TY - JOUR

T1 - An endoplasmic reticulum-engineered yeast platform for overproduction of triterpenoids

AU - Arendt, Philipp

AU - Miettinen, Karel

AU - Pollier, Jacob

AU - De Rycke, Riet

AU - Callewaert, Nico

AU - Goossens, Alain

N1 - Funding Information: We thank Annick Bleys for help in improving the manuscript. This work was financially supported by the VIB International PhD Fellowship Programme (predoctoral fellowship to P.A.), the Research Foundation Flanders (postdoctoral fellowship to J.P.), and the European Union Seventh Framework Programme FP7/2007?2013 under grant agreement number 613692?TriForC. Publisher Copyright: © 2017 International Metabolic Engineering Society

PY - 2017

Y1 - 2017

N2 - Saponins are a structurally diverse family of triterpenes that are widely found as main constituents in many traditional plant-based medicines and often have bioactivities of industrial interest. The heterologous production of triterpene saponins in microbes remains challenging and only limited successful pathway engineering endeavors have been reported. To improve the production capacities of a Saccharomyces cerevisiae saponin production platform, we assessed the effects of several hitherto unexplored gene knockout targets on the heterologous production of triterpenoids. Here, we show that the disruption of the phosphatidic acid phosphatase-encoding PAH1 through CRISPR/Cas9 results in a dramatic expansion of the endoplasmic reticulum (ER), which stimulated the production of recombinant triterpene biosynthesis enzymes and ultimately boosted triterpenoid and triterpene saponin accumulation. Compared to the wild-type starter strain, accumulation of the oleanane-type sapogenin β-amyrin, of its oxidized derivative medicagenic acid, and its glucosylated version medicagenic-28-O-glucoside was respectively increased by eight-, six- and 16-fold in the pah1 strain. A positive effect of pah1 could also be observed for the production of other terpenoids depending on ER-associated enzymes for their biosynthesis, such as the sesquiterpenoid artemisinic acid, which increased by twofold relative to the wild-type strain. Hence, this report demonstrates that pathway engineering in yeast through transforming the subcellular morphology rather than altering metabolic fluxes is a powerful strategy to increase yields of bioactive plant-derived products in heterologous hosts.

AB - Saponins are a structurally diverse family of triterpenes that are widely found as main constituents in many traditional plant-based medicines and often have bioactivities of industrial interest. The heterologous production of triterpene saponins in microbes remains challenging and only limited successful pathway engineering endeavors have been reported. To improve the production capacities of a Saccharomyces cerevisiae saponin production platform, we assessed the effects of several hitherto unexplored gene knockout targets on the heterologous production of triterpenoids. Here, we show that the disruption of the phosphatidic acid phosphatase-encoding PAH1 through CRISPR/Cas9 results in a dramatic expansion of the endoplasmic reticulum (ER), which stimulated the production of recombinant triterpene biosynthesis enzymes and ultimately boosted triterpenoid and triterpene saponin accumulation. Compared to the wild-type starter strain, accumulation of the oleanane-type sapogenin β-amyrin, of its oxidized derivative medicagenic acid, and its glucosylated version medicagenic-28-O-glucoside was respectively increased by eight-, six- and 16-fold in the pah1 strain. A positive effect of pah1 could also be observed for the production of other terpenoids depending on ER-associated enzymes for their biosynthesis, such as the sesquiterpenoid artemisinic acid, which increased by twofold relative to the wild-type strain. Hence, this report demonstrates that pathway engineering in yeast through transforming the subcellular morphology rather than altering metabolic fluxes is a powerful strategy to increase yields of bioactive plant-derived products in heterologous hosts.

KW - Combinatorial biosynthesis

KW - Cytochrome P450

KW - Metabolic engineering

KW - Saponins

KW - Terpenoids

UR - http://www.scopus.com/inward/record.url?scp=85014353157&partnerID=8YFLogxK

U2 - 10.1016/j.ymben.2017.02.007

DO - 10.1016/j.ymben.2017.02.007

M3 - Journal article

C2 - 28216107

AN - SCOPUS:85014353157

VL - 40

SP - 165

EP - 175

JO - Metabolic Engineering

JF - Metabolic Engineering

SN - 1096-7176

ER -