEQ ID NO.4, and the genearoBThe nucleotide sequence of (2) is shown as SEQ ID NO.5, and the genetktAThe nucleotide sequence of (2) is shown as SEQ ID NO. 6.

2. A method for producing shikimic acid, characterized in that the genetically engineered bacterium of claim 1 is used for fermentation production of shikimic acid.

3. The method of claim 2, wherein the conditions of shake flask fermentation are 35-38deg.C, 200-220 rpm, and the initial OD of strain fermentation ₆₀₀ And (3) fermenting for 70-75 h by illuminating for 48-72 h with the light of 0.04-0.1.

4. The method according to claim 2, wherein the fermentation conditions in the fermenter are 35-38deg.C, 480-530 rpm, the inoculum size is 5-10%, the liquid loading is 30-50%, the pH is 6.0-7.0, and the initial OD of the strain fermentation is obtained ₆₀₀ 0.04-0.3, ventilation of 1-2 vvm, fermentation of 72-100 h.

5. The use of the genetically engineered bacterium of claim 1 in the production of shikimic acid.