Combinatorial Fine-Tuning of GNA1 and GlmS Expression by 5’-Terminus Fusion Engineering Leads to Overproduction of N-Acetylglucosamine inBacillus subtilis

Abstract

Glucosamine-6-phosphate N-acetyltransferase (GNA1) that catalyzes acetyl transfer from acetyl-coenzyme A to glucosamine-6-phosphate (GlcN-6P), and glutamine-fructose-6-phosphate aminotransferase (GlmS) that catalyzes the formation of GlcN-6P from fructose-6-phosphate (Fru-6P), are two key enzymes in Bacillus subtilis for the bioproduction of N-acetylglucosamine (GlcNAc), a nutraceutical that has various applications in healthcare. In this study, the expression of GNA1 and GlmS is fine-tuned by 5'-terminus fusion engineering to improve GlcNAc production. Specifically, the expression level of GNA1 is enhanced at the translational level via fusion of an epitope tag to the 5'-terminus of GNA1 gene and ribosome binding site (RBS) sequence engineering. Next, enhanced expression of GlmS is achieved at the transcriptional and translational levels by fusing an mRNA stabilizer to the 5'-terminus of GlmS gene. Under the control of GNA1 (fusion with cMyc tag and with the optimum RBS M-Rm) and GlmS (fusion with mRNA stabilizer ΔermC+14/7A), the GlcNAc titer and yield in the shake flask increase to 18.5 g L and 0.37 g GlcNAc/g glucose, which are 2.9-fold and 2.3-fold that of the control, respectively. This synthetic pathway fine-tuning method at the transcriptional and translational levels by combinatorial modulation of regulatory elements, including epitope tag, RBS sequence, and mRNA stabilizer, might represent a general and effective approach for the construction of microbial cell factories.

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

“…2a). Specific activity analysis of Ce GNA1 in the lysis supernatant found that it increased by 21.7% to 1060 U/mg [6]. However, the decreased pyruvate concentration had little impact on pH in (Fig.…”

“…The primers are listed in the Additional file 1: Table S1. BSGN12 (Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh ΔptaΔ ackA::lox72 , Δ alsRSD:: Pveg-Δ ermC +14/7A- ecglm ), which secreted pyruvate into the medium during fermentation, was used as the host strain [6]. During the construction of the strains and plasmids, all strains were grown at 37 °C in standard Luria–Bertani broth (LB) (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) or LB agar plates, with an appropriate concentration of antibiotics used for selection (100 μg/mL ampicillin, 25 μg/mL kanamycin, or 30 μg/mL zeocin).…”

“…After process optimization, we found that 1 ng of template and 20 cycles of amplification were suitable for the production of one or two amino acids containing mutants. After the amplification under the suitable conditions using the primer pair er-ceN-F1/er-ceN-R1, the PCR products were purified and ligated with the linearized plasmid pP43-cMyc (M-Rm)- Ce GNA1 [6], which had been PCR amplified using the primer pair er-ceN-F2/er-ceN-R2 to remove the wild type cegna1 gene. The ClonExpress™ II kit (Vazyme Biotech Co., Ltd) was used for the ligation, and then the ligation products were used to transform Escherichia coli JM109 cells.…”

“…The concentration of urea in the fermentation medium was quantified by high-pressure liquid chromatography with fluorescence detection after automated derivatization with xanthydrol [20]. The concentration of glucose, GlcNAc, and pyruvate in the fermentation broth was analyzed by HPLC as described before [6]. Cell growth was monitored by measuring the absorbance at 600 nm (OD 600 ).…”

“…However, the fact that overflow of metabolic by-products acetoin and acetate had been blocked by mutations in alsRSD and ackA , meant that pyruvate accumulated as an overflow metabolite in this strain (Fig. 1) [5, 6].…”