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Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae.

Author(s): Fossati E, Ekins A, Narcross L, Zhu Y, Falgueyret JP, Beaudoin GA, Facchini PJ, Martin VJ

Nat Commun. 2014;5:3283 Authors: Fossati E, Ekins A, Narcross L, Zhu Y, Falgueyret JP, Beaudoin GA, Facchini PJ, Martin VJ

Article GUID: 24513861
Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.

Author(s): Fossati E, Narcross L, Ekins A, Falgueyret JP, Martin VJ

PLoS One. 2015;10(4):e0124459 Authors: Fossati E, Narcross L, Ekins A, Falgueyret JP, Martin VJ

Article GUID: 25905794
Microbial Factories for the Production of Benzylisoquinoline Alkaloids.

Author(s): Narcross L, Fossati E, Bourgeois L, Dueber JE, Martin VJJ

Trends Biotechnol. 2016 Mar;34(3):228-241 Authors: Narcross L, Fossati E, Bourgeois L, Dueber JE, Martin VJJ

Article GUID: 26775900
Reconstituting Plant Secondary Metabolism in Saccharomyces cerevisiae for Production of High-Value Benzylisoquinoline Alkaloids.

Author(s): Pyne ME, Narcross L, Fossati E, Bourgeois L, Burton E, Gold ND, Martin VJ

Methods Enzymol. 2016;575:195-224 Authors: Pyne ME, Narcross L, Fossati E, Bourgeois L, Burton E, Gold ND, Martin VJ

Article GUID: 27417930
Mining Enzyme Diversity of Transcriptome Libraries through DNA Synthesis for Benzylisoquinoline Alkaloid Pathway Optimization in Yeast.

Author(s): Narcross L, Bourgeois L, Fossati E, Burton E, Martin VJ

ACS Synth Biol. 2016 12 16;5(12):1505-1518 Authors: Narcross L, Bourgeois L, Fossati E, Burton E, Martin VJ

Article GUID: 27442619
A Combinatorial Approach To Study Cytochrome P450 Enzymes for De Novo Production of Steviol Glucosides in Baker's Yeast.

Author(s): Gold ND, Fossati E, Hansen CC, DiFalco M, Douchin V, Martin VJJ

ACS Synth Biol. 2018 Dec 21;7(12):2918-2929 Authors: Gold ND, Fossati E, Hansen CC, DiFalco M, Douchin V, Martin VJJ

Article GUID: 30474973
Title:A Combinatorial Approach To Study Cytochrome P450 Enzymes for De Novo Production of Steviol Glucosides in Baker's Yeast.
Authors:Gold ND,  Fossati E,  Hansen CC,  DiFalco M,  Douchin V,  Martin VJJ
Link:https://www.ncbi.nlm.nih.gov/pubmed/30474973?dopt=Abstract
DOI:10.1021/acssynbio.8b00470
Category:ACS Synth Biol
PMID:30474973
Dept Affiliation: GENOMICS
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.

Description:

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]