title: An endoplasmic reticulum-engineered yeast platform for overproduction of triterpenoids.
abstract: 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.
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium; Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, B-9000 Ghent, Belgium; VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.
authors.forename: Philipp
authors.initials: P
authors.lastname: Arendt
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.
authors.forename: Karel
authors.initials: K
authors.lastname: Miettinen
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.
authors.forename: Jacob
authors.initials: J
authors.lastname: Pollier
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.
authors.forename: Riet
authors.initials: R
authors.lastname: De Rycke
authors.affiliation: Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, B-9000 Ghent, Belgium; VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium. Electronic address: alain.goossens@ugent.vib.be.
authors.forename: Alain
authors.initials: A
authors.lastname: Goossens
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000502:physiology*; D000072669:Gene Editing / Q000379:methods; D024861:Genetic Enhancement / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D010711:Phosphatidate Phosphatase / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D012503:Saponins / Q000096:biosynthesis* / Q000302:isolation & purification; D014315:Triterpenes / Q000302:isolation & purification / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D012503:Saponins; D014315:Triterpenes; D010711:Phosphatidate Phosphatase
keywords: Combinatorial biosynthesis; Cytochrome P450; Metabolic engineering; Saponins; Terpenoids