Efficient Production Of Human Serum Albumin And Novel Genome Editing Technology In Pichia Pastoirs

Human serum albumin has important applications in the clinical,and current production capacity of albumin is unable to meet the increasing demand in medical market.Since the industrial production of recombinant alhumin is not still acheieved up to now,it is very important to develop the enabling technology of high albumin expression and build the corresponding genomic editing technology in Pichia pastoris.Firstly,the gene of human serum albumin was codon optimized and integrated into P.pastoris at HIS4 locus to construct recombinant yeast strain.After primary optimization of fermentation conditions,the expression of albumin was improved up to 0.52 g/L with this strain.Then,in order to facilitate the industrialization of recombination human serum albumin by metabolic engeneering,further efforts were paid to improve the editing efficiency of CRISPR/Cas9 system by improving the transformation efficiency,stability and copy numbers of plasmids by harboring various origins(PARS 1,panARS,mitoARS,PpARS2 and ScARS)in the plasmid construction.Compared with the system using the endogenous and most commonly used ARS(PARS 1),the employment of panARS brought about the best stability and high copy numbers.With this new system,this CRISPR/Cas9 genome editing efficiency was increased for more than 10-fold.The Cas9 gene was further integrated into the genome of P.pastoris.It was observed that the endogenous promoters(RNA pol II or RNA pol III)to express sgRNA would maintain high efficiency of gene editing in P.pastoris.Finally,one efficient editing system was constructed to have more than 80%deletion efficiency and 70%integration efficiency.In the conclusion,one recombinant system was constructed to produce recombinant album efficiently,and the corresponding genomic editing system was also built in P.pastoris.The genomic editing technology would greatly facilitate the development of novel technologices for high-level production of recombinant medical proteins,including album,in P.pastor is in the future.