Zada et al. Biotechnol Biofuels (2018) 11:210 https://doi.org/10.1186/s13068-018-1210-0 Biotechnology for Biofuels RESEARCH Open Access Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives Bakht Zada1†, Chonglong Wang2†, Ji‑Bin Park1, Seong‑Hee Jeong1, Ju‑Eon Park1, Hawaibam Birla Singh1 and Seon‑Won Kim1* Abstract Background: Current petroleum-derived fuels such as gasoline ­(C5–C12) and diesel ­(C15–C22) are complex mixtures of hydrocarbons with diferent chain lengths and chemical structures. Isoprenoids are hydrocarbon-based compounds with diferent carbon chain lengths and diverse chemical structures, similar to petroleum. Thus, isoprenoid alcohols such as isopentenol ­(C5), geraniol ­(C10), and farnesol ­(C15) have been considered to be ideal biofuel candidates. NudB, a native phosphatase of Escherichia coli, is reported to dephosphorylate isopentenyl diphosphate (IPP) and dimethy‑ lallyl diphosphate (DMAPP) into isopentenol. However, no attention has been paid to its promiscuous activity toward longer chain length ­(C10–C15) prenyl diphosphates. Results: In this study, the promiscuous activity of NudB toward geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) was applied for the production of isoprenoid alcohol mixtures, including isopentenol, geraniol, and farnesol, and their derivatives. E. coli was engineered to produce a mixture of ­C5 and ­C15 alcohols by overexpressing NudB (dihy‑ droneopterin triphosphate diphosphohydrolase) and IspA (FPP synthase) along with a heterologous MVA pathway, which resulted in a total of up to 1652 mg/L mixture of ­C5 and ­C15 alcohols and their derivatives. The production was further increased to 2027 mg/L by overexpression of another endogenous phosphatase, AphA, in addition to NudB. Production of DMAPP- and FPP-derived alcohols and their derivatives was signifcantly increased with an increase in the gene dosage of idi, encoding IPP isomerase (IDI), indicating a potential modulation of the composition of the alcohols mixture according to the expression level of IDI. When IspA was replaced with its mutant IspA*, generating GPP in the production strain, a total of 1418 mg/L of the isoprenoid mixture was obtained containing ­C10 alcohols as a main component. Conclusions: The promiscuous activity of NudB was newly identifed and successfully used for production of isopre‑ noid-based alcohol mixtures, which are suitable as next-generation biofuels or commodity chemicals. This is the frst successful report on high-titer production of an isoprenoid alcohol-based mixture. The engineering approaches can provide a valuable platform for production of other isoprenoid mixtures via a proportional modulation of IPP, DMAPP, GPP, and FPP syntheses. Keywords: Isoprenoid mixture, Isopentenol, Geraniol, Farnesol, Escherichia coli, NudB *Correspondence: [email protected] †Bakht Zada and Chonglong Wang contributed equally to this work 1 Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 52828, Republic of Korea Full list of author information is available at the end of the article © The Author(s) 2018. 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. Zada et al. Biotechnol Biofuels (2018) 11:210 Page 2 of 12 Background however, only a tiny amount (5.3 mg/L) of geraniol was Petroleum-derived fuels used for transportation are com- produced by PhoA in a fask culture [29]. Farnesyl phos- plex mixtures of carbon compounds with diferent chain phatases from Homo sapiens and Drosophila mela- lengths and chemical structures. For example, gasoline, nogaster can hydrolyze FPP to farnesol [30, 31]. Farnesol diesel, and jet fuels are mixture of hydrocarbons with synthase from Oryza sativa and sesquiterpene synthase carbon number distribution from 5 to 12, 15–22, and from Zea mays catalyze the conversion of FPP to farnesol 8–16, respectively. With the increasing concern about [32, 33]. Several phosphatases from S. cerevisiae such as the limited supply of petroleum and the efects of climate a truncated alkaline phosphatase, diacylglycerol diphos- change [1], interest in researching microbial production phate phosphatase, and lipid phosphate phosphatase of sustainable and renewable biofuels has increased [2]. show specifcity toward FPP [34, 35]. E. coli native phos- Advanced biofuels are required to have similar fuel prop- phatases, PhoA, AphA, BacA, PgpA, and PgpB, have also erties to petroleum-derived gasoline, diesel, or jet fuels in been assessed for farnesol synthesis [36, 