Dataset Information

¹³C-Metabolic Flux Analysis Reveals the Metabolic Flux Redistribution for Enhanced Production of Poly-?-Glutamic Acid in dlt Over-Expressed Bacillus licheniformis.

ABSTRACT: Poly-?-glutamic acid (?-PGA) is an anionic polymer with various applications. Teichoic acid (TA) is a special component of cell wall in gram-positive bacteria, and its D-alanylation modification can change the net negative charge of cell surface, autolysin activity and cationic binding efficiency, and might further affect metabolic production. In this research, four genes (dltA, dltB, dltC, and dltD) of dlt operon were, respectively, deleted and overexpressed in the ?-PGA producing strain Bacillus licheniformis WX-02. Our results implied that overexpression of these genes could all significantly increase ?-PGA synthetic capabilities, among these strains, the dltB overexpression strain WX-02/pHY-dltB owned the highest ?-PGA yield (2.54 g/L), which was 93.42% higher than that of the control strain WX-02/pHY300 (1.31 g/L). While, the gene deletion strains produced lower ?-PGA titers. Furthermore, ¹³C-Metabolic flux analysis was conducted to investigate the influence of dltB overexpression on metabolic flux redistribution during ?-PGA synthesis. The simulation data demonstrated that fluxes of pentose phosphate pathway and tricarboxylic acid cycle in WX-02/pHY-dltB were 36.41 and 19.18 mmol/g DCW/h, increased by 7.82 and 38.38% compared to WX-02/pHY300 (33.77 and 13.86 mmol/g DCW/h), respectively. The synthetic capabilities of ATP and NADPH were also increased slightly. Meanwhile, the fluxes of glycolytic and by-product synthetic pathways were all reduced in WX-02/pHY-dltB. All these above phenomenons were beneficial for ?-PGA synthesis. Collectively, this study clarified that overexpression of dltB strengthened the fluxes of PPP pathway, TCA cycle and energy metabolism for ?-PGA synthesis, and provided an effective strategy for enhanced production of ?-PGA.

SUBMITTER: He P

PROVIDER: S-EPMC6367249 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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13C-Metabolic Flux Analysis Reveals the Metabolic Flux Redistribution for Enhanced Production of Poly-γ-Glutamic Acid in dlt Over-Expressed Bacillus licheniformis.

He Penghui P Wan Ni N Cai Dongbo D Hu Shiying S Chen Yaozhong Y Li Shunyi S Chen Shouwen S

Frontiers in microbiology 20190201

Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with various applications. Teichoic acid (TA) is a special component of cell wall in gram-positive bacteria, and its D-alanylation modification can change the net negative charge of cell surface, autolysin activity and cationic binding efficiency, and might further affect metabolic production. In this research, four genes (dltA, dltB, dltC, and dltD) of dlt operon were, respectively, deleted and overexpressed in the γ-PGA pr ...[more]

PMID: 30774627

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Project description:BackgroundBioprocessing 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.ResultsWe 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.ConclusionsThis 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.

Dataset Information

¹³C-Metabolic Flux Analysis Reveals the Metabolic Flux Redistribution for Enhanced Production of Poly-?-Glutamic Acid in dlt Over-Expressed Bacillus licheniformis.

Publications

<sup>13</sup>C-Metabolic Flux Analysis Reveals the Metabolic Flux Redistribution for Enhanced Production of Poly-γ-Glutamic Acid in <i>dlt</i> Over-Expressed <i>Bacillus licheniformis</i>.

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Dataset Information

13C-Metabolic Flux Analysis Reveals the Metabolic Flux Redistribution for Enhanced Production of Poly-?-Glutamic Acid in dlt Over-Expressed Bacillus licheniformis.

Publications

<sup>13</sup>C-Metabolic Flux Analysis Reveals the Metabolic Flux Redistribution for Enhanced Production of Poly-γ-Glutamic Acid in <i>dlt</i> Over-Expressed <i>Bacillus licheniformis</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

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¹³C-Metabolic Flux Analysis Reveals the Metabolic Flux Redistribution for Enhanced Production of Poly-?-Glutamic Acid in dlt Over-Expressed Bacillus licheniformis.