Development of Efficient Expression Systems for Bacteriolytic Proteases L1 and L5 of XL1.

Secreted bacteriolytic proteases L1 and L5 of the Gram-negative bacterium XL hydrolyze peptide bridges in bacterial peptidoglycans. Such specificity of action determines the prospects of these enzymes for medicine with the view of creating new antimicrobial drugs to combat antibiotic-resistant strains of pathogens. This research concerns the development of successful expression systems for producing active enzymes L1 and L5 in sufficient amounts for comprehensive studies. Based on XL strains with deletions in the (enzyme L1) and (enzyme L5) genes and the constructed expression vectors pBBR1-MCS5 P- and pBBR1-MCS5 P-, we obtained expression strains P- and P-, respectively. The yields of enzymes L1 and L5 in the developed strains increased by 4 and 137 times, respectively, as compared to the wild-type strain. The cultivation of the expression strains was successfully scaled up under non-selective conditions in a 10-L bioreactor. After fermentation, the yields of enzymes L1 and L5 were 35.48 mg/L and 57.11 mg/L, respectively. The developed homologous expression systems of bacteriolytic proteases L1 and L5 have biotechnological value as compared to those obtained by us earlier based on heterologous expression systems, which have lower yields and labor-intensive purification schemes.