TY - JOUR
T1 - Plasmid Design for Tunable Two-Enzyme Co-Expression Promotes Whole-Cell Production of Cellobiose.
AU - Schwaiger, Katharina N.
AU - Voit, Alena
AU - Dobiašová, Hana
AU - Luley, Christiane
AU - Wiltschi, Birgit
AU - Nidetzky, Bernd
PY - 2020/11
Y1 - 2020/11
N2 - Catalyst development for biochemical cascade reactions often follows a “whole-cell-approach” in which a single microbial cell is made to express all required enzyme activities. Although attractive in principle, the approach can encounter limitations when efficient overall flux necessitates precise balancing between activities. This study shows an effective integration of major design strategies from synthetic biology to a coherent development of plasmid vectors, enabling tunable two-enzyme co-expression in E. coli, for whole-cell-production of cellobiose. An efficient transformation of sucrose and glucose into cellobiose by a parallel (countercurrent) cascade of disaccharide phosphorylases requires the enzyme co-expression to cope with large differences in specific activity of cellobiose phosphorylase (14 U mg
−1) and sucrose phosphorylase (122 U mg
−1). Mono- and bicistronic co-expression strategies controlling transcription, transcription-translation coupling or plasmid replication are analyzed for effect on activity and stable producibility of the whole-cell-catalyst. A key role of bom (basis of mobility) for plasmid stability dependent on the ori is reported and the importance of RBS (ribosome binding site) strength is demonstrated. Whole cell catalysts show high specific rates (460 µmol cellobiose min
−1 g
−1 dry cells) and performance metrics (30 g L
−1; ∼82% yield; 3.8 g L
−1 h
−1 overall productivity) promising for cellobiose production.
AB - Catalyst development for biochemical cascade reactions often follows a “whole-cell-approach” in which a single microbial cell is made to express all required enzyme activities. Although attractive in principle, the approach can encounter limitations when efficient overall flux necessitates precise balancing between activities. This study shows an effective integration of major design strategies from synthetic biology to a coherent development of plasmid vectors, enabling tunable two-enzyme co-expression in E. coli, for whole-cell-production of cellobiose. An efficient transformation of sucrose and glucose into cellobiose by a parallel (countercurrent) cascade of disaccharide phosphorylases requires the enzyme co-expression to cope with large differences in specific activity of cellobiose phosphorylase (14 U mg
−1) and sucrose phosphorylase (122 U mg
−1). Mono- and bicistronic co-expression strategies controlling transcription, transcription-translation coupling or plasmid replication are analyzed for effect on activity and stable producibility of the whole-cell-catalyst. A key role of bom (basis of mobility) for plasmid stability dependent on the ori is reported and the importance of RBS (ribosome binding site) strength is demonstrated. Whole cell catalysts show high specific rates (460 µmol cellobiose min
−1 g
−1 dry cells) and performance metrics (30 g L
−1; ∼82% yield; 3.8 g L
−1 h
−1 overall productivity) promising for cellobiose production.
UR - http://europepmc.org/abstract/med/32668097
UR - http://www.scopus.com/inward/record.url?scp=85089081795&partnerID=8YFLogxK
U2 - 10.1002/biot.202000063
DO - 10.1002/biot.202000063
M3 - Article
C2 - 32668097
SN - 1860-6768
VL - 15
JO - Biotechnology Journal
JF - Biotechnology Journal
IS - 11
M1 - 2000063
ER -