An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

Abstract

Membrane-associated Cytochromes P450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compounds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here, we explore a small library of N-terminal expression tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalyzed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than twofold. The developed toolbox serves as a platform to tune P450 performance in microbial cells, thereby facilitating recombinant production of complex plant P450-derived biochemicals. Biotechnol. Bioeng. 2017;114: 751-760. © 2016 Wiley Periodicals, Inc.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii

“…The constraints to achieve high yields are connected to expression of multiple CYPs and reconstruction of pathways with multiple functionally divergent steps (Brown et al, 2015; Li and Smolke, 2016; Zhou et al, 2015). Strategies addressing these issues are for example the development of synthetic microbial consortia of S. cerevisiae and E. coli , optimization of CYPS for functional expression in E. coli , optimization of interactions between the CYPs and their reductase partner and N-terminal modifications (Biggs et al, 2016; Laursen et al, 2016; Vazquez-Albacete et al, 2017; Zhou et al, 2015). …”

Recombinant production of eukaryotic cytochrome P450s in microbial cell factories

“…Another strategy, namely fusing the N-terminus of the P450 to sequences of bacterial proteins, has been extensively studied by Vazquez-Albacete et al [ 27 ], who used C-terminal GfP-fusions of plant cytochrome P450 CYP79A1 for analyzing expression levels. They created P450 fusions with bacterial membrane anchors with the C-terminus facing the cytoplasmic site, bacterial membrane proteins with the C-terminus facing the periplasmic site, bacterial signal peptides, and bacterial transporters.…”

Production of the cyanogenic glycoside dhurrin in yeast

“…Microsomes prepared from yeast expressing individual P450s have been used to characterize enzymes in cyanogenic glycoside pathways in vitro (Forslund et al., 2004; Yamaguchi et al., 2014; Hamann and Møller, 2007). Strategies involving re-engineering the N-terminal domains of the dhurrin pathway P450s have also recently been applied to functionally co-express SbCYP79A1 and SbCYP71E1 in Escherichia coli (Vazquez-Albacete et al., 2017).…”