CN114438005B - Construction method and application of recombinant bacterium for synthesizing indigo pigment - Google Patents

Construction method and application of recombinant bacterium for synthesizing indigo pigment Download PDF

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CN114438005B
CN114438005B CN202111576815.0A CN202111576815A CN114438005B CN 114438005 B CN114438005 B CN 114438005B CN 202111576815 A CN202111576815 A CN 202111576815A CN 114438005 B CN114438005 B CN 114438005B
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CN114438005A (en
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杨欣伟
柯崇榕
唐雅倩
向梦杰
黄建忠
陶勇
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Fujian Normal University
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Abstract

The invention discloses a construction method and application of recombinant bacteria for synthesizing indigo pigment, which are characterized in that coding genes of indigo synthetase, 4' -phosphopantetheinyl transferase and glutamine synthetase are introduced into mutant escherichia coli through a recombinant vector to construct recombinant bacteria for synthesizing indigo pigment; the recombinant bacteria can weaken the branch paths of glutamic acid and glutamine, and effectively improve the yield of indigo pigment and the conversion efficiency of glutamate. The recombinant bacteria constructed by the method provided by the invention are used for producing the indigo pigment, and have the advantages of low raw materials, mild reaction conditions of a bioconversion method, simple process, high production efficiency and the like, and have good industrial application prospects.

Description

Construction method and application of recombinant bacterium for synthesizing indigo pigment
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a construction method and application of recombinant bacteria for synthesizing indigo pigment.
Background
Indigo pigment is one of the oldest pigments, and is mainly applied to dyeing of blue jeans, jeans and the like; as a colorant, indigo and its derivatives are also widely used in industries such as foods and cosmetics, and at the same time, indigo and its derivatives can be used in pharmaceutical industries for treating some human diseases such as inflammation, bacterial infection, epilepsy, antitumor therapy, and chronic myeloleukemia. In recent years, the use of the blue pigment is becoming more and more widespread, and the blue pigment is also becoming a useful and sensitive index in biochemical research. The carbon-carbon double bond in the indigo structure is conjugated with carbonyl, so that the indigo structure becomes a powerful free radical scavenger, and plant pathogens can resist oxidative stress.
The traditional indigo synthesis is mainly extracted from plants such as sophora blue, woad, polygonum blue, indian blue and wood blue, and the yield of the indigo obtained by the method is extremely low. With the increasing demand, there is a strong need for a method of rapidly synthesizing blue pigment from blue pigment directly obtained from plants. In the 80 s of the 19 th century, a group of chemists, including bayer, of german chemists, tried to synthesize indigo by chemical methods, but the synthesis of blue pigment by chemical methods was gradually eliminated because of the huge environmental damage caused by the large amount of aniline, nitrobenzene, and other raw materials used in the chemical synthesis route. With the continuous development of biotechnology, the synthesis of indigo by microorganisms is a method of synthesis that has been emphasized by scientists.
Indigo (indigoid) is formed by the condensation of two molecules of L-glutamine catalyzed by indigo synthase, a Non-ribosomal polypeptide synthase (Non-ribosomal peptide synthase, NRPS). The indigo synthetases of different sources have high amino acid sequence homology, organized in a similar fashion to an assembly line, and all contain four domains of C (condensation) -A (adenylation) -Ox (oxidation) -T (sulfhydrylation) -TE (sulfhydrylation). Indigo synthetases also require a phosphopantetheinyl transferase to transfer 4' -phosphopantetheinyl diamine from coenzyme A to serine residues conserved in the thiol domain for activation. Chinese patent application No. CN109722401a discloses that the genes encoding the indigo synthase from Streptomyces lavendulae and the phosphopantetheinyl transferase from Bacillus subtilis are co-expressed in corynebacterium glutamicum and that after an additional amount of L-glutamine of 11.68g/L, an additional cultivation of 0.8mM IPTG is induced for 48h at 18 ℃ the indigo yield is 1.75g/L. The method for synthesizing the indigo pigment uses glutamine as a substrate, has simple reaction, can synthesize the product indigo pigment only by one-step reaction, but has higher substrate cost, is not suitable for industrial production, and has relatively lower yield and substrate conversion rate in the synthesis process.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a construction method and application of recombinant bacteria for synthesizing indigo pigment, and the constructed recombinant bacteria can catalyze L-glutamate to react, so that conversion is realized, and the yield of indigo and the conversion efficiency of glutamate are effectively improved.
The technical scheme of the invention is as follows:
the invention discloses a construction method for synthesizing indigo pigment recombinant bacteria, which comprises the steps of introducing coding genes of indigo synthetase, 4' -phosphopantetheinyl transferase and glutamine synthetase into a recipient bacteria through a recombinant vector to obtain bacteria for synthesizing indigo pigment;
the protein with the amino acid sequence of SEQ ID No.1 encoded by the indigo synthase gene or the derivative protein with the indigo synthase activity obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1;
the 4 '-phosphopantetheinyl transferase gene codes protein with an amino acid sequence of SEQ ID No.2 or derivative protein with 4' -phosphopantetheinyl transferase activity, which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2;
the glutamine synthetase gene codes protein with an amino acid sequence of SEQ ID No.3 or derivative protein with glutamine synthetase activity, which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 3;
the receptor bacteria are mutant escherichia coli or wild-type escherichia coli.
Further, the mutant escherichia coli is a mutant of the wild-type escherichia coli obtained by carrying out any one of the following gene modifications d1, d2, d3 and d4, any two gene modification combinations, any three gene modification combinations or four gene modification combinations on the wild-type escherichia coli;
d1, knocking out glutamate decarboxylase GadA;
d2, knocking out glutamate decarboxylase GadB;
d3, knocking out glutaminase GlsA;
d4, knocking out glutaminase GlsB;
thus, the mutant E.coli D1 is a mutant obtained by modifying the wild E.coli with D1, D2, D3 and D4 as described above; specifically, the escherichia coli mutant K12 delta gadA delta gadB delta glsA delta glsB is obtained by knocking out coding genes of glutamate decarboxylase GadA, glutamate decarboxylase GadB, glutaminase GlsA and glutaminase GlsB simultaneously;
the mutant escherichia coli D2 is a mutant of wild escherichia coli obtained by carrying out the D1 and D2 modification on the wild escherichia coli; specifically, the Escherichia coli mutant K12ΔgadA ΔgadB obtained by knocking out the coding genes of the glutamate decarboxylase GadA and the glutamate decarboxylase GadB simultaneously;
the mutant escherichia coli D3 is a mutant of wild escherichia coli obtained by modifying the wild escherichia coli with D3 and D4; specifically, colibacillus mutant K12ΔglsA ΔglsB with simultaneous knockout of glutaminase GlsA and glutaminase GlsB coding genes;
the mutant escherichia coli D4 is a mutant of the wild escherichia coli obtained by carrying out the D1 and D3 modification on the wild escherichia coli; specifically, a mutant K12ΔgadA ΔglsA of the escherichia coli, from which coding genes of glutamate decarboxylase GadA and glutaminase GlsA are knocked out simultaneously;
the mutant escherichia coli D5 is a mutant of the wild escherichia coli obtained by carrying out the D1 and D4 modification on the wild escherichia coli; specifically, colibacillus mutant K12ΔgadA ΔglsB obtained by knocking out coding genes of glutamate decarboxylase GadA and glutaminase GlsB simultaneously;
the mutant escherichia coli D4 is a mutant of the wild escherichia coli obtained by carrying out the D1 and D3 modification on the wild escherichia coli; specifically, a mutant K12ΔgadA ΔglsA of the escherichia coli, from which coding genes of glutamate decarboxylase GadA and glutaminase GlsA are knocked out simultaneously;
the mutant escherichia coli D6 is a mutant of wild escherichia coli obtained by modifying the wild escherichia coli with D2 and D3; specifically, colibacillus mutant K12ΔgadbΔglsA obtained by knocking out coding genes of glutamate decarboxylase GadB and glutaminase GlsA simultaneously;
the mutant escherichia coli D7 is a mutant of wild escherichia coli obtained by modifying the wild escherichia coli with D2 and D4; specifically, the method can be an escherichia coli mutant K12ΔgadbΔglsB obtained by knocking out coding genes of glutamate decarboxylase GadB and glutaminase GlsB simultaneously;
the mutant escherichia coli D8 is a mutant of wild escherichia coli obtained by modifying the wild escherichia coli with D1, D2 and D3; specifically, colibacillus mutants K12ΔgadA, ΔgadB, ΔglsA obtained by knocking out coding genes of glutamate decarboxylase GadA, glutamate decarboxylase GadB and glutaminase GlsA simultaneously;
the mutant escherichia coli D9 is a mutant of wild escherichia coli obtained by modifying the wild escherichia coli with D1, D2 and D4; specifically, colibacillus mutants K12ΔgadA, ΔgadB, ΔglsB with coding genes of glutamate decarboxylase GadA, glutamate decarboxylase GadB and glutaminase GlsB knocked out simultaneously;
the mutant escherichia coli D10 is a mutant of wild escherichia coli obtained by modifying the wild escherichia coli with D1, D3 and D4; specifically, the mutant can be an escherichia coli mutant in which the coding genes of glutamate decarboxylase GadA, glutaminase GlsA and glutaminase GlsB are knocked out simultaneously
K12ΔgadAΔglsAΔglsB；
The mutant escherichia coli D11 is a mutant of wild escherichia coli obtained by modifying the wild escherichia coli with D2, D3 and D4; specifically, the coding genes of glutamate decarboxylase GadB, glutaminase GlsA and glutaminase GlsB are knocked out simultaneously to obtain an escherichia coli mutant K12ΔgagdΔglsA ΔglsB;
wherein, the gene knockdown of the coding genes of the glutamate decarboxylase GadA, the glutamate decarboxylase GadB, the glutaminase GlsA and the glutaminase GlsB can be realized by homologous recombination.
