Volke et al. Microb Cell Fact (2019) 18:192 https://doi.org/10.1186/s12934-019-1235-5 Microbial Cell Factories RESEARCH Open Access Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis Daniel Christoph Volke1,2, Johann Rohwer3, Rainer Fischer1,4 and Stefan Jennewein1* Abstract Background: Terpenoids are of high interest as chemical building blocks and pharmaceuticals. In microbes, terpe- noids can be synthesized via the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways. Although the MEP pathway has a higher theoretical yield, metabolic engineering has met with little success because the regulation of the pathway is poorly understood. Results: We applied metabolic control analysis to the MEP pathway in Escherichia coli expressing a heterologous isoprene synthase gene (ispS). The expression of ispS led to the accumulation of isopentenyl pyrophosphate (IPP)/ dimethylallyl pyrophosphate (DMAPP) and severely impaired bacterial growth, but the coexpression of ispS and iso- pentenyl diphosphate isomerase (idi) restored normal growth and wild-type IPP/DMAPP levels. Targeted proteomics and metabolomics analysis provided a quantitative description of the pathway, which was perturbed by randomizing the ribosome binding site in the gene encoding 1-deoxyxylulose 5-phosphate synthase (Dxs). Dxs has a fux control coefcient of 0.35 (i.e., a 1% increase in Dxs activity resulted in a 0.35% increase in pathway fux) in the isoprene-pro- ducing strain and therefore exerted signifcant control over the fux though the MEP pathway. At higher dxs expres- sion levels, the intracellular concentration of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) increased substantially in contrast to the other MEP pathway intermediates, which were linearly dependent on the abundance of Dxs. This indicates that 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), which consumes MEcPP, became saturated and therefore limited the fux towards isoprene. The higher intracellular concentrations of MEcPP led to the efux of this intermediate into the growth medium. Discussion: These fndings show the importance of Dxs, Idi and IspG and metabolite export for metabolic engineer- ing of the MEP pathway and will facilitate further approaches for the microbial production of valuable isoprenoids. Keywords: MEP pathway, Metabolic control analysis, Recombineering, Isoprene, E. coli Highlights • Metabolomic and proteomic analysis of the MEP pathway. • Metabolic control analysis of the MEP pathway in E. • Reduction of MEcPP to HMBPP is a limiting step coli. for fux through the MEP pathway and promotes the • Dxs has high fux and concentration control over efux of MEcPP at higher fux rates. MEP pathway intermediates. Background *Correspondence: [email protected] Microbes can synthesize a broad spectrum of valuable 1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany compounds and precursors, providing an inexpensive Full list of author information is available at the end of the article and sustainable source of industrially-relevant chemicals © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/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. Volke et al. Microb Cell Fact (2019) 18:192 Page 2 of 15 including terpenoids. More than 60,000 terpenoids purifcation, makes isoprene a promising product for fer- have been described [1], ranging from pharmacologi- mentation process [9]. Te MCA framework was applied cally active molecules such as paclitaxel and artemisinin, to the concentrations of MEP pathway metabolites and which are used for the treatment of cancer and malaria, the fux towards isoprene according to diferent expres- respectively [2, 3], to promising biofuels such as sion levels of the dxs gene. To our knowledge, this is the farnesene [4]. All terpenoids originate from the iso- frst time that this approach was combined with recom- meric precursors dimethylallyl pyrophosphate (DMAPP) bineering, quantitative proteomics and metabolomics. and isopentenyl pyrophosphate (IPP), both of which are Tis combination yielded a quantitative description of produced via one of two natural metabolic routes: the the control of Dxs on the fux through and metabolite 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway concentrations in the MEP pathway, which will guide fur- and the mevalonate (MVA) pathway [5]. ther engineering eforts. Te MVA pathway has been engineered to produce large quantities of terpenoids [6, 7] but attempts to engi- Materials and methods neer the MEP pathway have been less successful [8, 9]. In Bacterial strains and culture conditions part, this refects our incomplete knowledge of the reg- Standard cloning and metabolic engineering were carried ulation and