Characterizing and engineering promoters for metabolic engineering of

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 (PPDH, PPYK, PFBA, PPGM, PGLK, PTRI, PGPI, PADH1, PTEF1 and PGCW14) were obtained with the activity range of 13%–130% of the common promoter PGAP (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which PPDH and PGCW14 were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced PICL1, rhamnose-induced PLRA3 and PLRA4, and a bidirectional promoter (PMal-PPer) 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 PLRA3 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.