Protoplast fusion as a strategy to increase ploidy in Rhodotorula toruloides for strain development

Authors: DIAS LOPES, DAIANE - Orise Fellow; Dien, Bruce; Hector, Ronald; Thompson, Stephanie; SINGH, VIJAY - University Of Illinois; JAGTAP, SUJIT - University Of Illinois; Nichols, Nancy; RAO, CHRISTOPHER - University Of Illinois; Skory, Christopher; Slininger, Patricia

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Rhodotorula are pigmented yeast known for producing high amounts of natural oils when grown on sugars from agricultural crops. These oils are valuable for their applications in renewable fuels, food ingredients, and biobased consumer care products. To maximize production of these oils, ARS researchers in Peoria, Illinois, developed improved breeding methods that selectively cross different yeast strains to create new hybrids with desired traits, such as rapid growth, robustness, and high oil yield, which are important for efficient and affordable production from agricultural crops. This advancement contributes to a more sustainable bioeconomy and provides farmers with new markets for their crops and agricultural waste products.

Technical Abstract: R. toruloides is a red oleaginous yeast with growing commercial interest because of its hardiness and exceptional lipid production capacity. Because it is a basidiomycete yeast with a complex life cycle, many of the classical breeding methods used with ascomycetes are unavailable for strain improvement. However, we have been able to construct polyploid yeast by fusing protoplasts of parents with the same mating type. Fusing of Y-6985 (A2) and Y- 41890 (A2), which had been transformed with complementary antibiotic markers, led to the recovery of two diploids and one triploid. The stability of the fusion yeasts was tested by plating them on non-selective medium and testing five colonies per strain for nuclear DNA contents using flow cytometry and standard cell cycle analysis: the triploid and one diploid were stable. Fusants inherited their mitochondria from a single parent, which was demonstrated using restriction fragment length polymorphism (RFLP) of mitochondrial DNA. The phenotypic properties of the parents and fusants were compared in glucose fed-batch bioreactor studies and cellulosic sugar batch cultures. The final lipid titers for the fed-batch cultures were 24.9 – 39.7 g/L with Y-6985 and the diploid and triploid performing the best and worst, respectively. The fusants demonstrated intermediate hardiness for growth on hydrolysate prepared with dilute-acid pretreated switchgrass. Unlike one of the haploid parents, the fusants grew in 70%v/v concentrated hydrolysate. However, they did not grow as fast as the other haploid. In this study, a modernized protoplast fusion method resurrected a useful tool for strain development in this yeast, which is complementary with other available methods.