Details

Autor(en) / Beteiligte

• Chen, Hui
• Huang, Rui
• Zhang, Y.-H. Percival

Titel

Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3)

Ist Teil von

• Applied microbiology and biotechnology, 2017-06, Vol.101 (11), p.4481-4493

Ort / Verlag

Berlin/Heidelberg: Springer Berlin Heidelberg

Erscheinungsjahr

2017

Quelle

MEDLINE

Beschreibungen/Notizen

• The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).