5-aminolevulinic acid producing strain as well as construction method and application thereof
A technology for producing aminolevulinic acid and strains, which is applied in the fields of genetic engineering and microbial fermentation, and can solve the problems of aggravating the metabolic burden of engineering bacteria, long metabolic pathways, and low yield.
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Embodiment 1
[0071] Embodiment 1. alanine: the construction of glyoxylate transaminase (AGT) expression vector
[0072] In order to verify the role of alanine: glyoxylate transaminase (AGT) in the synthesis of glycine and ALA, first use the pZPA26 vector (p15A ori, Cm resistance, preserved in our laboratory, its structure is as follows figure 1 shown) and the strong promoter J23100 to construct the AGT expression plasmid. Primers AGT-F and AGT-R were designed according to the genome sequence of Saccharomyces cerevisiae published by NCBI (Table 1), and the AGT coding gene fragment with the J23100 promoter was amplified by PCR using the yeast genome as a template. At the same time, primers p26-F and p26-R (Table 1) were used to amplify the fragments of the pZPA26 vector, and the above fragments were recovered and recombined to obtain the AGT expression vector pAGT.
Embodiment 2
[0073] Embodiment 2. Construction of isocitrate lyase (AceA) and AGT co-expression vector
[0074] In order to construct the co-expression vector of isocitrate lyase (AceA) and AGT, primers AceA-F and AceA-R (Table 1) were designed according to the sequence sequence of the E. coli aceA gene published by NCBI, and the genome of E. coli BW25113 was used as a template by PCR The aceA gene fragment was amplified. After the fragment was recovered, it was recombined with pAGT treated with BamHI digestion to obtain the vector pAGT-AceA co-expressing AGT and AceA.
[0075] Table 1. Primer List
[0076]
Embodiment 3
[0077] Embodiment 3. Construction of malate synthase (AceB) deletion mutant
[0078] In order to open up the synthesis pathway of glyoxylate to glycine, it is necessary to delete the gene aceB encoding malate synthase in the glyoxylate cycle in the host bacteria, so that glyoxylate becomes the final product. The aceB:kan strain JW3974 was picked from the keio collection, prepared competently, and transformed into pCP20 (expressing the recombinase FLP). After heat induction, the kan marker gene and the pCP20 plasmid were removed to obtain the aceB-deleted strain BW25113ΔaceB. And the above vectors and pZGA24 (ALAS expression vector) or pZPA6 (ALAS and PPC co-expression vector) were transferred into the strain to obtain recombinant engineering strains BW25113 / pZGA24 / pZPA26, BW25113 / pZGA24 / pAGT, BW25113 / pZGA24 / pAGT-AceA 、BW25113 / pZPA6 / pZPA26、BW25113 / pZPA6 / pAGT、BW25113 / pZPA6 / pAGT-AceA、BW25113ΔaceB / pZGA24 / pZPA26、BW25113ΔaceB / pZGA24 / pZPA26、BW25113ΔaceB / pZGA24 / pAGT-AceA、BW25113ΔaceB / ...
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