Further, the glutamic acid decarboxylase GadA gene codes for a protein with an amino acid sequence of SEQ ID No. 4; the glutamic acid decarboxylase GadB gene codes protein with an amino acid sequence of SEQ ID No. 5; the glutaminase GlsA gene codes for a protein with an amino acid sequence of SEQ ID No. 6; the glutaminase GlsB gene codes protein with an amino acid sequence of SEQ ID No. 7;
wherein the glutamic acid decarboxylase GadA gene is any one DNA molecule of D11-D13:
d11, a cDNA molecule or genomic DNA having the coding sequence SEQ ID No. 8;
d12, a cDNA molecule or genomic DNA which hybridizes under stringent conditions with the DNA molecule defined by D11 and which encodes the glutamate decarboxylase GadA;
d13, a cDNA molecule or genomic DNA having 75% or more identity to a DNA molecule defined by D11 or D12 and encoding the glutamate decarboxylase GadA;
wherein the glutamic acid decarboxylase GadB gene is any DNA molecule in D21-D23:
d21, a cDNA molecule or genomic DNA having the coding sequence SEQ ID No. 9;
d22, a cDNA molecule or genomic DNA which hybridizes under stringent conditions with the DNA molecule defined by D21 and which encodes said glutamate decarboxylase GadB;
d23, a cDNA molecule or genomic DNA having 75% or more identity to a DNA molecule defined by D21 or D22 and encoding said glutamate decarboxylase GadB;
wherein the glutaminase GlsA gene is any one DNA molecule of D31-D33:
d31, a cDNA molecule or genomic DNA having the coding sequence SEQ ID No. 10;
d32, a cDNA molecule or genomic DNA which hybridizes under stringent conditions to a DNA molecule defined by D31 and which encodes the glutaminase GlsA;
d33, a cDNA molecule or genomic DNA having 75% or more identity to a DNA molecule defined by D31 or D32 and encoding the glutaminase GlsA;
wherein the glutaminase GlsB gene is any one DNA molecule of D41-D43:
d41, cDNA molecule or genomic DNA whose coding sequence is SEQ ID No. 11;
d42, a cDNA molecule or genomic DNA which hybridizes under stringent conditions to a DNA molecule defined by D41 and which encodes the glutaminase GlsB;
d43, a cDNA molecule or genomic DNA having 75% or more identity to a DNA molecule defined by D41 or D42 and encoding the glutaminase GlsB;
the term "identity" as used herein refers to sequence similarity to a native nucleotide sequence; "identity" includes a nucleotide sequence having 75% or more identity to the DNA molecule or cDNA molecule shown in SEQ ID No.8 of the present invention; a nucleotide sequence having 75% or more identity to the DNA molecule or cDNA molecule shown in SEQ ID No.9 of the present invention; a nucleotide sequence having 75% or more identity to the DNA molecule or cDNA molecule shown in SEQ ID No.10 of the present invention; a nucleotide sequence having 75% or more identity to the DNA molecule or cDNA molecule shown in SEQ ID No.11 of the present invention; wherein, the identity of 75% or more is 80%, 85%, 90% or more than 95%.
Further, the recombinant vector contains the coding genes of indigo synthase, phosphopantetheinyl transferase and glutamine synthase; the recombinant vector expresses three enzymes under the same or different promoters of the same vector or expresses three enzymes under the same or different promoters of two vectors or expresses three enzymes on the three vectors respectively.
Further, the recombinant vector is obtained by replacing the DNA sequence between recognition sites of the vector XhoI and BglII with the DNA sequence shown in SEQ ID No.12 for encoding the indigo synthase; replacing the DNA sequence between PstI and KpnI recognition sites with the DNA sequence shown in SEQ ID No.13 for encoding 4' -phosphopantetheinyl transferase; the DNA sequence between EcoRI and HindIII recognition sites was replaced with the DNA sequence shown in SEQ ID No.14 for encoding glutamine synthetase, and the other DNA sequences were kept unchanged.
The invention also provides recombinant bacteria constructed by the construction method for synthesizing the indigo pigment recombinant bacteria.
The invention also provides an application of the recombinant bacterium in synthesizing indigo pigment.
Furthermore, the recombinant bacteria are used for preparing the indigo pigment through catalytic conversion, and specifically comprise the following steps:
(1) Inducing recombinant bacteria by inducing and culturing the recombinant bacteria with arabinose;
(2) The induced recombinant bacteria are used for catalyzing L-glutamate and/or glucose and/or glycerin to carry out catalytic reaction, and ATP (NH) is added into a catalytic system 4 ) 2 HPO 4 、(NH 4 ) 2 SO 4 And NH 4 Cl to obtain conversion solution, and collecting indigo pigment from the conversion solution.
Further, the temperature of the induction culture in the step (1) is 20-37 ℃, and the induction culture time is 12-24 hours.
Further, the temperature of the catalytic reaction in the step (2) is 20-40 ℃, the catalytic reaction time is 10-28h, and the L-glutamate is sodium L-glutamate.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention constructs novel high-yield recombinant bacteria of the indigo pigment, and the encoding genes of the indigo synthetase, the 4' -phosphopantetheinyl transferase and the glutamine synthetase are introduced into mutant escherichia coli through the recombinant vector to construct the recombinant bacteria, so that the recombinant bacteria can weaken the branch paths of glutamic acid and glutamine to the maximum extent on the premise of not influencing the growth of bacteria, and the yield of the indigo and the conversion efficiency of glutamate are effectively improved.
2. The recombinant bacteria are used for preparing the indigo pigment, the induced recombinant bacteria are obtained after the recombinant bacteria are induced and cultured by the inducer, the L-glutamate is used as a substrate by the recombinant bacteria for inducing expression, the indigo is prepared by bioconversion, the yield of the indigo reaches 5.38g/L after 24h of conversion, and the preparation of the indigo pigment by utilizing the recombinant bacteria provided by the invention has the advantages of simple process, high synthesis efficiency and low production cost, and has good industrial application prospect.
Drawings
FIG. 1 is a schematic diagram of the synthesis of indigo pigment in recombinant bacteria constructed in accordance with the present invention;
FIG. 2 is a schematic diagram showing the yield of indigo pigment synthesized by transforming L-sodium glutamate with the recombinant strain in example 2 according to the present invention;
FIG. 3 shows the addition of ATP, (NH) in a catalytic reaction according to example 3 of the present invention 4 ) 2 HPO 4 、(NH 4 ) 2 SO 4 And NH4Cl post-synthesis indigo pigment yields schematic;
FIG. 4 shows the time-varying curve of the conversion of sodium L-glutamate by recombinant bacterial columns EI01 and EI12 to indigo according to the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified;
the experimental methods in the following examples are all conventional methods unless otherwise specified;
the wild escherichia coli is escherichia coli K12; coli K12 (Tomoya Baba, takeshi Ara, miki Hasegawa, yuki Takai, yoshiko Okumura, miki Baba, kirilla Datsenko, masaru Tomita, barry L Wanner and Hirotada Mori1.Construction of Escherichia coli K-12in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems biology (2006): 1-11) public in the examples described below is available from the university of Fujian, and this biomaterial is used only for repeated experiments relating to the present invention and is not available for other uses;
in the following examples, vector pBADhisB is a product of the company Invitrogen, and the catalog number is V430-01;
the T4 ligase in the following examples is a product of Thermo company, and the catalog number is EL0011;
the restriction enzymes XhoI, bglII, pstI, kpnI, ecoRI, hind III and DpnI in the following examples are NEB company products with catalog numbers of R0146, R0144, R0140, R3142, R3101, R3104 and R0176, respectively;
the pCas Plasmid in the examples below was purchased from Addgene under the product number Plasmid #62225 (Jiang Y, chen B, duan C, sun B, yang J, yang S: multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514);
the pTargetF Plasmid in the examples described below was purchased from Addgene under the product number Plasmid #62226 (Jiang Y, chen B, duan C, sun B, yang J, yang S: multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.appl Environ Microbiol 2015, 81:2506-2514);
CRISPR techniques applied in the examples below are described in the prior art (Jiang Y, chen B, duan C, sun B, yang J, yang S: multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514);
coli K12 glutamate decarboxylase encoding gene deletion strains (K12ΔgadA and K12ΔgadA) and E.coli K12 glutaminase encoding gene deletion strains (K12ΔglsA and K12ΔglsB) are described in "Tomoya Baba, takeshi Ara, miki Hasegawa, et al construction of Escherichia coli K-12in-frame, single-gene knockout mutants: the Keio collection molecular Biology (2006), doi:10.1038/msb4100050", which is available to the public from the applicant, and can only be used for repeated experiments of the present invention.
