Acetate as alternative carbon source for production of mono- and di-rhamnolipids in Pseudomonas putida KT2440

The bacterium Pseudomonas putida KT2440 is used as a safe platform for rhamnolipid production via expression of the rhlABC genes, yielding mono- and di-rhamnolipid congeners. While glucose is commonly used as a carbon source, more sustainable alternatives such as acetate from waste streams or electrolysis are gaining interest. These two carbon sources are assimilated into the core carbon metabolism through distinct entry points. The impact of this on the proteome and rhamnolipid congener composition remain unclear. In this study, P. putida JAG1 was used to compare rhamnolipid production and composition during growth on glucose and acetate. Additionally, the impact of formate as a non-assimilable electron donor and the potential of modeled electrochemically derived CO₂-based carbon mixtures were evaluated.

Rhamnolipid precursor synthesis involves the rhamnose pathway and de-novo fatty acid synthesis. While glucose feeds directly into the rhamnose pathway and acetate into fatty acid synthesis, both carbon sources resulted in a similar di-rhamnolipid fraction of approximately 75 mol%. Although the carbon source significantly influenced the proteome profile, the carbon-to-nitrogen consumption ratio remained constant (C/N ≈ 15). Proteomic differences in P. putida JAG1 comparing the two carbon sources suggest ATP independent acetyl-CoA recycling as energy saving strategy. Enzyme abundances moreover suggest a coupling of the glyoxylate shunt and glycerol metabolism as strategy for gluconeogenesis. Growth rates, as well as biomass and product yields, were slightly lower on acetate compared to glucose. Co-feeding formate with acetate did not cause significant changes relative to acetate alone. Simultaneous consumption of acetate, formate, and ethanol only slightly affect product-per-biomass yields but strongly shifted the composition toward mono-rhamnolipids. Furthermore, comparison of the di-rhamnolipid producer with a mono-rhamnolipid producer indicates that di-rhamnolipid formation is associated with a higher molar product yield.

Acetate resembles a viable alternative carbon source for rhamnolipid production with only slight drawbacks to glucose. The metabolism of the microbial cell factory P. putida JAG1 appears homeostatic in presence of a single assimilable carbon source without affecting the rhamnolipid congener composition. The availability of a second assimilable carbon source, however, shifted the composition in favor of the mono-rhamnolipid. The molar product yield per carbon atom was higher during production of di-rhamnolipids compared to sole production of mono-rhamnolipids, highlighting it as a promising target product for future studies.