MEP pathway Pyruvate + NADP NADPH2 Glucose Isopentenol NADP+ + 2NADPH 2NADP G3P 2 NADPH2 Pentose Phosphate pathway MEP pathway-mediated isopentenol production in metabolically engineered Escherichia coli Liu et al. Liu et al. Microbial Cell Factories 2014, 13:135 http://www.microbialcellfactories.com/content/13/1/135 Liu et al. Microbial Cell Factories 2014, 13:135 http://www.microbialcellfactories.com/content/13/1/135 RESEARCH Open Access MEP pathway-mediated isopentenol production in metabolically engineered Escherichia coli Huaiwei Liu1,2†, Yang Wang1,2†, Qiang Tang1,2, Wentao Kong1,2, Wook-Jin Chung3 and Ting Lu1,2,4* Abstract Background: Isopentenols, such as prenol and isoprenol, are promising advanced biofuels because of their higher energy densities and better combustion efficiencies compared with ethanol. Microbial production of isopentenols has been developed recently via metabolically engineered E. coli. However, current yields remain low and the underlying pathways require systematic optimization. Results: In this study, we targeted the E. coli native 2-methyl-(D)-erythritol-4-phosphate (MEP) pathway and its upstream glycolysis pathway for the optimization of isopentenol production. Two codon optimized genes, nudF and yhfR from Bacillus subtilis, were synthesized and expressed in E. coli W3110 to confer the isopentenol production of the strain. Two key enzymes (IspG and Dxs) were then overexpressed to optimize the E. coli native MEP pathway, which led to a significant increase (3.3-fold) in isopentenol production. Subsequently, the glycolysis pathway was tuned to enhance the precursor and NADPH supplies for the MEP pathway by activating the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (ED), which resulted in additional 1.9 folds of increase in isopentenol production. A 5 L-scale batch cultivation experiment was finally implemented, showing a total of 61.9 mg L−1 isopentenol production from 20 g L−1 of glucose. Conclusion: The isopentenol production was successfully increased through multi-step optimization of the MEP and its upstream glycolysis pathways. It demonstrated that the total fluxes and their balance of the precursors of the MEP pathway are of critical importance in isopentenol production. In the future, an elucidation of the contribution of PPP and ED to MEP is needed for further optimization of isopentenol production. Keywords: Isopentenol, MEP pathway, Entner-Doudoroff pathway, Pentose phosphate pathway (PPP), Escherichia coli, Metabolic engineering Background Recently, microbial production of advanced biofuels, The growing global demand for energy, continuing con- such as butanol and isobutanol, is being increasingly cerns about the environment and the increasing cost of explored due to the facts that advanced biofuels have fossil fuels have called for the development of new forms higher energy contents and, more importantly, are more of energy [1,2]. The microbial production of bio-based compatible with existing engines and fuel distribution fuels and chemicals from biomass has recently become as infrastructures [7-9]. One class of such advanced biofuels a promising, sustainable solution to this need [3,4]. Early is isopentenols, including prenol and isoprenol, which efforts in biofuel production have focused on improving have better combustion efficiencies than ethanol and the yield of ethanol made from the fermentation of plant better octane numbers that are more similar to that of sugars, and have shown a great success [5,6]. gasoline compared with ethanol [10]. Isopentenol production using engineered microbes was first reported in 2007 by Withers and co-workers [11]. * Correspondence: [email protected] Through the enrichment of a library of genomic DNA †Equal contributors 1Department of Bioengineering, University of Illinois at Urbana-Champaign, from Bacillus subtilis, the authors discovered two genes Urbana IL61801, USA (yhfR and nudF) that encode enzymes that function di- 2 Institute for Genomic Biology, University of Illinois at Urbana-Champaign, rectly on the prenyl diphosphate precursors, isopentenyl Urbana IL61801, USA Full list of author information is available at the end of the article pyrophosphate (IPP) and dimethylallyl pyrophosphate © 2014 Liu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. Liu et al. Microbial Cell Factories 2014, 13:135 Page 2 of 7 http://www.microbialcellfactories.com/content/13/1/135 (DMAPP) and convert them to isopentenols. In 2012, Chou and Keasling reported another gene nudB which Glucose encodes an enzyme capable of converting prenyl diphos- glycolysis phate precursors to isopentenols [10]. Overexpression of these genes along with the introduction of a heterologous G3P Pyruvate mevalonate-dependent pathway (MVA) in E. coli has resulted in successful isopentenol production [10-12]. dxs In addition to the MVA pathway, the E. coli native MEP pathway