Acidification by nitrogen metabolism triggers extracellular biopolymer production in an oleaginous yeast.

The oleaginous yeast Rhodotorula toruloides is a natural producer of lipids and carotenoids. However, its potential as a producer of extracellular biopolymers remains unexplored. Hence, we aimed to evaluate the R. toruloides CCT0783 for extracellular biopolymer production. We found that the carbon-to-nitrogen ratio influenced exopolysaccharide (EPS) production, reaching 5.84 ± 0.45 g/L under glucose-rich conditions. We characterized the crude biopolymer using FTIR and GC-MS, identifying polysaccharide peaks, wherein EPS consisted of (%) glucose (88.83 ± 4.87), galactose (5.50 ± 1.50), mannose (4.80 ± 1.57), and xylose and arabinose (0.87 ± 0.04) monomers. The dried EPS also contained a fractional presence of protein (1.0%). Interestingly, inorganic, but not organic, nitrogen metabolism was associated with the acidification of the culture environment and simultaneous EPS production. A comparison of cultivation conditions revealed that EPS was produced when metabolic activity contributed to the culture media acidification to a pH of approximately 2. Finally, a comparative bioinformatics analysis allowed us to map the putative EPS biosynthesis and transport pathways, as well as regulators of intracellular pH maintenance. In conclusion, our study demonstrates R. toruloides' potential as an extracellular microbial biopolymer producer, enabling its consideration for extracellular bioproduction, besides its typically reported intracellular products.IMPORTANCEMicrobial biopolymers have been extensively studied for their impact on the environment and on health. However, developing a biotechnology process for producing such biopolymers remains challenging despite their potential for valuable applications. Considering this opportunity, we investigated the oleaginous yeast Rhodotorula toruloides as a producer of extracellular biopolymers, which, to date, is mostly used as a cell factory for intracellular bioproducts, namely, lipids and carotenoids. Our study identifies the conditions and maps pathways that allow exopolysaccharide (EPS) production in this yeast. These biopolymers, besides highlighting R. toruloides potential for extracellular production, could be deployed in diverse applications, from gelling agents in pharmaceuticals to emulsifiers in the food industry. Furthermore, our comparative bioinformatics analysis provides a foundational resource that could enable the development of Rhodotorula cell factories for extracellular bioproduction.