Heterologous Expression Of Trichoderma Reesei Cel5A And Cel6A In Pichia Pastoris

Deficient activity of Cel5 A and Cel6 A secreted by Trichoderma reesei is one of the challenges involved in effective cellulase saccharification of cellulosic substrates. To cope with it in this work, considerable efforts were addressed in efficient expression of the Cel5 A and Cel6 A in Pichia pastoris by constructing a recombinant. With the gene optimization based on codon bias, GC content and mRNA secondary structure, as well as the construction of expression vectors pPIC9K-eg2, pPIC9K-cbh2, pGAPZαA-eg2 and p GAPZαA-cbh2, the optimized gene were electro-transformed into P. pastoris to form transformants. Then, high Cel5 A activity producing recombinants, namely P. pastoris GS115-Cel5 A and P. pastoris X-33-Cel5 A, were selected on resistant plates, followed by shake-flask cultivation. And high Cel6 A activity producing recombinants, namely P. pastoris GS115-Cel6 A and P. pastoris X-33-Cel6 A, were selected in the same way.Analysis of the basic enzyme property showed that the recombinant Cel5 A reacted optimally at pH 4.5 and 60 °C, with 50 kDa of molecular weight, preferentially degrading amorphous cellulose. And the recombinant Cel6 A reacted optimally at p H 5.5 and 60 °C, with 60 kDa of molecular weight, preferentially degrading crystalline cellulose and amorphous cellulose. The analysis indicated that the recombinants Cel5 A and Cel6 A were not significantly different from the native T. reesei Cel5 A and Cel6 A.Moreover, a shake-flask fermentation of the recombinant strains was optimized as below: bottled fluid volume 50 mL, shaking speed 250 r×min^-1, incubation temperature 28 °C, initial pH 5.0, inoculum volume 2%, methanol addition（per 24 h） 1.5%(v×v^-1), sorbitol addition（per 24 h） 4 g×L^-1 and Tween-80 4 g×L^-1. Under above optimized condition, P. pastoris GS115-Cel5 A produced 24.0 U×m L^-1 of the Cel5 A at 192 h fermentation. Under the condition of bottled fluid volume 50 mL, shaking speed 250 r×min^-1, incubation temperature 28 °C, initial pH 6.0, inoculum volume 2%, glycerol addition 30 g×L^-1 and Tween-80 6 g×L^-1, the P. pastoris X-33-Cel5 A produced 14.8 U×mL^-1 of the Cel5 A at 96 h fermentation. Under the condition of bottled fluid volume 50 m L, shaking speed 250 r×min^-1, incubation temperature 26 °C, initial pH 7.0, inoculum volume 3%, methanol addition（per 24 h） 1.5%(v×v^-1), sorbitol addition（per 24 h） 12 g×L^-1 and Tween-80 4 g×L^-1, P. pastoris GS115-Cel6 A produced 0.28 U×m L^-1 of the Cel6 A at 192 h fermentation. Under the condition of bottled fluid volume 50 mL, shaking speed 250 r×min^-1, incubation temperature 28 °C, initial pH 6.0, inoculum volume 3%, glycerol addition 30 g×L^-1 and Tween-80 6 g×L^-1, P. pastoris X-33-Cel6 A produced 0.15 U×m L^-1 of the Cel6 A at 96 h fermentation.Further the recombinant strains were incubated in a 5 L fermentation, the Cel5 A enzyme activity produced by P. pastoris GS115-Cel5 A enhanced high up to 270.9 U×m L^-1 at 180 h, with 4.16 g×L^-1 of the total protein. The Cel5 A enzyme activity produced by P. pastoris X-33-Cel5 A enhanced high up to 129.5 U×m L^-1 at 96 h, with 2.6 g×L^-1 of the total protein. The Cel6 A enzyme activity produced by P. pastoris GS115-Cel6 A enhanced high up to 1.08 U×mL^-1 at 204 h, with 2.01 g×L^-1 of the total protein. The Cel6 A enzyme activity produced by P. pastoris X-33-Cel6 A enhanced high up to 0.38 U×m L^-1 at 96 h, with 1.25 g×L^-1 of the total protein.Genes coding Vitreoscilla hemoglobin（VHb） was inserted in the expression vector pPICZαA and the target proteins（Cel5A and Cel6A） were co-expressed in P. pastoris under the AOX1 promoter, respectively. Then, high Cel5 A and Cel6 A activity producing recombinants, namely P. pastoris GS115-Cel5A+VHb and P. pastoris GS115-Cel6A+VHb, were selected on resistant plates, followed by shake-flask cultivation. And the recombinant strains were at incubation in a 5 L fermentor, the Cel5 A enzyme activity producing by P. pastoris GS115-Cel5A+VHb enhanced high up to 366.8 U×mL^-1 at 180 h, with 4.3 g×L^-1 of the total protein. The Cel6 A enzyme activity produced by P. pastoris GS115-Cel6A+VHb enhanced high up to 1.3 U×m L^-1 at 204 h, with 2.23 g×L^-1 of the total protein. The enzyme activity of co-expression strains increased 35% and 20%, respectively. The study indicates that the recombinant strains P. pastoris GS115-Cel5A+VHb and P. pastoris GS115-Cel6A+VHb are extremely suitable for heterologous expression of T. reesei cellulase Cel5 A and Cel6 A, respectively. And the recombinants Cel5 A and Cel6 A can be used as an alternative to the native T. reesei Cel5 A and Cel6 A in development of a commercially relevant enzyme based biorefinery process.