Exploring proteomes of robust Yarrowia lipolytica isolates cultivated in biomass hydrolysate reveals key processes impacting mixed sugar utilization, lipid accumulation, and degradation

Authors: WALKER, CALEB - University Of Tennessee; Dien, Bruce; GIANNONE, RICHARD - Oak Ridge National Laboratory; Slininger, Patricia - Pat; Thompson, Stephanie; TRINH, CONG - University Of Tennessee

Submitted to: mSystems
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2021
Publication Date: 8/3/2021
Citation: Walker, C., Dien, B.S., Giannone, R., Slininger, P.J., Thompson, S.R., Trinh, C. 2021. Exploring proteomes of robust Yarrowia lipolytica isolates cultivated in biomass hydrolysate reveals key processes impacting mixed sugar utilization, lipid accumulation, and degradation. mSystems. 6(4). Article e00443-21. https://doi.org/10.1128/mSystems.00443-21.
DOI: https://doi.org/10.1128/mSystems.00443-21

Interpretive Summary: Some yeast can be grown in such a way as to produce oil that is similar to vegetable oil. The oil is stored within the yeast cell in the form of droplets, which can be extracted following growth. The oil has utility for use in making biofuels suitable to run heavy equipment and trucks. The oil-producing yeast most commonly used is Yarrowia lipolytica. ARS researchers are investigating using this yeast for production of oil by growing the yeast on sugars extracted from bioenergy crops bred by ARS plant researchers. Potentially, this approach could produce more oil for biodiesel production than is currently available from oil seed crops (e.g., soybeans). Our approach has been to evaluate Y. lipolytica yeast isolated from various locations to find the one that is best at growing and producing oil on sugars extracted from these crops. During the course of our studies, an unexpected discovery was made. Some of our yeast maintained their oil content throughout the fermentation while the yeast strain typically used in other labs depleted its oil towards the end. Working with collaborators, we were able to use advanced analytical techniques to peek within the cell metabolism and understand this phenomenon. It appears that our superior yeast strains maintain their oil content because they are better able to utilize sugars later on in the fermentation. Scientifically, this is of additional interest because the observed difference is predicated on use of a type of sugar that this yeast is commonly thought not to use. Practically, this will aid us in engineering better Yarrowia strains for oil production. This work will be of interest to stakeholders interested in growing bioenergy crops on non-crop lands and to processors interested in producing further products from agricultural resources. It is of general interest to the public because biodiesel production is hampered by a shortage of available oil and this approach can fill that gap without withdrawing crop lands.

Technical Abstract: Yarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504 (or W29), which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing nonpreferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica. To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels, while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes, and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins involved in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, and ß-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation, and degradation in biomass hydrolysates.