CN109897809B - Escherichia coli recombinant bacterium for synthesizing cobalt (II) quinoline acid a, c-diamide and construction method and application thereof - Google Patents

Abstract

The invention provides an escherichia coli recombinant bacterium for synthesizing CBAD and a construction method and application thereof. Specifically, the invention uses escherichia coli as an original strain, constructs an engineering bacterium which comprises an uroporphyrinogen III gene module and has the expression of an endogenous heme synthesis gene reduced, and the engineering bacterium can improve the accumulation of precursor uroporphyrinogen III of HBA and reduce heme pathway flux, thereby further improving the yield of HBA.

Description

Escherichia coli recombinant bacterium for synthesizing cobalt (II) quinoline acid a, c-diamide and construction method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an escherichia coli recombinant bacterium for synthesizing cobalt (II) quinoline acid a, c-diamide and a construction method and application thereof.

Background

Cobalt (II) quinolinic acid a, c-diamide (cob (II) yrinic acid a, c-diamide, CBAD) is a synthetic vitamin B₁₂A precursor of (2). The cobalt (II) quinolinic acid a, c-diamide can be obtained by expressing the adenosylcobalamin synthesis genes cobAIGJMFKLH, cobB, cobNST and cbiMNQO in Escherichia coli. However, the low yield of cobalt (II) quinolinic acid a, c-diamide in the prior art inevitably leads to vitamin B₁₂The yield of (2) is low. LC-MS did not detect hydrogenogenic acid (HBA), indicating that the low yield of HBA may be a direct cause of the low yield of cobalt (II) quinolinic acid a, c-diamide.

Delta-aminolevulinate (ALA) is a synthetic vitamin B₁₂The E.coli itself may pass through C₅The ALA is synthesized by the way. The synthesis of ALA by E.coli has been studied more often, and most of them are obtained by constructing heterologous C₄(ii) a pathway. Expression of the ALA synthase genes hemA, hemO of photosynthetic bacteria such as Rhodobacter sphaeroides, Rhodopseudomonas palustris all makes it possible to express E.coliALA is accumulated.

At present, the yield of the cobalt (II) quinoline acid a, c-diamide synthesized by using escherichia coli in the prior art is not high, and the requirement of industrial production cannot be met. Therefore, the development of an escherichia coli recombinant bacterium suitable for industrial production and used for synthesizing cobalt (II) quinoline acid a, c-diamide, and a construction method and application thereof are urgently needed in the field.

Disclosure of Invention

The invention aims to provide an escherichia coli recombinant bacterium for synthesizing cobalt (II) quinoline acid a, c-diamide and a construction method and application thereof.

In a first aspect of the present invention, there is provided an engineered bacterium for the production of CBAD (cobalt (II) oxoacid a, c-diamide) or a precursor thereof, the engineered bacterium being escherichia coli and containing an exogenous gene module (genemodule) comprising:

(a) an uroporphyrinogen III gene module expressing a gene for the biosynthesis of uroporphyrinogen III;

preferably, the genes for biosynthesis of uroporphyrinogen III include: hemA or hemO gene, hemB gene, hemC gene, and hemD gene;

and, the expression of the endogenous heme synthesis gene of the engineered bacterium is down-regulated;

preferably, the heme synthesis gene comprises: hemE gene, hemF gene, hemG gene, and/or hemH gene.

In another preferred example, the endogenous gene endA of the engineered bacterium is down-regulated or deleted.

In another preferred embodiment, the uroporphyrinogen III gene module is integrated into the genome, preferably into the arabinose-inducible promoter P_BADA site.

In another preferred embodiment, the hemA gene, hemO gene, hemB gene, hemC gene, and hemD gene are each independently derived from: a Rhodobacter capsulatus strain, a Sinorhizobium meliloti strain, a Rhodopseudomonas palustri strain, a Brucella melitensis strain, a Rhodobacter sphaeroides strain, or a Brucella melitensis strain;

preferably, the hemA gene is derived from a Sinorhizobium meliloti strain, or a R.palustri strain,

hemO gene is from R.palustri strain;

hemB gene, hemC gene and hemD gene are derived from S.meliloti strain.

In another preferred embodiment, the hemA gene from a strain of Sinorhizobium meliloti has the sequence shown in SEQ ID No. 1.

In another preferred example, the hemA gene derived from the R.palustri strain has the sequence shown in SEQ ID No. 2.

In another preferred embodiment, the hemO gene has the sequence shown in SEQ ID No. 3.

In another preferred embodiment, the hemB gene has the sequence shown in SEQ ID No. 4.

In another preferred embodiment, the sequence of hemC gene is shown in SEQ ID No. 5.

In another preferred embodiment, the hemD gene has the sequence shown in SEQ ID No. 6.

In another preferred embodiment, the gene module further comprises:

(b) an HBA gene module expressing a gene for biosynthesis of HBA using uroporphyrinogen III as a raw material;

preferably, the gene for biosynthesis of HBA comprises: cobA gene, cobI gene, cobG gene, cobJ gene, cobM gene, cobF gene, cobK gene, cobL gene, and cobH gene;

(c) an HBAD gene module that expresses a gene for biosynthesis of HBAD using HBA as a raw material;

preferably, the gene for biosynthesis of HBAD comprises: the cobB gene;

(d) a CBAD gene module expressing a gene for biosynthesis of CBAD using HBAD as a raw material;

preferably, the genes for biosynthesis of CBAD include: (i) a cobN gene, a cobS gene, a cobT gene, a cobW gene, or (ii) a cobN gene, a chlI gene, a chlD gene, a cobW gene, or a combination thereof;

(e) a cobalt-uptake gene module expressing a gene encoding a transporter for transporting cobalt ions into the cell;

preferably, the gene encoding a transporter for transporting cobalt ions into cells comprises a cbinmnqo operon comprising: tandem expression of the cbiM gene, cbiN gene, cbiQ gene and cbiO gene; and

(f) a heme pathway attenuation module for down-regulating (by sRNA) expression of a heme synthesis gene;

preferably, the heme synthesis gene comprises: hemE gene, hemF gene, hemG gene, and/or hemH gene.

In another preferred embodiment, the cobalt uptake gene module further comprises a cbtAB operon comprising: a cbtA gene and a cbtB gene expressed in tandem.

In another preferred embodiment, when the gene module contains more than 2 genes, some or all of the genes are expressed in tandem.

In another preferred embodiment, the genes in the HBA gene module are expressed in tandem.

In another preferred embodiment, the HBAD gene module and the CBAD gene module are expressed in tandem.

In another preferred embodiment, each gene in each gene module is driven by a constitutive or inducible promoter.

In another preferred embodiment, each gene in each gene module is driven by an inducible promoter.

In another preferred embodiment, the promoter is selected from the group consisting of: a T7 promoter, a tac promoter, a trc promoter, a lac promoter, an arabinose inducible promoter, or a combination thereof.

In another preferred embodiment, the expression module is partially or completely integrated in the genome.

In another preferred embodiment, the expression module is partially or completely located on an expression vector.

In another preferred embodiment, the vector is a plasmid and/or a nucleic acid fragment.

In another preferred embodiment, the vector further comprises a resistance gene element.

In another preferred embodiment, the resistance gene is selected from the group consisting of: a tetracycline resistance gene, a kanamycin resistance gene, an ampicillin resistance gene, a chloramphenicol resistance gene, a streptomycin sulfate resistance gene, or a combination thereof.

