Overexpression and structural studies on AA13 and AA10 LPMOs

Lytic polysaccharide monooxygenases (LPMOs) are copper dependent metalloenzymes which oxidatively cleave recalcitrant polysaccharides. With the biofuel industry developing new ways of processing lignocellulosic biomass, LPMOs could play and important role in reducing production cost and increasing saccharification yield. This study reports the expression testing of two ancestral reconstructions, N231 and N237, from the starch degrading AA13 LPMO family and the characterisation of SliLPMO10G from Streptomyces lividans in cellulolytic/chitinolytic AA10 LMPO family. N231 and N237 were both tested using the Tat and Sec signalling pathways and tested for expression in a variety of Escherichia coli cell lines. Results from the test expressions showed bands of expected mass for N231 with the Tat pathway in BL21-RIL and the Sec pathway in both BL21-DE3 and T7 Shuffle. However, no expression was seen for N237. SliLPMO10G has a carbohydrate-binding module (CBM) from CBM5 appended to its LPMO domain via a linker. PCR was used to truncate the SliLPMO10G and the LPMO domain, CBM5 domain and full SliLPMO10G were all expressed and purified using ion exchange chromatography, immobilized metal affinity chromatography and gel filtration. Crystallographic trials were prepared for the CBM5 domain, whilst trials had previously been carried out for the LPMO domain. The structure of the SliLPMO10G LPMO domain was analysed, and results indicated the same β- sandwich fold and Loop 2 region consistent with other AA10 structures. The conserved copper active site was also identified, with copper binding to the amino group Nt and side chain Nδ1 of the N-terminal histidine and the Nε2 of a secondary histidine side chain, in a T-shaped brace.