Elucidation of the co-metabolism of glycerol and glucose in Escherichia coli by genetic engineering, transcription profiling, and 13 C metabolic flux analysis

Yao et al. Biotechnol Biofuels (2016) 9:175 DOI 10.1186/s13068-016-0591-1 Biotechnology for Biofuels Open Access RESEARCH Elucidation of the co‑metabolism of glycerol and glucose in Escherichia coli by genetic engineering, transcription profiling, and 13C metabolic flux analysis Ruilian Yao1, Dewang Xiong1, Hongbo Hu1, Masataka Wakayama2, Wenjuan Yu3, Xuehong Zhang1* and Kazuyuki Shimizu2* Abstract Background: Glycerol, a byproduct of biodiesel, has become a readily available and inexpensive carbon source for the production of high-value products. However, the main drawback of glycerol utilization is the low consumption rate and shortage of NADPH formation, which may limit the production of NADPH-requiring products. To overcome these problems, we constructed a carbon catabolite repression-negative ΔptsGglpK* mutant by both blocking a key glucose PTS transporter and enhancing the glycerol conversion. The mutant can recover normal growth by co-utilization of glycerol and glucose after loss of glucose PTS transporter. To reveal the metabolic potential of the ΔptsGglpK* mutant, this study examined the flux distributions and regulation of the co-metabolism of glycerol and glucose in the mutant. Results: By labeling experiments using [1,3-13C]glycerol and [1-13C]glucose, 13C metabolic flux analysis was employed to decipher the metabolisms of both the wild-type strain and the ΔptsGglpK* mutant in chemostat cultures. When cells were maintained at a low dilution rate (0.1 h−1), the two strains showed similar fluxome profiles. When the dilution rate was increased, both strains upgraded their pentose phosphate pathway, glycolysis and anaplerotic reactions, while the ΔptsGglpK* mutant was able to catabolize much more glycerol than glucose (more than tenfold higher). Compared with the wild-type strain, the mutant repressed its flux through the TCA cycle, resulting in higher acetate overflow. The regulation of fluxomes was consistent with transcriptional profiling of several key genes relevant to the TCA cycle and transhydrogenase, namely gltA, icdA, sdhA and pntA. In addition, cofactor fluxes and their pool sizes were determined. The ΔptsGglpK* mutant affected the redox NADPH/NADH state and reduced the ATP level. Redox signaling activated the ArcA regulatory system, which was responsible for TCA cycle repression. Conclusions: This work employs both 13C-MFA and transcription/metabolite analysis for quantitative investigation of the co-metabolism of glycerol and glucose in the ΔptsGglpK* mutant. The ArcA regulatory system dominates the control of flux redistribution. The ΔptsGglpK* mutant can be used as a platform for microbial cell factories for the production of biofuels and biochemicals, since most of fuel molecule (e.g., alcohols) synthesis requires excess reducing equivalents. Keywords: Glycerol, 13C metabolic flux analysis, Carbon catabolite repression, Cofactor, PTS, Transcriptional regulation *Correspondence: ; 1 State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China 2 Institute for Advanced Biosciences, Keio University, 246‑2, Mizukami, Kakuganji, Tsuruoka, Yamagata 997‑0052, Japan Full list of author information is available at the end of the article © 2016 The Author(s). 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. Yao et al. Biotechnol Biofuels (2016) 9:175 Background With increasing production of biodiesel, a large amount of glycerol is produced as an inevitable byproduct [1]. Significant glycerol surplus has led to a drastic decrease in glycerol prices over the past few years, which makes it an ideal feedstock for the production of high-value products [1–3]. The main drawback of glycerol utilization in Escherichia coli is the relatively low carbon source consumption rate, cell growth, and productivity [4]. The main reason for this is the allosteric inhibition of the rate-limiting GLPK by FBP and EIIAGlc under aerobic conditions [5–8]. FBP and EIIAGlc both act to reduce vmax of GLPK, which displays a dimer–tetramer equilibrium in solution [6]. FBP acts both to promote dimer–tetramer assembly and to inactivate the tetramers [7]. The crystal structure of the EIIAGlc:GLPK complexes has been determined [8]. GLPK with bound glycerol and ADP forms tetramers in which each GLPK subunit interacts with one EIIAGlc molecule, and the association of the two proteins forms a novel intermolecular binding site for Zn (II) [9]. Genetic modification of the glpK gene has resulted in the change in this enzyme that is insensitive to FBP and EIIAGlc, allowing improved the glycerol consumption rate [10, 11]. Another obstacle to glycerol utilization is the shortage of NADPH formation because minimal glycolytic flux is reverted from GAP upwards to the oxidative pentose phosphate (PP) pathway. Because NADPH is an important cofactor needed for the production of useful metabolites, co-fermentation of glycerol with glucose has been proposed as an efficient process [4, 12, 13], especially for promoting 1,3-propanediol fermentation for the industrial scale production by DuPont [14]. However, glucose utilization prevents the metabolism of glycerol because of carbon catabolite repression (CCR) [15]. The central players in CCR in E. coli are the transcriptional activator Crp, cAMP receptor protein, the signal metabolite cAMP, Cya, and the phosphorylation system (PTS); these systems are involved in transport and/ or phosphotransferase reactions of carbohydrates [15]. The PTS in E. coli consists of two common cytoplasmic proteins, EI, encoded by ptsI, and HPr, encoded by ptsH, as well as carbohydrate-specific EII complexes encoded by crr and ptsG [16]. One metabolic engineering strategy to relax CCR is the inactivation of PTS genes [13, 17, 18]. The part PEP not consumed in glucose transport was canalized to shikimate pathway [17], which is a very important route for the synthesis of aromatic amino acids and natural products [19]. Therefore, we constructed a CCR-negative ΔptsGglpK* mutant by both blocking a key glucose transporter gene ptsG and replacing the native glpK with glpK22 from E. coli Lin 43 to enhance the glycerol conversion [12]. This mutant can co-consume glycerol and glucose with a faster glycerol assimilation rate Page 2 of 14 than glucose assimilation rate [12]. To reveal the metabolic potential of the ΔptsGglpK* mutant, it is necessary to understan (...truncated)