EXAMPLE 1 construction of recombinant bacteria producing indigo
1.Construction of recombinant vectors
The DNA sequence between the XhoI and BglII recognition sites of the pBADhisB vector is replaced by the DNA sequence shown in SEQ ID No.12 for encoding indigo synthase; replacing the DNA sequence between PstI and KpnI recognition sites with the DNA sequence shown in SEQ ID No.13 for encoding 4' -phosphopantetheinyl transferase; replacing the DNA sequence between EcoRI and HindIII recognition sites with a DNA sequence shown in SEQ ID No.14 for encoding glutamine synthetase, and keeping other DNA sequences unchanged to obtain a recombinant vector PEI; from the restriction enzyme identification, it was demonstrated that the IndB, sfp and GlnA genes were successfully inserted between the XhoI and HindIII recognition sites of the pBADhisB vector; the recombinant vector is expressed as PEI, and PEI can express indigo synthetase shown as SEQ ID No.1, 4' -phosphopantetheinyl transferase shown as SEQ ID No.2 and glutamine synthetase shown as SEQ ID No. 3.
2. Construction of mutant E.coli
First, a mutant E.coli D2 was constructed
(1) Preparation of electrotransformation competent cells: the pCas plasmid is transformed into Escherichia coli K12Δgadb by a chemical transformation method, positive clones are cultured and screened on an LB plate containing kanamycin (the kanamycin concentration is 50 mu g/mL) at 30 ℃, and the positive clones are inoculated in an LB liquid culture medium containing 2g/L arabinose and cultured at 30 ℃ until the OD600 is about 0.6, so that electrotransformation competent cells are prepared;
(2) Construction of pTarget plasmid: selecting N20 of a knocked-out gadA gene by using a website https:// crispy. Second symetabolitites. Org, designing a primer to construct a corresponding pTarget plasmid, carrying out PCR amplification by using pTarget-gadA-F and pTarget-gadA-R as templates to obtain a fragment with the size of about 2100bp, digesting for about 3 hours by using DpnI methylase, directly using a chemical conversion method to convert DH5 alpha competence of escherichia coli, screening positive clones on LB plates containing streptomycin (the streptomycin concentration is 50 mu g/mL), and using the primer pTarget-cexu-F for sequencing verification, and naming the positive clones as pTarget-gadA after sequencing is correct; the primer sequences are as follows (the sequence of N20 is underlined):
pTarget-gadA-F：5’-GCACTGATCGATTTCACACGgttttagagctagaaata-3’；
pTarget-gadA-R：5’-CGTGTGAAATCGATCAGTGCactagtattatacctagga-3’；
pTarget-cexu-F：5’-ctttcctgcgttatcccctg-3’；
(3) Amplifying the targeting fragment: PCR amplification is carried out on the gadA-up-F and the gadA-up-R, the gadA-down-F and the gadA-down-R by using primer pairs respectively, so that fragments with the size of about 500bp are obtained respectively; PCR amplification is carried out on the mixture of the two fragments serving as a template by using primer pairs gadA-up-F and gadA-down-R to obtain a delta gadA targeting fragment with the size of about 1000bp, and the targeting fragment is recovered; the primer sequences used were as follows:
gadA-up-F：5’-cttgcatccgcaaaaaccagg-3’；
gadA-up-R：5’-aaacacacctgataacataacgttgtaaaaacc-3’；
gadA-down-F：5’-gtccatttcgaactccttaaatttatttgaaggc-3’；
gadA-down-R：5’-agactttaactttggggaaattacggc-3’；
(4) Electric conversion: 200ng of pTarget-gadA plasmid, 400ng of ΔgadA targeting fragment and 100 mu L of electrotransformation competent cells prepared in the step (1) are mixed, placed in a 2mm electrorotating cup, subjected to 2.5kV electric shock, added with 1mL of LB liquid medium, resuscitated at 30 ℃ and coated on LB plates containing kanamycin and streptomycin (the kanamycin concentration is 50 mu g/mL, the streptomycin concentration is 50 mu g/mL), cultured at 30 ℃, positive clones are screened, PCR amplification is carried out on gadA-up-F and gadA-down-R by using primers, and sequencing of amplified fragments is verified;
(5) Elimination of the pTarget plasmid: positive clones, which were sequenced to verify correct, were inoculated in LB liquid medium containing 0.1mM IPTG and kanamycin and cultured overnight at 30℃to eliminate the pTarget-gadA plasmid. The strain after overnight culture is streaked on LB solid plates containing kanamycin, and is cultured overnight at 30 ℃ to obtain mutant escherichia coli D2 strain K12ΔgadA ΔgadB containing pCas plasmid;
then, constructing mutant E.coli D3 strain K12ΔglsA ΔglsB, mutant E.coli D4 strain K12ΔgadA ΔglsA, mutant E.coli D5 strain K12ΔgadA ΔglsB, mutant E.coli D6 strain K12ΔgadB ΔglsA and mutant E.coli D7 strain K12ΔgadB according to the steps (1) to (5) of constructing mutant E.coli D2 strain, respectively, using different single mutant strains as starting strains; constructing mutant E.coli D8 strain K12 delta gadA delta gadB delta glsA, mutant E.coli D9 strain K12 delta gadA delta gadB delta glsB, mutant E.coli D10 strain K12 delta gadA delta glsA delta glsB and mutant E.coli D11 strain K12 delta gadB delta glsA delta glsB according to steps (1) - (5) of constructing mutant E.coli D2 strain; constructing mutant E.coli D1 strain K12ΔgadA ΔgadB ΔglsA ΔglsB according to steps (1) - (5) of constructing mutant E.coli D2 by using mutant E.coli D11 strain as an initial strain;
the primer sequences used are as follows (underlined as the sequence of N20):
pTarget-glsA-F：5’-GTTGGTTTACCGGGCAAAAGgttttagagctagaaat-3’；
pTarget-glsA-R：5’-CTTTTGCCCGGTAAACCAACactagtattatacctagga-3’；
pTarget-glsB-F：5’-GCATTAATGAACGGATTACGgttttagagctagaaat-3’；
pTarget-glsB-R：5’-CGTAATCCGTTCATTAATGCactagtattatacctag-3’；
glsA-up-F：5’-gattgatgactccgccagcg-3’；
glsA-up-R：5’-catcttttgttaactcctttttatagatgcgggag-3’；
glsA-down-F：5’-gtgtttaagggctgatcatgatgaacac-3’；
glsA-down-R：5’-acgaggatcacttccagccg-3’；
glsB-up-F：5’-accagtccgcaagcaaagg-3’；
glsB-up-R：5’-cgttcggtttattaatgcagtctctcg-3’；
glsB-down-F：5’-cactttctactcctggaccgcag-3’；
glsB-down-R：5’-tgcatcatcaggtggagaaaaccc-3’；
3. recombinant bacterium for constructing synthetic indigo pigment
The obtained recombinant vector PEI was introduced into the constructed mutant E.coli D1-D11 strain by the calcium chloride method, positive clones were selected on plates containing ampicillin, the obtained positive clones were designated as PEI/K12. DELTA. GadA. DELTA. GlsA. DELTA. GlsB, PEI/K12. DELTA. GadA. DELTA. GadB, PEI/K12. DELTA. GlsA. DELTA. GlsB, PEI/K12. DELTA. GadA. DELTA. GlsA, PEI/K12. DELTA. GadA. GlsA. DELTA. GLsB, PEI/K12. DELTA. GlsA. DELTA. GlsB and PEI/K12. DELTA. GlsA. DELTA. GlsB, PEI 01, EI02 EI03 EI, EI05, EI 8, EI06 and EI 11.
EXAMPLE 2 conversion of L-sodium glutamate by recombinant bacteria to indigo
1. Induction culture of recombinant bacteria
Marking recombinant bacteria EI01, EI02, EI03, EI04, EI05, EI06, EI07, EI08, EI09, EI10, EI11, EI12 and escherichia coli K12 for generating indigo respectively on an LB plate containing agar with the mass concentration of 1.5% and ampicillin with the mass concentration of 100 mug/mL, picking single colonies on the plate after culturing for 12 hours at 37 ℃, inoculating the single colonies into a liquid LB culture medium containing ampicillin with the mass concentration of 100 mug/mL, and culturing overnight at 37 ℃ under shaking at 220rpm; the overnight culture was inoculated into the ZYM as an auto-induction medium at an inoculum size of 1% by volume, and cultured with shaking at a rotation speed of 200rpm and 30℃for 16 hours to obtain the induced EI01-EI12 strain and K12 strain, respectively.
2. Synthesis of indigo by converting recombinant bacterium into L-sodium glutamate
Centrifuging the obtained induced EI01-EI12 strain and K12 strain at 4deg.C and 8000rpm for 10min, and collecting thallus; washing the thalli for 1 time by using a sodium chloride aqueous solution with the concentration of 10mM, and then collecting thalli again under the same centrifugation condition to obtain each washed strain; the washed strains were resuspended in PBS buffer (pH 8.0) containing 50mM sodium L-glutamate and glucose to obtain the transformation solutions of EI01-EI12 strain and K12 strain, respectively, wherein the thallus content in the transformation solutions is 15g/L in terms of wet weight; converting the conversion solution of each strain into indigo at 30 ℃ and the rotating speed of 100rpm to obtain conversion solutions of EI01-EI12 strain and K12 strain in 8h, 16h and 24h respectively, wherein the conversion solution is shown in FIG. 1 and is the synthesis path of indigo pigment in recombinant bacteria;
indigo yields of the different strains described above, see FIG. 2, EI01 strain transformed for 24h with indigo yield of 18.59mM; the EI02 strain was transformed for 24 hours with an indigo yield of 16.35mM; the EI03 strain was transformed for 24 hours with an indigo yield of 14.68mM; the EI04 strain was transformed for 24 hours with an indigo yield of 15.17mM; the EI05 strain is transformed for 24 hours, and the indigo yield reaches 15.46mM; the EI06 strain was transformed for 24h with an indigo yield of 16.65mM; the EI07 strain is transformed for 24 hours, and the indigo yield is up to 17.33mM; the EI08 strain was transformed for 24 hours, and the indigo yield was only 18.03mM; the EI09 strain is transformed for 24 hours, and the yield of the indigo reaches 18.12mM; the EI10 strain was transformed for 24 hours with an indigo yield of 17.84mM; EI11 strain was transformed for 24h with indigo yields up to 18.26mM; the EI12 strain was transformed for 24 hours with an indigo yield of only 14.13mM; k12 strain was transformed for 24 hours, and indigo production was still undetectable; as can be seen from FIG. 2, the indigo yield of the EI01-EI11 strain is significantly higher than that of the EI12 strain, wherein the EI01 strain has the highest yield and the best conversion rate, and the conversion efficiency per unit time of the indigo is improved.