also holds the potential to be exploited DXP for isopentenol production. Indeed, several attempts NADPH+H + have been made for successful biosynthesis of other ispC NADP+ forms of isoprenoids including taxadiene through the E. coli native MEP pathway [13-15]. Up to date, reported MEP CTP isopentenol production via the MVA pathway is much ispD PPi higher than that via the MEP pathway. On the other CDP-ME hand, there are advantages in using the MEP pathway: ATP MEP According to the stoichiometry and redox balance ana- ispE ADP Pathway lysis by a previous study [16], MEP is energetically more CDP-MEP balanced and theoretically more efficient than MVA in converting sugars or glycerol to isoprenoid. In addition, CMP ispF due to the inherent presence of the MEP pathway, it MECPP requires fewer heterologous genes to be introduced. 2H ++2e We were therefore motivated in this work to increase ispG H2O the isopentenol production of engineered E. coli by opti- HMBPP mizing its inherent MEP pathway (Figure 1). We first NAD(P)H+H + synthesized two codon optimized genes, nudF and yhfR ispH from B. subtilis, and expressed them in our expression host NAD(P)+ E. coli W3110 to confer its production of isopentenols IPP idi including both prenol and isoprenol. We then over- DMAPP expressed two key enzymes (IspG and Dxs) to optimize nudF the native E. coli MEP pathway, leading to a significant increase in isopentenol titer. To further maximize the Isoprenol Prenol isopentenol productivity, we enhanced the precursor Figure 1 The isopentenol biosynthesis pathway in engineered and NADPH supplies needed for the MEP pathway by E. coli. Pathway intermediates: G3P, glyceraldehyde-3-phosphate; activating the pentose phosphate pathway (PPP) and DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-D-erythritol Entner-Doudoroff (ED) pathway through the disruption 4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methyl-D-erythritol; of the E. coli phosphoglucose isomerase gene (pgi). To CDP-MEP, 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; MECPP, 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate; HMBPP, evaluate the efficacy of our multi-step optimization of 1-hydroxy-2-methyl-2-(E)-butenyl 4-pyrophosphate; IPP, isopentenyl isopentenol production, we finally implemented a 5 L- pyrophosphate; DMAPP, dimethylallyl pyrophosphate; DHAP, scale laboratory batch fermentation using the optimal dihydroxyacetone 3-phosphate. strain identified. E. coli host, the ORF sequences of these two genes were Results and discussion optimized to achieve a CAI of 1.0 for both (See Additional Expression of nudF and yhfR in E. coli W3110 file 1). The optimized yhfR and nudF genes were then To enable a successful isopentenol production in E. coli, synthesized, ligated into the expression vector pACYC- two genes, nudF and yhfR from Bacillus subtilis, were duet, and subsequently introduced into E. coli W3110 identified as heterologous enzyme genes for direct con- (DE3) for isopentenol production. GC analysis results version of IPP and DMAPP into isoprenol and prenol showed that, after 48 h of cultivation in M9 medium con- respectively. However, due to the different codon usages taining yeast extract and glucose, the strain E. coli W3110 of E. coli and B. subtilis, the codon adaption indexes (DE3)/pACYC-yhfR produced a tiny amount of isoprenol − (CAI) of the wild-type yhfR (GeneBank: CP002906.1) and prenol (<1 mg L 1) (Figure 2, EWIP1). In contrast, the and nudF (GeneBank: CP003329.1) genes for the host strain E. coli W3110 (DE3)/pACYC-nudF produced − − E. coli W3110 are only 0.31 and 0.25 respectively [17]. 2.6 mg L 1 prenol and 2.2 mg L 1 isoprenol (Figure 2, To address the potential low expression issue in the EWIP2). No isoprenol or prenol production was detected Liu et al. Microbial Cell Factories 2014, 13:135 Page 3 of 7 http://www.microbialcellfactories.com/content/13/1/135 in the control strain that harbors the empty vector pACYC- Tuning the glycolysis pathway to maximize the duet (Figure 2, Control). These results supported the isopentenol production earlier shown feasibility of producing isopentenol in To further increase the isopentenol production of our E. coli and also suggested that NudF is a more effective engineered strains, we traced back to the precursor enzyme than yhfR in isopentenol production. supplies of the MEP pathway and examined their roles in influencing the overall isopentenol production. As shown in the first step of MEP pathway in Figure 1, Optimization of MEP pathway for increased isopentenol one mole of deoxyxylulose-5-phosphate (DXP) is formed production from the equimolar condensation of glyceraldehyde-3- To elevate isopentenol production, the upstream MEP phosphate (G3P) and pyruvate. This suggests that limita- pathway was targeted for optimization. In a recent study, tions in G3P or pyruvate or their imbalance may reduce Zhou et al.