Expression of Hemoglobin Gene in the Pichia pastoris Engineering Strain of Producing CBHII

Objective:In order to improve the extracellular secretion of CBHII from P.pastoris-CBHⅡon liquid-state fermentation.Methods:Recombinant vectors pPICZαA-vhb(for extracellular expression) and pPICZαA(-s)-vhb(for intracellular expression) were constructed.These two recombinant vectors were then transformed into the P.pastoris-CBHⅡ with the method of electroporation respectively,resulting in the recombinant strains P.pastoris-CBHⅡ-vhb(extracellular secretion) and P.pastoris-CBHⅡ-vhb(-s)(intracellular expression) which can correctly produce VHb protein with biological activity.The shake flask fermentation experiments showed that the presence of VHb can efficiently enhanced the recombinant strains' secretive expression of CBHII on the condition of oxygen deficiency.Results:The CMC activities in the culture supernatant of two vhb harboring strains were 2.04U/ml(intracellular) and 1.93U/ml(extracellular),higher than the P.pastoris-CBHⅡ(1.81U/ml).These results indicated that intracellular expression of VHb was more beneficial to the expression of CBHⅡ than that of extracellular expression.