In another preferred embodiment, the vector further comprises a terminator element.

In another preferred embodiment, the Escherichia coli further comprises an exogenous T7RNA polymerase expression cassette.

In another preferred embodiment, the T7RNA polymerase expression cassette is integrated on the genome, preferably at the lacZ site.

In another preferred embodiment, the cbiM gene, cbiN gene, cbiQ gene and cbiO gene are each independently derived from: a strain of capsulatus, a strain of Salmonella typhimurium, a strain of Propionibacterium, a strain of freudenreichii subsp.Shermanii, a strain of Klebsiella pneumoniae, a strain of Yersinia enterica, a strain of Bacillus stearothermophilus, a strain of Listeria monocytogenes, a strain of Clostridium acetobutylicum, a strain of Clostridium perfringens, a strain of Clostridium botulinum, a strain of Clostridium botulitum, a strain of Clostridium difficile, a strain of Desutobacterium halobacterium, a strain of Streptomyces coelicolor, a strain of Propionibacterium freudenreichii subsp.Shermanii, a strain of Chrotium tedus, a strain of Metasphaerella, a strain of Metallubacterium lacticum, or a strain of Geotrichu.

In another preferred embodiment, the cbinmq operon is integrated on the genome, preferably at the ldhA locus.

In another preferred embodiment, the cbtA gene and cbtB gene are each independently derived from: pseudomonas strains, Mesorhizobium loti strains, Brucella melitensis strains, Agrobacterium tumefaciens strains, Pseudomonas putida strains, Pseudomonas fluorescens strains, Pseudomonas syringae strains, or Pseudomonas aeruginosa strains.

In another preferred example, each gene in the HBA gene module is derived from r.capsulatus strain, s.meliloti strain, b.melitensis strain, or Pseudomonas dentificans strain; preferably from the r.

In another preferred embodiment, the cobB gene, cobN gene, cobS gene, cobT gene and cobW gene are each independently derived from: a capsulatus strain, a s.meliloti strain, a b.melitensis strain, a sinorhizobium meliloti strain, a Mesorhizobium loti strain, a Bradyrhizobium japonicum strain, an Agrobacterium tumefaciens strain, or a Rhodopseudomonas palustris strain;

preferably, the cobN gene, cobS gene, cobT gene and cobW gene are derived from a B.melitensis strain;

preferably, the cobB gene is derived from r.

In another preferred example, the cobN gene, the chlI gene, the chlD gene and the cobW gene are each independently derived from a Pseudomonas denitificans strain, a Burkholderia pseudolei strain, a Ralstoniasenapearum strain, a Pseudomonas aeruginosa strain, a Pseudomonas putida strain, a Pseudomonas fluorescens strain, a Pseudomonas syringine strain, a Corynebacterium diphyteria strain, a Mycobacterium tuberculosis strain, a Thermobifida fusca strain, a Rhodococcus strain, a Streptomyces coelicolor strain, a Treponema dentolola strain, a Chrobium tepidum strain, or a Halobacterium strain;

preferably, the cobN gene, the chlI gene, the chlD gene and the cobW gene are derived from Pseudomonas dentificas strain.

In another preferred embodiment, the engineering bacteria are used for synthesizing CBAD from the beginning.

In another preferred example, the engineering bacteria can be used for synthesizing vitamin B from head₁₂。

In another preferred example, the engineering bacteria can be used for synthesizing vitamin B by an aerobic synthetic pathway₁₂。

In another preferred embodiment, the yield of CBAD of the engineering bacteria is more than or equal to 0.17mg/g cell dry weight, preferably more than or equal to 0.5mg/g cell dry weight.

In a second aspect of the invention, there is provided a method of producing CBAD or a precursor thereof, comprising the steps of:

(i) culturing the engineered bacterium of the first aspect of the invention, thereby obtaining a fermentation product comprising CBAD or a precursor thereof; and

(ii) isolating CBAD or a precursor thereof from the fermentation product.

In a third aspect of the present invention, there is provided a method for constructing the engineered bacteria of the first aspect of the present invention, comprising the steps of:

(a) constructing a vector containing an uroporphyrinogen III gene module expressing a gene for biosynthesis of uroporphyrinogen III or integrating it into a genome;

preferably, the genes for biosynthesis of uroporphyrinogen III include: hemA or hemO gene, hemB gene, hemC gene, and hemD gene;

(b) constructing a vector for down-regulating the expression of the heme synthesis gene,

preferably, the heme synthesis gene comprises: a hemE gene, a hemF gene, a hemG gene, and/or a hemH gene; and

(c) and (c) respectively transferring the vectors obtained in the step (a) and the step (b) into escherichia coli to obtain the engineering bacteria which contain the gene modules and have the expression of the endogenous heme synthesis gene down-regulated.

In another preferred embodiment, the method further comprises the steps of:

(I) constructing a vector containing an HBA gene module, wherein the HBA gene module expresses a gene for biologically synthesizing HBA by using uroporphyrinogen III as a raw material;

(II) constructing a vector containing an HBAD gene module which expresses a gene for biosynthesis of HBAD using HBA as a raw material;

(III) constructing a vector containing a CBAD gene module, wherein the CBAD gene module expresses a gene for biosynthesis of CBAD by taking HBAD as a raw material;

(IV) constructing a vector or integrating into the genome a cobalt uptake gene module for expression of a transporter that transports cobalt ions into the cell; and

(V) construction of an endA knock-out strain.

In another preferred example, in the step (c), the vectors obtained in the steps (a), (b), (I), (II), (III) and (IV) are respectively transferred into the escherichia coli obtained in the step (V), so as to obtain the engineering bacteria containing the gene module.

In another preferred embodiment, all or part of the module is integrated into the E.coli genome.

In another preferred embodiment, the Escherichia coli includes, but is not limited to, strains with an endA gene knockout or downregulation.

In another preferred example, the method further comprises step (d): PCR verifying the genotype of the recombinants obtained in step (c); and/or

A step (e): fermentation testing the recombinant obtained in step (c) for the production of CBAD or a precursor thereof.

In a fourth aspect of the invention there is provided the use of an engineered bacterium according to the first aspect of the invention, wherein the engineered bacterium is used as a strain for the fermentative production of CBAD or a precursor thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows OD during fermentation of recombinant bacteria expressing heterologous hemABCD₆₀₀。

FIG. 2 shows the ALA production variation during fermentation of recombinant bacteria expressing heterologous hemABCD.

FIG. 3 shows the variation of HBA production during fermentation of recombinant bacteria expressing heterologous hemABCD.

FIG. 4 shows OD during fermentation of recombinant bacteria of genes hemE, hemF, hemG, hemH that attenuate the hemE synthesis pathway₆₀₀。

FIG. 5 shows the ALA production variation during fermentation of recombinant bacteria with the genes hemE, hemF, hemG, hemH, which attenuate the hemE synthesis pathway.

FIG. 6 shows the variation of HBA production during fermentation of recombinant bacteria of genes hemE, hemF, hemG, hemH which attenuate the hemE synthesis pathway.

FIG. 7 shows OD during fermentation of recombinant bacteria combining genes hemEF, hemEG, hemEH, hemFG, hemFH, hemGH attenuating the heme synthesis pathway₆₀₀。

FIG. 8 shows the ALA production changes during fermentation of recombinant bacteria combining genes hemEF, hemEG, hemEH, hemFG, hemFH, hemGH attenuating the heme synthesis pathway.

