Yarrowia lipolytica oleochemicals based on odd chain fatty acids – YaLiOl

1) Construction of strains producing odd-chain fatty acids:
A first strain overexpressing Y. lipolytica hexokinase to improve fructose consumption has been developed (JMY7877; Obese-LPSH). Two auxotrophic strains were obtained. From this starting strain, several genetic modifications were introduced; in particular the overexpression of genes involved in the lipid accumulation pathway. These genes were introduced separately or in combination.

2/ Optimization of Y. lipolytica fermentation conditions:
A screening study by a design of experiments approach was carried out on the JMY7877 strain with the aim of studying the influence of the constituents of the culture medium (factors) on growth as well as on the accumulation of lipids and OCFAs, including C17:1, (reponses). It has been noted that the influential factors are sucrose, glycerol, yeast extract, propionate, and acetate. Those with no significant influence are the saline mixture and ammonium chloride. An optimization study by a composite design of experiments was then carried out in order to determine the optimal concentrations maximizing the growth and accumulation of C17:1.
Tests were then carried out to replace sucrose with beet molasses, and pure glycerol with crude glycerol (from the vegetable oil transformation process). This will have made it possible to reduce the cost attributed to the culture medium.

1) Odd-chain fatty acid-producing strains:
According to the modifications made, respective increases of 77%, 80%, and 30% were observed for the total fatty acids, the odd chain fatty acids, and the % of C17:1. The ratio of unsaturated fatty acids to total fatty acids went from 74% to ˜80.3-83.4% depending on the clone. The objective is to have an unsaturated fatty acid/total fatty acid ratio > 90%.

2) Optimization of fermentation conditions:
The optimal conditions obtained with the JMY7877 strain were as follows: [sucrose] = 52.4 g/L, [glycerol] = 26.9 g/L, [sodium acetate] = 10.4 g/L, [sodium propionate] = 5 g /L, and [yeast extract] = 4 g/L. Under these conditions, and in a 5 L bioreactor, the respective lipid and C17:1 contents were 2.52 ± 0.05 and 0.82 ± 0.01 g/L of culture medium, after 96 h of fermentation.
The production of lipids by the yeasts on a medium containing molasses (20 g/L of sucrose as optimal concentration) and crude glycerol (30 g/L) reached a quantity of 4.63 ± 0.95 g/L of culture medium. The content of odd-chain fatty acids represented 58% of the total fatty acids in lipids, which corresponds to ˜2.69 ± 0.03 g/L of culture medium. Fermentation in 5 L bioreactors with co-feeding increased the accumulation of OCFAs by 56% compared to cultures carried out without co-feeding.

Work over the next period will focus on:
1) the study of the genes involved in the transport of acetate and propionate;
2) engineering by deletion and overexpression of new targets to increase the lipid content and the ratio of unsaturated fatty acids to saturated fatty acids;
3) Test the optimized fermentation conditions on new strains producing odd-chain fatty acids;
4) Develop lipid hydrolysis conditions during their extraction;
5) Fractionate the lipids of Y. lipolytica into a fraction enriched in unsaturated fatty acids, and a fraction enriched in saturated fatty acids.
6) Transform the fractions obtained into oleochemical products with higher added value.

1. Al Sahyouni W., El Kantar S., Khelfa A., Park Y.-K., Nicaud J.-M., Louka N., Koubaa M. (2022). Optimization of cis-9-heptadecenoic acid production from the oleaginous yeast Yarrowia lipolytica. Fermentation. 8, 245. doi.org/10.3390/fermentation8060245

2. Park Y.-K., González-Fernández C., Robles-Iglesias R., Vidal L., Fontanille P., Kennes C., Tomás Pejó E., Nicaud J.-M., Fickers P. (2021). Bioproducts generation from carboxylate platforms by the non-conventional yeast Yarrowia lipolytica. FEMS Yeast Res. 21(6):foab047. doi.org/10.1093/femsyr/foab047

3. El Kantar S., Koubaa M. (2022). Valorization of low-cost substrates for the production of odd chain fatty acids by the oleaginous yeast Yarrowia lipolytica. Fermentation. doi.org/10.3390/fermentation8060284

4. El Kantar S., Khelfa A., Vorobiev E., Koubaa M. (2021). Strategies for increasing lipid accumulation and recovery from Y. lipolytica: A review. Oilseeds and fats, Crops and Lipids, 28, 51. doi.org/10.1051/ocl/2021038

5. El Kantar S., Koubaa M. (2022). Pulsed electric field treatment for the stimulation of microorganisms: Applications in food production. Research in Agricultural Engineering. 68, doi.org/10.17221/78/2021-RAE

One of the promising alternatives that can substitute fossil resources is the use of microbial oils. The strategies that can reduce their production costs include the engineering of high value-added fatty acids, the fermentation on a low-cost carbon substrate, and the transformation of oils into higher-value products. In this context, the YaLiOl project aims to propose a new oleochemical route based on odd-chain fatty acids (OCFA) produced by the yeast Yarrowia lipolytica. The core objectives of this project consist of 1) engineering Y. lipolytica for the production of OCFA, 2) the development of a fermentation medium based on sugar beet molasses, and 3) the recovery of OCFA, their characterization and their transformation into oleochemical derivatives for viscosity modifiers of lubricants and thermal energy storage. Pilot-scale production of oil and odd fatty acid esters will help rounding-up the financial study.