Engineering strain, preparation method thereof and method for producing glycollic acid by efficiently utilizing ethylene glycol
A technology for engineering strains and strains, applied in microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of unclear genetic background and genetic modification methods, and achieve the effect of improving utilization.
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Embodiment 1
[0046] Example 1 Wild-type Escherichia coli MG1655 cannot utilize EG
[0047] The wild-type Escherichia coli MG1655 was cultured in LB medium, and 10g / L EG was added to it as the sole carbon source, and the shake flask fermentation experiment at 37°C and 220rpm proved that the wild-type Escherichia coli MG1655 could grow in LB containing 10g / L EG. growth medium, but cannot effectively utilize EG in the medium ( figure 1 ).
Embodiment 2
[0048] Example 2 Screening for ethylene glycol Using foreign genes to obtain recombinant Escherichia coli with ethylene glycol utilization ability
[0049] The pedE and PQQ genes from Pseudomonas putida KT2440 were introduced into Escherichia coli MG1655, cultured using M9 without glucose, and 10 g / L ethylene glycol was added to the medium as a substrate. The experiment verified that E. coli that only introduced pedE and PQQ genes could not utilize ethylene glycol, and the concentration of ethylene glycol increased slightly during the culture process, which may be caused by the volatilization of the medium during the shaker fermentation process. In order to prevent the accumulation of glycolaldehyde and inhibit the utilization of ethylene glycol, the endogenous gene aldA was overexpressed on the basis of introducing exogenous genes pedE and PQQ, and the concentration of ethylene glycol was reduced, but the effect was not obvious ( figure 2 ). In view of the fact that the end...
Embodiment 3
[0050] Example 3 Constructing a heterologous pathway for ethylene glycol utilization in Escherichia coli MG1655
[0051] The exogenous pathway of introducing Pseudomonas putida into Escherichia coli could not fully utilize ethylene glycol in the medium, and the Gox0313 gene from Gluconobacter oxydans and the mutant enzyme fucO(I7L / L8V) of Escherichia coli MG1655, respectively It was introduced into Escherichia coli MG1655 and cultured using M9 without glucose, and 10 g / L of ethylene glycol was added to the medium as a substrate. Among them, the Escherichia coli introduced into the Gox0313 gene of Gluconobacter oxidans can fully utilize 10g / L ethylene glycol in 72h, OD 600 Eventually it gets to 11. However, E. coli introduced with the mutant enzyme fucO(I7L / L8V) had a poor ability to utilize ethylene glycol. On this basis, the ability of E. coli to utilize ethylene glycol after overexpressing the endogenous gene aldA was not improved ( Figure 4 , Figure 5 ). It has been r...
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