FIG. 9 shows the variation of HBA production during fermentation of recombinant bacteria combining genes hemEF, hemEG, hemEH, hemFG, hemFH, hemGH attenuating the heme synthesis pathway.

FIG. 10 shows OD in the fermentation process of FH228 recombinant bacteria₆₀₀And HBA production variation.

FIG. 11 shows LC-MS verification of cobalt (II) quinoline acid a, c-diamide synthesized by FH274 recombinant bacteria.

FIG. 12 shows LC-MS verification of cobalt (II) quinoline acid a, c-diamide synthesized by FH275 recombinant bacteria.

Detailed Description

The inventor of the present invention has extensively and deeply studied, and by using Escherichia coli as an original strain and screening and testing a large amount of exogenous genes and combinations thereof, surprisingly found for the first time that the yield of HBA can be increased by strengthening the uroporphyrinogen III synthesis pathway and weakening the heme synthesis pathway, thereby increasing CBAD and vitamin B₁₂The yield of (2). Experiments show that the expression of heterologous hemA/hemO, hemB, hemC and hemD can improve the accumulation of precursor uroporphyrinogen III of HBA, and attenuation of hemE, hemF, hemG and hemH by sRNA can reduce the flux of a hemE synthesis pathway, thereby further improving the flux of the hemE synthesis pathwayHBA production. In addition, when the HBA gene module, the uroporphyrinogen III gene module, and the heme pathway attenuation module are integrated into one plasmid, and the HBAD gene module, the CBAD gene module, and the cobalt uptake gene module are integrated into another plasmid, plasmid instability is caused. And after the uroporphyrinogen III gene module and the cobalt absorption gene module are integrated on a genome by knocking out the endA gene of the host bacterium, the instability of plasmids is solved. The present invention has been completed based on this finding.

The genes involved in the invention are shown in the following table:

name of Gene	Login number	Remarks for note
			hemA	9004270	ALA synthetase
hemO	JQ048722.1	ALA synthetase
			hemE	948497	Uroporphyrinogen decarboxylase
hemF	946908	Coproporphyrinogen III oxidase
			hemG	948331	Protoporphyrin oxidase
hemH	947532	Protoporphyrin ferrous chelatases
			cobB	31490895	Hydrocorrinic acid a, c-diamide synthase
cobN	29593490	Cobalt chelatase
			cobS	29594978	Cobalt chelatase
cobT	29594975	Cobalt chelatase
			cobW	29593484	Cobalt chelating helper proteins
chlI	32563000	Magnesium chelatase
			chlD	32564605	Magnesium chelatase
cbiM	31490899	Cobalt transport system permeases
			cbiN	31490898	Cobalt transporters
cbiQ	31490897	Cobalt transport system permeases
			cbiO	31490896	ATP-binding protein of cobalt transport system
cbtA	32564928	Cobalt transporters
			cbtB	32562082	Cobalt transporters

The meanings of some abbreviations referred to in the present invention are as follows:

ALA: 5-aminolevulinic acid (delta-aminolevulinate); HBA: hydrogen corrinic acid (hydrogenobutyric acid); urogen III: uroporphyrinogen iii (uroporphyrinogen iii); CBAD: cobalt (II) quinolinic acid a, c-diamide (cob (II) hydrazinic acid a, c-diamide); LC-MS: liquid chromatography-massspectrometry;

the main experimental process of the invention is as follows:

(a) constructing uroporphyrinogen III gene modules, including p15ASI-RphemOBCD, p15ASI-RphemABCD, p15ASI-SmhemABCD, p15ASI-RchemABCD and p15ASI-hemOBCD plasmids. The plasmid is transformed into HBA synthetic starting bacterium FH001, and the influence of the expression uroporphyrinogen III gene module on HBA is evaluated after fermentation of a TYG culture medium.

(b) The hemE pathway attenuation module is cloned to a BamHI enzyme cutting site of a pET28-HBA plasmid to obtain plasmids pET28-HBA-anti-hemE, pET28-HBA-anti-hemF, pET28-HBA-anti-hemG, pET28-HBA-anti-hemH, pET28-HBA-anti-hemEF, pET28-HBA-anti-hemEG, pET 28-HBA-anti-hemEHE, pET28-HBA-anti-hemFG, pET28-HBA-anti-hemFH and pET 28-HBA-anti-hemGH. The plasmids and p15ASI-RphemOBCD plasmid are respectively transformed into FH001 together, and the obtained strain is fermented by a TYG culture medium to evaluate the influence of the weakening heme monogene and the weakening heme bigene on HBA synthesis.

(c) The uroporphyrinogen III gene module is cloned to SacI and HindIII restriction enzyme cutting sites of pET28-HBA-anti-hemFG plasmid to obtain pET 28-HBA-anti-hemFG-RhemOBCD plasmid, the plasmid is transformed into Escherichia coli MG1655(DE3), and HBA yield is evaluated after TYG culture medium fermentation.

(d) Constructing an integration plasmid pCDF-RccobB-BmcobN-his-BmcobS-BmcobT-cbiNQO plasmid of the HBA gene module, the CBAD gene module and the cobalt absorption gene module. Constructing plasmids pCDF-RccobbmcobNSTW and pCDF-BWNID integrating HBAD gene module and CBAD gene modules from different sources.

(e) Coli MG1655(DE3) endA gene was first knocked out to obtain FH224, and then cobalt-absorbed gene module, P_TacThe cbinmqo expression cassette integrates into the ldhA site of FH224, resulting in FH 225. Finally, the uroporphyrinogen III gene module, i.e., P_TacIntegration of the RphemOBCD expression cassette into the arabinose-induced promoter site of FH225, yielded FH236 as a underplate cell for CBAD synthesis.

(f) pET28-HBA-anti hemFG and pCDF-RccobbmcobNSTW plasmid were co-transformed into FH236, resulting in FH 274. pET28-HBA-anti hemFG and pCDF-BWNID plasmid were co-transferred to FH236, resulting in FH 275. FH274 and FH275 were fermented in TYG medium and then evaluated for CBAD production.

In particular, the uroporphyrinogen III gene module related by the invention is used for synthesizing HBA and vitamin by escherichia coliElement B₁₂And precursors thereof. Weakening hemE pathway synthesis genes hemE, hemF, hemG and hemH for synthesizing HBA and vitamin B by using escherichia coli₁₂And precursors thereof. The CBAD gene module and the cobalt absorption gene module are commonly applied to escherichia coli for synthesizing CBAD and vitamin B₁₂And precursors thereof. FH236, FH274 and FH275 were applied to synthesis of CBAD and vitamin B₁₂And precursors thereof.

The main advantages of the invention include:

(a) the invention greatly improves the yield of HBA by expressing heterologous hemA/hemO, hemB, hemC and hemD genes and weakening hemE synthesis pathway genes hemE, hemF, hemG and hemH.

(b) The invention solves the problem of plasmid instability by knocking out the endA gene on the genome and integrating the uroporphyrinogen III synthesis module and the cobalt absorption and transportation module on the plasmid on the genome and reducing the plasmid

(c) The invention obtains the optimized recombinant bacterium for synthesizing the cobalt (II) quinoline acid a, c-diamide by fermenting and evaluating the recombinant bacterium for expressing chelatases from different sources, which is the recombinant bacterium for synthesizing vitamin B₁₂The basis of (1).

