Expression and Activities Experiment of DNA Transduction Motif Based on GAL4 in Pichia Pastoris

The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

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.