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. IMPORTANCE: Microbial 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.

Polysaccharides are often used to mimic physiological environments such as for cancer research models. However, established polysaccharides can display limited long-term stability and high batch-to-batch variability. To overcome this, biomanufactured polysaccharides are increasingly utilized in biomaterials. Here, we produced and characterized Rhodotorula toruloides yeast exopolysaccharides (EPS) and used it to engineer hydrogel for culturing cancer cells. Yeast fermentation of glucose, mannose, and xylose yielded varying EPS amounts (1.68, 1.44, and 0.48 g/L, respectively) with similar compositions, suggesting a common biosynthetic pathway. The glucose-derived EPS characterization identified multiple linkage types and three molecular weight fractions (1.75, 30.0, and 1000 kDa), and its solutions exhibited Newtonian behavior, indicating minimal chain-chain interactions. Solubilizing this polydisperse EPS with polyethylene glycol diacrylate and UV-crosslinking it enabled the engineering of semi-interpenetrating polymer network hydrogel that efficiently embedded cancer spheroids. Our study introduces an integrated biomanufacturing strategy to generate stable and consistent biomaterials, applicable for tissue engineering. Graphical

Additive manufacturing (3D printing) of engineered living materials (ELMs) enables the spatial patterning of cells within three-dimensional (3D) polymer matrices. However, a critical barrier to deploying ELMs is the lack of universal and reliable strategies to contain cells within 3D printed matrices. While synthetic biology renders cell-specific containment, a materials-centric approach can provide a cell-agnostic biocontainment platform. Therefore, this study presents a hydrogel-based additive manufacturing strategy to fabricate ELMs with physicochemical biocontainment. The 3D printed ELMs are fabricated by copolymerizing Pluronic F127-bisurethane methacrylate (F127-BUM) with methacrylic acid (MAA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA). Varying concentrations of these comonomers allow modulation of the ELM functionalities, namely, biocompatibility or antimicrobial properties. In particular, MAA-containing ELMs enable tunable biocontainment of the yeast Saccharomyces cerevisiae after immersion in 70 vol.% ethanol, leading to the formation of a time-dependent cell-free zone. The resulting yeast-laden ELMs enhance cell retention up to 300% (98 ± 12 h) and provide an average 38% fermentation increase compared to ELMs composed of only F127-BUM. In conclusion, this hydrogel-based spatiotemporal control strategy improves cell retention and metabolic activity performance in 3D-printed ELMs. These capabilities are promising for several applications, including 3D printed bioreactors and living therapeutic materials.

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 between non-buffered and buffered media indicated that EPS was produced when the final pH was around or below 2, which was observed in the former but not in the latter. Finally, a comparative bioinformatics analysis allowed us to map a 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 development as a cell factory. Importance: Microbial biopolymers are extensively investigated for their impact on the environment and 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. Our study describes the conditions of exopolysaccharide (EPS) biosynthesis in R. toruloides . This extracellular biopolymer could have a wide range of applications, from gelling agents in pharmaceuticals to emulsifiers in the food industry. Furthermore, our comparative bioinformatics analysis results could be useful for metabolic engineering and developing Rhodotorula cell factories.

The increasing demand for sustainable material production has fueled extensive research on alternative polysaccharide sources