control of the MEP pathway, despite recent out using E. coli strains 10β and BL21 (DE3), respectively reports that have revealed a number of regulatory mech- (both supplied by New England Biolabs, Ipswich, MA, anisms [10–14]. Even so, the MEP pathway is a promis- USA). For general cloning, the bacteria were cultivated ing target for metabolic engineering because of its higher in lysogenic broth (LB) medium, whereas for all other theoretical yield (~ 20% more than the MVA pathway experiments the bacteria were cultivated in M9 medium under aerobic conditions in Escherichia coli) and its bal- supplemented with 0.5% (w/v) glucose and appropri- anced use of reducing equivalents [15, 16]. Attempts to ate antibiotics (50 μg mL−1 kanamycin, 100 μg mL−1 engineer the MEP pathway have thus far included rand- ampicillin and/or 25 μg mL−1 chloramphenicol) at 37 °C omization approaches, the combinatorial expression of [24]. Strains carrying the plasmids pSIM5 or pSIM6 [25] a subset of MEP pathway genes [17–20] and the opti- were grown at 30 °C and cured of the plasmids at 37 °C. mization of precursor supply [21]. Eforts to circumvent Cultures were grown in bafed Erlenmeyer fasks flled regulation by expressing the complete MEP pathway in to one-ffth of their nominal volume and agitated at heterologous hosts such as the yeast Saccharomyces cer- 180 rpm. Cell growth in liquid medium was monitored evisiae have not been successful [22]. Several studies have by spectrophotometry to determine the optical density at investigated the rate-limiting steps in the MEP pathway, 600 nm (OD600). but have failed to deliver a consistent picture [17, 18, 20]. Others have found indications for the intrinsic regulation General cloning and amplifcation of the MEP pathway at the steps catalyzed by 1-deoxy- General cloning procedures and plasmid purifcation d-xylulose-5-phosphate synthase (Dxs) and 1-deoxy- were carried out according to standard laboratory prac- d-xylulose-5-phosphate reductoisomerase (Dxr) [10], tice [24]. Herculase II Fusion DNA Polymerase (Agilent 2-C-methyl-d-erythritol-4-phosphate cytidyltransferase Technologies, Santa Clara, CA, USA) was used for the (IspD) [11] or 2-C-methyl-d-erythritol-2,4-cyclopyroph- amplifcation of DNA fragments by the polymerase chain osphate synthase (IspF) [12]. Te MEP pathway interme- reaction (PCR) according to the manufacturer’s instruc- diate 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate tions. All plasmids and altered genomic regions were (MEcPP) is found at higher concentrations in the extra- verifed by Sanger sequencing carried out by Eurofns cellular milieu in E. coli overexpressing MEP pathway GmbH (Ebersberg, Germany). genes [14]. However, the behavior of the MEP pathway has not been described in a precise and quantitative Construction of plasmids for the production of isoprene manner either in wild-type or engineered microbes. Te ispS gene from Populus alba was codon optimized We therefore set out to establish a quantitative descrip- for E. coli (Additional fle 1: Table S1) (Termo Fisher Sci- tion of the MEP pathway for terpenoid production using entifc, Waltham, MA, USA) and the gene was amplifed metabolic control analysis (MCA), a form of sensitivity using forward primer 5′-AAT AAT TTT GTT TAA CTT analysis that quantifes the efect of small changes in a TAA TAA GGA GAT ATA CCA TGG AAG CTC GTC given parameter (such as enzyme activity) on overall sys- GTT CTG C-3′ and reverse primer 5′-TTA GCG TTC tem characteristics such as metabolite concentration and AAA TGG CAG TAG CAA GCT TGT CGA CCA CGT fux [23]. Here we used the MEP pathway for the produc- TCG AAC GGC AGG ATC-3′ (start codon in bold). Vec- tion of isoprene. Besides having broad industrial appli- tor pCOLA was amplifed using forward primer 5′-GGT cations, its low boiling point, and therefore its ease of ATA TCT CCT TAT TAA AGT TAA ACA-3′ and reverse Volke et al. Microb Cell Fact (2019) 18:192 Page 3 of 15 primer 5′-GGT CGA CAA GCT TGC GGC CG-3′. GCC AAC GCG CTT CAA TG-3′ and reverse primer Te products were joined by Gibson assembly yielding 5′-TCG TGG TGA AGC AGA ACA AG-3′ for the dxr pCOLA::IspS, which was then amplifed using forward expression library. Te amplicons were sequenced using primer 5′-GGC CGC ATA ATG CTT AAG TCG-3′ and the same primers. reverse primer 5′-GCA AGC TTG TC GAC CTT AGC- 3′. Te isopentenyl diphosphate isomerase (idi) gene was Metabolite quantifcation amplifed from the genome of E. coli strain BL21 using A 10-mL volume of M9 medium supplemented with forward primer 5′-TAC TGC CAT TTG AAC GCT AAG 0.5% (w/v) glucose was inoculated with 100 µL of over- GTC GAC AAG CTT GCA AGG AGA TAT ACC ATG night culture in 200-mL Erlenmeyer fasks.