Efficient expression and secretion of two co-produced xylanases from Aspergillus niger in Pichia pastoris directed by their native signal peptides and the Saccharomyces cerevisiae alpha-mating factor

摘要

The two genes encoding precursors of co-produced endo-1,4-beta-D-xylanases,Xyn6 and XynB,were isolated from Aspergillus niger IBT-90 by using RT-PCR technique and expressed in Pichia pastoris under the control of the alcohol oxidase I promoter.The secretion was driven by the Saccharomyces cerevisiae alpha-mating factor fused to mature xylanases and by the native 27-amino acid leader peptide of Xyn6 or 37-amino acid signal of XynB when the entire open reading frames of proteins were cloned.The secretion level of XynB directed by alpha-factor estimated at 140 mg l~(-1) was comparable to 150 mg l~(-1) with its own leader peptide;whereas in case of Xyn6,the yield was up to 180 and 220mg l~(-1),respectively.The N-termini of active recombinant Xyn6 and XynB indicated that their endogenous pre(pro)signals are effectively recognised and correctly processed in P.pastoris,like alpha-factor.These findings should contribute to develop the possibilities of application of the alternative secretion signals in P.pastoris.The initial studies revealed the different pH optima of Xyn6 (3.5) and XynB (5.0).The further analysis of individual gene products should enable to clarify the role of a particular enzyme in a complex xylanase system of A.niger.