Determining mating type and ploidy in Rhodotorula toruloides and its effect on growth on sugars from lignocellulosic biomass

Authors: DIAS LOPES, DAIANE - Orise Fellow; Dien, Bruce; Hector, Ronald - Ron; SINGH, VIJAY - University Of Illinois; Thompson, Stephanie; Slininger, Patricia - Pat; BOUNDY-MILLS, KYRIA - University Of California, Davis; JAGTAP, SUJIT - University Of Illinois; RAO, CHRISTOPHER - University Of Illinois

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/13/2023
Publication Date: 11/21/2023
Citation: Dias Lopes, D., Dien, B.S., Hector, R.E., Singh, V., Thompson, S.R., Slininger, P.J., Boundy-Mills, K., Jagtap, S.S., Rao, C.V. 2023. Determining mating type and ploidy in Rhodotorula toruloides and its effect on growth on sugars from lignocellulosic biomass. Journal of Industrial Microbiology and Biotechnology. https://doi.org/10.1093/jimb/kuad040.
DOI: https://doi.org/10.1093/jimb/kuad040

Interpretive Summary: Yeast fermentations are favored for production of chemicals by the industrial biotechnology community because they have several advantages compared to other microorganisms. Typically, the industry has relied on a few yeast species, most famously Saccharomyces cerevisiae. However, the large increase in the types and number of targeted products, as well as use of new sources of sugars or other feeds recommend the use of non-conventional yeast. One of those gaining increased popularity is Rhodotorula toruloides. This red-pigmented yeast grows quickly, achieves high yeast concentrations, grows on a wide variety of sugars and waste products, and has the ability to produce to oil as droplets. The oil it produces is similar in composition to vegetable oils and is expected to find use in manufacturing sustainable aviation fuel and biodiesel. In this study, we compare different strains of this yeast for the ability to produce yeast oil from unrefined sugars. The unrefined sugars were prepared in house from field grown switchgrass. Switchgrass is a perennial warm season grass naturally occurring in the North American prairie that is being developed by ARS researchers as a future bioenergy crop. This study identifies specific strains that are better than others for growth on switchgrass based sugars. It also lays out a strategy to develop other R. toruloides yeast strains that might be superior to those reported here. This study is of interest to the biodiesel and emerging sustainable aviation fuel industries because their current production is limited by a scarcity of affordable sources of plant oil. It should also be of interest to groups promoting rural development because switchgrass production is suitable for cultivation on land that does not support row crops.

Technical Abstract: Rhodotorula toruloides is gaining popularity for industrial biotechnology applications because of its favorable physiology. This includes its ability to produce and store large amounts of lipids in the form of droplets. Seventeen strains were characterized for mating type, ploidy, robustness for growth, and accumulation of lipids on inhibitory switchgrass hydrolysate (SGH). Mating type was determined using a novel PCR-based assay, which was validated using the classical microscopic test. Three of the strains were diploids (A1/A2), ten haploids (A1 or A2), and the remainder single mating types with greater than haploid DNA contents. Two of the diploids were compared to their haploid parents for growth on 75%v/v concentrated SGH. The diploids were much more robust than the parental strains, which either did not grow or had extended lag times. The entire set was evaluated in 60%v/v SGH batch cultures for growth kinetics and biomass and lipid production. Lipid titers were 2.33 - 9.40 g/L with a median of 6.12 g/L. Four strains had significantly higher lipid yields and contents. One of these strains, which had the highest lipid yield, has not been previously described in the literature.