Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis

Abstract

BackgroundGlucosamine-6-phosphate N-acetyltransferase (GNA1) is the key enzyme that causes overproduction of N-acetylglucosamine in Bacillus subtilis. Previously, we increased GlcNAc production by promoting the expression of GNA1 from Caenorhabditis elegans (CeGNA1) in an engineered B. subtilis strain BSGN12. In this strain overflow metabolism to by-products acetoin and acetate had been blocked by mutations, however pyruvate accumulated as an overflow metabolite. Although overexpression of CeGNA1 drove carbon flux from pyruvate to the GlcNAc synthesis pathway and decreased pyruvate accumulation, the residual pyruvate reduced the intracellular pH, resulting in inhibited CeGNA1 activity and limited GlcNAc production.ResultsIn this study, we attempted to further overcome pyruvate overflow by enzyme engineering and host engineering for enhanced GlcNAc production. To this end, the key enzyme CeGNA1 was evolved through error-prone PCR under pyruvate stress to enhance its catalytic activity. Then, the urease from Bacillus paralicheniformis was expressed intracellularly to neutralize the intracellular pH, making it more robust in growth and more efficient in GlcNAc production. It was found that the activity of mutant CeGNA1 increased by 11.5% at pH 6.5–7.5, with the catalytic efficiency increasing by 27.5% to 1.25 s−1 µM−1. Modulated expression of urease increased the intracellular pH from 6.0 to 6.8. The final engineered strain BSGN13 overcame pyruvate overflow, produced 25.6 g/L GlcNAc with a yield of 0.43 g GlcNAc/g glucose in a shake flask fermentation and produced 82.5 g/L GlcNAc with a yield of 0.39 g GlcNAc/g glucose by fed-batch fermentation, which was 1.7- and 1.2-times, respectively, of the yield achieved previously.ConclusionsThis study highlights a strategy that combines pathway enzyme engineering and host engineering to resolve overflow metabolism in B. subtilis for the overproduction of GlcNAc. By means of modulated expression of urease reduced pyruvate burden, conferred bacterial survival fitness, and enhanced GlcNAc production, all of which improved our understanding of co-regulation of cell growth and metabolism to construct more efficient B. subtilis cell factories.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1049-x) contains supplementary material, which is available to authorized users.

Development of Processes for Recombinant L-Asparaginase II Production by Escherichia coli Bl21 (De3): From Shaker to Bioreactors

“…The complex medium presented the largest µ x . It is known that there is a linear relationship between µ x and acetate production: the higher the µ x the greater the acetate production [ 29 , 30 ]. The production of acetate must be controlled because in high concentrations acetate inhibits cell growth [ 31 ].…”

Metabolic engineering of Aspergillus niger via ribonucleoprotein-based CRISPR–Cas9 system for succinic acid production from renewable biomass

“…Furthermore, when we overexpressed both the Acdct gene and the frd gene in strain SAP-3, a synergistic effect of these two genetic modification led to the highest succinic acid production. The soluble NADH-dependent fumarate reductase was expressed to continue the carbon flux towards succinic acid via reduction of the cytosolic precursor fumarate [ 33 ], which can normally be produced via the dehydration of malate by fumarase in the rTCA branch [ 34 ]. Although fumaric acid was not found at detectable amounts in this study, malic acid was produced by all the engineered strains.…”

Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography

“…Hence, we recommend that for every refolding trial, this critical point can be determined to attain optimum yield and efficiency. Mild refolding conditions involving the use of detergents [ 12 ], reversed micelles, and an intermediate or low concentration of denaturant [ 60 ] as well as physical conditions such as alkaline pH and/or high hydrostatic pressure have been reported [ 16 , 21 , 22 , 61 ] as strategies for high yields. HHP refolding has particularly been used successfully to refold several proteins including an oligomeric [ 17 ] as well as a disulphide rich protein [ 15 ] both of which demonstrated reasonably high yields of well folded protein.…”