Proteolytic degradation has been a severe problem when Pichia pastoris is employed to express recombinant proteins.One alternative method to circumvent this problem is to construct protease gene disruptant. However, the main study of gene disruption is focused on nonrecombinant Pichia pastoris rather than recombinant strain. In our study, we established two different methods to directly disrupt PRC1 and KEX1 gene in recombinant Pichia pastoris. On the basis of this, we further discussed and compared the application and advantages of both methods.