ABSTRACT. Acetivibrio thermocellus (formerly Clostridium thermocellum) is a potential platform for lignocellulosic ethanol production. Its industrial application is hampered by low product titres, resulting from a low thermodynamic driving force of its central metabolism. It possesses both a functional ATP- and a functional PPi-dependent 6-phosphofructokinase (PPi-Pfk), of which only the latter is held responsible for the low driving force. Here we show that, following the replacement of PPi-Pfk by cytosolic pyrophosphatase and transaldolase, the native ATP-Pfk is able to carry the full glycolytic flux. Interestingly, the barely-detectable in vitro ATP-Pfk activities are only a fraction of what would be required, indicating its contribution to glycolysis has consistently been underestimated. A kinetic model demonstrated that the strong inhibition of ATP-Pfk by PPi can prevent futile cycling that would arise when both enzymes are active simultaneously. As such, there seems to be no need for a long-sought-after PPi-generating mechanism to drive glycolysis, as PPi-Pfk can simply use whatever PPi is available, and ATP-Pfk complements the rest of the PFK-flux. Laboratory evolution of the ΔPPi-Pfk strain, unable to valorize PPi, resulted in a mutation in the GreA transcription elongation factor. This mutation likely results in reduced RNA-turnover, hinting at transcription as a significant (and underestimated) source of anabolic PPi. Together with other mutations, this resulted in an A. thermocellus strain with the hitherto highest biomass-specific cellobiose uptake rate of 2.2 g/gx/h. These findings are both relevant for fundamental insight into dual ATP/PPi Pfk-nodes, which are not uncommon in other microorganisms, as well as for further engineering of A. thermocellus for consolidated bioprocessing.

ABSTRACT. Clostridium kluyveri is a microorganism capable of biosynthesise medium chain fatty acids (MCFA), such as butyrate and caproate by the r-βOx pathway, concurrently releasing hydrogen. This study investigated the impact of initial ethanol and acetate concentrations and ethanol-to-acetate (E:A) molar ratios on batch culture performance. A central composite design was used to comprehensively analyse ethanol and acetate concentration influence on product distribution and hydrogen generation. At a constant ethanol to-acetate (E:A) ratio, increasing initial ethanol concentrations reduces the caproate-to-butyrate ratio, which contrasts with the general observation that higher E:A ratios lead to a greater proportion of carbon directed toward caproate. The highest specific productivity for both butyrate and caproate was observed at an initial ethanol concentration of 340 mM. Hydrogen production demonstrated a balance between carboxylate elongation and biomass formation, with maximum specific hydrogen productivity achieved at initial concentrations of 340 mM ethanol and 170 mM acetate. The C. kluyveri strain in this study exhibited a specific hydrogen productivity of 9.56 mmol H2·gCDW−1·h−1, a rate that exceeds those reported for other Clostridium species. This work provides insights into tailoring initial substrate conditions for targeted product formation in C. kluyveri batch fermentations.

ABSTRACT. Global climate change driven by anthropogenic carbon emissions and food security represent two intertwined grand challenges of the 21st century. China, accounting for approximately one-third of global CO₂ emissions, simultaneously imports over 250 million tons of soybeans and palm oil annually, confronting severe external supply dependence and associated carbon footprints. Here, we present a disruptive gas fermentation technology that converts industrial CO₂ and by-product hydrogen into high-value microbial protein and oil, breaking foreign technological monopolies in this field. Our engineered strains achieved a record-breaking acetate productivity of 55 g/L (1.9× the global benchmark) and lipid yields matching international leading levels. Building on these advances, we established the world’s first pilot-scale cascade system for integrated CO₂-to-protein/oil production. Scaled deployment of this technology could yield 50 million tons of microbial protein and oil annually, replacing 50% of China’s current soybean and palm oil imports, saving 300 million mu of arable land, and reducing 250 million tons of CO₂ emissions. This work establishes a transformative pathway toward a sustainable, carbon-negative future food and feed supply chain.

ABSTRACT. Climate change and poor recycling of waste is threatening global biosustainability. Humankind is thus facing an urgent need for sustainable production of chemicals and fuels, and improved waste recycling. Acetogens have become attractive biocatalysts for converting inexpensive and abundant solid and gaseous waste feedstocks into high-value products using gas fermentation. While bioengineering methods allow to create novel acetogen cell factories, metabolic engineering is hindered by our limited understanding of acetogen metabolism. We thus previously used adaptive laboratory evolution (ALE) to improve autotrophic growth of the model-acetogen Clostridium autoethanogenum. In this presentation, I will provide an overview of our recent efforts in exploring the metabolic regulatory landscape behind these enhanced phenotypes. Firstly, we used reverse genetic engineering to reproduce mutations obtained through ALE that potentially realised better autotrophy. Indeed, reverse-engineered strains recovered the superior phenotypes of ALE isolates as they could grow faster, without complex nutrients, and were robust for operating continuous cultures. Notably, bioinformatics suggested that the targeted genes may be involved in overlapping regulatory networks. Secondly, we aimed to identify genome-wide protein-RNA interactions potentially involved in realising autotrophy by combining steady-state chemostat cultivation, functional genomics, and computational methods. While we predicted 14 regulatory RNA-binding proteins interacting with genes differentially regulated between autotrophy and heterotrophy, none were linked to known genes key for autotrophy. Our work contributes towards better understanding of regulation of acetogen metabolism and offers engineering targets in an industrially-relevant acetogen.

