z-logo
Premium
Plasmid Design for Tunable Two‐Enzyme Co‐Expression Promotes Whole‐Cell Production of Cellobiose
Author(s) -
Schwaiger Kathari.,
Voit Alena,
Dobiašová Hana,
Luley Christiane,
Wiltschi Birgit,
Nidetzky Bernd
Publication year - 2020
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.202000063
Subject(s) - cellobiose , plasmid , chemistry , biochemistry , specific activity , enzyme , transcription (linguistics) , microbiology and biotechnology , biology , dna , linguistics , philosophy , cellulase
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.

This content is not available in your region!

Continue researching here.

Having issues? You can contact us here