(d) Compared with the prior art, the yield of the cobalt (II) quinoline acid a, c-diamide synthesized by the recombinant bacterium is improved by at least 12.6 times.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Versatile materials and methods

Materials: in the examples, all plasmids and strains are conventional or commercially available unless otherwise specified. Specifically, the starting strain in the examples is Escherichia coli, one example of which is Escherichia coli MG1655, which has integrated into its genome a T7RNA polymerase derived from an expression phage, designated MG1655(DE 3).

The Gibson assembly method is described in Gibson DG et al, 2009, Nature Methods, 6(5): 343-; coli gene knock-out by the CRISPR/Cas9 method is described in Zhao D et al, 2016, Microbial Cell industries, 15: 205.

Example 1

HBA (human hepatitis B A) synthetic recombinant bacterium construction and fermentation evaluation for expressing uroporphyrinogen III gene module

1. HBA (human hepatitis B A) synthetic recombinant bacterium construction for expressing uroporphyrinogen III gene module

Construction of p15ASI-hemOBCD plasmid: the gene with hemO optimized according to E.coli codon was synthesized in Beijing Kinzhi Biotechnology GmbH and cloned into puc57 (Kinzhi Gene Synthesis Standard vector), the plasmid was named puc 57-hemO. Using this plasmid as a template, a linker hemO-Gibson fragment was amplified. Using the genome of S.meliloti 320 as a template, and amplifying to obtain hemB fragment and hemCD fragment for Gibson assembly. The p15ASI plasmid is used as a template, a p15ASI plasmid skeleton is obtained through amplification, and the p15ASI-hemOBCD plasmid is obtained through Gibson assembly with the hemO-Gibson fragment, the hemB-Gibson fragment and the hemCD-Gibson fragment.

Construction of p15ASI-SmhemABCD, p15ASI-RchemABCD plasmids: using the S.meliloti 320 genome as a template to amplify a SmhemA fragment, and using a p15ASI-hemOBCD plasmid as a template to amplify to obtain a fragment p15ASI-hemBCD, wherein the fragment is used as a plasmid framework. The fragment SmhemA and the fragment p15ASI-hemBCD are connected by Gibson assembly to obtain a plasmid p15 ASI-SmhemABCD. And amplifying the RchemA fragment by using the R. capsulatus genome as a template, and connecting the RchemA fragment with the fragment p15ASI-hemBCD through Gibson assembly to obtain the plasmid p15 ASI-RChemABCD.

And (3) constructing p15ASI-RphemOBCD and p15ASI-RphemABCD plasmids. The Rhodopseudomonas palustris genome is used as a template, RPhemA-F1 and RPhemA-R1 primers are used for amplifying RPhemA and upstream and downstream fragments thereof, and RPhemO-F1 and RPhemO-R1 primers are used for amplifying RPhemO and upstream and downstream fragments thereof. Cloning the amplified RphemA and upstream and downstream fragments thereof, and RphemO and upstream and downstream fragments thereof to a pEASY-blunt vector, and sequencing to obtain CDS of the RphemA and the RphemO. The fragments of RphemO for Gibson assembly were amplified using primers RPhemO-p15ASI-F-GBS and RPhemO-p15ASI-R-GBS, and ligated to fragment p15ASI-hemBCD by Gibson assembly to give plasmid p15 ASI-RPhemOBCD. The fragment of RphemA for Gibson assembly was amplified using primers RPhemA-p15ASI-F-GBS and RPhemA-p15ASI-R-GBS, and ligated to fragment p15ASI-hemBCD by Gibson assembly to give plasmid p15 ASI-RphemABCD.

The primers used in this section were as follows:

TABLE 1 primers for construction of HBA synthetic recombinant bacteria expressing uroporphyrinogen III gene modules

The plasmids p15ASI, p15ASI-RphemOBCD, p15ASI-RphemABCD, p15ASI-SmhemABCD, p15ASI-RchemABCD and p15ASI-hemOBCD are respectively transformed into FH001 strain (from the patent' de novo synthesis of vitamin B)₁₂The recombinant Escherichia coli strain, and a construction method and application thereof) to obtain FH167, FH168, FH177, FH178, FH179, and FH 180.

The strains and plasmids used in this section were as follows:

TABLE 2 HBA-synthesizing recombinant bacteria expressing uroporphyrinogen III gene module and plasmids used therein

2. Fermentation of recombinant bacteria

From the plate picking FH167, FH168, FH177, FH178, FH179, FH180 single colony, in the test tube overnight culture. Then, the cells were inoculated into a 250mL Erlenmeyer flask containing 50mL of LB medium at an inoculum size of 1%, and cultured for 12 hours. Then inoculated into a medium containing 600ml of TYG medium (5g/L yeast extract, 10g/L tryptone, 5g/L KH) at an inoculum size of 5%₂PO₄2g/L glucose, 2g/L glycine, 10g/L succinic acid, 15g/L betaine with initial pH adjusted to 6.5 with NaOH) in a 3L Erlenmeyer flask at 37 ℃ at 200 r.min^-1Cultured under conditions of OD₆₀₀When it reaches 0.6IPTG was added to a final concentration of 0.4mM, and the temperature was changed to 28 ℃ for culture.

3. Separation and purification of HBA

After fermentation, a certain amount of fermentation liquor is taken for centrifugation, thalli are collected, high-pressure crushing and centrifugation are carried out to obtain intracellular products, and HBA is obtained through DEAE-Sephadex A-25 anion exchange chromatography column purification. The resulting HBA sample was filtered through a 0.22 μm filter and subjected to quantitative HPLC analysis.

4. Quantification of ALA

The ALA is measured by a colorimetric method. Taking fermentation liquor at 12000 r.min^-1Centrifuging for 2min under the condition, collecting supernatant 200 μ L, adding 100 μ L sodium acetate (pH 4.6, 1M) buffer solution, mixing with 50 μ L acetylacetone solution, boiling in water bath for 15min, and cooling to room temperature. Mixing 100 mu L of the reaction solution after water bath with 100 mu L of modified Ehrlich's reagent (1g of p-dimethylaminobenzaldehyde, 30mL of glacial acetic acid is added, 8mL of 70% perchloric acid is added, and the mixture is filled to 50mL by glacial acetic acid) in an enzyme label plate, measuring the absorbance light value of the mixture at 554 nm by using an enzyme label instrument after the mixture is stabilized for 15min, and calculating the ALA content in the sample according to the standard curve of ALA.

5. Quantification of HBA

HBA was quantified by HPLC under the following conditions: the analysis was carried out on an Agilent 1260 instrument fitted with an Agilent TC C18 column (4.6X 250 mm). The amount of the sample was 20. mu.L. Phase A was 0.10M potassium phosphate (pH 6.5), and phase B was 50% 0.10M potassium phosphate (pH 8), 50% acetonitrile. Flow rate: 1.0mL/min, column temperature: the detection wavelength was 329nm at 35 ℃. Gradient elution conditions: 0-5min, 0-2% buffer B; 5-10min, 2-5% buffer B; 10-20min, 5-20% buffer B; 20-30min, 20% buffer B; 30-40min, 20-100% buffer B; 40-45min, 100-0% buffer B.

