Zhao et al. Microb Cell Fact (2017) 16:17 DOI 10.1186/s12934-017-0641-9 Microbial Cell Factories RESEARCH Open Access Dynamic control of ERG20 expression combined with minimized endogenous downstream metabolism contributes to the improvement of geraniol production in Saccharomyces cerevisiae Jianzhi Zhao1, Chen Li1, Yan Zhang1, Yu Shen1, Jin Hou1* and Xiaoming Bao1,2* Abstract Background: Microbial production of monoterpenes provides a promising substitute for traditional chemical-based methods, but their production is lagging compared with sesquiterpenes. Geraniol, a valuable monoterpene alcohol, is widely used in cosmetic, perfume, pharmaceutical and it is also a potential gasoline alternative. Previously, we constructed a geraniol production strain by engineering the mevalonate pathway together with the expression of a high-activity geraniol synthase. Results: In this study, we further improved the geraniol production through reducing the endogenous metabolism of geraniol and controlling the precursor geranyl diphosphate flux distribution. The deletion of OYE2 (encoding an NADPH oxidoreductase) or ATF1 (encoding an alcohol acetyltransferase) both involving endogenous conversion of geraniol to other terpenoids, improved geraniol production by 1.7-fold or 1.6-fold in batch fermentation, respectively. In addition, we found that direct down-regulation of ERG20 expression, the branch point regulating geranyl diphos- phate flux, does not improve geraniol production. Therefore, we explored dynamic control of ERG20 expression to redistribute the precursor geranyl diphosphate flux and achieved a 3.4-fold increase in geraniol production after optimizing carbon source feeding. Furthermore, the combination of dynamic control of ERG20 expression and OYE2 deletion in LEU2 prototrophic strain increased geraniol production up to 1.69 g/L with pure ethanol feeding in fed- batch fermentation, which is the highest reported production in engineered yeast. Conclusion: An efficient geraniol production platform was established by reducing the endogenous metabolism of geraniol and by controlling the flux distribution of the precursor geranyl diphosphate. The present work also provides a production basis to synthesis geraniol-derived chemicals, such as monoterpene indole alkaloids. Keywords: Monoterpene, Geraniol, Geranyl diphosphate, ERG20, Saccharomyces cerevisiae Background 2C-methyl-D-erythrtiol 4-phosphate (MEP) pathway in In nature, isoprenoids are a large group of diverse com- plants and most bacteria, or the mevalonate (MVA) path- pounds, that are biosynthesized via both the cyto- way in animals and eukaryotes [1]. The universal precur- solic mevalonate (MVA) pathway and the plastidial sors of isoprenoids, isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are derived from both pathways. Among them, monoterpenes are a par- *Correspondence: [email protected]; [email protected] ticularly interesting subset of this family, which has been 1 State Key Laboratory of Microbial Technology, School of Life Science, widely used as flavors, fragrances and pharmaceuticals as Shandong University, Jinan 250100, China Full list of author information is available at the end of the article well as potential fuels [2, 3]. Traditionally, monoterpenes © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhao et al. Microb Cell Fact (2017) 16:17 Page 2 of 11 and their derivatives are produced from plants in low amounts, and the extraction process from plants is costly and highly dependent on the availability of raw materi- als. Engineering microbial organisms for monoterpene synthesis provides a potential effective route for their production. Monoterpene geraniol (trans-3,7-dimethyl-2,6-octa- dien-1-ol; C10H18O), an acyclic monoterpene, is typically generated from aromatic plants and has many applica- tions in perfume, pharmaceutical, and other chemical industries [4]. Geraniol is also considered as a promis- ing biofuel due to its high energy content, low hygrosco- picity and low volatility in comparison with ethanol [5]. In addition, a recent study reported that geraniol can undergo an 11-step heterologous biosynthetic pathway to form strictosidine, which is the common intermedi- ate for production of the anticancer agent monoterpene indole alkaloids (MIAs) [6]. Given its low yield in aro- matic plants, geraniol has been successfully synthesized in Escherichia coli and Saccharomyces cerevisiae through Fig. 1 Schematic overview of geraniol biosynthesis based on the metabolic