Engineering Cell Wall Integrity Enables Enhanced Squalene Production in Yeast

Microbial production of many lipophilic compounds is often limited by product toxicity to host cells. Engineering cell walls can help mitigate the damage caused by lipophilic compounds by increasing tolerance to those compounds. To determine if the cell wall engineering would be effective in enhancing lipophilic compound production, we used a previously constructed squalene-overproducing yeast strain (SQ) that produces over 600 mg/L of squalene, a model membrane-damaging lipophilic compound. This SQ strain had significantly decreased membrane rigidity, leading to increased cell lysis during fermentation. The SQ strain was engineered to restore membrane rigidity by activating the cell wall integrity (CWI) pathway, thereby further enhancing its squalene production efficiency. Maintenance of CWI was associated with improved squalene production, as shown by cell wall remodeling through regulation of Ecm33, a key regulator of the CWI pathway. Deletion of ECM33 in the SQstrain helped restore membrane rigidity and improve stress tolerance. Moreover, ECM33 deletion suppressed cell lysis and increased squalene production by approximately 12% compared to that by the parent SQ strain. Thus, this study shows that engineering of the yeast cell wall is a promising strategy for enhancing the physiological functions of industrial strains for production of lipophilic compounds.