Promoter engineering of cascade biocatalysis for α-ketoglutaric acid production by coexpressing l-glutamate oxidase and catalase
Enzymatic transformation is now an attractive alternative for α-ketoglutaric acid (α-KG) production, but the oxidative deamination from l-glutamic acid to α-KG is along with H2O2 accumulation. To remove the effect of H2O2 on α-KG production, in vitro cascade biocatalysis was designed using the purified l-glutamate oxidase (LGOX) from Streptomyces ghanaensis and catalase (KatG) from Escherichia coli W3110, and the optimal ratio of LGOX:KatG (2.0:1250) was detected in this system. To achieve this ratio, in vivo cascade biocatalysis was constructed by varying promoters and ribosome binding sites (RBSs), and optimized by promoter engineering, such as adjusting the junctions between the SD sequence and start codon ATG and tuning the strengths of RBSs. When the final ratio of LGOX:KatG (2.1:1185) in strain E. coli-(T7)LGOX-(rbs2)KatG was used, α-KG concentration and its conversion rate were increased to 106 g L−1 and 96%, respectively. This strategy described here paves the way to the development of cascade biocatalysis for enzymatic production of other chemicals.
Keywordsl-Glutamate oxidase Catalase α-Ketoglutaric acid Promoter engineering Cascade biocatalysis
This work was financially supported by the Key Technologies R&D Program of Jiangsu Province (BE2015307), the National Natural Science Foundation of China (21576117), and the Fundamental Research Funds for the Central Universities (JUSRP51611A).
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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