Abstract
The micronutrient zinc plays vital roles in ABE fermentation by Clostridium acetobutylicum. In order to elucidate the zinc-associated response for enhanced glucose utilization and earlier solventogenesis, transcriptional analysis was performed on cells grown in glucose medium at the exponential growth phase of 16 h without/with supplementary zinc. Correspondingly, the gene glcG (CAC0570) encoding a glucose-specific PTS was significantly upregulated accompanied with the other two genes CAC1353 and CAC1354 for glucose transport in the presence of zinc. Additionally, genes involved in the metabolisms of six other carbohydrates (maltose, cellobiose, fructose, mannose, xylose and arabinose) were differentially expressed, indicating that the regulatory effect of micronutrient zinc is carbohydrate-specific with respects to the improved/inhibited carbohydrate utilization. More importantly, multiple genes responsible for glycolysis (glcK and pykA), acidogenesis (thlA, crt, etfA, etfB and bcd) and solventogenesis (ctfB and bdhA) of C. acetobutylicum prominently responded to the supplementary zinc at differential expression levels. Comparative analysis of intracellular metabolites revealed that the branch node intermediates such as acetyl-CoA, acetoacetyl-CoA, butyl-CoA, and reducing power NADH remained relatively lower whereas more ATP was generated due to enhanced glycolysis pathway and earlier initiation of solventogenesis, suggesting that the micronutrient zinc-associated response for the selected intracellular metabolisms is significantly pleiotropic.
Highlights
The micronutrient zinc plays vital roles in ABE fermentation by Clostridium acetobutylicum
As high as ~6.5 g/L butanol and ~10.5 g/L ABE were produced efficiently with ~30 g/L glucose utilized while only ~3.5 g/L butanol and ~6.5 g/L ABE produced with ~20 g/L glucose utilized in the control, suggesting the solventogenesis was prominently initiated by supplementary zinc at this time point
Cells grown in the glucose medium without/with supplementary zinc at the exponential growth phase of 16 h were used for target total cellular RNA isolation and transcriptional analysis with RNA-Seq to elucidate the zinc-associated response for enhanced glucose utilization, acid re-assimilation and earlier initiation of solventogenesis by C. acetobutylicum
Summary
Transcriptional analysis of genes for glucose-specific PTS. According to our previous study[30], micronutrient zinc at milligram level could contribute to rapid glucose utilization, enhanced cell growth, acids re-assimilation and butanol production and earlier initiation of solventogenesis by C. acetobutylicum. Much lower acids and higher butanol levels due to zinc supplementation were present in the growing environment at 16 h, leading to differentially upregulated expression of genes eno, pgm and hydA by supplementary zinc This phenomenon suggested that zinc was convincingly involved in the transcriptional regulation of these selected genes in C. acetobutylicum. Combined with the comparative analysis of another four intermediate metabolites involved in acidogenesis and solventogenesis (see Fig. 3c,d), pyruvate (Pyr) was accumulated relatively higher (1.91 vs 0.57 μ mol/g-DCW) at 8 h of fermentation followed by rapid utilization while acetyl-CoA (AcCoA), acetoacetyl-CoA (AcAcCoA), and butyl-CoA (BuCoA) remained much lower levels within the first 32 h of fermentation compared to those detected in the control, respectively, indicating that the rapid ABE production was highly ascribed to the transcriptional regulation on glycolytic, acidogensic and solventogenic genes by supplementary zinc, which contributed to the earlier initiation of solventogenesis in C. acetobutylicum. The results above demonstrated that the micronutrient zinc could play crucial roles facilitating the efficiency of clostridial fermentation and has great potentials as a simple supplement applied in the renewable feedstocks-based industrial ABE fermentation at large scale, and the micronutrient zinc-associated response for these selected intracellular metabolisms of C. acetobutylicum is significantly pleiotropic, provoking further studies on the systematic mechanism on how zinc acts as an important factor to reprogram metabolic network of C. acetobutylicum
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