Rewiring glycerol metabolism for enhanced production of poly-γ-glutamic acid in Bacillus licheniformis

Abstract

BackgroundPoly-γ-glutamic acid (γ-PGA) is a natural polymer with great potential applications in areas of agriculture, industry, and pharmaceutical. The biodiesel-derived glycerol can be used as an attractive feedstock for γ-PGA production due to its availability and low price; however, insufficient production of γ-PGA from glycerol is limitation.ResultsThe metabolic pathway of Bacillus licheniformis WX-02 was rewired to improve the efficiency of glycerol assimilation and the supply of NADPH for γ-PGA synthesis. GlpK, GlpX, Zwf, and Tkt1 were found to be the key enzymes for γ-PGA synthesis using glycerol as a feedstock. Through combinational expression of these key enzymes, the γ-PGA titer increased to 19.20 ± 1.57 g/L, which was 1.50-fold of that of the wild-type strain. Then, we studied the flux distributions, gene expression, and intracellular metabolites in WX-02 and the recombinant strain BC4 (over-expression of the above quadruple enzymes). Our results indicated that over-expression of the quadruple enzymes redistributed metabolic flux to γ-PGA synthesis. Furthermore, using crude glycerol as carbon source, the BC4 strain showed a high productivity of 0.38 g/L/h, and produced 18.41 g/L γ-PGA, with a high yield of 0.46 g γ-PGA/g glycerol.ConclusionsThe approach to rewiring of metabolic pathways enables B. licheniformis to efficiently synthesize γ-PGA from glycerol. The γ-PGA productivity reported in this work is the highest obtained in glutamate-free medium. The present study demonstrates that the recombinant B. licheniformis strain shows significant potential to produce valuable compounds from crude glycerol.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1311-9) contains supplementary material, which is available to authorized users.

Sustainable biosynthesis of chemicals from methane and glycerol via reconstruction of multi‐carbon utilizing pathway in obligate methanotrophic bacteria

“…S1 ). Previously, the poor growth on glycerol of some bacteria was also reported, and it was suggested that this glycerol utilization pathway might be toxic for cells due to unbalanced redox power (Kang et al., 2014 ), allosteric inhibition (Applebee et al., 2011 , Zhan et al., 2018 ) or intermediate toxicity (Rittmann et al., 2008 ). In E. coli , the glpFK operon is tightly controlled by the Glp repressor, which is not present in M. alcaliphilum 20Z, and expression of two‐gene cluster glpFK may not be the main reason for poor growth on glycerol in this engineered strain (Weissenborn et al., 1992 ).…”

Recent Advances in Microbial Synthesis of Poly-γ-Glutamic Acid: A Review

“…Based on the anti-freezing activity, γ-PGA is also a cryoprotectant to preserve the viability of probiotics during freeze-drying [ 130 ]. γ-PGA, with a molecular weight of lower than 20,000 Da, has higher anti-freezing activity than glucose and is more effectively protective to Lactobacillus paracasei than sucrose, trehalose, and sorbitol [ 29 , 81 ]. Furthermore, γ-PGA can increase the Ca 2+ bioavailability and effectively prompt intestinal absorption [ 131 ].…”

“…γ-PGA, a natural, non-toxic, and non-immunogenic biomacromolecule, can significantly decrease the toxic effects and improve the efficiency of drugs when combined with other matters [ 11 ]. Especially, biodegradable γ-PGA has a bright potential in drug-delivery platforms and as suitable carriers for gene therapy [ 81 ]. Molecular weight of γ-PGA was the decisive factor for considering the drug-delivery properties, including controlling or delaying the release [ 11 ].…”

“…γ-PGA is expected to be the most useful flocculating agent due to its biodegradability and non-toxicity toward humans and the environment, which is superior to other conventional agents [ 19 , 138 ]. γ-PGA will be utilized not only in biomass waste and wastewater treatments in the industry but also in domestic water processing [ 81 ]. Besides, numerous chemically modified derivatives of γ-PGA have been developed so far as biopolymers utilized as novel biomaterials [ 12 ]; e.g., esterified γ-PGA can be used as an excellent thermoplastic for its capability to form biodegradable fibers and film [ 44 , 81 ].…”

“…γ-PGA will be utilized not only in biomass waste and wastewater treatments in the industry but also in domestic water processing [ 81 ]. Besides, numerous chemically modified derivatives of γ-PGA have been developed so far as biopolymers utilized as novel biomaterials [ 12 ]; e.g., esterified γ-PGA can be used as an excellent thermoplastic for its capability to form biodegradable fibers and film [ 44 , 81 ].…”