6. HBA synthetic recombinant bacterium fermentation result for expressing uroporphyrinogen III gene module

HBA can be synthesized after expression of cobAIGJMFKLH in E.coli. Escherichia coli may pass through C₅ALA is synthesized in a way, and is catalyzed by HemB, HemC and HemD to generate Urogen III. The low production of HBA may be the reason for the poor genomic expression of hemA, hemB, hemC, hemD in the synthetic pathway. To increase the supply of precursors, bacteria FH001 were usedGenes hemA, hemB, hemC, hemD synthesized by Urogen III were expressed in the strain. HemA is a key enzyme for the synthesis of ALA. Whereas ALA is a precursor of HBA, the concentration of ALA directly affects the production of HBA. The production of HBA can be controlled by screening different sources of ALA synthase to modulate ALA production. Therefore, S.meliloti 320, R.capsulatus-derived hemA, R.palustris-derived ALA synthesis isozyme encoding gene hemA or hemO gene, and 5 operons consisting of S.meliloti 320-derived hemB, hemC and hemD genes are expressed in FH001 strain. Meanwhile, a control bacterium FH167 is constructed, which is a strain expressing p15ASI empty plasmid in FH 001.

OD of each recombinant bacterium was measured at 8, 20, 32, and 45h after induction with IPTG₆₀₀ALA and HBA yields. The results of the measurement are shown in FIGS. 1 to 3. Recombinant bacterium OD for expressing exogenous hemA/hemO gene and hemB, hemC and hemD genes of S.meliloti 320₆₀₀All were reduced, but HBA production was significantly improved (fig. 1, fig. 3). The batch of fermentation TYG culture medium contains 2g/L glucose. The glucose is used up when the fermentation is carried out for 8 hours, which may also be a reason for the low biomass of the recombinant bacteria. The highest HBA yield of the recombinant strain FH168 expressing the non-named hemO gene (RhemO) of the R.palustris strain and hemB, hemC and hemD genes of S.meliloti 320 in the recombinant strain reaches 9.02mg/g DCW, while the maximum HBA yields of the two control strains FH001 and FH167 are only 0.54mg/g DCW and 1.24mg/g DCW respectively. At the same time, the recombinant extracellular ALA maximum yield of the Urogen III precursor synthesis module exceeds 0.69g/L, and FH001 and FH167 have only endogenous C₅Synthetic pathway (FIG. 2), maximum ALA production was only 0.10g/L and 0.077g/L, respectively. In addition, FH168, FH177 and FH179 all showed a reduction in biomass in the late stage of fermentation from 32h to 45 h.

The HBA yield of all strains in the later fermentation period is obviously reduced, probably because the enzyme exists in an inclusion body form due to long induction time, the production amount of the HBA is reduced, and the originally produced HBA is gradually decomposed due to instability. Another possibility is plasmid instability. The plasmid is gradually lost in the late fermentation stage, resulting in reduced HBA synthesis.

Example 2

HBA (hepatitis B nucleic acid) synthetic recombinant bacterium construction and fermentation evaluation of gene weakening heme synthetic pathway

1. Construction of HBA synthetic recombinant bacterium of gene weakening heme synthetic pathway

A module for attenuating genes was synthesized in Kingson, containing the Pr promoter, MicC Scaffold, B0015double terminator. In addition, in order to weaken a plurality of genes simultaneously, a Biobrick prefix (including EcoR I and Xba I enzyme cutting sites) and a suffix (including Spe I and Pst I enzyme cutting sites) are added in front and back respectively. This sequence was cloned into the EcoRV cleavage site of the pUC57 plasmid and the new plasmid was named pUCS.

Construction of a heme synthesis pathway single gene attenuation plasmid: and (3) using the pUCS plasmid as a template, performing reverse PCR amplification by using a designed primer to obtain a full-length plasmid fragment, and performing self-ligation to obtain plasmids of pUCS-anti-hemE, pUCS-anti-hemF, pUCS-anti-hemG and pUCS-anti-hemH.

Construction of a heme synthesis pathway double-gene weakening plasmid: the fragment was purified by digesting plasmid pUCS-anti-hemE with Spe I and Pst I, and the smaller fragment was recovered by digesting pUCS-anti-hemF plasmid with Xba I and Pst I. The two fragments were ligated with T4 ligase to generate plasmid pUCS-anti-hemEF, which was used to attenuate hemE and hemF simultaneously. Plasmids pUCS-anti-hemEG, pUCS-anti-hemEH, pUCS-anti-hemFG, pUCS-anti-hemFH and pUCS-anti-hemGH can be obtained by the same method.

Constructing plasmid combining a heme synthesis pathway single gene weakening module and an HBA synthesis module: the plasmids of pUCS-anti-hemE, pUCS-anti-hemF, pUCS-anti-hemG and pUCS-anti-hemH are used as templates, elements of hemE, hemF, hemG and hemH weakened by sRNA are respectively amplified by SRNA-HBA-F-GBS and SRNA-HBA-R-GBS primers, and are inserted into BamHI sites of pET28-HBA plasmids through GibsonassembY to obtain plasmids of pET28-HBA-anti-hemE, pET28-HBA-anti-hemF, pET28-HBA-anti-hemG and pET 28-HBA-anti-hemH.

Constructing plasmid combining a heme synthesis pathway double-gene weakening module and an HBA synthesis module: the plasmids of pUCS-anti-hemEF, pUCS-anti-hemEG, pUCS-anti-hemEH, pUCS-anti-hemFG, pUCS-anti-hemFH and pUCS-anti-hemGH are used as templates, elements weakening hemEF, hemEG, hemEH, hemFG, hemFH and hemGH by sRNA are respectively amplified by SRNA-HBA-F-GBS and SRNA-HBA-R-GBS primers, and are inserted into pET28-HBA plasmid BamHI sites through Gibsassemby to obtain the plasmids of pET28-HBA-anti-hemEF, pET28-HBA-anti-hemEG, pET 28-HBA-anti-hempE, pET 28-anti-hemFG, pET 46T-29-HBA-hemFH and HBS-anti-59GH 25.

Construction of pET 28-HBA-anti-hemFG-RhemOBCD plasmid: the plasmid pET 28-HBA-anti-hemFG-RhemOBCD is obtained by cloning to the SacI and HindIII sites of the plasmid pET28-HBA-anti-hemFG by Gibson assembly, using a p15 ASI-RhemOBCD plasmid as a template, and using primers RPhemOBCD-F-pET28-Sac I and RPhemOBCD-R-pET28-Hind III to amplify the plasmid RhemOBCD and a promoter part thereof. The primers used in this section were as follows:

TABLE 3 primers used in construction of recombinant HBA-synthesizing bacteria that attenuate genes of the heme synthesis pathway

Plasmids pET28-HBA-anti-hemE, pET28-HBA-anti-hemF, pET28-HBA-anti-hemG, pET28-HBA-anti-hemH, pET28-HBA-anti-hemEF, pET28-HBA-anti-hemEG, pET28-HBA-anti-hemEH, pET28-HBA-anti-hemFG and pET28-HBA-anti-hemFH are respectively transformed into Escherichia coli MG1655(DE3) to obtain FH189, FH190, FH191, FH192, FH193 and FH 194. The strains and plasmids used in this section were as follows:

TABLE 4 recombinant bacteria for HBA synthesis of genes weakening heme synthesis pathway and plasmids thereof

2. Fermentation of the recombinant strain, separation, purification and quantification of HBA, and quantification of ALA were performed in the same manner as in example 1. The only change was to increase the amount of glucose added to the TYG medium from 2g/L to 10 g/L.

