|Title||A comparative summary of expression systems for the recombinant production of galactose oxidase|
|Publication Type||Journal Article|
|Year of Publication||2010|
|Authors||Spadiut, O, Olsson, L, Brumer, H|
|Journal||MICROBIAL CELL FACTORIES|
|Date Published||SEP 13|
|Type of Article||Article|
Background: The microbes Escherichia coli and Pichia pastoris are convenient prokaryotic and eukaryotic hosts, respectively, for the recombinant production of proteins at laboratory scales. A comparative study was performed to evaluate a range of constructs and process parameters for the heterologous intra-and extracellular expression of genes encoding the industrially relevant enzyme galactose 6-oxidase (EC 188.8.131.52) from the fungus Fusarium graminearum. In particular, the wild-type galox gene from F. graminearum, an optimized variant for E. coli and a codon-optimized gene for P. pastoris were expressed without the native pro-sequence Results: The intracellular expression of a codon-optimized gene with an N-terminal His(10)-tag in E. coli, using the pET16b(+) vector and BL21DE3 cells, resulted in a volumetric productivity of 180 U.L(-1).h(-1). The intracellular expression of the wild-type gene from F. graminearum, using the pPIC3.5 vector and the P. pastoris strain GS115, was poor, resulting in a volumetric productivity of 120 U.L(-1).h(-1). Furthermore, this system did not tolerate an N-terminal His(10)-tag, thus rendering isolation of the enzyme from the complicated mixture difficult. The highest volumetric productivity (610 U.L(-1).h(-1)) was achieved when the wild-type gene from F. graminearum was expressed extracellularly in the P. pastoris strain SMD1168H using the pPICZ alpha-system. A C-terminal His(6)-tag did not significantly affect the production of the enzyme, thus enabling simple purification by immobilized metal ion affinity chromatography. Notably, codon-optimisation of the galox gene for expression in P. pastoris did not result in a higher product yield (g protein.L(-1) culture). Effective activation of the enzyme to generate the active-site radical copper complex could be equally well achieved by addition of CuSO(4) directly in the culture medium or post-harvest. Conclusions: The results indicate that intracellular production in E. coli and extracellular production in P. pastoris comprise a complementary pair of systems for the production of GalOx. The prokaryotic host is favored for high-throughput screening, for example in the development of improved enzymes, while the yeast system is ideal for production scale-up for enzyme applications.