4 research outputs found
Molecular tool development for conditional protein degradation in the model bacterium <i>Escherichia coli</i>
CRISPR/Cas9-based genome editing for simultaneous interference with gene expression and protein stability
CRISPR/Cas9-based genome editing for simultaneous interference with gene expression and protein stability
De-bugging and maximizing plant cytochrome P450 production in <i>Escherichia coli</i> with C-terminal GFP fusions
Cytochromes P450 (CYP) are attractive enzyme targets in biotechnology as they catalyze stereospecific C-hydroxylations of complex core skeletons at positions that typically are difficult to access by chemical synthesis. Membrane bound CYPs are involved in nearly all plant pathways leading to the formation of high-value compounds. In the present study, we systematically maximize the heterologous expression of six different plant-derived CYP genes in Escherichia coli, using a workflow based on C-terminal fusions to the green fluorescent protein. The six genes can be over-expressed in both K- and B-type E. coli strains using standard growth media. Furthermore, sequences encoding a small synthetic peptide and a small bacterial membrane anchor markedly enhance the expression of all six genes. For one of the CYPs, the length of the linker region between the predicted N-terminal transmembrane segment and the soluble domain is modified, in order to verify the importance of this region for enzymatic activity. The work describes how membrane bound CYPs are optimally produced in E. coli and thus adds this plant multi-membered key enzyme family to the toolbox for bacterial cell factory design