3. Results of fermentation

After the HBA producing strain expresses the Urogen III precursor synthesis module, the obvious change of the color of thalli is found in the fermentation process, and the reason is that the Urogen III flows to a heme synthesis way. Too much heme is not only toxic to cells but also results in a decrease in HBA yield. To inhibit hemE synthesis, metabolic flux was directed to HBA, genes hemE, hemF, hemG, hemH of FH168 hemE synthesis pathway were inhibited by sRNA, respectively, and the strains were named FH185, FH186, FH187, FH188, respectively. To further increase the level of the engineered bacteria HBA, we attenuated the strains of genes hemEF, hemEG, hemEH, hemFG, hemFH, hemGH for heme synthesis by sRNA combinations, which are referred to as FH189, FH190, FH191, FH192, FH193, FH194, respectively.

The results of the fermentation experiments are shown in FIGS. 4-6. The HBA unit cell yield of control FH168 was 6.43mg/g dry cell weight. The yields of HBA after fermentation of FH185, FH186, FH187 and FH188 after gene attenuation of the heme synthesis pathway were improved to different extents, and the maximum yields per cell reached 6.68 mg/g, 8.66mg/g, 9.34mg/g and 8.66mg/g of dry cell weight, respectively (FIG. 6). Although the HBA production of FH168 was reduced as a proportion of dry cell weight after increasing glucose concentration, the maximum OD was reduced₆₀₀The total HBA production increased slightly from 3.11 to 4.50 (FIG. 4). The ALA yield of all recombinant bacteria is close to that of 0.65g/L to 0.70g/L (FIG. 5).

Recombinant bacteria with combined attenuation of heme synthesis genes all had different degrees of improvement except for slight decrease in HBA yield of FH190 strain (FIG. 9). Therefore, the HBA yield reaches the highest 20h after fermentation induction, wherein the HBA yield of FH192 unit cells reaches the highest 14.09mg/g DCW, and the OD is at the moment₆₀₀At 4.00, maximum OD₆₀₀Was 4.78 of 32h of fermentation induction (FIG. 7). Maximum OD of all other strains₆₀₀All increased, and possibly reduced heme synthesis, resulting in reduced cytotoxicity. In addition, there was no significant change in ALA production by all strains (FIG. A)8)。

In order to reduce the metabolic burden on cells caused by multiple plasmids, a precursor synthesis module is cloned from a p15ASI-RPhemOBCD plasmid onto a pet28-HBA-anti-hemFG plasmid to obtain a pet28-HBA-anti-hemFG-RPhemOBCD plasmid. Coli MG1655(DE3) (from the same patent as the co-pending patent "DE novo vitamin B synthesis₁₂The recombinant Escherichia coli, and a construction method and application thereof ") to obtain the FH228 strain. The biomass of the strain is not reduced to 38h, and the maximum OD₆₀₀5.93 (FIG. 10), an increase over the previous recombinant bacteria, indicating that the reduction of metabolic burden increases biomass. The maximum HBA yield was 12.88mg/g DCW (FIG. 10).

Example 3

Construction and fermentation of optimized CBAD synthetic recombinant bacteria

1. Construction of optimized CBAD synthetic recombinant bacteria

Construction of pCDF-RccobB-BmcobN-his-BmcobS-BmcobT-cbiMNQO plasmid: p15ASI-cbiMNQO plasmid is used as a template, and Rccbi-F-pac I-gbs and Rccbi-R-pac I-gbs primers are used for amplifying P_TacThe cbiMNQO expression cassette is cloned to the pacI enzyme cutting site of the pCDF-RccobB-BmcobN-his-BmcobS-BmcobT plasmid through Gibson assembly to obtain the plasmid.

pET 28-HBA-anti-hemFG-RpheOBCD plasmid and pCDF-RccobB-BmcobN-his-BmcobS-BmcobT-cbiMNQO plasmid were co-transferred to E.coli MG1655(DE3), and no corrin compound was detected after fermentation, presumably due to plasmid instability. For this purpose, the end a gene of e.coli MG1655(DE3) was first knocked out by the CRISPR/Cas9 method to give FH 224. The cobalt-uptake gene module, i.e., P, was then inserted by the CRISPR/Cas9 method_TacThe cbinmqo expression cassette integrates into the ldhA site of FH224, resulting in FH 225. Finally, the uroporphyrinogen III gene module, P, was transformed by the CRISPR/Cas9 method_Tac-integration of the RphemOBCD expression cassette into the arabinose-inducible promoter site of FH225, resulting in FH236 as a underpan cell for the synthesis of cobalt (II) quinolinic acid a, c-diamide.

Construction of pCDF-RccobbmcobNSTW plasmid: the genome B.melitensis bv.1str.16M is used as a template, BMcobW-F-Ecor I and BMcobW-R-Xba I primers are used for amplifying BmcobW, and the BmcobW is cloned to the Ecor I and Xba I enzyme cutting sites of pTrc99a plasmid to obtain pTrc99a-BmcobW plasmid. Using this plasmid as a template, the BmcobW expression cassette was amplified using primers PTrc-BMcobW-Xho I-F-gbs and PTrc-BMcobW-Xho I-R-gbs, and cloned into the Xho I cleavage site of the pCDF-RccobB-BmcobN-his-BmcobS-BmcobT plasmid by Gibson assembly, thereby obtaining the pCDF-RccobBBmcobNSTW plasmid.

Construction of pCDF-BWNID plasmid: the genome of Pseudomonas denticifica ATCC 13867 is used as a template, PdcobW fragments are amplified by PDcobW-F-Gibson and PDcobW-R-Gibson primers, PdcobN fragments are amplified by PDcobN-F-Gibson and PDcobN-R-Gibson primers, and the PdcobWN fragments are obtained by fusing the PdcobWN fragments by SOE-PCR. And (3) taking the pCDF-RccobB plasmid as a template, amplifying the backbone of the pCDF-RccobB plasmid by using primers PCDF-RccobB-gbs-2-F2 and PCDF-RccobB-gbs-2-R2, and obtaining the pCDF-RccobB-PdcobWN plasmid by Gibsassembly with a PdcobWN fragment. The chlID fragment was amplified using the chlID-pet28-F-Gibson, and the chlID-pet28-R-Gibson primers, using the P.dentrificans ATCC 13867 genome as a template. The pET28a plasmid backbone was amplified using pET28-chlID-F-Gibson and pET28-chlID-R-Gibson primers using pET28a plasmid as a template, and Gibson assembly with the chlID fragment to give pET28-chlID plasmid. The plasmid pET28-chlID is used as a template, a chlID expression cassette is amplified by using primers T7-chlID-F-Gibson and T7-chlID-R-Gibson, and is cloned to a Bgl II enzyme cutting site of a pCDF-RccobB-PdcobWN plasmid through Gibson assembly, so as to obtain the pCDF-BWNID plasmid.

Table 5 primers for construction of optimized CBAD-synthesizing recombinant bacteria

pET28-HBA-anti hemFG and pCDF-RccobbmcobNSTW plasmid were co-transformed into FH236, resulting in FH 274. pET28-HBA-anti hemFG and pCDF-BWNID plasmid were co-transferred to FH236, resulting in FH 275.

