Format

Send to

Choose Destination
Metab Eng. 2017 Nov;44:117-125. doi: 10.1016/j.ymben.2017.09.006. Epub 2017 Sep 20.

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae.

Author information

1
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, United States.
2
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, United States.
3
Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, United States; Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, United States.
4
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, United States; Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, United States. Electronic address: yitang@ucla.edu.

Abstract

Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous 'ene'-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.

KEYWORDS:

Iridoids; Monoterpene indole alkaloids; Old yellow enzyme; Saccharomyces cerevisiae

PMID:
28939278
PMCID:
PMC5705256
DOI:
10.1016/j.ymben.2017.09.006
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center