Developing an Integrated Systems and Synthetic Biology Platform for Gas Fermenting Acetogens

Gas fermentation offers numerous advantages for the production of sustainable fuels and chemicals without compromising food security or being dependent on the availability of arable land. Advantages over the use of conventional (sugar, oil or algae) feedstocks also include minimal water and nutrient demand and the capability to capture greenhouse gases that would otherwise be emitted into the atmosphere. Practically all carbon-containing feedstocks, including industrial off-gases and gasified agricultural and municipal waste, can be readily utilised or converted to syngas for subsequent transformation into fuels and chemicals.

Acetogens such as Clostridium Ljundahlii or Clostridium autoethanogenum use the reductive acetyl-CoA (Wood-Ljungdahl) pathway as a terminal electron-accepting, energy-conserving, CO2-fixing process. This pathway is speculated to be the first biochemical pathway in existence on Earth and continues to play a key role in the global acetate cycle with annual acetogenesis in sediments and termite hindguts estimated to amount to several trillion kg of acetate. Energy metabolism in acetogens is complex and many aspects  are only partly understood.

While all acetogens use the Wood-Ljungdahl pathway to fix CO2, they vary in terms of redox coupling (cytochromes, sodium translocating Rnf or proton translocating Rnf) and co-factor use in the bifurcating hydrogenase (NADPH or NADH). Understanding the complex energy metabolism is critical, since ATP availability is a fundamental limitation in engineering acetogen metabolism. C. autoethanogenum offers a robust engineering system and a flexible platform for syngas fermentation. Fermentation of C. autoethanogenum has high product selectivity, tolerates a broad range of gas compositions and accepts contaminants well, making it the preferred microorganism for industrial gas fermentation.

Using C. autoethanogenum as the system for this study, we used a multi-omics approach in order to gain a measure of enzyme capacity (transcriptomics and proteomics) and thermodynamic driving force (metabolomics). By comparing gene expression and transcription when cells are grown autotrophically and heterotrophically (CO CO2 and H2 vs fructose fermentation) we found that, compared to C. ljungdahlii, in C. autoethanogenum the RNF complex is extremely efficient and is able to maintain high ATPase activity at the transcriptional (RNA-seq) and translational (iTRAQ) levels even when cells are fermented exclusively using gas. Furthermore, our RNA-sequencing data show that under autotrophic conditions, genes in the RNF complex are highly transcribed, resulting in equi-molar quantities of ATP (metabolomics data). This high efficiency of the electron chain transfer, coupled to the recently discovered electron bifunctional hydorgenases is potentially what makes C. autoethanogenum so unique and a great industrial platform for the conversion of syngas into valuable fuels and chemicals.