ABSTRACT. Clostridium thermocellum is a promising organism for sustainable biofuel production and consolidated bioprocessing (CBP) due to its ability to efficiently breakdown lignocellulosic biomass under anaerobic conditions. Despite its capacity to utilize cellulose, it lacks the native ability to metabolize pentose sugars derived from hemicellulose, limiting complete biomass conversion. To address this limitation, C. thermocellum has been engineered to co-utilize cellulose- and hemicellulose-derived sugars.

Here, we investigate the metabolic consequences of engineered xylose utilization in C. thermocellum under xylose-only and mixed-sugar (cellobiose and xylose) conditions. We combine LC–MS-based metabolomics with 13C stable isotope tracing using positionally labeled xylose to resolve carbon flow through central metabolism and primary biosynthetic pathways. Metabolic flux analysis (MFA) shows increased flux through the pentose phosphate pathway under xylose supplementation, consistent with activation of heterologous xylose catabolism. Additionally, isotopic labeling further demonstrates carbon routing under both single- and co-substrate conditions. Integration of these datasets enables construction of a quantitative flux map, revealing shifts in central carbon partitioning, potential pathway-level constraints and identifying metabolic conflicts during simultaneous sugar utilization.

Together, these results provide a systems-level view of engineered hemicellulose metabolism in C. thermocellum and establish a foundation for improving carbon co-utilization in CBP applications.

ABSTRACT. Clostridium ljungdahlii has attracted considerable interest due to its carbon-negative fermentation metabolism for ethanol production. During fructose and syngas fermentation, this species utilizes a metabolic network linking acetyl-CoA, ethanol, and acetate biosynthesis via bifunctional alcohol-acetaldehyde dehydrogenases (AdhEs) and acetaldehyde:ferredoxin oxidoreductases (AORs). This study investigates the metabolic functions of the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) domain of AdhE across distinct fermentation processes. ALDH and ALDH dynamically modulate catalytic pathways in response to varying carbon and energy sources, thereby coordinating cellular growth and product composition and maintaining intracellular metabolic homeostasis. During fructose fermentation, ALDH deficiency enhances ethanol synthesis by rebalancing nicotinamide adenine dinucleotide (NADH) metabolism. Conversely, under CO fermentation, ALDH disruption severely impairs ethanol synthesis by inhibiting acetaldehyde-to-acetyl-CoA conversion, a critical step for NADH regeneration. Whereas a double amino acid substitution inactivating the ADH domain of AdhE completely abolishes ethanol production, the wildtype strain produces 19 g/L ethanol under the same fermentation conditions using CO₂/H₂ as the sole carbon and energy source. These results confirm that the bifunctional aldehyde/alcohol dehydrogenase (AdhE) plays a key regulatory role in ethanol metabolism. Overall, these findings reveal the regulation of acetate and ethanol metabolism in C. ljungdahlii, advancing metabolic engineering in syngas fermentation.

ABSTRACT. Clostridium tyrobutyricum is a promising anaerobic chassis for the biosynthesis of butyryl-CoA-derived chemicals due to its strong carbon flux toward butyrate formation and high tolerance to organic acids. In this study, we investigated the role of Rex in regulating C2/C4 carbon flux partitioning in an AdhE2-expressing C. tyrobutyricum background. The wild-type strain and the Δrex mutant were compared under glucose and mannitol conditions, which represent different reducing environments. Under glucose fermentation, rex deletion redirected product formation from C2 products toward C4 products, with reduced acetate and ethanol accumulation and increased butyrate production. In contrast, under mannitol fermentation, the Δrex mutant displayed impaired growth and acid production, indicating that Rex deficiency caused more severe metabolic disturbance under highly reducing conditions. Time-course RT-qPCR analysis showed that rex deletion broadly increased the transcription of butyrate synthesis genes, including thl, hbd, crt, bcd, etfB1, etfA1, and cat1. Intracellular cofactor measurements further validated that Δrex did not simply maintain a higher NADH/NAD⁺ ratio; instead, rex deletion reshaped both NADH and NAD+ pools and caused a disturbed redox profile during fermentation. Meanwhile, ATP levels in the Δrex mutant were markedly lower than those in the wild-type strain, indicating that Rex is also involved in maintaining energy homeostasis. Electrophoretic mobility shift assays were performed using purified Rex protein and promoter regions of the butyrate synthesis gene cluster. Rex was found to bind multiple promoters associated with butyryl-CoA formation and butyrate conversion, and the Rex-DNA interaction was modulated by NADH/NAD⁺. These results support a direct link between redox sensing, transcriptional regulation of the butyrate pathway, and C2/C4 carbon flux redistribution. Together, this work demonstrates that Rex coordinates butyrate pathway expression with intracellular redox and energy states in C. tyrobutyricum.