3.4 Production of Z11-14:OAc by fed-batch fermentation
and alcohol acetylation
The engineered strain ST9253 (MATa ku70 ∆ Cas9 hfd4 ∆hfd1 ∆ pex10 ∆ fao1 ∆ hfd2 ∆ hfd3 ∆
GPAT_100bpPr Fas2pI1220F 2xLbo_PPTQ2xHarFAR ↑FAS1 ) 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.