Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae

Abstract

Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T 73 was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.Monoterpenes are a class of isoprenoids of increasing industrial and clinical interest usually produced by plants. They are used as aromatic additives in the food and cosmetics industries and are also important components in wine aroma. Moreover, certain monoterpenes display antimicrobial, antiparasitic, and antiviral properties as well as a plethora of promising health benefits (for recent reviews, see references 2, 7, 15, 28, and 30 and references cited therein). To date, many studies have focused on plant metabolic engineering of monoterpene production (for selected reviews, see references 1, 14, 19, 29, and 35 and references cited therein), and few studies have been carried out on microorganisms (9,21,22,34,38). Efficient microbial production of these metabolites could provide an alternative to the current methods of chemical synthesis or extraction from natural sources. In this regard, a considerable number of studies have shown the utility of Saccharomyces cerevisiae as a valuable platform for sesquiterpene, diterpene, triterpene, and carotene production (references 5, 10, 23, 26, 30, 31, 32, and 33 and references cited therein). However, all the efforts dedicated to the improvement of isoprenoid yields in S. cerevisiae have been performed using conventional laboratory strains, and there are no studies concerning natural or industrially relevant isolates.In recent years, many genes that encode plant monoterpene synthases (MTS), a family of enzymes which specifically catalyze the conversion of the ubiquitous C 10 intermediate of isoprenoid biosynthesis geranyl pyrophosphate (GPP) to monoterpenes, have been characterized. Such is the case with the LIS gene (codes for S-linalool synthase) of Clarkia breweri, the first MTS-encoding gene to be isolated (13). In contrast to plants, S. cerevisiae cannot produce monoterpenes efficiently, mainly due to the lack of specific pathways involving MTS. However, GPP is formed as a transitory intermediate in the two-step synthesis of farnesyl pyrophosphate (FPP), catalyzed by FPP synthase (FPPS) (Fig. 1), and some natural S. cerevisiae strains have been shown to possess the ability to produce small amounts of monoterpenes (8). Whether this occurs through unspecific dephosphorylation of a more available endogenous pool of GPP and subsequent bioconversions is not known. In addition, it has recently been established that S. cerevisiae has enough free GPP to...

Influence of Edible Coatings on Biochemical Fruit Quality and Storage Life of Bell Pepper cv. “Yolo Wonder”

“…Moreover, the linear increase in pH might be ascribed to biochemical, structural, and physiological alterations taking place during respiration. The increase in pH could be attributed to the accumulation of dry matter content and depolymerization of pepper polysaccharides under cold storage [51]. Meanwhile, membrane leakage was increased with the progression of cold storage period; however, coated bell pepper fruit exhibited reduced membrane leakage compared to the uncoated fruit.…”

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

“…Constitutive overexpression of HMGR gene highly increases accumulation of monoterpenes (27), sesquiterpenes (2,28), diterpenes (4), and tetraterpenes (43). Mutations of the HMGR gene also decrease triterpenoid levels in Arabidopsis thaliana (24).…”

Metabolic Engineering for Advanced Biofuels Production and Recent Advances Toward Commercialization

“…[49] Efforts to produce linalool in S. cerevisiae were initiated by incorporating a linalool synthase (lis) from Clarkia breweri, but the strain produced linalool at titers of less than 1 mg L À1 due to issues with hmgr overexpression upstream within the MVA pathway. [50] Further attempts to improve S. cerevisiae production by down regulating squalene synthase (erg9) also resulted in similarly low titers of linalool, which was ultimately attributed to its high toxicity in yeast. [51] Another new target, sabinene, has been produced at similar levels in E. coli (82 mg L À1 ), with fed-batch fermentation levels reaching as high as 2.7 g L À1 at a yield of 3.5% using glycerol as a carbon source.…”