EXAMPLE 3 addition of ATP, (NH) 4 ) 2 HPO 4 、(NH 4 ) 2 SO 4 And NH 4 Cl promotes synthesis of indigo pigment
Referring to FIG. 3, ATP and (NH) are added in the catalytic reaction during the process of producing indigo pigment by recombinant bacteria 4 ) 2 HPO 4 、(NH 4 ) 2 SO 4 And NH 4 Cl promotes the synthesis of indigo pigment due to ATP, (NH) 4 ) 2 HPO 4 、(NH 4 ) 2 SO 4 And NH 4 Cl is the cosubstrate in the reaction of indigo pigment and indigo, so that the synthesis amount of indigo can be effectively increased by adding a small amount of Cl. When the recombinant strain EI01 is used as a whole-cell catalyst and 5mM ATP is added, the concentration of indigo in a reaction system with 50mM sodium L-glutamate as a substrate can reach about 21.28mM after 24 hours, and the conversion rate is 85.12 percent, which is improved by nearly 10 percent compared with that of the reaction system without adding the catalyst. Respectively adding 5mM (NH) 4 ) 2 HPO 4 、(NH 4 ) 2 SO 4 And NH 4 After Cl, the indigo yield was increased by adding (NH 4 ) 2 HPO 4 At a maximum of about 20.85mM, ATP and (NH) are added in a similar amount to the amount of ATP added 4 ) 2 HPO 4 The yield of the indigo can reach about 22.04mM, and the conversion rate is 88.16 percent; wherein, the addition of ATP can provide the energy required by L-glutamic acid to synthesize glutamine; (NH) 4 ) 2 HPO 4 、(NH 4 ) 2 SO 4 And NH 4 The addition of Cl and the like can provide an amino donor for synthesizing glutamine from L-glutamic acid.
EXAMPLE 4 Synthesis of indigo pigment by Whole-cell catalysis of recombinant strain EI01
The EI01 single colony is picked and inoculated into a liquid LB culture medium containing ampicillin with the mass concentration of 100 mu g/mL, and the culture medium is subjected to shaking culture at 37 ℃ overnight with the rotating speed of 220rpm; inoculating the overnight culture into a 2L fermentation tank containing 1.5L self-induction culture medium ZYM at an inoculum size of 1% by volume, fermenting and culturing at 30 ℃ for 18h under the condition that the aeration ratio is 1.2-1.5vvm and the rotating speed is 700rpm to obtain a fermentation liquor; centrifuging the fermentation liquor by using a centrifuge, collecting EI01 bacterial cells into a 1L conversion tank, adding about 600mL of PBS buffer solution (pH 8.0) to resuspend bacterial strains to obtain a resuspend bacterial strain solution, enabling the bacterial cell content in the resuspend bacterial strain solution to be 15g/L based on the bacterial cell wet weight, adding L-sodium glutamate and glucose into the resuspend bacterial strain solution to enable the concentration of the L-sodium glutamate in the resuspend bacterial strain solution to be 50mM, converting the conversion solution into indigo blue under the condition that the temperature of 30 ℃ and the rotating speed of 150rpm are carried out on the conversion solution, obtaining the conversion solution of the EI01 bacterial strain under the condition that the L-sodium glutamate is taken as a substrate, wherein the conversion time is 24 hours (sampling every 3 hours), and after 9 hours, feeding the feed solution containing the L-sodium glutamate and glucose is started, and the flow rate is 0.1mL/min; referring to FIG. 4, the EI01 strain is transformed for 24 hours by using L-sodium glutamate as a substrate, the indigo yield reaches 53.23mM, the total consumption of the L-sodium glutamate is 132.15mM, and the transformation rate is 80.59%; the control strain EI12 was transformed with the same culture and transformation conditions for 24h with an indigo yield of only 40.34mM and a transformation rate of 61.05%.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> university of Fujian
<120> construction method and application of recombinant bacterium for synthesizing indigo pigment
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Thr Thr Leu Gln Cys Val Pro Thr Leu Leu Gln Gly Leu Ile Asp Thr
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Gly Arg Ala Leu Ile Asn Val Tyr Gly Pro Thr Glu Thr Thr Ile Asn
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Ser Ile Ser Ile Gly Ala Pro Val His Gly Thr Thr Tyr His Ile Leu
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Asp Lys Glu Thr Leu Lys Pro Val Gly Val Gly Glu Ile Gly Glu Leu
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Pro Val Arg Leu Tyr Lys Thr Gly Asp Leu Gly Gln Trp Asn Ala Asp
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Gly Thr Val Gln Phe Ala Gly Arg Ala Asp Asn Gln Val Lys Leu Arg
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Gly Tyr Arg Val Glu Leu Asp Glu Ile Ser Leu Ala Ile Glu Asn His
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Asp Trp Val Arg Asn Ala Ala Val Ile Val Lys Asn Asp Gly Arg Thr
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Gly Phe Gln Asn Leu Ile Ala Cys Ile Glu Leu Ser Glu Lys Glu Ala
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Asp Ala Glu Leu Ala Ala Arg Pro Ala Phe Asp Leu Glu Gly Ser Glu
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Phe Tyr Glu Gly Gly Ala Val Thr Gln Asp Asp Leu Ile Lys Leu Leu
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Glu Glu Arg Leu Leu Pro Lys Tyr Gly Tyr Ala Ser Pro Gly Ala Leu
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Tyr Ala Thr Gln Met Tyr Phe Glu Leu Glu Gly Val Gly Gly Leu Lys
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Val Arg Glu Leu Leu Asp Val Ala Asp Glu Asp Tyr Tyr Leu Gly Thr
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Phe Glu Leu Val Pro Asn Glu Gly Pro Arg Glu Asp His Ala Glu Val
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Tyr Val Gln Thr His Gly Gly Lys Val Ala Gly Leu Pro Glu Gly Gln
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Tyr Arg Tyr Glu Asn Gly Ala Leu Thr Arg Phe Ser Asp Asp Ile Val
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Asn Leu Gly Phe Met Ser Ser Gly Tyr Ser Ser Lys Thr Gly Asn Pro
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Leu Pro Ala Ser Arg Arg Met Asp Ala Val Leu Ala Glu Asn Gly Val
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Glu Ala Gly Pro Met Tyr Phe Phe Val Gly Gly Arg Val Ser Asp Glu
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Gln Leu Gly His Glu Gly Met Arg Glu Asp Ser Val His Met Arg Gly
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Pro Tyr Glu Thr Ile Thr Glu Met Ala Lys Lys Asp Ile Glu Ala Leu
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Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro Cys Tyr Ile Lys
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Gln His Phe Ser Ile Pro Ala Ala Ala Phe Asp Glu Asp Ala Ile Glu
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Asp Glu Ser Asp Met Asn Leu Leu Pro Asp Leu Thr Thr Ala Thr Leu
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Asp Pro Phe Arg Lys Ala Lys Thr Leu Asn Val Lys Phe Phe Val His
