Mediator Engineering of Saccharomyces cerevisiae To Improve Multidimensional Stress Tolerance

Saccharomyces cerevisiae is a well-performing workhorse in chemical production, which encounters complex environmental stresses during industrial processes. We constructed a multiple stress tolerance mutant, Med15^V76R/R84K, that was obtained by engineering the KIX domain of Mediator tail subunit Med15. Med15^V76R/R84K interacted with transcription factor Hap5 to improve ARV1 expression for sterol homeostasis for decreasing membrane fluidity and thereby enhancing acid tolerance. Med15^V76R/R84K interacted with transcription factor Mga2 to improve GIT1 expression for phospholipid biosynthesis for increasing membrane integrity and thereby improving oxidative tolerance. Med15^V76R/R84K interacted with transcription factor Aft1 to improve NFT1 expression for inorganic ion transport for reducing membrane permeability and thereby enhancing osmotic tolerance. Based on this Med15 mutation, Med15^V76R/R84K, the engineered S. cerevisiae strain, showed a 28.1% increase in pyruvate production in a 1.0-L bioreactor compared to that of S. cerevisiae with its native Med15. These results indicated that Mediator engineering provides a potential alternative for improving multidimensional stress tolerance in S. cerevisiae. IMPORTANCE This study identified the role of the KIX domain of Mediator tail subunit Med15 in response to acetic acid, H₂O₂, and NaCl in S. cerevisiae. Engineered KIX domain by protein engineering, the mutant strain Med15^V76R/R84K, increased multidimensional stress tolerance and pyruvate production compared with that of S. cerevisiae with its native Med15. The Med15^V76R/R84K could increase membrane related genes expression possibly by enhancing interaction with transcription factor to improve membrane physiological functions under stress conditions.