Chloroplast engineering of the green microalgae Chlamydomonas reinhardtii for the production of HAA, the lipid moiety of rhamnolipid biosurfactants

Hydroxyalkanoyloxyalkanoates (HAA) are lipidic surfactants with a number of potential applications, but more remarkably, they are the biosynthetic precursors of rhamnolipids (RL), which are preferred biosurfactants thanks to their excellent physicochemical properties, biological activities, and environmental biodegradability. Because the natural highest producer of RLs is the pathogenic bacterium Pseudomonas aeruginosa, important efforts have been dedicated to transfer production to heterologous non-pathogenic microorganisms. Unicellular photosynthetic microalgae are emerging as important hosts for sustainable industrial biotechnology due to their ability to transform CO₂ efficiently into biomass and bioproducts of interest. Here, we have explored the potential of the eukaryotic green microalgae Chlamydomonas reinhardtii as a chassis to produce RLs. Chloroplast genome engineering allowed the stable functional expression of the gene encoding RhlA acyltransferase from P. aeruginosa, an enzyme catalyzing the condensation of two 3-hydroxyacyl acid intermediaries in the fatty acid synthase cycle, to produce HAA. Four congeners of varying chain lengths were identified and quantified by UHPLC-QTOF mass spectrometry and gas chromatography, including C₁₀-C₁₀ and C₁₀-C₈, and the less abundant C₁₀-C₁₂ and C₁₀-C₆ congeners. HAA was present in the intracellular fraction, but also showed increased accumulation in the extracellular medium. Moreover, HAA production was also observed under photoautotrophic conditions based on atmospheric CO₂. These results establish that RhlA is active in the chloroplast and is able to produce a new pool of HAA in a eukaryotic host. Subsequent engineering of microalgal strains should contribute to the development of an alternative clean, safe and cost-effective platform for the sustainable production of RLs.