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Asp Pro Phe Thr Arg Glu Ala Phe Ser Arg Asp Pro Arg Asn Val Ala
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Arg Lys Ala Glu Gln Tyr Leu Ala Ser Thr Gly Ile Ala Asp Thr Cys
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Asn Phe Gly Ala Glu Ala Glu Phe Tyr Leu Phe Asp Lys Val Arg Tyr
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Ser Thr Glu Ile Asn Thr Gly Phe Tyr Glu Val Asp Thr Asn Glu Gly
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Gly Ser Lys Asn Arg Val Lys Gly Gly Tyr Phe Pro Val Ala Pro Tyr
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Asp Gln Ala Val Asp Val Arg Asp Asp Met Val Arg Asn Leu Thr Gln
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Ala Gly Phe Asn Leu Glu Arg Phe His His Glu Val Gly Gly Gly Gln
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Gln Glu Ile Asn Tyr Arg Phe Asn Thr Leu Leu His Ala Ala Asp Asp
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Ile Gln Thr Phe Lys Tyr Ile Val Lys Asn Thr Ala Arg Gln His Gly
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Gln Ser Ala Thr Phe Met Pro Lys Pro Leu Ala Gly Asp Asn Gly Ser
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Gly Met His Ala His Gln Ser Leu Trp Lys Asp Gly Lys Pro Leu Phe
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His Asp Glu Ser Gly Tyr Ala Gly Leu Ser Asp Ile Ala Arg Tyr Tyr
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Ile Gly Gly Ile Leu His His Ala Gly Ala Val Leu Ala Phe Thr Asn
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Ala Thr Leu Asn Ser Tyr His Arg Leu Val Pro Gly Phe Glu Ala Pro
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Ile Asn Leu Val Tyr Ser Gln Arg Asn Arg Ser Ala Ala Val Arg Ile
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Pro Ile Thr Gly Ser Asn Pro Lys Ala Lys Arg Ile Glu Phe Arg Ala
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Pro Asp Pro Ser Gly Asn Pro Tyr Leu Gly Phe Ala Ala Met Met Met
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Ala Gly Leu Asp Gly Ile Lys Asn Arg Ile Glu Pro His Ala Pro Val
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Asp Lys Asp Leu Tyr Glu Leu Pro Pro Glu Glu Ala Ala Ser Ile Pro
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Gln Ala Pro Thr Ser Leu Glu Ala Ser Leu Lys Ala Leu Gln Glu Asp
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Thr Asp Phe Leu Thr Glu Ser Asp Val Phe Thr Glu Asp Leu Ile Glu
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Ala Tyr Ile Gln Tyr Lys Tyr Asp Asn Glu Ile Ser Pro Val Arg Leu
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Arg Pro Thr Pro Gln Glu Phe Glu Leu Tyr Phe Asp Cys
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<212> PRT
<213> Escherichia coli (Escherichia coli)
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Met Asp Gln Lys Leu Leu Thr Asp Phe Arg Ser Glu Leu Leu Asp Ser
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Arg Phe Gly Ala Lys Ala Ile Ser Thr Ile Ala Glu Ser Lys Arg Phe
20 25 30
Pro Leu His Glu Met Arg Asp Asp Val Ala Phe Gln Ile Ile Asn Asp
35 40 45
Glu Leu Tyr Leu Asp Gly Asn Ala Arg Gln Asn Leu Ala Thr Phe Cys
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Gln Thr Trp Asp Asp Glu Asn Val His Lys Leu Met Asp Leu Ser Ile
65 70 75 80
Asn Lys Asn Trp Ile Asp Lys Glu Glu Tyr Pro Gln Ser Ala Ala Ile
85 90 95
Asp Leu Arg Cys Val Asn Met Val Ala Asp Leu Trp His Ala Pro Ala
100 105 110
Pro Lys Asn Gly Gln Ala Val Gly Thr Asn Thr Ile Gly Ser Ser Glu
115 120 125
Ala Cys Met Leu Gly Gly Met Ala Met Lys Trp Arg Trp Arg Lys Arg
130 135 140
Met Glu Ala Ala Gly Lys Pro Thr Asp Lys Pro Asn Leu Val Cys Gly
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Pro Val Gln Ile Cys Trp His Lys Phe Ala Arg Tyr Trp Asp Val Glu
165 170 175
Leu Arg Glu Ile Pro Met Arg Pro Gly Gln Leu Phe Met Asp Pro Lys
180 185 190
Arg Met Ile Glu Ala Cys Asp Glu Asn Thr Ile Gly Val Val Pro Thr
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Phe Gly Val Thr Tyr Thr Gly Asn Tyr Glu Phe Pro Gln Pro Leu His
210 215 220
Asp Ala Leu Asp Lys Phe Gln Ala Asp Thr Gly Ile Asp Ile Asp Met
225 230 235 240
His Ile Asp Ala Ala Ser Gly Gly Phe Leu Ala Pro Phe Val Ala Pro
245 250 255
Asp Ile Val Trp Asp Phe Arg Leu Pro Arg Val Lys Ser Ile Ser Ala
260 265 270
Ser Gly His Lys Phe Gly Leu Ala Pro Leu Gly Cys Gly Trp Val Ile
275 280 285
Trp Arg Asp Glu Glu Ala Leu Pro Gln Glu Leu Val Phe Asn Val Asp
290 295 300
Tyr Leu Gly Gly Gln Ile Gly Thr Phe Ala Ile Asn Phe Ser Arg Pro
305 310 315 320
Ala Gly Gln Val Ile Ala Gln Tyr Tyr Glu Phe Leu Arg Leu Gly Arg
325 330 335
Glu Gly Tyr Thr Lys Val Gln Asn Ala Ser Tyr Gln Val Ala Ala Tyr
340 345 350
Leu Ala Asp Glu Ile Ala Lys Leu Gly Pro Tyr Glu Phe Ile Cys Thr
355 360 365
Gly Arg Pro Asp Glu Gly Ile Pro Ala Val Cys Phe Lys Leu Lys Asp
370 375 380
Gly Glu Asp Pro Gly Tyr Thr Leu Tyr Asp Leu Ser Glu Arg Leu Arg
385 390 395 400
Leu Arg Gly Trp Gln Val Pro Ala Phe Thr Leu Gly Gly Glu Ala Thr
405 410 415
Asp Ile Val Val Met Arg Ile Met Cys Arg Arg Gly Phe Glu Met Asp
420 425 430
Phe Ala Glu Leu Leu Leu Glu Asp Tyr Lys Ala Ser Leu Lys Tyr Leu
435 440 445
Ser Asp His Pro Lys Leu Gln Gly Ile Ala Gln Gln Asn Ser Phe Lys
450 455 460
His Thr
465
<210> 5
<211> 466
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<213> Escherichia coli (Escherichia coli)
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Met Asp Lys Lys Gln Val Thr Asp Leu Arg Ser Glu Leu Leu Asp Ser
1 5 10 15
Arg Phe Gly Ala Lys Ser Ile Ser Thr Ile Ala Glu Ser Lys Arg Phe
20 25 30
Pro Leu His Glu Met Arg Asp Asp Val Ala Phe Gln Ile Ile Asn Asp
35 40 45
Glu Leu Tyr Leu Asp Gly Asn Ala Arg Gln Asn Leu Ala Thr Phe Cys
50 55 60
Gln Thr Trp Asp Asp Glu Asn Val His Lys Leu Met Asp Leu Ser Ile
65 70 75 80
Asn Lys Asn Trp Ile Asp Lys Glu Glu Tyr Pro Gln Ser Ala Ala Ile
85 90 95
Asp Leu Arg Cys Val Asn Met Val Ala Asp Leu Trp His Ala Pro Ala
100 105 110
Pro Lys Asn Gly Gln Ala Val Gly Thr Asn Thr Ile Gly Ser Ser Glu
115 120 125
Ala Cys Met Leu Gly Gly Met Ala Met Lys Trp Arg Trp Arg Lys Arg
130 135 140
Met Glu Ala Ala Gly Lys Pro Thr Asp Lys Pro Asn Leu Val Cys Gly
145 150 155 160
Pro Val Gln Ile Cys Trp His Lys Phe Ala Arg Tyr Trp Asp Val Glu
165 170 175
Leu Arg Glu Ile Pro Met Arg Pro Gly Gln Leu Phe Met Asp Pro Lys
180 185 190
Arg Met Ile Glu Ala Cys Asp Glu Asn Thr Ile Gly Val Val Pro Thr
195 200 205
Phe Gly Val Thr Tyr Thr Gly Asn Tyr Glu Phe Pro Gln Pro Leu His
210 215 220
Asp Ala Leu Asp Lys Phe Gln Ala Asp Thr Gly Ile Asp Ile Asp Met
225 230 235 240
His Ile Asp Ala Ala Ser Gly Gly Phe Leu Ala Pro Phe Val Ala Pro
245 250 255
Asp Ile Val Trp Asp Phe Arg Leu Pro Arg Val Lys Ser Ile Ser Ala
260 265 270
Ser Gly His Lys Phe Gly Leu Ala Pro Leu Gly Cys Gly Trp Val Ile
275 280 285
Trp Arg Asp Glu Glu Ala Leu Pro Gln Glu Leu Val Phe Asn Val Asp
290 295 300
Tyr Leu Gly Gly Gln Ile Gly Thr Phe Ala Ile Asn Phe Ser Arg Pro
305 310 315 320
Ala Gly Gln Val Ile Ala Gln Tyr Tyr Glu Phe Leu Arg Leu Gly Arg
325 330 335
Glu Gly Tyr Thr Lys Val Gln Asn Ala Ser Tyr Gln Val Ala Ala Tyr
