Recombinant Escherichia coli for producing l-lactic acid and its application
A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of industrial microorganisms, can solve the problems of failing to meet the requirements of polylactic acid, refractory treatment, and a large amount of calcium sulfate waste.
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Embodiment 1
[0067] Example 1. Integration of L-lactate dehydrogenase encoding gene into Escherichia coli Dlac-206
[0068] The L-lactate dehydrogenase gene (abbreviated as ldhL) was integrated into the fumarate reductase coding gene (abbreviated as frd) site of Escherichia coli Dlac-206 by homologous recombination to obtain recombinant engineering bacteria Slac002, which specifically included the following steps:
[0069] (1) Extraction of Genomic DNA of Lactobacillus plantarum ATCC 14917
[0070] There are a variety of microorganisms in nature that can directly synthesize L-lactic acid. In order to efficiently produce L-lactic acid in Escherichia coli, according to the activity of L-lactate dehydrogenase reported in the literature, the L-lactate dehydrogenase encoding gene ldhL from Lactobacillus plantarum was selected for constructing recombinant engineering bacteria. Among them, Lactobacillus plantarum ATCC14917 was purchased from China General Microbiological Culture Collection Cente...
Embodiment 2
[0083] Example 2, using artificial regulatory elements to control the expression of the gene encoding L-lactate dehydrogenase (ldhL for short)
[0084] Using artificial regulatory elements to control the expression intensity of the ldhL gene, the specific steps include:
[0085] In the first step, using the pXZ-CS plasmid (Tan, et al., Appl Environ Microbiol, 2013, 79:4838-4844) DNA as a template, using the primer Frd-cs-up / 14917-M93CS-down (from Table 2 frd-cs-up and 14917-m93cs-down) to amplify a 2719bp DNA fragment I (including the 50bp nucleotide sequence of the upstream homology arm of the frd gene, the cat-sacB expression cassette 2619bp and the ldhL gene initial 50bp), For the first step of homologous recombination. The amplification system and amplification conditions are consistent with those described in the first step of Example 1(2).
[0086] DNA Fragment I was used for the first homologous recombination: first, the pKD46 plasmid (Datsenko and Wanner2000, Proc Na...
Embodiment 3
[0090] Example 3, Knockout of D-lactate dehydrogenase encoding gene (ldhA for short)
[0091] The two-step homologous recombination method was used to knock out the D-lactate dehydrogenase encoding gene (ldhA for short) of Escherichia coli Slac004 to realize the efficient production of L-lactic acid by the engineered strain. The specific steps include:
[0092] In the first step, using the pXZ-CS plasmid (Tan, et al., Appl Environ Microbiol, 2013, 79:4838-4844) DNA as a template, using primers ldhA-delCS-up / ldhA-delCS-down (from Table 2 ldhA-delcs-up and ldhA-delcs-down) to amplify 2719bp DNA fragment I (ldhA gene upstream homology arm 50bp, cat-sacB expression cassette 2619bp and ldhA gene downstream homology arm 50bp), used for One-step homologous recombination. The amplification system and amplification conditions are consistent with those described in the first step of Example 1(2).
[0093] DNA Fragment I was used for the first homologous recombination: first, the pKD46...
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