Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

Unravelling phenotypic changes in gas-fermenting acetogen Clostridium autoethanogenum through proteomics (#167)

Karen Rodriguez Martinez 1 , Esteban Marcellin 1
  1. Australian Institute for Bioengineering and Nanotechnology, West End, QLD, Australia

Concerns over high greenhouse gases emissions from transportation have made us consider lower-carbon fuels. An alternative to petroleum-based fuels is engineered microorganisms that use renewable carbon sources to produce biofuels. Acetogens represent an attractive platform for production of biofuels due to their ability to metabolise carbon oxides from waste gases into high-value compounds such as acetate, ethanol and 2,3-butanediol through the Wood-Ljungdahl pathway. Here, we aim to develop a recombinant strain of the model acetogen Clostridium autoethanogenum to produce isobutanol, a next-generation biofuel that can be used as jet fuel and has properties that closely resemble to gasoline, including high energy density and calorific value as well as low volatility and miscibility with water. Fist, we designed an isobutanol recombinant pathway derived from pyruvate including a combinatorial assembly of enzymes from several organisms. To identify the optimal chassis strain to carry this pathway, we studied two C. autoethanogenum strains exhibiting different phenotypes and quantified their proteome aiming to understand the regulatory networks and genotype-phenotype relationships that could potentially lead to improved isobutanol production. Using adaptative laboratory evolution, these two strains have been adapted to grow in autotrophically solely on CO, CO2 and H2. One of these strains, hereafter named ALE4, shows a higher production of 2,3-butanediol, one of the secondary products from the Wood-Ljungdahl pathway. The other strain, named EvoBrini, grows more robustly, has increased specific gas uptakes and lower 2,3-butanediol production. Proteome quantification from continuous cultures of both strains at steady-state elucidated key differences in protein expression leading to these phenotypes. Proteins involved in production of 2,3-butanediol from pyruvate and Nfn transhydrogenase complex involved in generation of redox cofactors, were overexpressed in the ALE4 strain. In EvoBrini, we identified overexpression of proteins from the ethanol metabolism and biomass production. Insights into the proteome of these strains provides a better understanding of metabolic networks that could be used for improved isobutanol production.