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

Applied Microbiology and Biotechnology (2018) 102:6923–6934 https://doi.org/10.1007/s00253-018-9154-7 BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Upregulating the mevalonate pathway and repressing sterol synthesis in Saccharomyces cerevisiae enhances the production of triterpenes Jan Niklas Bröker 1 & Boje Müller 2 & Nicole van Deenen 1 & Dirk Prüfer 1,2 & Christian Schulze Gronover 2 Received: 18 April 2018 / Revised: 30 May 2018 / Accepted: 2 June 2018 / Published online: 15 June 2018 # The Author(s) 2018 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 pushand-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. Keywords Metabolic engineering . MVA pathway . Sterol biosynthesis . tHMGR . Pentacyclic triterpenes . Saccharomyces cerevisiae Introduction Isoprenoids are a diverse group of natural compounds found in all living organisms, with at least 50,000 different structures already reported (Hemmerlin et al. 2012; Liao et al. 2016). In plants, these products are derived from the plastididial 2Cmethyl-d-erythritol 4-phosphate (MEP) and the cytosolic mevalonate (MVA) pathway. In the latter, acetyl-CoA is converted to the isoprenoid precursor isopentenyl diphosphate (IPP) via six enzymatic steps. Two important MVA pathway enzymes are the sequentially acting 3-hydroxy-3- * Christian Schulze Gronover 1 Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48143 Münster, Germany 2 Fraunhofer Institut für Molekularbiologie und Angewandte Oekologie, Schlossplatz 8, 48143 Münster, Germany methylglutaryl-coenzyme A (HMG-CoA) synthase (HMGS) and HMG-CoA reductase (HMGR), the latter representing the rate-limiting step (Demierre et al. 2005). IPP is isomerized to form dimethylallyl pyrophosphate (DMAPP), and together, IPP and DMAPP can act as substrates for various isoprenoid-derived pathways. For example, two molecules of IPP and one of DMAPP can be converted into farnesyl pyrophosphate (FPP) which in turn can be converted into squalene by squalene synthase (SQS). The oxidized form of squalene (2,3-oxidosqualene) is a precursor for the synthesis of sterols (leading to the production of lanosterol in fungi and animals, or cycloartenol in plants) and also pentacyclic triterpenes (Fig. 1a), the latter involving various oxidosqualene cyclases (OSCs) such as lupeol synthase in the dandelion Taraxacum officinale and β-amyrin synthase in the wormwood plant Artemisia annua (Shibuya et al. 1999; Kirby et al. 2008). The products of these enzymes can be further metabolized by acylation or oxidation. The efficient triterpene oxidation of, e.g., lupeol to betulin and betulinic acid by P450 enzymes could be demonstrated in yeast (Zhou 6924 Appl Microbiol Biotechnol (2018) 102:6923–6934 a sterols IPP AACT HMGS HMGR SQS SQE acetyl-CoA squalene 2,3-oxidosqualene OSCs pentacyclic triterpenes DMAPP b TkLUP PGAL1 TCYC1 c d lupeol 0.25 300 0.20 200 lupeol standard mg/g CDW ion count [m/z 218] TkLUP 0.15 0.10 100 β-amyrin standard 0.05 vector control 0 17.8 WT 18.0 18.2 18.4 18.6 0.00 vector control TkLUP ret. time [min.] Fig. 1 Triterpene accumulation in the yeast S. cerevisiae expressing TkLUP. a Schematic representation of the MVA pathway leading to the synthesis of sterols and pentacyclic triterpenes via oxidosqualene cyclases (OSCs). Dashed arrows represent multiple enzymatic reactions. AACT = acetyl-CoA C-acetyltransferase; DMAPP = dimethylallyl pyrophosphate; IPP = isopentenyl diphosphate; HMGS = 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) synthase; HMGR = HMG-CoA reductase; SQS = squalene synthase; SQE = squalene epoxidase. b Schematic representation of the TkLUP coding sequence under the control of the GAL1 promoter (PGAL1) and CYC1 terminator (TCYC1). c Yeast cells carrying the TkLUP coding sequence showed two additional peaks in the GC-MS spectrum (m/z = 218, arrows), probably representing βamyrin (retention time = 17.95 min) and lupeol (retention time = 18.25 min) because they match the corresponding standards. d Yeasts carrying the TkLUP coding sequence accumulated 0.16 mg/g CDW of the putative lupeol but the quantification of the β-amyrin peak was not possible. Wild-type (WT) and pAG424_PGAL1-ccdb vector control CEN.PK2-1C cells served as controls. The standard deviation was calculated from n = 3 individual transformants; CDW = cell dry weight et al. 2016). FPP is also a precursor for the synthesis of sesquiterpenes, e.g., farnesene or amorpha-4,11-diene, a precursor of the anti-malarial drug artemisinin (Martin et al. 2003). The value of isoprenoids, particularly as pharmaceuticals, has prompted the development of heterologous production systems including the yeast Saccharomyces cerevisiae (reviewed by Liao et al. 2016 and Vickers et al. 2017). The potential of the yeast MVA pathway for the production of isoprenoids was first demonstrated by overexpressing the catalytic domain of HMGR (tHMGR), which increased the yield of squalene (Donald et al. 1997). The consequences of overexpressing other MVA pathway genes were determined by combinatorial library screening for the overexpression of ERG10 (acetoacetyl CoA thiolase; AACT), ERG13 (HMGS), and ERG12 (mevalonate kinase) which enhanced the production of amorpha-4,11-diene (Yuan and Ching 2014). The MVA pathway has also been targeted using the CRISPR/Cas9 system, revealing loci that trigger the accumulation of mevalonate and triterpenes when knocked out (Jakočiūnas et al. 2015; Arendt et al. 2017). The targets included (...truncated)