37]. However, order to replace the current fuels [3–5]. the enzymes investigated for farnesol synthesis have low Isoprenoids, also known as terpenoids, are a large substrate specifcity, low catalytic activity, or poor pro- and diverse class of natural compounds used as phar- tein expression in microbial hosts. Tus, the discovery of maceuticals, fragrances and favors, solvents, and more a promising candidate enzyme that can efectively cata- recently, potential advanced biofuels [6–9]. Isopentenol lyze dephosphorylations of prenyl diphosphates to their (C5H10O, also known as isoprenol and prenol), geraniol corresponding alcohols is still needed. (C10H18O), and farnesol (C15H26O) are important acy- Nudix hydrolases, which are distributed among all clic hemiterpene, monoterpene, and sesquiterpene alco- kingdoms of life, potentially recognize and dephospho- hols, respectively. Tese alcohols are considered to be rylate phosphate moieties of a wide range of phosphoryl- advanced biofuels due to their high-energy content, low ated compounds [38, 39]. Nudix hydrolases from Bacillus hygroscopicity, and low volatility [2, 10–13]. Addition- subtilis (BsNudF) and E. coli (NudB, NudF, NudI, NudJ, ally, they have a wide range of commercial applications and NudM) convert IPP and DMAPP into isopentenol as favor, fragrance, pharmaceuticals, biopesticides, and [13, 40]. As Nudix hydrolases have been suspected to precursors to important industrial chemicals [7, 14–23]. dephosphorylate isopentenyl diphosphates to isoprenoid Isopentenol, geraniol, and farnesol are biologically gener- alcohols, the highly active NudB is used here for produc- ated from prenyl diphosphates precursors, IPP/DMAPP, tion of a mixture of isoprenoid alcohols including geran- GPP, and FPP, respectively. IPP and DMAPP are synthe- iol, farnesol, and isopentenol (Fig. 1a). sized via the methylerythritol phosphate (MEP) pathway, starting from condensation of pyruvate and glyceralde- Results and discussion hyde-3-phosphate (G-3-P) in most prokaryotes including Engineering a synthetic pathway for a mixture E. coli, and the mevalonate (MVA) pathway, commencing of isoprenoid‑based C5 and C15 alcohols with condensation of three acetyl-CoA in eukaryotes [24, In order to convert IPP/DMAPP to isopentenol and FPP 25]. GPP and FPP are synthesized by an isoprenyl diphos- to farnesol in E. coli, it is necessary to overexpress endog- phate synthase such as IspA via a sequential head-to-tail enous or exogenous phosphatases for dephosphorylation condensation of IPP and DMAPP. of these prenyl diphosphates. Te endogenous phos- Te dephosphorylation of IPP/DMAPP, GPP, and FPP phatase NudB efectively hydrolyzes IPP and DMAPP to to isopentenol, geraniol, and farnesol, respectively can isopentenol [13, 41–43]. As Nudix hydrolases generally be catalyzed by various phosphatases, diphosphatases, show broad substrate specifcities [38], we hypothesized and terpene synthases. Some of E. coli endogenous that NudB could also have catalytic activity toward FPP. phosphatases efciently catalyze the dephosphorylation Tus, nudB was cloned with ispA encoding FPP synthase of IPP and DMAPP to isopentenol [13], but an efec- into a strong expression vector, pTrc99A, resulting in tive native enzyme, catalyzing GPP and FPP to geraniol plasmid pT-MBF1.0 (Fig. 1b). Plasmid pSNA, express- and farnesol, respectively, has not yet been identifed. ing an exogenous mevalonate (MVA) pathway, was used Exogenous geraniol synthase from Ocimum basilicum to efectively supply prenyl diphosphate precursors has been expressed for geraniol production in E. coli for synthesis of mixture isoprenoid alcohols (Fig. 1b) [26–28]. Bifunctional diacylglycerol diphosphate phos- [44]. Both of the plasmids were transformed into E. coli phatase (DPP1) and lipid phosphate phosphatase (LPP1) DH5α, resulting in strain NA-MBF1.0 (Additional fle 1: from Saccharomyces cerevisiae have been evaluated Table S1). After culture of the strain for 48 h, the cul- for geraniol biosynthesis [29]. Te endogenous E. coli ture broth was analyzed by gas chromatography (GC), ADP-ribose pyrophosphatase (NudF) and alkaline phos- which showed seven peaks that were subsequently iden- phatase (PhoA) were also tested for geraniol production; tifed through gas chromatography–mass spectrometry Zada et al. Biotechnol Biofuels (2018) 11:210 Page 3 of 12 Fig. 1 Schematic overview of synthesis pathway (a) and expression constructs containing the pathway genes (b). GPP and FPP