Abstract
Natural methylotrophs are attractive methanol utilization hosts, but lack flexible expression tools. In this study, we developed yeast transcriptional device libraries for precise synthesis of value-added chemicals from methanol. We synthesized transcriptional devices by fusing bacterial DNA-binding proteins (DBPs) with yeast transactivation domains, and linking bacterial binding sequences (BSs) with the yeast core promoter. Three DBP-BS pairs showed good activity when working with transactivation domains and the core promoter of PAOX1 in the methylotrophic yeast, Pichia pastoris. Fine-tuning of the tandem BSs, spacers and differentiated input promoters further enabled a constitutive transcriptional device library (cTRDL) composed of 126 transcriptional devices with an expression strength of 16-520% and an inducible TRDL (iTRDL) composed of 162 methanol-inducible transcriptional devices with an expression strength of 30-500%, compared with PAOX1. Selected devices from iTRDL were adapted to the dihydromonacolin L biosynthetic pathway by orthogonal experimental design, reaching 5.5-fold the production from the PAOX1-driven pathway. The full factorial design of the selected devices from the cTRDL was adapted to the downstream pathway of dihydromonacolin L to monacolin J. Monacolin J production from methanol reached 3.0-fold the production from the PAOX1-driven pathway. Our engineered toolsets ensured multilevel pathway control of chemical synthesis in methylotrophic yeasts.