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Lysobacter is a genus of Gram-negative gliding bacteria that are emerged as novel biocontrol agents and new sources of bioactive natural products. The bacteria are naturally resistant to many antibiotics commonly used in transformant selection, which has hampered the genetic manipulations. Here, we described a facile method for quick-and-easy identification of the target transformants from a large population of the wild type and nontarget transformants. The method is based on a distinct yellow-to-black color change as a visual selection marker for site-specific integration of the gene of interest. Through transposon random mutagenesis, we identified a black-colored strain from the yellow-colored L. enzymogenes . The black strain was resulted from a disruption of hmgA, a gene required for tyrosine/phenylalanine metabolism. The disruption of hmgA led to accumulation of dark brown pigments. As proof of principle, we constructed a series of expression vectors for a regulator gene found within the WAP-8294A biosynthetic gene cluster. The yield of WAP-8294A in the black strains increased by 2 fold compared to the wild type. Interestingly, the yield of another antibiotic (HSAF) increased up to 7 fold in the black strains. WAP-8294A is a family of potent anti-MRSA antibiotics and is currently in clinical studies, and HSAF is an antifungal compound with distinct structural features and a novel mode of action. This work represents the first successful metabolic engineering in Lysobacter. The development of this facile method opens a way toward manipulating antibiotic production in the largely unexplored sources.

acs synthetic biology

Facile Method for Site-specific Gene Integration in Lysobacter enzymogenes for Yield Improvement of the Anti-MRSA Antibiotics WAP-8294A and the Antifungal Antibiotic HSAF