Systems-based Saccharomyces cerevisiae strain design for improved squalene synthesis.

Constraint-based flux balance analysis of S. cerevisiae has led to the identification of a novel
gene deletion targets, LYS1 and ADK1, for enhancement of squalene flux. LYS1 deletion
resulted in 2-fold improvement in squalene when compared to reference strain BY4741 with a
maximum yield of 33.1 mg/g DCW. A double mutant of ADK1 and LYS1 genes has increased
the squalene yield to 38 mg/g DW which is 2.38-fold higher over the control strain.
Furthermore, single copies of tHMG1 and POS5 (with mitochondrial signal sequence) genes
have been integrated into this double mutant in order to enhance the precursor pool and the
cofactor regeneration capacity, respectively, for enhanced squalene synthesis. The improved
strain, SK22 has resulted in squalene yield of 65 mg/g DW which is 4-folds higher than the
control strain. Finally, the engineered strain was cultivated in a bioreactor using fed-batch
strategy to improve the titer and productivity of squalene. Exponential feeding (open-loop
strategy) using high residual glucose (~40-60 g/L) has increased the squalene titer to a
maximum of 1.9 g/L with a yield of 0.15 g/g DCW, which is several folds higher than the
shake-flask results. Redirecting the lysine synthesis by external supplementation could
potentially improve squalene flux in S. cerevisiae.