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

Area of Science:

• Biotechnology
• Microbial Genetics
• Industrial Microbiology

Background:

• Rhodotorula toruloides, a red oleaginous yeast, is commercially valuable for its lipid production and hardiness.
• Its complex life cycle as a basidiomycete limits traditional breeding methods for strain improvement.

Purpose of the Study:

• To develop and assess a protoplast fusion method for creating polyploid strains of Rhodotorula toruloides.
• To evaluate the stability and phenotypic characteristics of the resulting fusant strains for industrial applications.

Main Methods:

• Protoplast fusion was performed between Rhodotorula toruloides strains Y-6985 (A2) and Y-48190 (A2) transformed with antibiotic markers.
• Polyploid stability was assessed using flow cytometry and cell cycle analysis after growth on selective and non-selective media.
• Mitochondrial inheritance was determined using restriction fragment length polymorphism (RFLP) of mitochondrial DNA.
• Phenotypic traits were compared in glucose fed-batch bioreactor cultures and cellulosic sugar batch cultures.

Main Results:

• Stable diploid and triploid fusants were successfully generated.
• Fusants exhibited intermediate hardiness on switchgrass hydrolysate, with some strains growing in concentrated hydrolysate.
• Lipid titers in fed-batch cultures ranged from 24.9-39.7 g/L, with the diploid strain showing high performance.

Conclusions:

• Protoplast fusion is a viable and complementary tool for strain development in Rhodotorula toruloides.
• The generated polyploid strains offer potential for enhanced lipid production and utilization of lignocellulosic feedstocks.
• This method overcomes limitations associated with the complex life cycle of this industrially relevant yeast.