High-level production of linalool by engineered Saccharomyces cerevisiae harboring dual mevalonate pathways in mitochondria and cytoplasm

Highlights

• Overexpression of MVA pathway in both mitochondria and cytoplasm in S. cerevisiae.

• Down-regulation of ERG20 and overexpressing tHMG1 improved linalool production.

• Pyruvate and mevalonlactone were helpful to linalool production in S. cerevisiae.

• The maximum titer of linalool reached 23.45 mg/L in a 2 L fermenter.

Abstract

Linalool, a valuable monoterpene alcohol, is widely used in cosmetics and flavoring ingredients. However, its scalable production by microbial fermentation is not yet achieved. In this work, considerable increase in linalool production was obtained in Saccharomyces cerevisiae by dual metabolic engineering of the mevalonic acid (MVA) pathway in both mitochondria and cytoplasm. A farnesyl pyrophosphate synthase mutant ERG20^F96W/N127W and a linalool synthase from Cinnamomum osmophloeum (CoLIS) were introduced and meanwhile the endogenous ERG20 was down-regulated to prevent the competitive loss of precursor. In addition, overexpression of the proteins of CoLIS and ERG20^F96W/N127W and another copy of the same enzymes CoLIS/ERG20^F96W/N127W with mitochondrial localization signal (MLS) were carried out to further pull the flux to linalool. Finally, a maximum linalool titer of 23.45 mg/L was obtained in a batch fermentation with sucrose as carbon source. This combinatorial engineering strategy may provide hints for biosynthesis of other monoterpenes.

Introduction

Linalool, a valuable monoterpene, is widely used in cosmetics and flavoring ingredients as well as in traditional medicines [1]. Recent studies suggest that linalool has several important biological properties, such as anti-tumor [2], anti-inflammatory [3], anti-oxidant [4] and hypolipidemic activities [5]. Monoterpenes are a subclass of C10 terpenoids containing two isoprene units [isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)] [6]. Natural monoterpenes are mainly found in plants as the major constituent of essential oils [7]. In plants, monoterpenes are synthesized in plastids, whereas IPP and DMAPP are produced by two different pathways: the mevalonic acid (MVA) pathway in the cytoplasm and the methylerythritol phosphate (MEP) pathway in plastids [8]. Microbial production of monoterpenes enabled by recent progresses in metabolic engineering and synthetic biology provides a promising substitution for traditional chemical-based or plant extraction methods with a number of advantages, such as short cycle time, low energy consumption and environmental friendliness [9]. However, heterologous production of monoterpenes has been obtained at low levels, preventing their industrial application.

Recently, heterologous production of linalool in Saccharomyces cerevisiae has been reported, with the maximum titer of 240 μg/L achieved in a 3 L fermenter [10]. In S. cerevisiae, the native MVA pathway is employed to generate essential terpenoids [11]. To optimize the MVA pathway for enhanced terpenoids production, overexpression of the rate-limiting enzyme tHMG1 has been adopted as a common strategy [10,12,13]. In the oleaginous yeast Y. lipolytica, co-overexpression of tHMG1 with some other MVA pathway genes was shown to further enhance monoterpenes production, and the final linalool production reached 6.96 mg/L [13,14]. Therefore, an overall strengthened MVA pathway with sufficient precursor supply may be beneficial. Lv et al. [15] have constructed S. cerevisiae strain BY4742-M-04 for isoprene synthesis by assembling the whole MVA pathway in the mitochondria, and further constructed a mitochondria/cytoplasm dual-regulation strain BY4742-MC-01 by overexpressing tHMG1 and repressing ERG20. Considering the rich IPP/DMAPP stock in these strains, they would also be good starting strains for linalool synthesis. As the direct precursor of monoterpenes, geranyl diphosphate (GPP) is synthesized from IPP and DMAPP by the bifunctional enzyme ERG20, which can further catalyze the conversion of GPP into farnesyl pyrophosphate (FPP) [16]. Site-directed mutagenesis on the conserved region of ERG20 could hinder its FPP synthase activity without affecting its GPP synthase activity, leading to increase in available GPP for monoterpenes synthesis. Mutation of the K197 site of ERG20 has been proved to increase the yield of linalool [10]. Double mutations of F96W and N127W led to enhanced production of sabinene [17]. Overexpression of ERG20^K197G and ERG20^F96W-N127W resulted in a 2.1 and 3.5-fold increase in the production of geraniol, respectively [18]. It would therefore be interesting to investigate the effects of different combinations of these mutations on linalool biosynthesis.

Generally cytoplasmic engineering is used in yeast, whereas localization of heterologous pathways in other subcellular compartments for chemical production is rarely reported [15,19]. The aim of this study is to construct a strain with high yield of linalool based on dual metabolic engineering of the MVA pathway in both mitochondria and cytoplasm in S. cerevisiae. To improve the linalool biosynthetic capability, upregulating the mevalonate pathway and repressing FPP synthesis in S. cerevisiae were performed to enhance the supply of GPP as a key precursor. Mutation of ERG20 and its subsequent fusion with a linalool synthase gene from Cinnamomum osmophloeuma were carried out to pull the metabolic flux from FPP to linalool. Finally, the effects of carbon sources on linalool production were examined. The proposed combinational strategy successfully led to high-level production of linalool in engineered S. cerevisiae (Fig. 1).