(500e) Integrative Omics Analysis of Yarrowia Lipolytica for the Bioconversion of Lipid-Based Substrates

Major cornerstones for modern sustainability are centered around the reduction of waste and the conservation of world resources. The development of microbial cell factories has been leveraged to provide a solution to both concerns through synthetic biology. Yarrowia lipolytica has emerged as an important host for many industrial applications, such as production of organic acids and terpenoid compounds. Equally, it is also desired for its ability to metabolize hydrophobic substrates, like alkanes and lipids, and for its high chemical tolerance. The US alone generates billions of pounds of waste oils each year which are often overlooked as substrates for microbial biorefineries, and offer a potentially cheaper alternative to carbohydrate based feedstock. While the bioconversion of sugars has been widely studied in Y. lipolytica, its potential for catabolizing lipids is less explored. Due to this, there are critical knowledge gaps in Y. lipolytica lipid catabolism, including NADPH generation routes and flux compartmentalization of key metabolites. This study utilizes integrative omics analysis to address the critical knowledge gaps in Y. lipolytica lipid metabolism in order to enable effective metabolic and process engineering strategies.

Methods

Omics analysis of Y. lipolytica W29 was performed to provide insights into the cellular regulation of lipid assimilation. Namely, transcriptomics, metabolomics, and fluxomics were used for this study. RNA sequencing data was collected and compared for cells grown on glucose, glycerol, and oleic acid. Free metabolite analysis evaluated the metabolic pool sizes of cells grown on different substrates. Moreover, steady-state ¹³C metabolic flux analysis (MFA) was performed. Specifically, cells were grown in positionally labelled substrates (1,2-¹³C­ glucose; 1,3-¹³C glycerol; 1,2,3,7,8-¹³C oleic acid) and the resulting amino acid and free metabolite labelling profiles were collected and used for flux calculation. MFA was conducted using WUFlux [1] to simulate mass isotopomer distribution (MID) values using the input carbon transition matrices of pathway reactions. The objective function minimized the sum of square residuals between the experimentally measured and computationally simulated MIDs. Compartment-specific amino acid generation was used to constrain fluxes for parallel metabolic reactions in multiple compartments. In addition, biomass composition analysis was conducted to inform the biomass equation used to fit the model and illuminate differences in bulk macromolecule distribution within the cell.

Results

¹³C-MFA results showed the flux distribution response to oleic acid differs significantly from the flux results of glucose and glycerol metabolism. For instance, Y. lipolytica grown on oleic acid show higher activity in the TCA cycle than in the pentose phosphate or glycolysis pathways. Following β-oxidation, the generated acetyl-CoA enters into the TCA cycle or contributes to the process of lipogenesis. The flux results were used to constrain the genome scale model (GSM) for Y. lipolytica to provide an overall picture of the metabolism when oleic acid is used as the carbon source. Additionally, time-course dynamics of metabolite isotopologues were examined during lipid catabolism [2]. The isotopologue profiles suggests a segregated metabolic network in Y. lipolytica during growth on lipids (i.e. glycerol and fatty acid co-utilization). Biomass composition analysis shows protein content significantly reduced in oil and fatty acid conditions, however amino acid composition remains consistent. Transcription data analysis shows the differentially expressed genes between glucose and oleic acid-grown cells, pointing to unique genetic regulations in oleic acid-grown cells. Furthermore, metabolomics was performed on Y. lipolytica cultured on different substrates. Metabolite pool size data provide further insights into cell metabolic regulation. Based on metabolic analysis, strain engineering was performed to improve the productivity of β-carotene and astaxanthin producing strains.

Implications

Understanding the changes in cellular networks and regulation of Y. lipolytica during growth on lipid substrates provides insights to help establish this oleaginous yeast as an industrial host for the bioconversion of waste oils. This study evaluated the ability of Y. lipolytica to grow on lipid-based substrates and analyzed the cellular response at multiple levels. An important application of lipid catabolism in Y. lipolytica is for the biosynthesis of terpenoid compounds [3]. Terpenes are derived from the native mevalonate pathway, which is powered by acetyl-CoA. Catabolism of fatty acids can introduce an abundance of acetyl-CoA needed to power the mevalonate pathway and increase production of terpene compounds. This knowledge found from this work provides insights which can be applied to genetic engineering and process development strategies to use Y. lipolytica as a host for a microbial biorefinery generating value-added products from waste oils.

This work was supported by the USDA NIFA (award number 2020-67022-31146).

References

1. He L, Wu SG, Zhang M, Chen Y, Tang YJ. WUFlux: an open-source platform for ¹³C metabolic flux analysis of bacterial metabolism. BMC Bioinformatics. 2016; 17(1):444
2. Worland AM, Czajka JJ, Xing Y, Harper WF, Moore A, Xiao Z, Han Z, Wang Y, Su WW, Tang YJ. Analysis of Yarrowia lipolytica growth, catabolism, and terpenoid biosynthesis during utilization of lipid-derived feedstock. Metabolic Engineering Communications, Volume 11, 2020, e00130
3. Worland AM, Czajka JJ, Li Y, Wang Y, Tang YJ, Su WW. Biosynthesis of terpene compounds using the non-model yeast Yarrowia lipolytica: grand challenges and a few perspectives. Current Opinion in Biotechnology, Volume 64, 2020, Pages 134-140