ABSTRACT Bioethanol production from syngas using acetogenic bacteria has attracted considerable attention in recent years. However, low ethanol yield is the biggest challenge that prevents the commercialization of syngas fermentation into biofuels using microbial catalysts. The present study demonstrated that ethanol metabolism plays an important role in recycling NADH/NAD+ during autotrophic growth. Deletion of bifunctional aldehyde/alcohol dehydrogenase (adhE) genes leads to significant growth deficiencies in gas fermentation. Using specific fermentation technology in which the gas pressure and pH were constantly controlled at 0.1 MPa and 6.0, respectively, we revealed that ethanol was formed during the exponential phase, closely accompanied by biomass production. Then, ethanol was oxidized to acetate via the aldehyde ferredoxin oxidoreductase pathway in Clostridium ljungdahlii. A metabolic experiment using 13C-labeled ethanol and acetate, redox balance analysis, and comparative transcriptomic analysis demonstrated that ethanol production and reuse shared the metabolic pathway but occurred at different growth phases. IMPORTANCE Ethanol production from carbon monoxide (CO) as a carbon and energy source by Clostridium ljungdahlii and “Clostridium autoethanogenum” is currently being commercialized. During gas fermentation, ethanol synthesis is NADH-dependent. However, ethanol oxidation and its regulatory mechanism remain incompletely understood. Energy metabolism analysis demonstrated that reduced ferredoxin is the sole source of NADH formation by the Rnf-ATPase system, which provides ATP for cell growth during CO fermentation. Therefore, ethanol production is tightly linked to biomass production (ATP production). Clarification of the mechanism of ethanol oxidation and biosynthesis can provide an important reference for generating high-ethanol-yield strains of C. ljungdahlii in the future.

Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite -4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases.

Recalcitrance of biomass feedstock remains a challenge for microbial conversion of lignocellulose into biofuel and biochemicals. Clostridium cellulosi, one thermophilic bacterial strain dominated in compost, could hydrolyze lignocellulose at elevated temperature by secreting more than 38 glycoside hydrolases belong to 15 different families. Though one multi-modular endoglucanase CcCel9A has been identified from C. cellulosi CS-4-4, mechanism of synergistic degradation of cellulose by various cellulases from strain CS-4-4 remains elusive. In this study, CcCel9A, CcCel9B, and CcCel48A were characterized as processive endoglucanase, non-processive endoglucanase, and exoglucanase, respectively. To understand how they cooperate with each other, we estimated the approximate concentration ratio on the zymogram and optimized it using purified enzymes in vitro. Synergism between individual glycoside hydrolase during cellulose hydrolysis in the mixture was observed. CcCel9A and CcCel48A could degrade cellulose chain from non-reducing ends and reducing ends, respectively, to cello-oligosaccharide. CcCel9B could cut cellulose chain randomly and cello-oligosaccharides with varied length were released. In addition, a β-glucosidase BlgA from Caldicellulosiruptor sp. F32 which could cleave cello-oligosaccharides including G2-G6 to glucose was added to the enzyme mixture to remove the product inhibition of its partners. The combination and ratios of these cellulases were optimized based on the release rate of glucose. Hydrolysis of corn stalk was conducted by a four-component cocktail (CcCel9A:CcCel9B:CcCel48A:BlgA = 25:25:10:18), and only glucose was detected as main production by using high-performance anion-exchange chromatography. Processive endoglucanase CcCel9A, dominated in secretome of C. cellulosi, showed good potential in developing cellulase cocktail due to its exquisite cooperation with various cellulases.