Project description:The expression profile of C. autoethanogenum DSM 10061 grown autotrophically with H2:CO:CO2 under visible light at an intensity of 4200 lux versus the expression profile of C. autoethanogenum DSM 10061 grown autotrophically in the dark
Project description:Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO2 + H2 as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H2) and the other steel mill off-gas (2% H2). Results were characterised using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimisation of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens.
Project description:Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named “LAbrini”, exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain “LAbrini” to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.
Project description:This experiment includes RNA-seq and metabolomics from a pure microbial strain to examine differential gene expression as a result of pre-adaptation and exposure to multiple carbon sources. This data will provide molecular insight into gene regulation and potential metabolic activity of Clostridium autoethanogenum, an important microbe for the bioenergy industry. C. autoethanogenum plays a unique role in lignocellulosic-based bioproduct synthesis due to its ability to consume single carbon gases as well as pentose and hexose sugars. Our preliminary research has demonstrated the organism’s ability to co-metabolize the pentose and hexose sugars xylose and fructose. However, different responses were observed depending on pre-adaptation or culturing history - whether cultures were grown on either xylose or fructose prior to the dual-sugar experiment. Sugar consumption as well as ethanol and acetate production differed between cultures pre-adapted on xylose versus fructose prior to the xylose-fructose fermentation. Fructose pre-adaptation resulted in both a faster sugar consumption and greater total consumption of sugar. Fructose pre-adapted cultures also produced three times more ethanol and two times more acetate.
The work (proposal:https://doi.org/10.46936/10.25585/60000475) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231.