1 Centre for Applied Synthetic Biology , Concordia University , Montréal , Québec H4B 1R6 , Canada.
2 Plant Biochemistry Laboratory, Department of Plant and Environmental Science , University of Copenhagen , DK-1871 Frederiksberg C , Denmark.
3 Center for Synthetic Biology , University of Copenhagen , DK-1871 Frederiksberg C , Denmark.
4 Centre for Structural and Functional Genomics , Concordia University , Montréal , Québec H4B 1R6 , Canada.
5 Evolva , Lersø Parkallé 42-44 , DK-2100 Copenhagen Ø , Denmark.
6 Department of Biology, Centre for Structural and Functional Genomic , Concordia University , Montréal , Québec H4B 1R6 , Canada.

A Combinatorial Approach To Study Cytochrome P450 Enzymes for De Novo Production of Steviol Glucosides in Baker's Yeast.

ACS Synth Biol. 2018 Dec 21;7(12):2918-2929

Authors: Gold ND, Fossati E, Hansen CC, DiFalco M, Douchin V, Martin VJJ

Abstract

Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO and KAH function in succession with the support of a NADPH-dependent cytochrome P450 reductase (CPR) to convert kaurene to steviol. This work describes a platform for recombinant production of steviol glucosides (SGs) in Saccharomyces cerevisiae, demonstrating the full reconstituted pathway from the simple sugar glucose to the SG precursor steviol. With a focus on optimization of the KO-KAH activities, combinations of functional homologues were tested in batch growth. Among the CYPs, novel KO75 (CYP701) and novel KAH82 (CYP72) outperformed their respective functional homologues from Stevia rebaudiana, SrKO (CYP701A5) and SrKAH (CYP81), in assays where substrate was supplemented to culture broth. With kaurene produced from glucose in the cell, SrCPR1 from S. rebaudiana supported highest turnover for KO-KAH combinations, besting two other CPRs isolated from S. rebaudiana, the Arabidopsis thaliana ATR2, and a new class I CPR12. Some coexpressions of ATR2 with a second CPR were found to diminish KAH activity, showing that coexpression of CPRs can lead to competition for CYPs with possibly adverse effects on catalysis.

PMID: 30474973 [PubMed - in process]