File:T--goettingen--gat engineering.jpg

A. Sequence alignment showing the similarities of the wild type (wt) gat alleles from Bacillus licheniformis and Bacillus subtilis as well as the gat alleles R7 and R11 from B. licheniformis that were obtained by 11 rounds of gene shuffling. The red arrows indicate the 31 amino acid exchanges in the glyphosate N-acetyltransferase (Gat) variant R11. The optimized Gat variants shown an up to a 4,500-fold increase in catalytic efficiency (kcat/Km) relative to the native enzyme. The GenBank accession numbers for the sequences: B. licheniformis ST401 GAT, AX543338; R7 GAT, AY597417; R11 GAT, AY597418; B. subtilis YitI, CAA70664. B. Structure of the Gat R7 variant (PDBid: 2JDD) in ternary complex with acetyl-CoA and the competitive inhibitor 3-phosphoglycerate (3PG). The four active site residues (Arg-21, Arg-73, Arg-111, and His-138), which are labelled in black color, contribute to a positively charged substrate-binding site that is conserved throughout the GAT subfamily. C. Top view on the active site of Gat R7. D. Construction of a plasmid for the expression of the gat R11 variant in B. subtilis. Expression is driven by the constitutively active σA-dependent Palf4 promoter The plasmid pAC7::gat R11 integrates into the amyE locus of the B. subtilis chromosome in single copy. lacZ, beta-galactosidase; amyE-5' and amyE-3', fragments of the B. subtilis amyE for homologous recombination; amp, ampicillin resistance gene for the selection in E. coli; kan, kanamycin resistance gene for the selection in B. subtilis; ori pBR322, origin of replication for E. coli.