Recombinant mycobacterial strains which overproduce essential biosynthetic enzymes of pathogenic mycobateria are provided. These strains overproduce enzymes involved in the synthesis and incorporation of D-alanine into mycobacterial peptidoglycan, the backbone of the mycobacterial cell wall. These overproducing strains may be used as reference strains in in vitro screening methods to identify antimycobacterial agents.