Upregulating the mevalonate pathway and repressing sterol synthesis in Saccharomyces cerevisiae enhances the production of triterpenes

Abstract

Pentacyclic triterpenes are diverse plant secondary metabolites derived from the mevalonate (MVA) pathway. Many of these molecules are potentially valuable, particularly as pharmaceuticals, and research has focused on their production in simpler and more amenable heterologous systems such as the yeast Saccharomyces cerevisiae. We have developed a new heterologous platform for the production of pentacyclic triterpenes in S. cerevisiae based on a combinatorial engineering strategy involving the overexpression of MVA pathway genes, the knockout of negative regulators, and the suppression of a competing pathway. Accordingly, we overexpressed S. cerevisiae ERG13, encoding 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, and a truncated and deregulated variant of the rate-limiting enzyme HMG-CoA reductase 1 (tHMGR). In the same engineering step, we deleted the ROX1 gene, encoding a negative regulator of the MVA pathway and sterol biosynthesis, resulting in a push-and-pull strategy to enhance metabolic flux through the system. In a second step, we redirected this enhanced metabolic flux from late sterol biosynthesis to the production of 2,3-oxidosqualene, the direct precursor of pentacyclic triterpenes. In yeast cells transformed with a newly isolated sequence encoding lupeol synthase from the Russian dandelion (Taraxacum koksaghyz), we increased the yield of pentacyclic triterpenes by 127-fold and detected not only high levels of lupeol but also a second valuable pentacyclic triterpene product, β-amyrin.

Production of the bioactive plant‐derived triterpenoid morolic acid in engineered Saccharomyces cerevisiae

“…Furthermore, to improve the flux of the MVA pathway, we also introduced truncated HMG1 and the mutant transcription factor upc2‐1 , which has been successfully used to enhance terpenoid production in a yeast system (Bröker et al, 2018; Zhang et al, 2015).…”

“…This system allows the use of β‐estradiol to induce the expression of genes driven by GAL promoters, independent of the presence or absence of galactose or glucose. To boost triterpenoid production in yeast, the overexpression of influential enzymes in the endogenous MVA pathway, such as 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGR) or squalene epoxidase, enhances precursor availability for heterologous triterpenoid production (Bröker, Müller, van Deenen, Prüfer, & Gronover, 2018; Dong et al, 2018; Moses et al, 2014). Overexpression of the mutant transcription factor upc2‐1 , which upregulates the transcription level of the native MVA pathway (Scalcinati et al, 2012), can increase the β‐amyrin level up to 65‐fold (Zhang, Cao, Liu, Li, & Li, 2015).…”

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz – role of cis‐prenyltransferase‐like 1 protein

“…Proteins from yeast were extracted as already described in Bröker et al . (). Concentrations of either latex or yeast proteins were determined via Bradford assay (Bradford, ).…”

“…To extract triterpenes from the different latex fractions, the isolated phases were lyophilized and the protocol developed for triterpene measurement in yeast (Bröker et al ., ) was applied, but 100 μL of Betuline (2.5 mg/mL stock solution in acetone) was used as internal standard.…”

Overexpression of key sterol pathway enzymes in two model marine diatoms alters sterol profiles inPhaeodactylum tricornutum

“…Moreover, it has been reported that genetic manipulations on HMGR, including truncation of N-terminal domain, led to considerable accumulation of terpenes in transgenic plants and yeast (Bansal et al, 2018;Bröker et al, 2018;Lee et al, 2019). Although HMGR has been extensively characterized in model eukaryotic organisms, little is known about its features in diatoms.…”