Abstract
Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engineering. Ogataea polymorpha, one of the methylotrophic yeasts, possesses advantages in broad substrate spectrum, thermal-tolerance, and capacity to achieve high-density fermentation. However, a limited number of available promoters hinders the engineering of O. polymorpha for bio-productions. Here, we systematically characterized native promoters in O. polymorpha by both GFP fluorescence and fatty alcohol biosynthesis. Ten constitutive promoters (P (PDH) , P (PYK) , P (FBA) , P (PGM) , P (GLK) , P (TRI) , P (GPI) , P (ADH1) , P (TEF1) and P (GCW14) ) were obtained with the activity range of 13%-130% of the common promoter P (GAP) (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which P (PDH) and P (GCW14) were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced P (ICL1) , rhamnose-induced P (LRA3) and P (LRA4) , and a bidirectional promoter (P (Mal) -P (Per) ) that is strongly induced by sucrose, further expanded the promoter toolbox in O. polymorpha. Finally, a series of hybrid promoters were constructed via engineering upstream activation sequence (UAS), which increased the activity of native promoter P (LRA3) by 4.7-10.4 times without obvious leakage expression. Therefore, this study provided a group of constitutive, inducible, and hybrid promoters for metabolic engineering of O. polymorpha, and also a feasible strategy for rationally regulating the promoter strength.