摘要
Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engi-neering.Ogataea polymorpha,one of the methylotrophic yeasts,possesses advantages in broad substrate spec-trum,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 de-hydrogenase),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.
基金
National Natural Science Foundation of China(21808216,22161142008 and M-0246)
Key project at central government level:The ability establishment of sustainable use for valuable Chinese medicine resources(2060302)
DICP innovation grant(DICP I202021 and I201920)from Dalian Institute of Chemicals Physics,CAS.