340 345 350
Leu Ala Asp Glu Ile Ala Lys Leu Gly Pro Tyr Glu Phe Ile Cys Thr
355 360 365
Gly Arg Pro Asp Glu Gly Ile Pro Ala Val Cys Phe Lys Leu Lys Asp
370 375 380
Gly Glu Asp Pro Gly Tyr Thr Leu Tyr Asp Leu Ser Glu Arg Leu Arg
385 390 395 400
Leu Arg Gly Trp Gln Val Pro Ala Phe Thr Leu Gly Gly Glu Ala Thr
405 410 415
Asp Ile Val Val Met Arg Ile Met Cys Arg Arg Gly Phe Glu Met Asp
420 425 430
Phe Ala Glu Leu Leu Leu Glu Asp Tyr Lys Ala Ser Leu Lys Tyr Leu
435 440 445
Ser Asp His Pro Lys Leu Gln Gly Ile Ala Gln Gln Asn Ser Phe Lys
450 455 460
His Thr
465
<210> 6
<211> 310
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 6
Met Leu Asp Ala Asn Lys Leu Gln Gln Ala Val Asp Gln Ala Tyr Thr
1 5 10 15
Gln Phe His Ser Leu Asn Gly Gly Gln Asn Ala Asp Tyr Ile Pro Phe
20 25 30
Leu Ala Asn Val Pro Gly Gln Leu Ala Ala Val Ala Ile Val Thr Cys
35 40 45
Asp Gly Asn Val Tyr Ser Ala Gly Asp Ser Asp Tyr Arg Phe Ala Leu
50 55 60
Glu Ser Ile Ser Lys Val Cys Thr Leu Ala Leu Ala Leu Glu Asp Val
65 70 75 80
Gly Pro Gln Ala Val Gln Asp Lys Ile Gly Ala Asp Pro Thr Gly Leu
85 90 95
Pro Phe Asn Ser Val Ile Ala Leu Glu Leu His Gly Gly Lys Pro Leu
100 105 110
Ser Pro Leu Val Asn Ala Gly Ala Ile Ala Thr Thr Ser Leu Ile Asn
115 120 125
Ala Glu Asn Val Glu Gln Arg Trp Gln Arg Ile Leu His Ile Gln Gln
130 135 140
Gln Leu Ala Gly Glu Gln Val Ala Leu Ser Asp Glu Val Asn Gln Ser
145 150 155 160
Glu Gln Thr Thr Asn Phe His Asn Arg Ala Ile Ala Trp Leu Leu Tyr
165 170 175
Ser Ala Gly Tyr Leu Tyr Cys Asp Ala Met Glu Ala Cys Asp Val Tyr
180 185 190
Thr Arg Gln Cys Ser Thr Leu Leu Asn Thr Ile Glu Leu Ala Thr Leu
195 200 205
Gly Ala Thr Leu Ala Ala Gly Gly Val Asn Pro Leu Thr His Lys Arg
210 215 220
Val Leu Gln Ala Asp Asn Val Pro Tyr Ile Leu Ala Glu Met Met Met
225 230 235 240
Glu Gly Leu Tyr Gly Arg Ser Gly Asp Trp Ala Tyr Arg Val Gly Leu
245 250 255
Pro Gly Lys Ser Gly Val Gly Gly Gly Ile Leu Ala Val Val Pro Gly
260 265 270
Val Met Gly Ile Ala Ala Phe Ser Pro Pro Leu Asp Glu Asp Gly Asn
275 280 285
Ser Val Arg Gly Gln Lys Met Val Ala Ser Val Ala Lys Gln Leu Gly
290 295 300
Tyr Asn Val Phe Lys Gly
305 310
<210> 7
<211> 308
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 7
Met Ala Val Ala Met Asp Asn Ala Ile Leu Glu Asn Ile Leu Arg Gln
1 5 10 15
Val Arg Pro Leu Ile Gly Gln Gly Lys Val Ala Asp Tyr Ile Pro Ala
20 25 30
Leu Ala Thr Val Asp Gly Ser Arg Leu Gly Ile Ala Ile Cys Thr Val
35 40 45
Asp Gly Gln Leu Phe Gln Ala Gly Asp Ala Gln Glu Arg Phe Ser Ile
50 55 60
Gln Ser Ile Ser Lys Val Leu Ser Leu Val Val Ala Met Arg His Tyr
65 70 75 80
Ser Glu Glu Glu Ile Trp Gln Arg Val Gly Lys Asp Pro Ser Gly Ser
85 90 95
Pro Phe Asn Ser Leu Val Gln Leu Glu Met Glu Gln Gly Ile Pro Arg
100 105 110
Asn Pro Phe Ile Asn Ala Gly Ala Leu Val Val Cys Asp Met Leu Gln
115 120 125
Gly Arg Leu Ser Ala Pro Arg Gln Arg Met Leu Glu Val Val Arg Gly
130 135 140
Leu Ser Gly Val Ser Asp Ile Ser Tyr Asp Thr Val Val Ala Arg Ser
145 150 155 160
Glu Phe Glu His Ser Ala Arg Asn Ala Ala Ile Ala Trp Leu Met Lys
165 170 175
Ser Phe Gly Asn Phe His His Asp Val Thr Thr Val Leu Gln Asn Tyr
180 185 190
Phe His Tyr Cys Ala Leu Lys Met Ser Cys Val Glu Leu Ala Arg Thr
195 200 205
Phe Val Phe Leu Ala Asn Gln Gly Lys Ala Ile His Ile Asp Glu Pro
210 215 220
Val Val Thr Pro Met Gln Ala Arg Gln Ile Asn Ala Leu Met Ala Thr
225 230 235 240
Ser Gly Met Tyr Gln Asn Ala Gly Glu Phe Ala Trp Arg Val Gly Leu
245 250 255
Pro Ala Lys Ser Gly Val Gly Gly Gly Ile Val Ala Ile Val Pro His
260 265 270
Glu Met Ala Ile Ala Val Trp Ser Pro Glu Leu Asp Asp Ala Gly Asn
275 280 285
Ser Leu Ala Gly Ile Ala Val Leu Glu Gln Leu Thr Lys Gln Leu Gly
290 295 300
Arg Ser Val Tyr
305
<210> 8
<211> 1401
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 8
atggaccaga agctgttaac ggatttccgc tcagaactac tcgattcacg ttttggcgca 60
aaggccattt ctactatcgc ggagtcaaaa cgatttccgc tgcacgaaat gcgcgatgat 120
gtcgcatttc agattatcaa tgatgaatta tatcttgatg gcaacgctcg tcagaacctg 180
gccactttct gccagacctg ggacgacgaa aacgtccata aattgatgga tttgtcgatc 240
aataaaaact ggatcgacaa agaagaatat ccgcaatccg cagccatcga cctgcgttgc 300
gtaaatatgg ttgccgatct gtggcatgcg cctgcgccga aaaatggtca ggccgttggc 360
accaacacca ttggttcttc cgaggcctgt atgctcggcg ggatggcgat gaaatggcgt 420
tggcgcaagc gtatggaagc tgcaggcaaa ccaacggata aaccaaacct ggtgtgcggt 480
ccggtacaaa tctgctggca taaattcgcc cgctactggg atgtggagct gcgtgagatc 540
cctatgcgcc ccggtcagtt gtttatggac ccgaaacgca tgattgaagc ctgtgacgaa 600
aacaccatcg gcgtggtgcc gactttcggc gtgacctaca ccggtaacta tgagttccca 660
caaccgctgc acgatgcgct ggataaattc caggccgaca ccggtatcga catcgacatg 720
cacatcgacg ctgccagcgg tggcttcctg gcaccgttcg tcgccccgga tatcgtctgg 780
gacttccgcc tgccgcgtgt gaaatcgatc agtgcttcag gccataaatt cggtctggct 840
ccgctgggct gcggctgggt tatctggcgt gacgaagaag cgctgccgca ggaactggtg 900
ttcaacgttg actacctggg tggtcaaatt ggtacttttg ccatcaactt ctcccgcccg 960
gcgggtcagg taattgcaca gtactatgaa ttcctgcgcc tcggtcgtga aggctatacc 1020
aaagtacaga acgcctctta ccaggttgcc gcttatctgg cggatgaaat cgccaaactg 1080
gggccgtatg agttcatctg tacgggtcgc ccggacgaag gcatcccggc ggtttgcttc 1140
aaactgaaag atggtgaaga tccgggatac accctgtacg acctctctga acgtctgcgt 1200
ctgcgcggct ggcaggttcc ggccttcact ctcggcggtg aagccaccga catcgtggtg 1260
atgcgcatta tgtgtcgtcg cggcttcgaa atggactttg ctgaactgtt gctggaagac 1320
tacaaagcct ccctgaaata tctcagcgat cacccgaaac tgcagggtat tgcccagcag 1380
aacagcttta aacacacctg a 1401
<210> 9
<211> 1401
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 9
atggataaga agcaagtaac ggatttaagg tcggaactac tcgattcacg ttttggtgcg 60
aagtctattt ccactatcgc agaatcaaaa cgttttccgc tgcacgaaat gcgcgacgat 120
gtcgcattcc agattatcaa tgacgaatta tatcttgatg gcaacgctcg tcagaacctg 180
gccactttct gccagacctg ggacgacgaa aatgtccaca aattgatgga tttatccatt 240
aacaaaaact ggatcgacaa agaagaatat ccgcaatccg cagccatcga cctgcgttgc 300
gtaaatatgg ttgccgatct gtggcatgcg cctgcgccga aaaatggtca ggccgttggc 360
accaacacca ttggttcttc cgaggcctgt atgctcggcg ggatggcgat gaaatggcgt 420
tggcgcaagc gtatggaagc tgcaggcaaa ccaacggata aaccaaacct ggtgtgcggt 480
ccggtacaaa tctgctggca taaattcgcc cgctactggg atgtggagct gcgtgagatc 540
cctatgcgcc ccggtcagtt gtttatggac ccgaaacgca tgattgaagc ctgtgacgaa 600
aacaccatcg gcgtggtgcc gactttcggc gtgacctaca ctggtaacta tgagttccca 660
caaccgctgc acgatgcgct ggataaattc caggccgata ccggtatcga catcgacatg 720
cacatcgacg ctgccagcgg tggcttcctg gcaccgttcg tcgccccgga tatcgtctgg 780
gacttccgcc tgccgcgtgt gaaatcgatc agtgcttcag gccataaatt cggtctggct 840
ccgctgggct gcggctgggt tatctggcgt gacgaagaag cgctgccgca ggaactggtg 900
ttcaacgttg actacctggg tggtcaaatt ggtacttttg ccatcaactt ctcccgcccg 960
gcgggtcagg taattgcaca gtactatgaa ttcctgcgcc tcggtcgtga aggctatacc 1020
aaagtacaga acgcctctta ccaggttgcc gcttatctgg cggatgaaat cgccaaactg 1080
gggccgtatg agttcatctg tacgggtcgc ccggacgaag gcatcccggc ggtttgcttc 1140
aaactgaaag atggtgaaga tccgggatac accctgtatg acctctctga acgtctgcgt 1200
ctgcgcggct ggcaggttcc ggccttcact ctcggcggtg aagccaccga catcgtggtg 1260
