Engineering Clostridium cellulovorans for highly selective n‐butanol production from cellulose in consolidated bioprocessing

Abstract

Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlA CA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbd CT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P) + oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol.The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnr CA -thlA CAhbd CT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.

Recent advances in metabolic engineering of microorganisms for advancing lignocellulose-derived biofuels

“…Further exogenous supplementation of methyl viologen enhanced the butanol yield up to of 0.28 g/g from the corn stover [ 117 ]. In a different investigation, recombinant Clostridium cellulovorans adhE2 strain was engineered through the introduction of thiolase (thlACA) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbdCT) from C. tyrobutyricum [ 118 ]. The engineered C. cellulovorans strain was able to n-butanol from cellulose at a 50% higher yield (0.34 g/g).…”

Heterologous Gene Regulation in Clostridia: Rationally Designed Gene Regulation for Industrial and Medical Applications

“…However, most clostridia are completely benign . Beyond the domain of human health, solventogenic clostridia have been utilized in the industrial production of petrochemicals due to their ability to produce acetone–butanol–ethanol (ABE) by microbial fermentation (Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum), while cellulolytic, biofuel-producing strains, such as Clostridium cellulolyticum and Clostridium cellulovorans, are now attracting attention to satisfy the increasing demand for cleaner fuels. , Oncolytic strains, such as Clostridium sporogenes and Clostridium novyi, selectively germinate in the hypoxic/necrotic regions of tumors and their endogenous oncolytic activity can result in partial destruction of the tumor. , Subsequent studies have highlighted distinct advantages to using certain Clostridium species as a therapeutic vector in bacterial-mediated cancer therapy. − The addition, through genetic engineering, of clinically approved heterologous products, such as antibodies, proinflammatory cytokines, , and checkpoint inhibitors, could further increase their therapeutic value. An alternative approach is clostridia-directed enzyme prodrug therapy (CDEPT), which has seen significant advances in recent years. − Thus, demand for synthetic strains of these species has highlighted the need for genetic tools that will enable the precise control of synthetic products of industrial or therapeutic value.…”

An Experimental Study on Combustion and Cycle-by-Cycle Variations of an N-Butanol Engine with Hydrogen Direct Injection under Lean Burn Conditions

“…In addition, with the development of n-butanol production technology, the production cost of n-butanol has gradually reduced. N-butanol is prepared from a wide range of raw materials with low prices, mainly wheat, corn, pulp waste liquid, molasses and wild plants [ 9 , 10 , 11 , 12 ]. These characteristics make n-butanol more advantageous than methanol and ethanol as an alternative fuel for internal combustion engines and it has become one of the most promising alternative fuels for internal combustion engines.…”