title: Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3).
abstract: 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).
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, VA, 24061, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, VA, 24061, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Huang
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, VA, 24061, USA. ypzhang@vt.edu.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China. ypzhang@vt.edu.
authors.forename: Y-H Percival
authors.initials: YP
authors.lastname: Zhang
mesh_terms: D055162:Biocatalysis; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics; D060847:Metabolic Engineering; D009249:NADP; D010733:Phosphoglucomutase / Q000235:genetics; D010734:Phosphogluconate Dehydrogenase / Q000235:genetics; D006005:Phosphorylases / Q000235:genetics; D010957:Plasmids; D011134:Polysaccharides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D013728:Terminator Regions, Genetic; D014994:Xylose / Q000378:metabolism
chemical_list: D004798:Enzymes; D011134:Polysaccharides; D011994:Recombinant Proteins; D009249:NADP; C008315:maltodextrin; D014994:Xylose; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D006005:Phosphorylases; D010733:Phosphoglucomutase
keywords: Cascade biocatalysis; Co-expression; E. coli BL21(DE3); Expression control; T7 terminator