Dual-acting single-engineered hybrid-architectured promoters enhance and convert expressions into multi-carbon source-regulated systems in Komagataella phaffii.

Ethanol, glycerol, methanol, and acetate are sustainable carbon sources (SCSs) used as substrates for biochemical production. Cells are simultaneously exposed not to a single but multiple external stimuli. SCSs as substrates and co-substrates must be directed/redirected into fermentations. We need de novoengineered promoters inducible with multi-carbon sources. Core to this conceptual advance is the development of novel methodologies for integrating SCSs into fermentations through engineering transcriptional machinery-element interactions with multiple transcriptional switches, each designed with directed transcription factor (TF) binding site (TFBS)-TF interactions in Komagataella phaffii (Pichia pastoris). Dual-acting single-engineered promoters (DASEPs) were designed on alcohol dehydrogenase 2 (ADH2) hybrid-architectured promoter layout with two directed synthetic TFBS-TF interactions, function as transcriptional switches to drive SCS-induced upregulated- and/or rewired- transcription and expression. Using cross-yeast analogies, we predicted the master TFs (i) Cat8 on ethanol and methanol and (ii) Hap1 and Hap2/3/4/5 complex on the SCSs. Using single-acting single-engineered promoters (SASEPs) carrying synthetic TFBS-Cat8 transcriptional switch constructed on the base promoter ADH2 architecture, we generated DASEP1 and DASEP2 on the hybrid-architectured SASEP3 layout with synthetic TFBS-Hap1 and TFBS-Hap2/3/4/5 transcriptional switches, respectively. DASEP1 and DASEP2 performances tested by enhanced green fluorescent protein (eGFP) expression measurements in SCSs, outcompeted SASEPs and compared to SASEP3, respectively, (i) 8.2- and 6.5-fold on glycerol, (ii) 2.7- and 2.6-fold on 2 % (v/v) ethanol, (iii) 3.9- and 4.0-fold on 1 % (v/v) ethanol, (iv) 3.6- and 4.2-fold on 1 % (v/v) methanol, and (v) 3.7- and 2.8-fold on acetate. In contrast, lower cell concentrations indicated the metabolic burden of eGFP expression on the metabolic engineered K. phaffii cells constructed with DASEPs.