Enhanced isopropanol–butanol–ethanol mixture production through manipulation of intracellular NAD(P)H level in the recombinant Clostridium acetobutylicum XY16

Abstract

BackgroundThe formation of by-products, mainly acetone in acetone–butanol–ethanol (ABE) fermentation, significantly affects the solvent yield and downstream separation process. In this study, we genetically engineered Clostridium acetobutylicum XY16 isolated by our lab to eliminate acetone production and altered ABE to isopropanol–butanol–ethanol (IBE). Meanwhile, process optimization under pH control strategies and supplementation of calcium carbonate were adopted to investigate the interaction between the reducing force of the metabolic networks and IBE production.ResultsAfter successful introduction of secondary alcohol dehydrogenase into C. acetobutylicum XY16, the recombinant XY16 harboring pSADH could completely eliminate acetone production and convert it into isopropanol, indicating great potential for large-scale production of IBE mixtures. Especially, pH could significantly improve final solvent titer through regulation of NADH and NADPH levels in vivo. Under the optimal pH level of 4.8, the total IBE production was significantly increased from 3.88 to 16.09 g/L with final 9.97, 4.98 and 1.14 g/L of butanol, isopropanol, and ethanol. Meanwhile, NADH and NADPH levels were maintained at optimal levels for IBE formation compared to the control one without pH adjustment. Furthermore, calcium carbonate could play dual roles as both buffering agency and activator for NAD kinase (NADK), and supplementation of 10 g/L calcium carbonate could finally improve the IBE production to 17.77 g/L with 10.51, 6.02, and 1.24 g/L of butanol, isopropanol, and ethanol.ConclusionThe complete conversion of acetone into isopropanol in the recombinant C. acetobutylicum XY16 harboring pSADH could alter ABE to IBE. pH control strategies and supplementation of calcium carbonate were effective in obtaining high IBE titer with high isopropanol production. The analysis of redox cofactor perturbation indicates that the availability of NAD(P)H is the main driving force for the improvement of IBE production.

Phenotypic and genomic analysis of isopropanol and 1,3-propanediol producer Clostridium diolis DSM 15410

“…It is possible that NADP-dependent isopropanol dehydrogenase encoded by adh (F3K33_14815) can be expressed in more studied clostridial strains to obtain IBE producers. This was already performed for some C. acetobutylicum strains, however, expressed gene was cloned from C. beijerinckii DSM 6423 (= C. beijerinckii NRRL B-593) [71][72][73].…”

Trends in Systems Biology for the Analysis and Engineering of Clostridium acetobutylicum Metabolism

“…Therefore, as alternatives to gasoline, advanced biofuels like C3-C5 alcohols are believed to circumvent the current problems of ethanol. Clostridium acetobutylicum is a natural producer of industrially valuable chemicals like butanol, acetone and butyric acid and intensive efforts to engineer them for the efficient production of either kerosene precursors [1] or C2-C3-C4 alcohol mixtures as the only fermentation products have been undertaken [2][3][4][5][6][7]. Unfortunately, these bioprocesses are not economically viable for fuel applications due to high product toxicity for the producing bacteria, low yield and productivity and high cost of downstream processing [8].…”

Primary metabolites from overproducing microbial system using sustainable substrates

“…The highest production was observed at controlled pH (at 4.8) and supplementation of calcium carbonate (at 10 g⋅L −1 ). Engineering efforts finally improved the titer of isopropanol (from 4.98 to 6.0 g⋅L −1 ) and other alcohols, including butanol (from 9.97 to 10.5 g⋅L −1 ) or ethanol (1.14–1.24 g⋅L17 −1 ) [35]. Applying a CRISPR EnAbled Trackable genome Engineering (CREATE) strategy, E. coli PA14 was engineered for isopropanol production (titer ∼7.1 g⋅L −1 at 24 H, yield ∼0.75 mol /mol, substrate or maximum productivity ∼0.62 g⋅L −1 ⋅H −1 ) compared to the productivity of parent strain PA07 (0.22 g⋅L −1 ⋅H −1 ) [36].…”