Enhanced production of poly‐γ‐glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis

Abstract

Poly-γ-glutamic acid (γ-PGA) is an important multifunctional biopolymer with various applications, for which adenosine triphosphate (ATP) supply plays a vital role in biosynthesis. In this study, the enhancement of γ-PGA production was attempted through various approaches of improving ATP supply in the engineered strains of Bacillus licheniformis. The first approach is to engineer respiration chain branches of B. licheniformis, elimination of cytochrome bd oxidase branch reduced the maintenance coefficient, leading to a 19.27% increase of γ-PGA yield. The second approach is to introduce Vitreoscilla hemoglobin (VHB) into recombinant B. licheniformis, led to a 13.32% increase of γ-PGA yield. In the third approach, the genes purB and adK in ATP-biosynthetic pathway were respectively overexpressed, with the AdK overexpressed strain increased γ-PGA yield by 14.69%. Our study also confirmed that the respiratory nitrate reductase, NarGHIJ, is responsible for the conversion of nitrate to nitrite, and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia. Both NarGHIJ and NasBC were positively regulated by the two-component system ResD-ResE, and overexpression of NarG, NasC, and ResD also improved the ATP supply and the consequent γ-PGA yield. Based on the above individual methods, a method of combining the deletion of cydBC gene and overexpression of genes vgB, adK, and resD were used to enhance ATP content of the cells to 3.53 μmol/g of DCW, the mutant WX-BCVAR with this enhancement produced 43.81 g/L of γ-PGA, a 38.64% improvement compared to wild-type strain WX-02. Collectively, our results demonstrate that improving ATP content in B. licheniformis is an efficient strategy to improve γ-PGA production.

Enhanced Bacitracin Production by Systematically Engineering S-Adenosylmethionine Supply Modules in Bacillus licheniformis

“…In addition, SAM decarboxylase gene speD was deleted to block synthetic pathway of byproduct, meantime, based on our results, two other harmful by-products, cadaverine and putrescine (Li et al, 2019), were also decreased by 27.50 and 37.04%, due to the weakening of amino acid decarboxylase ( Figure 6C), and this result was positively correlated with our previous research (Wu et al, 2019). Furthermore, since SAM synthetase catalyzed the formation of SAM from Met and ATP, as well as the critical role of ATP supply in physiological metabolism (Cai et al, 2018), the ATP pool of DW2-KENPND should be enhanced in our future work.…”

“…All data were conducted to analyze the variance at P < 0.05 and P < 0.01, and a t-test was applied to compare the mean values using the software package Statistica 6.0, and bars represented the standard deviations, * P < 0.05; and * * P < 0.01 indicated the significance levels between recombinant strains and control (Cai et al, 2018).…”

“…To construct the strain which overexpressing SAM synthetase SAM2 from Saccharomyces cerevisiae, gene SAM2 mediated by P43 promoter was integrated into the chromosome of B. licheniformis DW2, following to previously reported protocol (Cai et al, 2018), diagnostic PCR and DNA sequence confirmed that the gene integrated overexpression strain was constructed successfully.…”

Increased Production of the Value-Added Biopolymers Poly(R-3-Hydroxyalkanoate) and Poly(γ-Glutamic Acid) From Hydrolyzed Paper Recycling Waste Fines

“…These properties make PGA suited for a variety of applications, including metal-ion binding and flocculation for wastewater treatment, composite materials for tissue engineering and drug delivery, and as a medicinal metal chelator for heavy metal removal (Yokoi et al, 1996;Shih et al, 2001;Ye et al, 2006;Siao et al, 2009;Inbaraj and Chen, 2012). Current research into improving PGA production for human use is focused on the metabolic engineering of various Bacillus species; notably, wild-type B. licheniformis WX-02 is capable of producing large amounts of PGA from glucose and glutamate and has been successfully engineered for enhanced biosynthesis (Cai et al, 2017(Cai et al, , 2018.…”

EngineeringBacillusfor efficient production of heterologous protein: current progress, challenge and prospect

“…), poly‐γ‐glutamic acid (Cai et al . ), N‐acetylglucosamine (Liu et al . ), hyaluronic acid (Westbrook et al .…”