Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae
Introduction
Geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) is an acyclic monoterpene alcohol with the chemical formula C10H18O, which is widely used in the perfume industry due to its pleasant odor (Chen and Viljoen, 2010). In addition, geraniol is known to exhibit insecticidal and insect repellent properties, and has also been developed as a chemo-prevention agent for the treatment of cancer (Kim et al., 2011). However, geraniol is naturally produced in limited quantities (Bakkali et al., 2008). Therefore, biosynthesis of such rare and valuable compounds in microorganisms by metabolic engineering strategies is a feasible route to fulfill the increasing commercial demand.
Monoterpenes are derived from two common building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized through the mevalonate pathway in Saccharomyces cerevisiae. Two molecules of IPP and one molecule of DMAPP can be condensed to one molecule of geranyl diphosphate (GPP) (Withers and Keasling, 2007) (Fig. 1). GPP is the precursor of monoterpenes such as (S)-linalool, geraniol, and cineole (Herrero et al., 2008), and the existence of intracellular GPP pool in S. cerevisiae has been verified in previous research (Oswald et al., 2007).
Although metabolic engineering of S. cerevisiae for monoterpene production has shown great potential, only a few monoterpene synthases have been characterized and expressed in S. cerevisiae, with very low productivity (Fischer et al., 2011, Oswald et al., 2007). The restrained monoterpene productivity is mainly due to the weak flux of the mevalonate pathway in S. cerevisiae (Rico et al., 2010). Acetyl-CoA is known to be the substrate of the mevalonate pathway. It has been reported that increased acetyl-CoA supply by overexpression of the key rate-limiting enzymes in the pyruvate dehydrogenase bypass could increase amorphadiene production by 2.8-fold (Shiba et al., 2007).
Metabolic engineering of the mevalonate pathway has been frequently carried out to increase the carbon flux in S. cerevisiae, and has been mainly focused on: (1) increasing mevalonate supply by overexpressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene (tHMGR) (Dai et al., 2012, Engels et al., 2008, Scalcinati et al., 2012) or a mutated HMG2K6R (Ignea et al., 2011); (2) reducing the biosynthesis of squalene by replacing its strong native promoter with an weaker or inducible one (Asadollahi et al., 2008, Paradise et al., 2008, Scalcinati et al., 2012); (3) increasing the intracellular GPP pool by mutating the conserved region of farnesyl diphosphate synthase (FPPS) (Fischer et al., 2011, Karsta et al., 2004); and (4) overexpressing upc2.1 allele (regulating ergosterol biosynthesis) to reduce the flux to sterol biosynthesis (Engels et al., 2008, Ro et al., 2006, Shiba et al., 2007).
Metabolic engineering of S. cerevisiae for increased monoterpene production has rarely been reported in the literature, and the rate-limiting points predominantly remain unclear. Therefore, further research to identify and optimize the key regulatory points could facilitate monoterpene production in S. cerevisiae. In the present study, microbial production of monoterpene geraniol was achieved through integrated expression of geraniol synthase and enhancement of the mevalonate pathway in S. cerevisiae. As a result of the genetic manipulations carried out, the final yield of geraniol increased to 36.04 mg L−1.
Section snippets
Strains and medium
Escherichia coli JM109 (Takara, Shiga, Japan) was used as the recipient strain for cloning manipulation and plasmid amplification. The cells were cultured at 37 °C in Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) with or without 100 μg L−1 of ampicillin.
S. cerevisiae CEN.PK2-1C (MAT a, ura3-52, trp1-289, leu2-3,112, his3Δ1, MAL2-8C, SUC2), used for genetic manipulation, were obtained from EUROSCARF (Frankfurt, Germany). The strains used in this study are listed in Table 1
Expression of geraniol synthase in S. cerevisiae
To evaluate the expression of geraniol synthase, the geraniol synthase encoding gene GES1 was expressed in S. cerevisiae CEN.PK2-1C using an episome plasmid pY26-TEF-GPD, generating the engineered S. cerevisiae strain JN02. The GC–MS results verified the production of intracellular and extracellular geraniol by JN02. No geraniol production was detected in the control strain JN01 carrying empty pY26-TEF-GPD (Fig. 2). The functional expression of wild-type geraniol synthase in JN02 yielded 0.52 mg L
Discussion
Wild-type S. cerevisiae cannot produce monoterpenes due to the deficiency of the enzyme monoterpene synthase with the exception of a few wine-making yeast strains have been found to be capable of producing less than 5 μg L−1 of monoterpenes such as (S)-linalool and α-terpineol (Carrau et al., 2005). While expression of geraniol synthase could result in geraniol accumulation in S. cerevisiae, increased copy number of the gene encoding geraniol synthase in S. cerevisiae (strain JN03 and JN04)
Acknowledgments
This work was supported by the Major State Basic Research Development Program of China (973 Program, 2012CB720802), the National Natural Science Foundation of China (31000807, 31130043, 21276109), the Natural Science Foundation of Jiangsu Province (BK2010150, BK2011004), the Fundamental Research Funds for the Central Universities (JUSRP51307A), the Open Project Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (KLIB-KF200907) and the 111 Project (111-2-06).
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2021, Metabolic EngineeringCitation Excerpt :The activity of GAL promoters can be induced after glucose depletion when GAL80 is deleted (Westfall et al., 2012). Since overexpression of tHMGR and IDI1 has been reported to increase the geraniol titer in yeast (Liu et al., 2013), S. cerevisiae strain yJGZ1 overexpressing tHMGR and IDI1 under the divergent promoter PGAL1/10 in the GAL80 locus was adopted as the parental strain for citronellol biosynthesis (Jiang et al., 2017). It has been demonstrated that tCrGES showed up to 4.5-fold increase in geraniol titer compared with CrGES, and fusion of tCrGES with Erg20F96W, N127W (tCrGES-Erg20F96W, N127W) led to a 15% increase in geraniol production compared to separate tCrGES and Erg20F96W, N127W (Jiang et al., 2017).