engineering strategies [7–11]. S. cerevisiae has mevalonate (MVA) pathway in S. cerevisiae. The endogenous genes WW been used widely as a cell factory to produce a diversity tHMG1, IDI1 and ERG20 and heterologous gene tVoGES encoding a truncated version of geraniol synthase from Valeriana officinalis were of terpenes, and many of them have achieved signifi- overexpressed to improve geraniol production in our previous work. cant yields [12–15]. However, monoterpene production The native promoter of ERG20 was replaced with the CTR3 promoter, has been achieved only at low levels thus far [8, 16, 17]. BTS1 promoter or HXT1 promoter. Other engineered genes in this There are two challenges for high level monoterpenes pathway: OYE2, NADPH oxidoreductase and ATF1, alcohol acetyl- production. One is the availability of intracellular geranyl transferase. HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A, IPP isopentenyl pyrophosphate, DMAPP dimethylallyl pyrophosphate, diphosphate (GPP) precursor, and the other is the toxicity GPP geranyl diphosphate, FPP farnesyl diphosphate of monoterpenes to cells [18, 19]. Most of monoterpenes are generated from the precursor GPP which is formed by condensation of one molecule of IPP with one mol- ecule of its isomer DMAPP (Fig. 1). Unlike plants, S. cer- production of the monoterpene sabinene by 10.4-fold evisiae does not supply enough GPP for production due to 0.53 mg/L in an industrial diploid strain [16]. In our to the lack of a specific GPP synthase (GPPS). In S. cerevi- previous work, we further improved geraniol production siae, the ERG20 gene of the endogenous MVA pathway through enhancing MVA pathway flux, screening differ- encodes a farnesyl pyrophosphate synthase (FPPS) that, ent sources of GESs and GPPSs, and designing fusion despite having both GPP and FPP synthase activity, only proteins of GES and GPPS. Geraniol of 293 mg/L pro- releases a very low amount of GPP from its catalytic site duction was achieved in fed-batch cultivation, which is [20]. To increase the flux to GPP, a set of ERG20 mutants the highest monoterpene production in S. cerevisiae ever was constructed to screen for GPPS preference mutants. reported [21]. However, it is still lower than the produc- ERG20K197G, the most effective mutant, improved geran- tion of many other terpenes in S. cerevisiae [12, 22, 23]. iol production to 5 mg/L while expressing geraniol syn- In addition to the low GPP pool, the toxicity to micro- thase (GES) from Ocimum basilicum in S. cerevisiae [8]. organisms of monoterpenes also largely limits their Liu et al. obtained 36 mg/L geraniol by overexpressing microbial production [2, 24]. To alleviate the toxicity of two key rate-limiting genes, tHMG1 encoding a trun- monoterpenes, a two-phase fermentation system has cated version of 3-hydroxy-3-methylglutaryl coenzyme A been extensively applied by adding a non-toxic extractive and IDI1 encoding IPP isomerase, to enhance MVA path- solvent (e.g., dodecane) [2]. In addition, monoterpenols, way flux and by overexpressing MAF1, a negative regula- usually undergo biotransformation to other terpenoids in tor of MOD5 encoding tRNA isopentenyltransferase, to aromatic plants, as well as in some wine yeasts [25, 26]. decrease the flux to tRNA biosynthesis [10]. In addition, A recent study demonstrated that geraniol is converted Ignea et al. demonstrated that the double mutant Erg20p into citronellol under the catalysis of the enzyme OYE2, (F96W-N127W) had a strong dominant negative abil- and it is acetylated by ATF1 encoding alcohol acetyltrans- ity to decrease the FPPS function of Erg20p, increasing ferase during wine fermentation by S. cerevisiae [27]. Zhao et al. Microb Cell Fact (2017) 16:17 Page 3 of 11 Intracellular bioconversion of geraniol may be a self- defense response to avoid the toxicity of monoterpenes. In addition, introduction of a heterologous biosynthetic pathway and the overexpression of an endogenous path- way often lead to the accumulation of some intermediate metabolites, that may interfere with the native regulation of metabolic flux and increase metabolic burden, result- ing in overproduction of some biosynthetic enzymes and accumulation of some toxic intermediate metabolites. Therefore, a dynamic control system that can sense envi- ronmental changes such as metabolic intermediates or nutrient concentrations can be used to avoid the toxicity of intermediate metabolites [28–30]. Previously, we achieved a significant increase in geran- iol production