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A yeast platform for high-level synthesis of tetrahydroisoquinoline alkaloids.

Author(s): Pyne ME, Kevvai K, Grewal PS, Narcross L, Choi B, Bourgeois L, Dueber JE, Martin VJJ

Nat Commun. 2020 Jul 03;11(1):3337 Authors: Pyne ME, Kevvai K, Grewal PS, Narcross L, Choi B, Bourgeois L, Dueber JE, Martin VJJ

Article GUID: 32620756
A Highly Characterized Synthetic Landing Pad System for Precise Multicopy Gene Integration in Yeast.

Author(s): Bourgeois L, Pyne ME, Martin VJJ

ACS Synth Biol. 2018 Nov 16;7(11):2675-2685 Authors: Bourgeois L, Pyne ME, Martin VJJ

Article GUID: 30372609
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
Title:A Highly Characterized Synthetic Landing Pad System for Precise Multicopy Gene Integration in Yeast.
Authors:Bourgeois L,  Pyne ME,  Martin VJJ
Link:https://www.ncbi.nlm.nih.gov/pubmed/30372609?dopt=Abstract
DOI:10.1021/acssynbio.8b00339
Category:ACS Synth Biol
PMID:30372609
Dept Affiliation: BIOLOGY
1 Department of Biology , Concordia University , Montréal , Québec H3G 1M8 , Canada.
2 Centre for Applied Synthetic Biology , Concordia University , Montréal , Québec H3G 1M8 , Canada.

Description:

A Highly Characterized Synthetic Landing Pad System for Precise Multicopy Gene Integration in Yeast.

ACS Synth Biol. 2018 Nov 16;7(11):2675-2685

Authors: Bourgeois L, Pyne ME, Martin VJJ

Abstract

A fundamental undertaking of metabolic engineering involves identifying and troubleshooting metabolic bottlenecks that arise from imbalances in pathway flux. To expedite the systematic screening of enzyme orthologs in conjunction with DNA copy number tuning, here we develop a simple and highly characterized CRISPR-Cas9 integration system in Saccharomyces cerevisiae. Our engineering strategy introduces a series of synthetic DNA landing pads (LP) into the S. cerevisiae genome to act as sites for high-level gene integration. LPs facilitate multicopy gene integration of one, two, three, or four DNA copies in a single transformation, thus providing precise control of DNA copy number. We applied our LP system to norcoclaurine synthase (NCS), an enzyme with poor kinetic properties involved in the first committed step of the production of high-value benzylisoquinoline alkaloids. The platform enabled rapid construction of a 40-strain NCS library by integrating ten NCS orthologs in four gene copies each. Six active NCS variants were identified, whereby production of ( S)-norcoclaurine could be further enhanced by increasing NCS copy number. We anticipate the LP system will aid in metabolic engineering efforts by providing strict control of gene copy number and expediting strain and pathway engineering campaigns.

PMID: 30372609 [PubMed - in process]