Engineered Deregulation of Expression in Yeast with Designed Hybrid-Promoter Architectures in Coordination with...

Engineered promoters are key components in the cell-factory design, allowing precise and enhanced expression of genes. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Here, the custom-designed AOX1 hybrid-promoter architectures in coordination with targeted transcription factors are shown, transcriptionally rewired the expression over methanol-free substrate-utilization pathway(s) and converted methanol-dependent Pichia pastoris alcohol oxidase 1(AOX1) promoter (P_AOX1 ) expression into a non-toxic carbon-source-regulated system. AOX1 promoter variants are engineered by replacing specified cis-regulatory DNA elements with synthetic Adr1, Cat8, and Aca2 cis-acting DNA elements for Mxr1, Cat8, and Aca1 binding, respectively. Applications of the engineered-promoters are validated for eGFP expression and extracellular human serum albumin production. The hybrid-promoter architecture designed with single Cat8 cis-acting DNA element deregulates the expression on ethanol. Compared with P_AOX1 on methanol, the expression on ethanol is increased with i) P_AOX1/Cat8-L3 (designed with single Cat8 cis-acting element) to 74%, ii) P_{AOX1/Adr1-L3/Cat8-L3} (designed with single- Cat8 and Adr1 cis-acting elements) to 85%, and for further consolidation of deregulated expression iii) P_eAOX1 (designed with triplet- Cat8 and Adr1 cis-acting elements) 1.30-fold, at t = 20 h of batch cultivations.