Metabolic process engineering of Clostridium tyrobutyricum Δack–adhE2 for enhanced n‐butanol production from glucose: Effects of methyl viologen on NADH availability, flux distribution, and fermentation kinetics

Abstract

Butanol biosynthesis through aldehyde/alcohol dehydrogenase (adhE2) is usually limited by NADH availability, resulting in low butanol titer, yield, and productivity. To alleviate this limitation and improve n-butanol production by Clostridium tyrobutyricum Δack-adhE2 overexpressing adhE2, the NADH availability was increased by using methyl viologen (MV) as an artificial electron carrier to divert electrons from ferredoxin normally used for H2 production. In the batch fermentation with the addition of 500 μM MV, H2 , acetate, and butyrate production was reduced by more than 80-90%, while butanol production increased more than 40% to 14.5 g/L. Metabolic flux analysis revealed that butanol production increased in the fermentation with MV because of increased NADH availability as a result of reduced H2 production. Furthermore, continuous butanol production of ∼55 g/L with a high yield of ∼0.33 g/g glucose and extremely low ethanol, acetate, and butyrate production was obtained in fed-batch fermentation with gas stripping for in situ butanol recovery. This study demonstrated a stable and reliable process for high-yield and high-titer n-butanol production by metabolically engineered C. tyrobutyricum by applying MV as an electron carrier to increase butanol biosynthesis.

Metabolic engineering of Clostridium tyrobutyricum for n‐butanol production through co‐utilization of glucose and xylose

“…The decline in acids production also contributed to improved butanol yield (0.24 g/g) and productivity (0.29 g/L · h). MV, as an artificial electron carrier, directed the electron toward NADH synthesis, instead of hydrogen production, and thus favored butanol biosynthesis, which required NADH (Du et al, ). However, the addition of MV showed negative effects on cell growth and xylose utilization, causing incomplete xylose consumption (65.2%) and decreased xylose uptake rate (0.76 g/L · h), probably because the expression and activities of XylABT were compromised by the reduced ATP as a result of less acids production.…”

“…Besides being stable without subjecting to biphasic metabolic shift, higher butanol tolerance and lack of acetone‐synthetic pathway in C. tyrobutyricum are also beneficial to butanol biosynthesis and downstream purification (Yu et al, ). Furthermore, butanol became the main fermentation product by C. tyrobutyricum ( Δack , adhE2 ) in the presence of methyl viologen (MV), an artificial electron carrier, which increased NADH availability and resulted in a high butanol titer and yield (Du et al, ).…”

Gas fermentation: cellular engineering possibilities and scale up

“…A metabolic flux analysis on the syngas species, Clostridium tyrobutyricum , correlated increase in NADH with increase in butanol production [129, 130]. Moreover, genome-scale metabolic flux balance analysis has been used to construct spatiotemporal metabolic models for Clostridium ljungdahlii [131].…”

High production of butyric acid by Clostridium tyrobutyricum mutant

“…A constraint-based metabolic model of the central metabolism of C. tyrobutyricum [31] was constructed to perform flux balance analysis (FBA) of carbon and energy. The data collected from butyric acid fermentations in the Table 1 Recent progresses of butyric acid production from sugar Strains Fermentation mode Sugar Concentration /(g$L -1 ) Ref.…”