Dataset Information

¹³C metabolic flux analysis-guided metabolic engineering of Escherichia coli for improved acetol production from glycerol.

ABSTRACT:

Background

Bioprocessing offers a sustainable and green approach to manufacture various chemicals and materials. Development of bioprocesses requires transforming common producer strains to cell factories. ¹³C metabolic flux analysis (¹³C-MFA) can be applied to identify relevant targets to accomplish the desired phenotype, which has become one of the major tools to support systems metabolic engineering. In this research, we applied ¹³C-MFA to identify bottlenecks in the bioconversion of glycerol into acetol by Escherichia coli. Valorization of glycerol, the main by-product of biodiesel, has contributed to the viability of biofuel economy.

Results

We performed ¹³C-MFA and measured intracellular pyridine nucleotide pools in a first-generation acetol producer strain (HJ06) and a non-producer strain (HJ06C), and identified that engineering the NADPH regeneration is a promising target. Based on this finding, we overexpressed nadK encoding NAD kinase or pntAB encoding membrane-bound transhydrogenase either individually or in combination with HJ06, obtaining HJ06N, HJ06P and HJ06PN. The step-wise approach resulted in increasing the acetol titer from 0.91 g/L (HJ06) to 2.81 g/L (HJ06PN). To systematically characterize and the effect of mutation(s) on the metabolism, we also examined the metabolomics and transcriptional levels of key genes in four strains. The pool sizes of NADPH, NADP⁺ and the NADPH/NADP⁺ ratio were progressively increased from HJ06 to HJ06PN, demonstrating that the sufficient NADPH supply is critical for acetol production. Flux distribution was optimized towards acetol formation from HJ06 to HJ06PN: (1) The carbon partitioning at the DHAP node directed gradually more carbon from the lower glycolytic pathway through the acetol biosynthetic pathway; (2) The transhydrogenation flux was constantly increased. In addition, ¹³C-MFA showed the rigidity of upper glycolytic pathway, PP pathway and the TCA cycle to support growth. The flux patterns were supported by most metabolomics data and gene expression profiles.

Conclusions

This research demonstrated how ¹³C-MFA can be applied to drive the cycles of design, build, test and learn implementation for strain development. This succeeding engineering strategy can also be applicable for rational design of other microbial cell factories.

SUBMITTER: Yao R

PROVIDER: S-EPMC6373095 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundGlycerol, 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.ResultsBy 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.ConclusionsThis 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.

Dataset Information

¹³C metabolic flux analysis-guided metabolic engineering of Escherichia coli for improved acetol production from glycerol.

Background

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

13C metabolic flux analysis-guided metabolic engineering of Escherichia coli for improved acetol production from glycerol.

Background

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

¹³C metabolic flux analysis-guided metabolic engineering of Escherichia coli for improved acetol production from glycerol.