The strains and plasmids used in this section were as follows:

table 6 optimized CBAD synthetic recombinant bacteria and plasmid used by same

2. Fermentation of synthetic recombinant bacteria for CBAD

Single colonies were picked from the plates and cultured overnight in tubes. Then, the cells were inoculated into a 250mL Erlenmeyer flask containing 50mL of LB medium at an inoculum size of 1%, and cultured for 12 hours. Then inoculated at 5% inoculum size to 600mL containing 20mg/LCoCl_2·6H₂TYG medium (5g/L yeast extract, 10g/L tryptone, 5g/L KH)₂PO₄10g/Lglucose, 2g/L glycine, 10g/L succinic acid, 15g/L betaine with initial pH adjusted to 6.5 with NaOH) in a 3L Erlenmeyer flask at 37 ℃ at 200 r.min^-1Cultured under conditions of OD₆₀₀When 0.6 was reached IPTG was added to a final concentration of 0.4mM, and the temperature was changed to 28 ℃ for cultivation.

3. LC-MS identification of CBAD

The analysis was carried out on an Agilent 1200/BrukermicrOTOF-Q II apparatus fitted with an Agilent TC C18 column (4.6X 250 mm). The amount of sample was 25. mu.L. The detection wavelength of HBA and HBAD was 329 nm. The detection wavelength of the cobalt (II) quinoline acid a, c-diamide is 314 nm. Phase A was water containing 0.1% formic acid and phase B was methanol containing 0.1% formic acid. The temperature of the chromatographic column is controlled at 30 ℃, the flow rate is 0.7ml/min, and the gradient elution conditions are as follows: maintaining 25% B for 0-5 min; 5-15min, 34% B; 15-19min, 100% B; 19-24min, 100% B; 24-25min, 25% B; 25-35min, 25% B.

The mass spectrum adopts a positive ion mode, and the parameters are set as follows: needle voltage, 4500V; spray pressure, 1.0 Bar; an ion source, electrospray ionization; atomizing airflow speed, 6.0L/min; the temperature of atomizing gas is 180 ℃. The scanning range is 400-2000 (m/z).

To increase HBA production and to resolve plasmid instability, e was knocked outndA integration of uroporphyrinogen III gene module and cobalt uptake gene module on the genome resulted in the bottom plate cell FH 236. CBAD was then synthesized in FH236 by expressing cobalt chelatases of the HBA module, HBAD module, b. Meanwhile, considering that CobW can play a role in transmitting cobalt ions, an expression cassette of the cobW is integrated on a CBAD gene module to obtain a pCDF-RccobbmcobNSTW plasmid. FH274 was a strain containing pET28-HBA-anti hemfg plasmid and expressing cobNSTW derived from b.melitensis, and FH275 was a strain containing pET28-HBA-anti hemfg plasmid and expressing cobN, chlID, cobW derived from p.denifican. To evaluate the yield of both bacteria, the two strains were treated with a solution containing 10g/L glucose and 20mg/L CoCl, due to the absence of CBAD standards₂·6H₂And performing LC-MS analysis after TYG culture medium fermentation. The CBAD yield of the two bacteria is greatly improved compared with that of the original bacteria FH164 strain. Fig. 11 and 12 are CBAD mass spectrograms of FH274 and FH275, respectively. Indicating that CBAD production can be improved by expressing uroporphyrinogen III gene modules and attenuating the heme pathway.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> Escherichia coli recombinant bacterium for synthesizing cobalt (II) quinoline acid a, c-diamide and construction method and application thereof

<130>P2017-2122

<160>46

<170>PatentIn version 3.5

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<213> Sinorhizobium meliloti (Sinorhizobium meliloti)

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<213> Rhodopseudomonas palustris (Rhodopseudomonas palustris)

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<213> Sinorhizobium meliloti (Sinorhizobium meliloti)

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gtcatcctct cgaccaaggg cgaccggatc accgaccgct cgctcgccga gatcggcggc 180

aagggcctgt tcaccgaaga gatcgagcaa caattgctgt cgggcgaact cgatttcgct 240

gtgcattcgt ccaaggacat gccgacgaaa ctgcctgacg gcctctgtct ttccgccttc 300

ctgccgcgtg aggacattcg agatgccttc atcggtcgga cggcaaaaaa actgatggaa 360

ctgccgcagg gcgtgaccgt cggctcgtcg tcgctgcgcc gccaggcgct gatccgccgg 420

ctgcgtccgg atatcaatgt catcacctat cgcggccagg tggaaacgcg gctgagaaag 480

ctcgccgagg gccaggtcga tgcgacactg cttgcctatg cgggcctcaa gcgtctcggc 540

atgaccgacg ttccgaccga actgctcgac ccccaggaat tcccgccggc gccggcccaa 600

ggcgcgatct gcatcgagag ccgcatcggc gacagccgca tcaacgacct gctcgcagcc 660

atcgacgatc cccgcaccca tgaggcagtc gcctgcgagc gtggcttcct cgcgacgctc 720

gacggctcgt gccgcacgcc gatcgccggt ctcgccacct cggacggcac ccatctcagc 780

ttttccggca tgatcctcac gcccgacggc cagacccatc accgggttac gatcgagggc 840

aaggccaccg acgccgaagc gcttgggcaa aaggccggcg aagagatccg cgccaaggcc 900

ggccccggct tctttgcaag ctggacttaa 930

<210>6

<211>714

<212>DNA

<213> Sinorhizobium meliloti (Sinorhizobium meliloti)

<400>6

atgcgcgtgc tcgtcacccg gccgcttccc gccgccgagg cgacggtacg ccggctggaa 60

gccgccggcc accggccgat cctgctgccg ctgatgcagg cgacacatct tgctgccgtc 120

tctgttgccg cgcttgaagt gccgcatgcg gcgatcgcgc tcaccagcgc cgaagccatt 180

cgggtgctcg tatcgtcaga tgcagacctg tcacagcatc tggcgacccc gtgcttctgc 240

gtgggcgccg ccacggcgca agcagcagcc gggctcggtt tctccgatct gcgcatagcg 300

gagggtaccg gccagtccct ggcggaactg atcggtgcga ccatcgacac gctgccgcca 360

cttcccctgc tctatctggc aggaacgccg cgctccgaag ggctggaaaa gggactgaga 420

caccgcggca tcgaacaccg gacggtcgag tgctaccgca tggagccgat cgcccattcg 480

cgcgccgcga tcgaagatct gcggcgaagc agccgtcccg acgccgtgct tttatactcg 540

cgagagaccg cgcgacagtt cgttcgcctg ctttccgaag ccggtgtcga tgctgcttcc 600

tttgcgccgc gctacctctg cctcagcccc gtggtggccg aggcactgcc gggtaacgtc 660

gtggcggaga ccgccgcaag caccgatgaa gacagccttt tcagtcttct ctaa 714

<210>7

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

tyaacgggag gacytcatga a 21

<210>8

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

cagcaacgag accatcaagc a 21

<210>9

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

caggtgctga gcaagatctc 20

<210>10

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

tcggcgatcg aggtgatgtt 20

<210>11

<211>48

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

gatccccaag gagatatacc atgcagtaca atcaattctt tcaagacg 48

<210>12

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

tccacattgt cacctcctta tcactccgca gcgatcg 37

<210>13

<211>45

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

gatccccaag gagatatacc atgaactacg aagcctattt caagc 45

<210>14

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

tccacattgt cacctcctta tcaggcttgc cttggcg 37

<210>15

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ttcgagctct gcgactcctg cattagg 27

