3.3. Metabolic engineering for improved production ofZ11-14:OH
After selecting Lbo_PPTQ and HarFAR as the biosynthetic enzymes forZ 11-14:OH production, we proceeded with further metabolic engineering strategies to increase the product titer (Figure 3A). First, we introduced two gene copies of HarFAR into the high myristate producing strain ST7982 to enable biosynthesis of fatty alcohols. The resulting strain ST8225 produced 318.3±52.4 mg/L total fatty alcohols, among which 14:OH constituted 67.5% and was the most abundant. In order to produce Z 11-14:OH, we expressed Lbo_PPTQ in ST8225 leading to ST8373, which yielded 2 1.5±2.2 mg/L of the target compound. Compared to the parental strain, this strain made 46.7% more of the total fatty alcohols. Then we introduced a second copy of Lbo_PPTQ and this led to a further 3-fold improvement of Z 11-14:OH titer.
We anticipated that further improvement could be achieved by overexpressing native FAS1 from Y. lipolytica . Previous studies in S. cerevisiae showed that the activity of FAS complex is enhanced when the copy number of FAS1 gene is increased [37]. Indeed, overexpression of FAS1 (strain ST9253) resulted in 60.7% improvement in the total fatty alcohol titer and increased production of Z 11-14:OH by 54.7%, resulting in 93.9±11.7 mg/L ofZ 11-14:OH in small-scale cultivation.