Enhance production of diterpenoids in yeast by overexpression of the fused enzyme of ERG20 and its mutant mERG20

Highlights

• Fusion of ERG20 and its mutant mERG20 significantly boosted diterpenoid production.

• The yeasts were achieved for producing nor-, syn- and ent-copalyl diphosphate.

• A yeast-based platform was built for characterizing all types of diterpene synthases.

Abstract

Yeast has been widely used for large-scale production of terpenoids. In yeast, modifications of terpenoid biosynthetic pathways have been intensively studied. tHMG1 (encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase of yeast) and UPC2-1 (the G888D mutant of UPC2 encoding a transcription factor) were integrated into yeast chromosome, and ERG9 (the squalene synthase gene of yeast) was knocked down to yield the chassis strain DH02. A F96C mutation in ERG20 (farnesyl diphosphate synthase of yeast) was conducted to obtain mERG20 which can function as a geranylgeranyl diphosphate synthase (GGPS). Then, three fused genes, including BTS1 (the yeast innate GGPS)-ERG20, ERG20-mERG20 and mERG20-ERG20, were constructed, and expressed either by the pESC-based plasmids in DH02, or by being integrated into DH02 chromosome. The highest geranylgeraniol (GGOH) content was observed in the extracts of DH12 integrated with ERG20-mERG20, corresponding to 3.2 and 2.3 folds of those of the strains integrated with BTS1 and mERG20, respectively. Finally, three genes encoding nor-copalyl diphosphate synthase (nor-CPS), ent-CPS and syn-CPS were integrated into the chromosome of DH12, respectively, to construct yeasts for producing corresponding copalyl diphosphates (CPPs). Thus, a yeast-based platform was built for characterizing all types of diterpene synthases using GGPP or various CPPs as their substrates.

Introduction

Over ten thousands of plant diterpenoids have been discovered (Mafu and Zerbe, 2018). They possess extremely diverse structures with acyclic and various cyclic skeletons (Mafu and Zerbe, 2018; Hanson, 2009, 2016). Many of them are highly valuable pharmaceuticals. Taxol (from Pacific yew), one of the best-known natural anticancer drugs, is used to treat many types of solid tumor cancers and Kaposi’s sarcoma (Rowinsky and Donehower, 1995; Saville et al., 1995). Ingenol mebutate from Euphorbia peplus was approved by FDA for treatment of actinic keratosis (Siller et al., 2010). Some other plant diterpenoids are undergoing clinical trials, such as an anti-HIV agent prostratin (Kulkosky et al., 2001), a platelet-activating factor receptor antagonist ginkgolide B (Stromgaard and Nakanishi, 2004) and an analgesic resiniferatoxin (Kissin and Szallasi, 2011).

Recently, synthetic biology approach has become a powerful tool for both clarification of terpenoid biosynthesis and large-scale production of highly valuable terpenoids (Zi and Peters, 2013; Zi et al., 2014b; Ignea et al., 2016; Bian et al., 2017; Li and Pfeifer, 2014). Although both Escherichia coli and Saccharomyces cerevisiae (yeast) are most widely used as microbial hosts for reconstruction of biosynthetic pathways of various plant metabolites, more successful examples were achieved in yeasts than in E. coli. One of the reasons that yeast is more widely used is that many membrane enzymes involved in plant metabolite biosynthesis, especially cytochrome P450 s, prefer to express and function in yeast (eukaryote) cells rather than in E. coli (prokaryote) cells (Facchini et al., 2012). Systematical optimization of yeast mevalonate pathway (MVA) has been intensively reported (Zhang et al., 2017). Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) synthesized through MVA pathway are subsequently tethered to yield farnesyl diphosphate (FPP) and then geranylgeranyl diphosphate (GGPP) under successive catalysis of ERG20 (yeast farnesyl diphosphate synthase, FPS) and BTS1 (yeast geranylgeranyl diphosphate synthase, GGPS) (Zhang et al., 2017). GGPP is the common precursor of diterpenoids (Zi et al., 2014a). Hence, engineering of the route from IPP and DMAPP towards GGPP may significantly enhance production of GGPP and its downstream products (i.e. various diterpenoid derivatives). Fusion of ERG20 and BTS1 can remarkably increase isoprenoid production in yeast (Zhou et al., 2012). And it has been reported that the F96C mutant of ERG20 (called mERG20 in this study) can act as a GGPS instead of FPS, and function more efficiently than BTS1 in yeast (Ignea et al., 2015). To investigate if the fused enzymes ERG20-mERG20 and mERG20-ERG20 are more efficient than BTS1-ERG20, the corresponding fused enzymes were constructed and overexpressed in yeasts. The results showed that overexpression of each of ERG20-mERG20 and mERG20-ERG20 indeed led to a significantly higher GGOH accumulation in yeast extracts than overexpression of BTS1-ERG20.