A kind of high-yield 5-aminolevulinic acid recombinant Escherichia coli strain and its application

A technology of aminolevulinic acid and recombinant Escherichia coli, which is applied in the fields of metabolic engineering and microbial fermentation, can solve the problems of many reaction steps, many by-products, and low yield, and achieves the effect of optimizing the synthesis path and improving the utilization efficiency.

Active Publication Date: 2022-06-28
北京道合成企业管理有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the chemical synthesis method has limited its popularization and application due to many reaction steps, many by-products, difficult separation and purification of products, and low yield, and the toxic reagents used in chemical synthesis will also cause environmental pollution.

Method used

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  • A kind of high-yield 5-aminolevulinic acid recombinant Escherichia coli strain and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Embodiment 1, construct the recombinant plasmid that expresses 5-ALA synthase

[0106] 1. Construction of recombinant plasmid pET28b-hML

[0107] (1) Extraction of E. coli genomic DNA and PCR amplification of heML gene.

[0108] The genomic DNA of Escherichia coli BW25113 or BL21(DE3) was extracted by bacterial genome extraction kit. Using the extracted genome as a template and heML-F and heML-R as primers, the gene fragment heML was amplified by high-fidelity Phanta max Super-Fidelity DNA polymerase PCR, and the target fragment was recovered by agarose gel electrophoresis.

[0109] (2) Construction of recombinant plasmid containing heML gene.

[0110] Using the pET28b empty plasmid as a template and V-pET28b-F and V-pET28b-R as primers, the vector fragment ET28b was amplified by high-fidelity Phantamax Super-Fidelity DNA polymerase, and the target fragment was recovered by agarose gel electrophoresis. ; The above-mentioned heML gene fragment was ligated with the ET2...

Embodiment 2

[0119] Embodiment two, construct the recombinant Escherichia coli strain that produces 5-ALA

[0120] 1. Construction of host bacteria

[0121] Using E. coli BW25113 as the starting strain, T7 was inserted into BW25113 using CRISPR / Cas9 technology (see the document "CRISPR / Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113makes T7 expression system work efficiently", Ye Changchuan). RNAP gene, construct BW25113-T7 strain.

[0122] Taking BW25113-T7 as the starting bacteria, a host bacteria including one or more of the following characters was constructed, and the genotypes of each host bacteria are shown in Table 1 below.

[0123] Table 1. Host strains and their genotypes

[0124] host bacteria genotype BW-T7 BW25113 int::(lacI::PlacUV5::T7 gene) ΔybhC 5-ALA01 BW-T7 △hemF 5-ALA02 BW-T7 △hemF △galR 5-ALA03 BW-T7 △hemF △galR::119-ppc 5-ALA04 BW-T7 △hemF △galR::119-ppc △poxB 5-ALA05 BW-T7 △hemF △galR::119-ppc △...

Embodiment 3

[0150] Embodiment 3. Utilize recombinant Escherichia coli engineering strain to produce 5-ALA

[0151] 1. Induction of 5-ALA-producing strains

[0152] The positive monoclonal bacteria stored at -80°C were streaked onto the corresponding resistant LB plates, and cultured upside down at 37°C for 12h. Single clones were picked, inoculated into liquid LB medium containing corresponding resistance, and cultured overnight at 37°C and 200 rpm with shaking. The production strains cultivated overnight were transferred to 30mL M9+yeast powder+gly medium at 2% inoculum, and cultivated to OD at 37°C and 200rpm. 600 =0.8, add IPTG with a final concentration of 0.1 mM for induction, and induce culture at 37°C and 200 rpm.

[0153] M9+yeast powder+gly medium formula: Na 2 HPO 4 ·12H 2 O 17.1g / L, KH 2 PO 4 3.0g / L, NaCl 0.5g / L, NH 4 Cl 1.0g / L, MgSO 4 2mM, CaCl 2 0.1mM, glucose 10g / L, yeast powder 2g / L, glycine 2g / L.

[0154] 2. Detection of 5-ALA

[0155] After adding the induce...

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Abstract

The invention discloses a recombinant Escherichia coli strain for producing 5-aminolevulinic acid (5-ALA); the invention also discloses a high-efficiency approach for synthesizing 5-aminolevulinic acid: strengthening the 5-aminolevulinic acid of Escherichia coli itself Aminolevulinic acid synthase (HemL and HemA), the strain initially has the ability to synthesize 5-aminolevulinic acid; strengthen the expression of the 5-aminolevulinic acid efflux protein eamA gene, and increase the 5-aminolevulinic acid efflux of the strain Removal ability; introduce exogenous 5-aminolevulinic acid synthase hemA gene to enhance the 5-aminolevulinic acid synthesis ability of the strain; transform galR, glk, ppc genes in the glucose utilization pathway to improve glucose utilization efficiency; knock out metabolism Bypassed genes (hemF, poxB, and aceB). The recombinant Escherichia coli strain constructed by the invention has the ability to efficiently utilize glucose and glycine to synthesize 5-aminolevulinic acid, making it useful for industrial production of 5-aminolevulinic acid.

Description

technical field [0001] The invention relates to the fields of metabolic engineering and microbial fermentation, in particular to a recombinant Escherichia coli strain with high production of 5-aminolevulinic acid (5-ALA) and application thereof. Background technique [0002] 5-aminolevulinic acid (5-ALA) is a non-protein amino acid widely present in bacteria, fungi, animals and plants, and is an important intermediate metabolism in the biosynthesis of pyrrole compounds. product. In the field of agriculture, because 5-ALA is easily degraded in the environment, it is non-toxic to crops, animals and humans, and can be used as plant growth regulators, green herbicides and pesticides. In the medical field, 5-ALA, as a photodynamic drug with few side effects and good permeability, has been widely used in the diagnosis and photodynamic therapy of acne, skin cancer, bladder cancer, breast cancer, upper gastrointestinal cancer, etc. . [0003] At present, the synthesis methods of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/54C12N15/60C12N15/51C12N15/31C12P13/00C12R1/19
CPCC12N9/90C12N9/0008C12N9/001C12N9/88C12N9/1205C12N9/1025C12N9/1029C07K14/245C12P13/005C12Y504/03008C12Y102/0107C12Y103/03003C12Y401/01031C12Y102/03003C12Y207/01002C12Y203/03009C12Y203/01037
Inventor 王春平杨梦杰
Owner 北京道合成企业管理有限公司
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