<210>16

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

cccaagcttt tagagaagac tgaaaaggct g 31

<210>17

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

gtcgacggag ctcgaattcg caggaaacag ctatgac 37

<210>18

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

aggaataggc actagttttg gtaaaacgac ggccagt 37

<210>19

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

ttaaaaacga gcaaccatta tcaccgcca 29

<210>20

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

gttcggtcat tttctgttgg gccattgcat tg 32

<210>21

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

cacaccaggt taagcaacca ttatcaccgc ca 32

<210>22

<211>35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

cgtcgggttt cattttctgt tgggccattg cattg 35

<210>23

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

tcttttctca acaagcaacc attatcaccg cca 33

<210>24

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

attaatgttt tcactttctg ttgggccatt gcattg 36

<210>25

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

aaaaccggta tcgcaaccat tatcaccgcc a 31

<210>26

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>26

agtctgacgc attttctgtt gggccattgc attg 34

<210>27

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>27

ttcgagctct gcgactcctg cattagg 27

<210>28

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>28

cccaagcttt tagagaagac tgaaaaggct g 31

<210>29

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>29

tctactagcg cagcttaatg cgactcctgc attaggttg 39

<210>30

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>30

tggcagcagc ctaggttaat ctaacgccgg gtccagc 37

<210>31

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>31

ggaattctaa ggaggtgaca atatgcaggg acagaaaatt cc 42

<210>32

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>32

gctctagagt cagtcggcat ctttcac 27

<210>33

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>33

gatcgctgac gtcggtaccc ttgacaatta atcatccggc 40

<210>34

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>34

cagcggtttc tttaccagac tcagtcggca tctttcac 38

<210>35

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>35

catatgtata tctccttctt atacttaact aatatac 37

<210>36

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>36

gcagatctca attggatatc gg 22

<210>37

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>37

aagaaggaga tatacatatg aaaaccctgg ccaaactcc 39

<210>38

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>38

atgcattaat tatacctcct ttacgccagg gcggtacg 38

<210>39

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>39

aggaggtata attaatgcat ctgctgcgca cc 32

<210>40

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>40

gatatccaat tgagatctgc tcactgctcc tcgctgtc 38

<210>41

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>41

atggtatatc tccttcttaa agttaaac 28

<210>42

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>42

aattcgagct ccgtcgac 18

<210>43

<211>35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>43

taagaaggag atataccatg agcgcccttc cccat 35

<210>44

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>44

ttgtcgacgg agctcgaatt tcacgagggt agcggagg 38

<210>45

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>45

acagcgagga gcagtgagca cgagatctcg atcccgcg 38

<210>46

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>46

gccggccgat atccaattga tcacgagggt agcggagg 38

Claims (13)

1. An engineered bacterium for producing CBAD (cobalt (II) oxoacid a, c-diamide) or a precursor thereof, wherein said engineered bacterium is escherichia coli, and said engineered bacterium contains an exogenous gene module (gene module) comprising:

(a) an uroporphyrinogen III gene module expressing a gene for the biosynthesis of uroporphyrinogen III,

wherein the gene for biosynthesis of uroporphyrinogen III comprises:hemAorhemOGene, gene,hemBGene, gene,hemCA gene, andhemDthe gene(s) is (are),

and, the above-mentionedhemAThe gene is derived fromS. melilotiA strain of, orR. palustriStrains of the specieshemOThe gene is derived fromR. palustriStrains of the specieshemBGene, gene,hemCGenes andhemDthe gene is derived fromS. melilotiA strain;

(b) an HBA gene module expressing a gene for biosynthesis of HBA using uroporphyrinogen III as a raw material;

(c) an HBAD gene module that expresses a gene for biosynthesis of HBAD using HBA as a raw material;

(d) a CBAD gene module expressing a gene for biosynthesis of CBAD using HBAD as a raw material;

(e) a cobalt-uptake gene module expressing a gene encoding a transporter for transporting cobalt ions into the cell; and

(f) a heme pathway weakening module for down-regulating expression of a heme synthesis gene, wherein the heme synthesis gene comprises:hemEgene, gene,hemFGene, gene,hemGA gene, and/orhemHA gene.

2. The engineered bacterium of claim 1, wherein the engineered bacterium has an endogenous geneendAIs down-regulated or deleted.

3. The engineered bacterium of claim 1, wherein said uroporphyrinogen III gene module is integrated into the genome.

4. The engineered bacterium of claim 3, wherein said uroporphyrinogen III gene module is integrated into arabinose-inducible promoter P_BADA site.

5. The engineered bacterium of claim 1, wherein said genes for biosynthesis of HBA comprise:cobAgene, gene,cobIGene, gene,cobGGene, gene,cobJGene, gene,cobMGene, gene,cobFGene, gene,cobKGene, gene,cobLA gene, andcobHa gene.

6. The engineered bacterium of claim 1, wherein said genes for biosynthesis of HBAD comprise:cobBa gene.

7. The engineered bacterium of claim 1, wherein said genes for biosynthesis of CBAD comprise: (i)cobNgene, gene,cobSGene, gene,cobTGene, gene,cobWA gene, or a combination thereof, or (ii)cobNGene, gene,chlIGene, gene,chlDGene, gene,cobWA gene, or a combination thereof.

8. The engineered bacterium of claim 1, wherein said gene encoding a transporter protein for intracellular transport of cobalt ions comprisescbiMNQOAn operator, saidcbiMNQOThe operator includes: expressed in tandemcbiMGene, gene,cbiNGene, gene,cbiQGenes andcbiOa gene.

9. The engineered bacterium of claim 1, which is characterized in thatCharacterized in that the cobalt absorption gene module also comprisescbtABAn operator, saidcbtABThe operator includes: expressed in tandemcbtAGenes andcbtBa gene.

10. The engineered bacterium of claim 1, wherein said engineered bacterium is used for de novo synthesis of CBAD.

11. A method of producing CBAD or a precursor thereof, comprising the steps of:

(i) culturing the engineered bacterium of claim 1, thereby obtaining a fermentation product comprising CBAD or a precursor thereof; and

(ii) isolating CBAD or a precursor thereof from the fermentation product.

12. A method for constructing the engineering bacteria of claim 1, which comprises the following steps:

(a) constructing a vector containing an uroporphyrinogen III gene module expressing a gene for biosynthesis of uroporphyrinogen III or integrating it into a genome;

wherein the genes for biosynthesis of uroporphyrinogen III comprise:hemAorhemOGene, gene,hemBGene, gene,hemCA gene, andhemDthe gene(s) is (are),

and, the above-mentionedhemAThe gene is derived fromS. melilotiA strain of, orR. palustriStrains of the specieshemOThe gene is derived fromR. palustriStrains of the specieshemBGene, gene,hemCGenes andhemDthe gene is derived fromS. melilotiA strain;

(b) constructing a vector for down-regulating the expression of the heme synthesis gene,

wherein, the heme synthesis gene comprises:hemEgene, gene,hemFGene, gene,hemGA gene, and/orhemHA gene; and

(c) and (c) respectively transferring the vectors obtained in the step (a) and the step (b) into escherichia coli to obtain the engineering bacteria which contain the gene modules and have the expression of the endogenous heme synthesis gene down-regulated.

13. Use of the engineered bacterium of claim 1 as a strain for the fermentative production of CBAD or a precursor thereof.

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