atgcgcatta tgtgtcgtcg cggcttcgaa atggactttg ctgaactgtt gctggaagac 1320
tacaaagcct ccctgaaata tctcagcgat cacccgaaac tgcagggtat tgcccaacag 1380
aacagcttta aacatacctg a 1401
<210> 10
<211> 933
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 10
atgttagatg caaacaaatt acagcaggca gtggatcagg cttacaccca atttcactca 60
cttaacggcg gacaaaatgc cgattacatt ccctttctgg cgaatgtacc aggtcaactg 120
gcggcagtgg ctatcgtgac ctgcgatggc aacgtctata gtgcgggtga cagtgattac 180
cgctttgcac tggaatccat ctcgaaagtc tgtacgttag cccttgcgtt agaagatgtc 240
ggcccgcagg cggtacagga caaaattggc gctgacccga ccggattgcc ctttaactca 300
gttatcgcct tagagttgca tggcggcaaa ccgctttcgc cactggtaaa tgctggcgct 360
attgccacca ccagcctgat taacgctgaa aatgttgaac aacgctggca gcgaatttta 420
catatccaac agcaactggc tggcgagcag gtagcgctct ctgacgaagt caaccagtcg 480
gaacaaacaa ccaacttcca taaccgggcc atagcctggc tgctgtactc cgccggatat 540
ctctattgtg atgcaatgga agcctgtgac gtgtataccc gtcagtgctc cacgctcctc 600
aatactattg aactggcaac gcttggcgcg acgctggcgg caggtggtgt gaatccgttg 660
acgcataaac gcgttcttca ggccgacaac gtgccgtaca ttctggccga aatgatgatg 720
gaagggctgt atggtcgctc cggtgactgg gcgtatcgtg ttggtttacc gggcaaaagc 780
ggtgtaggtg gcggtattct ggcggtcgtc cctggagtga tgggaattgc cgcgttctca 840
ccaccgctgg acgaagatgg caacagtgtt cgcggtcaaa aaatggtggc atcggtcgct 900
aagcaactcg gctataacgt gtttaagggc tga 933
<210> 11
<211> 927
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 11
gtggcagtcg ccatggataa tgcaatttta gaaaacatct tgcggcaagt gcggccgctc 60
attggtcagg gtaaagtcgc ggattatatt ccggcgctgg ctacagtaga cggttcccga 120
ttggggattg ctatctgtac cgttgacgga cagctttttc aggccggaga cgcgcaagaa 180
cgtttttcca ttcagtctat ttccaaagtg ctgagtctcg ttgtcgccat gcgtcattac 240
tccgaagagg aaatctggca acgcgtcggc aaagatccgt ctggatcacc gttcaattcc 300
ttagtgcaac tggaaatgga gcagggtata ccgcgtaatc cgttcattaa tgccggtgcg 360
ctggtggtct gcgatatgtt gcaagggcga ttaagcgcac cacggcaacg tatgctggaa 420
gtcgtgcgcg gcttaagcgg tgtgtctgat atttcctacg atacggtggt agcgcgttcc 480
gaatttgaac attccgcgcg aaatgcggct atcgcctggc tgatgaagtc gtttggcaat 540
ttccatcatg acgtgacaac cgttctgcaa aactactttc attactgcgc tctgaaaatg 600
agctgtgtag agctggcccg gacgtttgtc tttctggcta atcaggggaa agctattcat 660
attgatgaac cagtggtgac gccaatgcag gcgcggcaaa ttaacgcgct gatggcgacc 720
agtggtatgt accagaacgc gggggagttt gcctggcggg tggggctacc ggcgaaatct 780
ggcgttggtg gcggtattgt ggcgattgtt ccgcatgaaa tggccatcgc tgtctggagt 840
ccggaactgg atgatgcagg taactcgctt gcgggtattg ccgttcttga acaattgacg 900
aaacagttag ggcgttcggt ttattaa 927
<210> 12
<211> 3852
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
atgaccctgc aagagacttc tgttctggaa cctaccctgc gtggtaccac cactctgagc 60
ggtctgctgg cagaacgtgt agcagaacat ccggaagcga tcgctgttgc ctaccgtgac 120
gagaaactga ccttccgtga actggctagc cgttctgctg ctctggcaga ttacctgggt 180
cacctgggtg taagcgcgga ccagtgtgta ggcctgtttg tggaaccgag catcgatctg 240
atggtgggtg cttggggtat cctgggtgcg ggtgctgcat atctgccgct gtctcctgaa 300
tatccggagg accgtctgcg ttacatgatc gagaactctg agacgaagat catcctggca 360
cagcaacgtc tggtatctcg tctgcgcgaa ctggcaccgc aggatgtaac cattgtgacc 420
ctgcgtgaat ctgaagcttt cgttcgtcca gaaggccagg aagctccagc accgggtggt 480
gatgctcgtc ctgacactct ggcatacgtc atctacacga gcggttccac cggtaaaccg 540
aaaggtgtca tgatcgaaca ccactctatc gttaaccagc tgggttggct gcgtgaaacc 600
tacggcattg atcgtagcaa agtgatcctg cagaaaactc cgatgagctt cgacgcggcc 660
cagtgggaaa ttctgtcccc ggcaaacggt gcaaccgttg ttatgggtgc tcctggtgtt 720
tacgcagacc cggaaggtct gattgaaact atcgtgaaac acggtgtcac taccctgcag 780
tgcgtgccta ccctgctgca gggtctgatc gacactgaaa agttcccgga atgcgtttct 840
ctgcagcaga tcttctctgg tggcgaagcc ctgtctcgtc tgctggcaat ccaggcaact 900
caggaaatgc cgggtcgcgc cctgattaat gtgtacggtc cgaccgaaac gaccatcaac 960
tcttcttcct tcgttgtaga cccggctgaa ctggacgaag gtccgcagtc catctctatc 1020
ggtgctccgg ttcacggtac gacctaccat attctggata aagaaactct gaaaccggtt 1080
ggtgttggtg aaatcggcga actgtacatc ggcggtgttc aactggcacg tggctacctg 1140
caccgtgatg atctgaccgc ggaacgtttc ctggaaattg aactggaaga aggtgcagcg 1200
ccggttcgtc tgtacaaaac cggtgacctg ggccagtgga atgcagatgg tactgtgcag 1260
tttgctggtc gcgctgacaa ccaggtgaaa ctgcgtggtt atcgcgttga gctggacgaa 1320
atcagcctgg cgatcgaaaa ccatgattgg gttcgtaacg ctgctgttat cgtgaagaat 1380
gatggtcgta ccggcttcca gaacctgatc gcgtgcatcg aactgtccga aaaagaagcc 1440
gcgctgatgg atcagggtaa ccacggtagc caccacgcaa gcaaaaaatc caaactgcag 1500
gtgaaagcac agctgagcaa ccctggtctg cgtgatgacg ctgaactggc tgctcgcccg 1560
gcttttgacc tggaaggctc cgaaccgact ccagagcaac gtgcacgcgt tttcgcgcgt 1620
aaaacctatc gcttttacga aggtggcgcc gttactcaag atgatctgat taaactgctg 1680
ggtagcaaag tgaccgcggc ttattctcgt aaggctgcag atctggcgcc gtccgaactg 1740
ggtcagatcc tgcgttggtt tggtcagtac atctctgaag agcgtctgct gcctaaatac 1800
ggttatgcta gcccgggtgc tctgtatgca acgcaaatgt acttcgaact ggaaggcgtt 1860
ggtggtctga aaccgggcta ttactactac cagccggttc gccatcagct ggtcctgatt 1920
tccgagcgtg ctgccactgg tcgtcctacc gcacagatcc atttcattgg taaaaagtcc 1980
ggcatcgaac cggtctataa aaataacatc caggaagttc tggaaatcga aactggtcac 2040
atcctgggtc tgttcgaaca gatcctgccg gcttacggcc tggacgttca agatcgtgcg 2100
tatgatcctg cggtgcgtga actgctggat gttgcagacg aagattacta cctgggtacc 2160
ttcgaactgg tacctaacga aggtccgcgt gaagaccacg cggaggttta cgtgcagacc 2220
cacggtggca aggttgctgg tctgccggaa ggccagtacc gctatgaaaa cggtgcgctg 2280
acccgcttct ccgacgatat cgtgctgaaa aagcacgtaa ttgcaattaa ccagtccgtt 2340
tatcaagctg cgtccttcgg catctccgta tattctcgtg cagaagaaga atggctgaaa 2400
tatatcactc tgggtaaaaa gctgcaacac ctgatgatga acggtctgaa cctgggcttt 2460
atgtcctccg gctactcttc taaaactggt aacccgctgc cggcttctcg tcgtatggac 2520
gcagtactgg ctgaaaacgg cgtcgaagct ggtccgatgt acttctttgt tggcggtcgt 2580
gtttctgacg aacagctggg ccacgaaggt atgcgtgaag atagcgttca catgcgtggt 2640
ccggccgagc tgattcgtga cgacctggtt tccttcctgc cggactatat gattccaaac 2700
cgcgtagtag ttttcgatcg tctgccactg tccgctaatg gcaaaatcga cgtaaaggcg 2760
ctggcggttt ctgaccaagt gaacgcagaa ctggtcgaac gtccattcgt agctccgcgc 2820
accgaaacgg aaaaagaaat cgcggctgtt tgggaaaaat ctctgcgtcg tgaaaacgct 2880
tctgtacagg acgatttctt tgaatccggc ggcaactctc tgatcgcggt aggtctggtt 2940
cgtgaactga actcccgtct gggtgttagc ctgccgctgc aatctgttct ggaatccccg 3000
accatcgaaa aactggctcg tcgtctggaa cgcgaggtag cacaagaatc ctcccgtttc 3060
gtccgcctgc atgctgaaac cggtaaagct cgcccagtaa tctgctggcc tggcctgggt 3120
ggttatccga tgaacctgcg tagcctggcg ggtgaaattg gtctgggtcg ttctttctac 3180
ggtgtacaga gctacggcat taatgagggt gaaaccccgt atgagactat caccgagatg 3240
gcaaaaaaag atatcgaggc gctgaaagaa ctgcagccga ctggtccgta taccctgtgg 3300
ggctatagct ttggtgcacg tgttgctttc gaaaccgcat accaactgga gcaggccggt 3360
gagaaagtgg ataacctgtt cctgatcgct ccgggttctc cgaaagtgcg tgctgaaaac 3420
ggtaaagttt ggggccgtga agcgtctttc gcaaaccgtg gctataccac tattctgttc 3480
tctgtattta ccggtaccat cagcggcccg gatctggacc gttgcctgga aaccgtgact 3540
gacgaagcct ccttcgccga gttcatcagc gaactgaaag gtatcgatat cgatctggcg 3600
cgccgtatca tctccgttgt tggtcagacc tacgaattcg aatactcttt ccacgaactg 3660
gcagaacgca ccctgcaggc accgatctcc attttcaaag ccgttggcga cgattacagc 3720
