title: Elucidation of the co-metabolism of glycerol and glucose in Escherichia coli by genetic engineering, transcription profiling, and (13)C metabolic flux analysis.
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-(13)C]glycerol and [1-(13)C]glucose, (13)C 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 (13)C-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.
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240 China.
authors.forename: Ruilian
authors.initials: R
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240 China.
authors.forename: Dewang
authors.initials: D
authors.lastname: Xiong
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240 China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Hu
authors.affiliation: Institute for Advanced Biosciences, Keio University, 246-2, Mizukami, Kakuganji, Tsuruoka, Yamagata 997-0052 Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Wakayama
authors.affiliation: Instrumental Analysis Center, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240 China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240 China.
authors.forename: Xuehong
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Institute for Advanced Biosciences, Keio University, 246-2, Mizukami, Kakuganji, Tsuruoka, Yamagata 997-0052 Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Shimizu
keywords: 13C metabolic flux analysis; Carbon catabolite repression; Cofactor; Glycerol; PTS; Transcriptional regulation