Recombinant escherichia coli strain with high yield of 5-aminolevulinic acid and application of recombinant escherichia coli strain

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

Active Publication Date: 2022-04-22
北京道合成企业管理有限公司
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  • Abstract
  • Description
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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...

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  • Recombinant escherichia coli strain with high yield of 5-aminolevulinic acid and application of recombinant escherichia coli strain

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Experimental program
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Effect test

Embodiment 1

[0105] Embodiment 1. Construction of a recombinant plasmid expressing 5-ALA synthetase

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

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

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

[0109] (2) Construct a recombinant plasmid containing the hemL gene.

[0110] Using the pET28b empty plasmid as a template, using V-pET28b-F and V-pET28b-R as primers, use high-fidelity Phantamax Super-Fidelity DNA polymerase PCR to amplify the vector fragment ET28b, perform agarose gel electrophoresis, and recover the target fragment ; Use the In-Fusion seamless cloning ...

Embodiment 2

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

[0120] 1. Construct host bacteria

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

[0122] Using BW25113-T7 as the starting bacterium, construct host bacteria that include one or more of the following traits, and the genotypes of each host bacterium are shown in Table 1 below.

[0123] Table 1. Host bacteria 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 △poxB::11...

Embodiment 3

[0150] Embodiment three, utilize recombinant Escherichia coli engineering strain to produce 5-ALA

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

[0152] Streak the positive monoclonal bacterial solution stored at -80°C on the corresponding resistant LB plate, and incubate upside down at 37°C for 12 hours. Pick a single clone, inoculate it into the liquid LB medium containing the corresponding resistance, and shake it overnight at 37°C and 200rpm. The overnight cultured production strain was transferred to 30mL M9+yeast powder+gly medium according to 2% inoculum size, and cultivated to OD at 37°C and 200rpm 600 =0.8, add IPTG with a final concentration of 0.1mM for induction, and induce culture at 37°C and 200rpm.

[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 ...

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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 way for efficiently synthesizing the 5-aminolevulinic acid, wherein the 5-aminolevulinic acid synthetase (HemL and HemA) of the escherichia coli is enhanced, and the strain has the capability of synthesizing the 5-aminolevulinic acid preliminarily; the expression of the 5-aminolevulinic acid efflux protein eamA gene is enhanced, and the 5-aminolevulinic acid efflux capability of the strain is improved; an exogenous 5-aminolevulinic acid synthetase hemA gene is introduced, so that the 5-aminolevulinic acid synthesis capability of the strain is enhanced; galR, glk and ppc genes of a glucose utilization way are modified, and the utilization efficiency of glucose is improved; and genes (hemF, poxB and aceB) of metabolic bypasses are knocked out. The recombinant Escherichia coli strain constructed by the invention has the capability of efficiently synthesizing 5-aminolevulinic acid by using glucose and glycine, so that the recombinant Escherichia coli strain has the application of industrially producing 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 yield 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 easy to degrade in the environment and has no toxicity to crops, animals and humans, it can be used as a plant growth regulator, green herbicide and insecticide. In the medical field, 5-ALA, as a photodynamic drug with few side effects and good penetration, has been widely used in the diagnosis and photodynamic therapy of acne, skin cancer, bladder cancer, breast cancer, upper gastrointestinal cancer, etc. . [0003] Currently, the synthesis methods of 5-AL...

Claims

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Application Information

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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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