Cross-talk between engineered Clostridium acetobutylicum and Clostridium ljungdahlii in syntrophic cocultures enhances isopropanol and butanol production

There is a need for efficient and sustainable production of essential chemicals such as isopropanol and butanol – the focus of this study – from renewable sugar feedstocks. Microbial fermentations use glycolysis, and as result, a third of the sugar carbon is lost to CO ₂ through pyruvate decarboxylation to acetyl-CoA, the starting intermediate for the biosynthesis of most microbial metabolites. In nature, microbes exist in syntrophic consortia, allowing for mutually-beneficial interactions, the production of novel products and the realization of novel benefits – including better carbon conservation – not seen in monocultures. Here, for increased acetone production, we transformed Clostridium acetobutylicum with a plasmid (p95ace02a) expressing a synthetic acetone pathway made up of four native genes. This engineered C. acetobutylicum was cocultured with Clostridium ljungdahlii to capture the waste CO ₂ and H ₂ generated due to glucose catabolism by C. acetobutylicum , and to convert acetone into isopropanol. We examined the impact of starting cell densities, the gas atmosphere (N ₂ , H ₂ , or H ₂ /CO ₂ ) and coculture species ratios (using a recently-developed RNA-FISH flow cytometric assay) on metabolite production, yields and sugar-carbon utilization. Metabolic flux analysis identified the complex patterns by which the two species alter each other’s metabolism in a cell-density and gas-atmosphere dependent manner. For example, C. ljungdahlii activated the dormant acetate uptake in C. acetobutylicum , while coculture density dramatically impacted species ratios, electron management, and C. ljungdahlii’ s H ₂ utilization. We achieved exceptionally-high concentrations of our desired products – 246 mM isopropanol and 148 mM butanol – in 64 hours, with about 85% of the production occurring before 32 hours. We reached maximum productivities of 13.9 mM isopropanol/hour and 10.4 mM butanol/hour with 0.9 mol alcohol produced per mol of sugar consumed. Total product yields reached 84.7% on a C-mol basis, versus 65.6% that can be reached in a C. acetobutylicum monoculture.