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.