Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

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.