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

TY - JOUR

T1 - A Combinatorial Approach To Study Cytochrome P450 Enzymes for De Novo Production of Steviol Glucosides in Baker’s Yeast

AU - Gold, Nicholas D.

AU - Fossati, Elena

AU - Hansen, Cecilie Ida Cetti

AU - DiFalco, Marcos

AU - Douchin, Veronique

AU - Martin, Vincent J.J.

PY - 2018/11/26

Y1 - 2018/11/26

N2 - 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.

AB - 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.

KW - cytochrome P450 monooxygenase

KW - cytochrome P450 reductase

KW - diterpenoid biosynthesis

KW - steviol glucosides

U2 - 10.1021/acssynbio.8b00470

DO - 10.1021/acssynbio.8b00470

M3 - Journal article

C2 - 30474973

SN - 2161-5063

VL - 7

SP - 2918

EP - 2929

JO - A C S Synthetic Biology

JF - A C S Synthetic Biology

IS - 12

ER -