3.4 Production of Z11-14:OAc by fed-batch fermentation and alcohol acetylation
The engineered strain ST9253 (MATa ku70Cas9 hfd4hfd1pex10fao1hfd2hfd3 ∆ GPAT_100bpPr Fas2pI1220F 2xLbo_PPTQ2xHarFARFAS1 ) was cultivated in controlled bioreactors in fed-batch mode. The initial batch phase served as a biomass propagation phase. After the depletion of the initial carbon source supplied in the batch phase, the composition of the fed-batch feed solution was set to facilitate the production of fatty alcohols. In the first 42 h of fermentation, the accumulation of biomass was targeted, and stationary phase was reached after 49 h (Figure3B). The start of the fed-batch facilitated a shift towards nitrogen-limited conditions and thereby transition from the biomass build-up phase to production of fatty alcohols.
In the fatty alcohol production phase of the fermentation process, we observed a constant increase in specific yield. The specific production yield of Z 11-14:OH peaked at the very end of fermentation and reached 0.007 g product/g DW. At this time point, the titer ofZ 11-14:OH was 188.1±13.4 mg/L, while the cell dry weight reached 28.2±0.7 g/L (Figure 3B). The titer of 14:OH reached 1,350.1±188.1 mg/L. It was the most abundant fatty alcohol in the fermentation broth. Tight control of parameters such as pH, dissolved oxygen and glycerol levels in the bioreactor cultivations led to a 2-fold improvement in the target compound titer compared to the small-scale cultivations.
After 120 h of fed-batch fermentation, fatty alcohols were extracted with organic solvent and purified on a silica column. The resulting fatty alcohol mixture containing approximately 250 mg ofZ 11-14:OH was acetylated with acetic acid anhydride. It resulted in full conversion, where 320.1±13.4 mg of Z 11-14:OAc was obtained, and no traces of alcohols were left at the end of the reaction (Figure 4). The resulting product is referred to as BioPhe, for biologically-derived pheromone.