tttctggaga attctagcgg ctactccgca gaaccgccga ccgtaatcga cctggacgca 3780
gaccactata gcctgctgcg cgacgatatc ggtgagctgg ttaagcatat ccgttatctg 3840
ctgggcgaat aa 3852
<210> 13
<211> 675
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
atgaaaatct atggtatcta catggatcgt ccgctgtccc aggaggaaaa cgagcgtttc 60
atgtccttca tttccccgga aaaacgtgag aaatgtcgtc gtttctatca caaagaggac 120
gctcaccgca ccctgctggg tgacgtactg gtacgctctg tcatttcccg tcagtatcag 180
ctggataagt ctgacatccg tttctctacc caggagtacg gcaaaccttg tattccggac 240
ctgccggatg cgcactttaa catttctcac agcggtcgct gggtaatcgg cgctttcgat 300
tcccagccga tcggtatcga catcgaaaaa accaaaccga tcagcctgga aattgccaaa 360
cgttttttca gcaaaaccga atattctgat ctgctggcta aagataaaga cgaacagact 420
gactattttt accacctgtg gtccatgaaa gaatccttca ttaaacagga aggcaaaggc 480
ctgtctctgc cactggactc tttctctgtc cgtctgcacc aggatggcca agttagcatc 540
gaactgccgg actctcactc cccgtgttat attaaaacgt acgaaatcga cccgggctat 600
aaaatggcag tatgtgcggc gcacccggat ttcccggaag acatcaccat ggttagctat 660
gaagaactgc tgtaa 675
<210> 14
<211> 1434
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
atggcattcg agacgccgga agaggttacc aaattcatca aagatgagaa cgttgagttt 60
atcgacgtgc gtttcaccga tctgccgggc accgagcagc acttctccat tccggcggct 120
gctttcgacg aagacgctat cgaagaaggt ctggccttcg atggttcctc catccgtggt 180
ttcactacca tcgacgaaag cgacatgaat ctgctgccgg atctgaccac cgccaccctg 240
gatccgtttc gtaaagctaa aaccctgaat gtcaaattct tcgttcacga tcctttcacg 300
cgtgaagcat tctctcgtga cccacgcaac gtagcgcgca aagctgagca gtacctggct 360
tctaccggca tcgcagacac ctgcaatttc ggcgctgaag cagaatttta cctgttcgat 420
aaagtacgct attctaccga aatcaacacc ggtttttacg aagttgacac caatgagggt 480
tggtggaacc gcggtcgtga aactaacctg gacggtactc cgaacctggg ttctaaaaac 540
cgtgtgaaag gtggctactt ccctgtcgca ccgtatgatc aagctgtaga cgtgcgcgac 600
gacatggttc gtaacctgac tcaggctggt ttcaacctgg aacgttttca ccacgaggtt 660
ggtggtggcc agcaggagat caattaccgc ttcaacactc tgctgcatgc ggcagatgac 720
atccagactt ttaaatacat cgttaagaac actgcacgtc aacacggtca gtccgcgacc 780
ttcatgccga aaccgctggc tggtgataac ggttccggta tgcacgcgca ccaatccctg 840
tggaaagacg gtaaaccgct gtttcacgat gaatctggtt acgcgggcct gagcgacatc 900
gcgcgctatt acatcggcgg cattctgcac catgcaggcg cggtgctggc ttttacgaac 960
gcgaccctga actcttatca ccgtctggta ccgggttttg aagcaccgat caatctggtg 1020
tacagccagc gtaaccgctc tgcggctgtt cgcatcccga tcaccggctc taacccgaaa 1080
gcaaaacgca tcgaattccg cgctccagac ccatccggca acccgtacct gggcttcgcg 1140
gcgatgatga tggcgggtct ggacggtatc aaaaaccgta tcgaaccgca cgcgccggtg 1200
gacaaagacc tgtacgagct gccgccggaa gaagcggcct ctattccgca ggcaccgacc 1260
agcctggaag cgtccctgaa ggcactgcag gaagacactg acttcctgac tgaatccgat 1320
gtttttactg aggatctgat tgaagcatat atccagtaca aatacgacaa cgaaattagc 1380
ccggttcgtc tgcgtccgac cccgcaagaa ttcgaactgt acttcgactg ctaa 1434

Claims (8)

1. A construction method for synthesizing indigo pigment recombinant bacteria comprises the steps of introducing encoding genes of indigo synthetase, 4' -phosphopantetheinyl transferase and glutamine synthetase into a recipient bacteria through a recombinant vector to obtain bacteria for synthesizing indigo pigment;
the indigo synthetase gene codes protein with an amino acid sequence of SEQ ID No. 1;
the 4' -phosphopantetheinyl transferase gene codes for a protein with an amino acid sequence of SEQ ID No. 2;
the glutamine synthetase gene codes protein with an amino acid sequence of SEQ ID No. 3;
the receptor bacteria are mutant escherichia coli; the mutant escherichia coli is a mutant of the wild escherichia coli obtained by carrying out any one of the following gene modifications d1, d2, d3 and d4, any two gene modification combinations, any three gene modification combinations or four gene modification combinations on the wild escherichia coli;
d1, knocking out glutamate decarboxylase GadA;
d2, knocking out glutamate decarboxylase GadB;
d3, knocking out glutaminase GlsA;
d4, knocking out glutaminase GlsB;
wherein, the glutamic acid decarboxylase gadA gene codes protein with the amino acid sequence of SEQ ID No. 4; the glutamic acid decarboxylase gadB gene codes for protein with the amino acid sequence of SEQ ID No. 5; the glutaminase glsA gene codes for a protein with an amino acid sequence of SEQ ID No. 6; the glutaminase glsB gene codes for a protein with an amino acid sequence of SEQ ID No. 7.
2. The construction method for synthesizing the recombinant strain of indigo pigment according to claim 1, wherein the construction method comprises the following steps: the recombinant vector contains the coding genes of indigo synthetase, 4' -phosphopantetheinyl transferase and glutamine synthetase; the recombinant vector expresses three enzymes under the same or different promoters of the same vector or expresses three enzymes under the same or different promoters of two vectors or expresses three enzymes on the three vectors respectively.
3. The construction method for synthesizing the recombinant strain of indigo pigment according to claim 2, wherein: the recombinant vector is characterized in that a DNA sequence between recognition sites of the vector XhoI and BglII is replaced by a DNA sequence shown in SEQ ID No.12 and used for encoding indigo synthetase; replacing the DNA sequence between PstI and KpnI recognition sites with the DNA sequence shown in SEQ ID No.13 for encoding 4' -phosphopantetheinyl transferase; the DNA sequence between EcoRI and HindIII recognition sites was replaced with the DNA sequence shown in SEQ ID No.14 for encoding glutamine synthetase, and the other DNA sequences were kept unchanged.
4. A recombinant bacterium constructed by the construction method for synthesizing an indigo pigment recombinant bacterium according to any one of claims 1 to 3.
5. The use of the recombinant bacterium according to claim 4 for the synthesis of indigo pigments.
6. The use of the recombinant bacterium according to claim 4 for synthesizing indigo pigment, wherein: the recombinant bacteria are used for preparing the indigo pigment through catalytic conversion, and specifically comprise the following steps:
(1) The recombinant bacteria are subjected to induction culture by an induction culture medium ZYM to obtain induced recombinant bacteria;
(2) The induced recombinant bacteria are used for catalyzing sodium L-glutamate and/or glucose to carry out catalytic reaction, and ATP and (NH) are added into a catalytic system 4 ) 2 HPO 4 、(NH 4 ) 2 SO 4 And NH 4 Cl to obtain conversion solution, and collecting indigo pigment from the conversion solution.
7. The use of the recombinant bacterium according to claim 6 for synthesizing indigo pigment, wherein: the temperature of the induction culture in the step (1) is 20-37 ℃, and the induction culture time is 12-24h.
8. The use of the recombinant bacterium according to claim 6 for synthesizing indigo pigment, wherein: the temperature of the catalytic reaction in the step (2) is 20-40 ℃, and the catalytic reaction time is 10-28h.
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