title: Reconstituting Plant Secondary Metabolism in Saccharomyces cerevisiae for Production of High-Value Benzylisoquinoline Alkaloids.
abstract: Benzylisoquinoline alkaloids (BIAs) constitute a diverse class of plant secondary metabolites that includes the opiate analgesics morphine and codeine. Collectively, BIAs exhibit a myriad of pharmacological activities, including antimicrobial, antitussive, antispasmodic, and anticancer properties. Despite 2500 known BIA products, only a small proportion are currently produced though traditional crop-based manufacturing, as complex stereochemistry renders chemical synthesis of BIAs largely unfeasible. The advent of synthetic biology and sophisticated microbial engineering coupled with recent advances in the elucidation of plant BIA metabolic networks has provided growing motivation for producing high-value BIAs in microbial hosts. Here, we provide a technical basis for reconstituting BIA biosynthetic pathways in the common yeast Saccharomyces cerevisiae. Methodologies outlined in this chapter include fundamental techniques for expressing and assaying BIA biosynthetic enzymes, bioprospecting large libraries of BIA enzyme variants, and reconstituting and optimizing complete BIA formation pathways in yeast. To expedite construction of superior BIA-producing yeast strains, we emphasize high-throughput techniques. Finally, we identify fundamental challenges impeding deployment of yeast-based BIA production platforms and briefly outline future prospects to overcome such barriers.
authors.affiliation: Centre for Structural and Functional Genomics, Concordia University, Montréal, QC, Canada.
authors.forename: M E
authors.initials: ME
authors.lastname: Pyne
authors.affiliation: Centre for Structural and Functional Genomics, Concordia University, Montréal, QC, Canada.
authors.forename: L
authors.initials: L
authors.lastname: Narcross
authors.affiliation: Centre for Structural and Functional Genomics, Concordia University, Montréal, QC, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Fossati
authors.affiliation: Centre for Structural and Functional Genomics, Concordia University, Montréal, QC, Canada.
authors.forename: L
authors.initials: L
authors.lastname: Bourgeois
authors.affiliation: Centre for Structural and Functional Genomics, Concordia University, Montréal, QC, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Burton
authors.affiliation: Centre for Structural and Functional Genomics, Concordia University, Montréal, QC, Canada.
authors.forename: N D
authors.initials: ND
authors.lastname: Gold
authors.affiliation: Centre for Structural and Functional Genomics, Concordia University, Montréal, QC, Canada. Electronic address: vincent.martin@concordia.ca.
authors.forename: V J J
authors.initials: VJ
authors.lastname: Martin
mesh_terms: D044182:Benzylisoquinolines / Q000378:metabolism*; D053898:Biosynthetic Pathways; D017343:Genes, Plant; D060847:Metabolic Engineering / Q000379:methods*; D010944:Plants / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D064210:Secondary Metabolism; D058615:Synthetic Biology / Q000379:methods
chemical_list: D044182:Benzylisoquinolines
keywords: Alkaloids; Benzylisoquinoline alkaloids; Metabolic engineering; Opiates; Saccharomyces cerevisiae; Secondary metabolism; Synthetic biology; Yeast