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Methods Enzymol. 2016;575:143-78. doi: 10.1016/bs.mie.2016.03.023. Epub 2016 Apr 25.

Plug-and-Play Benzylisoquinoline Alkaloid Biosynthetic Gene Discovery in Engineered Yeast.

Author information

1
University of Calgary, Calgary, AB, Canada.
2
University of Calgary, Calgary, AB, Canada. Electronic address: hagelj@ucalgary.ca.
3
University of Calgary, Calgary, AB, Canada. Electronic address: pfacchin@ucalgary.ca.

Abstract

Benzylisoquinoline alkaloid (BIA) metabolism has been the focus of a considerable research effort over the past half-century, primarily because of the pharmaceutical importance of several compounds produced by opium poppy (Papaver somniferum). Advancements in genomics technologies have substantially accelerated the rate of gene discovery over the past decade, such that most biosynthetic enzymes involved in the formation of the major alkaloids of opium poppy have now been isolated and partially characterized. Not unexpectedly, the availability of all perceived biosynthetic genes has facilitated the reconstitution of several BIA pathways in microbial hosts, including yeast (Saccharomyces cerevisiae). Product yields are currently insufficient to consider the commercial production of high-value BIAs, such as morphine. However, the rudimentary success demonstrated by the uncomplicated and routine assembly of a multitude of characterized BIA biosynthetic genes provides a valuable gene discovery tool for the rapid functional identification of the plethora of gene candidates available through increasingly accessible genomic, transcriptomic, and proteomic databases. BIA biosynthetic gene discovery represents a substantial research opportunity largely owing to the wealth of existing enzyme data mostly obtained from a single plant species. Functionally novel enzymes and variants with potential metabolic engineering applications can be considered the primary targets. Selection of candidates from sequence repositories is facilitated by the monophyletic relationship among biosynthetic genes belonging to a wide range of enzyme families, such as the numerous cytochromes P450 and AdoMet-dependent O- and N-methyltransferases that operate in BIA metabolism. We describe methods for the rapid functional screening of uncharacterized gene candidates encoding potential BIA biosynthetic enzymes using yeast strains engineered to perform selected metabolic conversions. As an initial screening tool, the approach is superior to the in vitro characterization of recombinant enzyme candidates, and provides a standardized functional genomics opportunity for otherwise recalcitrant exotic plant species.

KEYWORDS:

Benzylisoquinoline alkaloid; Biochemical genomics; High-resolution liquid chromatography tandem mass spectrometry

PMID:
27417928
DOI:
10.1016/bs.mie.2016.03.023
[Indexed for MEDLINE]

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