Impact of glucose and propionic acid on even and odd chain fatty acid profiles of oleaginous yeasts

Abstract

Background: Odd chain fatty acids (OCFAs) are gaining attention for their valuable medical and nutritional applications. Microbial fermentation offers a sustainable and environmentally friendly alternative for OCFA production compared to traditional extraction or chemical synthesis methods. To achieve an economically feasible OCFA production process, it is essential to identify and develop microbial cell factories capable of producing OCFAs with high titers and yields.

Results: We selected 19 yeast species, including both oleaginous yeasts and representatives from the Ascomycota and Basidiomycota phyla, based on their known or potential ability to produce OCFAs. These species were screened under various growth conditions to evaluate their OCFA production potential. In glucose-based, nitrogen-limited media, the strains produced fatty acids to varying extents, with OCFAs comprising 0.5-5% of the total fatty acids. When using the OCFAs precursor propionic acid as the sole carbon source, only eight strains exhibited growth, with tolerance to propionic acid concentrations between 5 and 29 g/L. The strains also displayed varying efficiencies in converting propionic acid into fatty acids, yielding between 0.16 and 1.22 g/L of fatty acids, with OCFAs constituting 37-89% of total fatty acids. Among the top performing strains, Cutaneotrichosporon oleaginosus produced the highest OCFA titers and yields (0.94 g/L, 0.07 g/g), Yarrowia lipolytica demonstrated superior growth rates even at elevated propionic acid concentrations, and Rhodotorula toruloides achieved the highest proportion of OCFAs relative to total fatty acids (89%).

Conclusions: Our findings highlight the diverse capacities of the selected yeast species for OCFA production, identifying several promising strains for further optimization as microbial cell factories in sustainable OCFA production processes.

Keywords: Cutaneotrichosporon oleaginosus; Rhodotorula toruloides; Yarrowia lipolytica; Even chain fatty acid (ECFA); Fatty acids methyl ester; Non-conventional yeast; Odd chain fatty acid (OCFA); Propionic acid tolerance; Short chain fatty acid (SCFA).

Fig. 1

General overview of fatty acid metabolism in yeast under nitrogen limitation. A) Upon yeast’s growth in glucose-based medium under nitrogen starvation, the metabolism predominantly results in ECFAs, with only a negligible production of OCFAs; B) In a propionic acid-based medium, yeast produces elevated amounts of OCFAs. Additionally, the synthesis of ECFAs persists, albeit to a much lower extent with respect to glucose-based growth conditions. Double arrows represent multiple reactions; dotted arrows represent unknown pathways; MC, methyl citrate cycle; TCA, tricarboxylic acids cycle; TAGs, triacylglycerols; OCFAs, odd chain fatty acids; ECFAs, even chain fatty acids

Fig. 2

Comparison of growth profiles in glucose-based media with high and low C/N ratios. Yeast strain growth was analyzed in 20 g/L glucose at molar C/N ratios of 9 and 50. The graphs display data obtained from a Growth Profiler in a 96-well plate system. Data are shown as mean ± standard deviation for biological triplicates. The y-axis shows the biomass formed during cultivation on a logarithmic scale using green values, considered equivalent to OD, plotted against time (days) on the x-axis. A) strains with similar growth profiles at C/N ratios of 9 and 50. B) strains with distinct biphasic growth at C/N ratio 50, reaching the same or almost the same final ODeq as in C/N ratio 9. C) strains with non-biphasic growth at C/N ratio 50, lower maximum specific growth rates, and lower final ODeqs than those observed at C/N ratio 9

Fig. 3

Results from fatty acids analysis represented with different metrics. The 15 strains that produced high enough cell mass titers when cultivated in 20 g/L glucose at molar C/N ratio 50 are displayed. All plotted data are ordered by decreasing ECFAs g/L metric. A) Relative percentages ratio (%) of single fatty acids on total fatty acids profile; B) Histograms representation of yields on consumed substrate (S) in g/g of final biomass (CDW/S), final odd chain fatty acids (OCFAs/S) and final even chain fatty acids (ECFAs/S). Overlapping scatter point of final residual substrate (g/L) and final CDW (g/L) relative to each strain; C) titer in g/L of the final total amount of ECFAs; D) titer in mg/L of the final total amount of OCFAs

Fig. 4

Comparison of growth profiles in increasing concentrations of propionic acid. Yeast strain tolerance to PA were analyzed in 5, 10, 15, 19, 24, and 29 g/L of PA at molar C/N ratio of 9 following the growth using a Growth Profiler in a 96-well plate system. Data are shown as means of three biological replicates. The y-axis shows the biomass formed during cultivation on a logarithmic scale using green values, considered equivalent to OD, plotted against time (days) on the x-axis

Fig. 5

Fatty acids profiles of seven strains cultivated on 15 g/L propionic acid at molar C/N ratio 50. A) relative % fatty acid profiles. Other ECFAs: 7-C16:1, 9,12-C18:2. Other OCFAs: 10-C19:1, C21:0, C25:0; B) yields on substrate (S) of biomass (CDW/S), odd chain fatty acids (OCFAs/S) and even chain fatty acids (ECFAs/S), overlapping illustrations of residual substrate (g/L) and CDW (g/L) to show the full potential of every analyzed yeast; C) titer (g/L) of the total amount of odd chain and even chain fatty acids

Fig. 6

Comparison between the fatty acid profiles in 15 g/L of PA and 20 g/L of glucose at molar C/N ratio 50. The y-axis shows the µg fatty acids produced per mg of biomass (CDW). The x-axis shows the labels of the single fatty acid present in the profile. The orange and white bars represent the PA and glucose conditions, respectively