High-Level Expression of Alkaline Polygalacturonate Lyase in Pichia pastoris by Using a Combined Strategy

Abstract:

In this study，codon usage optimization and co-expression of molecular chaperone are used to improve the extracellular production of alkaline polygalacturonate lyase（PGL） in Pichia pastoris. According to the codon bias of P. pastoris，the gene of the stabilized mutant（K314M） of Bacillus sp. WSHB04-02 PGL is optimized and cloned into the EcoR I-Not I sites of pPIC9K-PGL，yielding the expression plasmid pPIC9K-PGL. pPIC9K-PGL is transformed into the P. pastoris GS115（his-），yielding the strain GS115/PGL 14#. GS115/PGL 14# produced 301.32 U/mL of extracellular PGL which is 25.1% higher than that of the strain with native PGL gene. On this basis，two molecular chaperones the protein folding factors endoplasmic reticulum oxidoreduction 1（ERO1） and ubiquitin-conjugating enzyme（UBC1） are co-expressed with the optimized K314M gene individually and simultaneously. It is showed that co-expression of ERO1 and UBC1 at the same time（GS115/PGLl-ERO1-UBC1 2#） increased the extracellular PGL by 49.3%，which is up to 450.12 U/mL. By using an exponential fed-batch strategy in 3L-fermenter，GS115/PGLl-ERO1- UBC1 2# produces 1 362.31 U/mL after the 96 h induction by methanol. The results here may benefit the industrial production and application of the PGL.