A metabolic engineering strategy for producing conjugated linoleic acids using the oleaginous yeast Yarrowia lipolytica.

Conjugated linoleic acids (CLAs) have been found to have beneficial effects on human health when used as dietary supplements. However, their availability is limited because pure, chemistry-based production is expensive, and biology-based fermentation methods can only create small quantities. In an effort to enhance microbial production of CLAs, four genetically modified strains of the oleaginous yeast Yarrowia lipolytica were generated. These mutants presented various genetic modifications, including the elimination of ß-oxidation (pox1-6∆), the inability to store lipids as triglycerides (dga1∆ dga2∆ are1∆ lro1∆), and the overexpression of the Y. lipolytica ∆12-desaturase gene (YlFAD2) under the control of the constitutive pTEF promoter. All strains received two copies of the pTEF-oPAI or pPOX-oPAI expression cassettes; PAI encodes linoleic acid isomerase in Propionibacterium acnes. The strains were cultured in neosynthesis or bioconversion medium in flasks or a bioreactor. The strain combining the three modifications mentioned above showed the best results: when it was grown in neosynthesis medium in a flask, CLAs represented 6.5% of total fatty acids and in bioconversion medium in a bioreactor, and CLA content reached 302 mg/L. In a previous study, a CLA degradation rate of 117 mg/L/h was observed in bioconversion medium. Here, by eliminating ß-oxidation, we achieved a much lower rate of 1.8 mg/L/h.

S. cerevisiae fermentation activity after moderate pulsed electric field pre-treatments.

The batch fermentation process, inoculated by Pulsed Electric Field (PEF) treated wine yeasts (Saccharomyces cerevisiae Actiflore F33), was studied. PEF treatment was applied to the aqueous yeast suspensions ([Y] = 0.012 g/L) at the electric field strengths of E = 100 and 6000 V/cm using the same treatment protocol (number of pulses n = 1000, pulse duration ti = 100 µs, and pulse repetition time Δt = 100 ms). Electrical conductivity was increasing during and after the PEF treatment, which reflected cell electroporation. Then, fermentation was run for 150 h in an incubator (30 °C) with synchronic agitation. Electro-stimulation was revealing itself by the improvement of fermentation characteristics, and thus increased yeast metabolism. At the end of the lag phase (t = 40 h), fructose consumption in samples with electrically activated inoculum exceeded that of the control samples by ≈ 2.33 times for E = 100 V/cm and by ≈ 3.98 for E = 6000 V/cm. At the end of the log phase (120 h of fermentation), ≈ 30% mass reduction was reached in samples with PEF-treated inocula (E = 6000 V/cm), whereas the same mass reduction of the control sample required approximately 20 extra hours of fermentation.