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

Abstract

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 producing strain Bacillus licheniformis WX-02. Our results implied that overexpression of these genes could all significantly increase γ-PGA synthetic capabilities, among these strains, the dltB overexpression strain WX-02/pHY-dltB owned the highest γ-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 γ-PGA titers. Furthermore, 13C-Metabolic flux analysis was conducted to investigate the influence of dltB overexpression on metabolic flux redistribution during γ-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 γ-PGA synthesis. Collectively, this study clarified that overexpression of dltB strengthened the fluxes of PPP pathway, TCA cycle and energy metabolism for γ-PGA synthesis, and provided an effective strategy for enhanced production of γ-PGA.

Identification and Characterization of a New Regulator, TagR, for Environmental Stress Resistance Based on the DNA Methylome of Streptomyces roseosporus

“…WTA has essential physiological functions in cell division, drug resistance, and biofilm formation ( 56 – 58 ). Moreover, it has been reported that the yield of different secreted products could be improved by altering WTA degradation in Escherichia and changing the d -alanylation modification of WTA in Bacillus ( 59 – 61 ). However, in actinomycetes the mechanisms of WTA synthesis and regulation are poorly understood ( 54 ).…”

Promoting Cell Growth And Poly-Y-Glutamic Acid Production By Boosting The Synthesis of Alanine And D-Alanyl-D-Alanine In Bacillus Licheniformis

“…While, the high viscosity of γ-PGA decreased the concentrations of dissolved oxygen and limited the absorption or utilization of nutrients during the fermentation, which property hindered the cell growth and the synthesis of γ-PGA (Hsueh et al 2017). Previous study in our lab found that the decrease of negative charge on cell wall surface could significantly improve cell growth and γ-PGA production in B. licheniformis (He et al 2019). So, cell wall properties are closely related to cell growth and γ-PGA synthesis.…”

“…Cell growth curve was determined by measuring the optical density at 600 nm (OD600) in LB medium. γ-PGA titer and biomass were detected by the method described in the previous research (He et al 2019). Concentrations of acetoin, 2,3-butanediol and acetic acid were quantified using Thermo Scientific GC-MS (Thermo, USA) equipped with TG-WAXMS column (30 m × 0.32 mm ID, 0.25 μm film) by an optimized method from previous research (Qi et al 2014).…”

Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production

“…While these foundational metabolic features have been clearly established, the metabolic rewiring of P. putida strains in response to complex engineering of central pathways is unknown. Many studies in microbes have examined flux changes in response to simple genetic edits ( i.e., deletion or overexpression ( He et al, 2019 ; Long and Antoniewicz, 2019b ; Long et al, 2016 ; Xu et al, 2021 ), however the impact of transcriptional downregulation due to multiplexed CRISPRi on flux topology has not been examined in-depth in any microbe. Due to the complexity and technical challenges needed to accurately quantify metabolic flux, previous researchers have used gene expression analysis (RNAseq) as a proxy in these engineered strains ( Reis et al, 2019 ; Silvis et al, 2021 ; Zhao et al, 2019 ).…”