Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae

doi:10.1016/j.jbiotec.2013.10.017

Journal of Biotechnology

Volume 168, Issue 4, December 2013, Pages 446-451

https://doi.org/10.1016/j.jbiotec.2013.10.017 Get rights and content

Highlights

•
IPP isomerase is one of the rate-limiting step in geraniol biosynthesis in yeast.
•
Overexpression of MAF1 is an effective route to increase geraniol production.
•
Geraniol production achieved to 36.04 mg L⁻¹ by metabolic engineering of yeast.

Abstract

Monoterpene geraniol, a compound obtained from aromatic plants, has wide applications. In this study, geraniol was synthesized in Saccharomyces cerevisiae through the introduction of geraniol synthase. To increase geraniol production, the mevalonate pathway in S. cerevisiae was genetically manipulated to enhance the supply of geranyl diphosphate, a substrate used for the biosynthesis of geraniol. Identification and optimization of the key regulatory points in the mevalonate pathway in S. cerevisiae increased geraniol production to 36.04 mg L⁻¹. The results obtained revealed that the IDI1-encoded isopentenyl diphosphate isomerase is a rate-limiting enzyme in the biosynthesis of geraniol in S. cerevisiae, and overexpression of MAF1, a negative regulator in tRNA biosynthesis, is another effective method to increase geraniol production in S. 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 HMG2^K6R (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, MAL2-8^C, 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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Citation Excerpt :
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Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae

Highlights

Abstract

Introduction

Section snippets

Strains and medium

Expression of geraniol synthase in S. cerevisiae

Discussion

Acknowledgments

Food Chem. Toxicol.

FEMS Microbiol. Lett.

S. Afr. J. Bot.

J. Biol. Chem.

J. Lipid Res.

Metab. Eng.

Trends Biochem. Sci.

Metab. Eng.

FEMS Yeast Res.

Biochem. Biophys. Res. Commun.

Metab. Eng.

Metab. Eng.

Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis

Biotechnol. Bioeng.

Saccharomyces cerevisiae contains two functional genes encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase

PNAS

Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae

Biotechnol. Bioeng.