Ethanol Metabolism Dynamics in Clostridium ljungdahlii Grown on Carbon Monoxide.

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.

Thermophilic Moorella thermoacetica as a platform microorganism for C1 gas utilization: physiology, engineering, and applications.

In the context of the rapid development of low-carbon economy, there has been increasing interest in utilizing naturally abundant and cost-effective one-carbon (C1) substrates for sustainable production of chemicals and fuels. Moorella thermoacetica, a model acetogenic bacterium, has attracted significant attention due to its ability to utilize carbon dioxide (CO2) and carbon monoxide (CO) via the Wood-Ljungdahl (WL) pathway, thereby showing great potential for the utilization of C1 gases. However, natural strains of M. thermoacetica are not yet fully suitable for industrial applications due to their limitations in carbon assimilation and conversion efficiency as well as limited product range. Over the past decade, progresses have been made in the development of genetic tools for M. thermoacetica, accelerating the understanding and modification of this acetogen. Here, we summarize the physiological and metabolic characteristics of M. thermoacetica and review the recent advances in engineering this bacterium. Finally, we propose the future directions for exploring the real potential of M. thermoacetica in industrial applications.

Metabolic Engineering of Gas-Fermenting Clostridium ljungdahlii for Efficient Co-production of Isopropanol, 3-Hydroxybutyrate, and Ethanol.

Rational design and modification of autotrophic bacteria to efficiently produce high-value chemicals and biofuels are crucial for establishing a sustainable and economically viable process for one-carbon (C1) source utilization, which, however, remains a challenge in metabolic engineering. In this study, autotrophic Clostridium ljungdahlii was metabolically engineered to efficiently co-produce three important bulk chemicals, isopropanol, 3-hydroxybutyrate (3-HB), and ethanol (together, IHE), using syngas (CO2/CO). An artificial isopropanol-producing pathway was first constructed and optimized in C. ljungdahlii to achieve an efficient production of isopropanol and an unexpected product, 3-HB. Based on this finding, an endogenous active dehydrogenase capable of converting acetoacetate to 3-HB was identified in C. ljungdahlii, thereby revealing an efficient 3-HB-producing pathway. The engineered strain was further optimized to reassimilate acetic acid and synthesize 3-HB by introducing heterologous functional genes. Finally, the best-performing strain was able to produce 13.4, 3.0, and 28.4 g/L of isopropanol, 3-HB, and ethanol, respectively, in continuous gas fermentation. Therefore, this work represents remarkable progress in microbial production of bulk chemicals using C1 gases.

Engineering Clostridium ljungdahlii as the gas-fermenting cell factory for the production of biofuels and biochemicals.

Clostridium ljungdahlii is a representative autotrophic gas-fermenting acetogen capable of converting CO2 and CO into biomass and multiple metabolites. The carbon fixation and conversion based on C. ljungdahlii have great potential for the sustainable production of bulk biochemicals and biofuels using industrial syngas and waste gases. With substantial recent advances in genetic manipulation tools, it has become possible to study and improve the metabolic capability of C. ljungdahlii in gas fermentation. The product scope of C. ljungdahlii has been expanded through the introduction of heterologous production pathways followed by the modification of native metabolic networks. In addition, progress has been made in understanding the physiological and metabolic mechanisms of this anaerobe, contributing to strain designs for expected phenotypes. In this review, we highlight the latest research progresses regarding C. ljungdahlii and discuss the next steps to comprehensively understand and engineer this bacterium for an improved bacterial gas bioconversion platform.

Rapid Generation of Universal Synthetic Promoters for Controlled Gene Expression in Both Gas-Fermenting and Saccharolytic Clostridium Species.

Engineering solventogenic clostridia, a group of important industrial microorganisms, to realize their full potential in biorefinery application is still hindered by the absence of plentiful biological parts. Here, we developed an effective approach for rapid generation of a synthetic promoter library in solventogenic clostridia based on a dual-reporter system (catP-lacZ) and a widely used strong thl promoter. The yielded artificial promoters, spanning 2 orders of magnitude, comprised two modular components (the core promoter region and the spacer between RBS and the translation-initiating code), and the strongest promoter had an over 10-fold-higher activity than the original expression part Pthl. The test of these synthetic promoters in controlled expression of sadh and danK in saccharolytic C. acetobutylicum and gas-fermenting C. ljungdahlii, respectively, gave the expected phenotypes, and moreover, showed good correlation between promoter activities and phenotypic changes. The presented wide-strength-range promoters here will be useful for synthetic biology application in solventogenic clostridia.