Discovery of a Polyamino Acid Antibiotic Solely Comprising l-β-Lysine by Potential Producer Prioritization-Guided Genome Mining

Abstract

While the genome mining approach has enabled the rational exploration of untapped bioactive natural products, in silico identifications of their biosynthetic genes are often unconnected to the actual production of the corresponding molecules in native strains due to the genetic dormancy. We report here the rational discovery of an unexplored cationic homo polyamino acid (CHPA) antibiotic by potential producer prioritization-guided genome mining. Mining the genome of γ-poly-D-diaminobutyric acid (poly-D-Dab)-producing Streptoalloteichus hindustanus NBRC 15115, which was selected based on the finding that the known CHPAs are universally co-produced in pairs, identified a putative CHPA synthetase, PblA, as a potential candidate being expressed actively. Bioinformatic and biochemical analyses of PblA provided the critical clue that its polymer product could be an unusual CHPA consisting of L-β-lysine. Instrumental analyses of the metabolites from S. hindastanus indeed revealed the production of an unprecedented linear CHPA, ε-poly-L-β-lysine, concomitantly with poly-D-Dab. The CHPA we discovered exerted excellent antimicrobial activity against a broad spectrum of microorganisms, including bacteria and fungi, and was revealed to show resistance against nonspecific proteolytic enzymes. This study marks the first report of the efficacy of the strain prioritization-guided genome mining strategy for the discovery of bioactive CHPAs.

Structural and functional insights into δ-poly-L-ornithine polymer biosynthesis from Acinetobacter baumannii

“…A second CHPA protein from bacteria Methylomarinum sp contains Stachelhaus code residues “D G E S I S V V N K” that showed the presence of Gly at Met218 position, which might activate a larger substrate. A recently characterized εPLs homolog from Streptoalloteichus hindustanus NBRC 15115 with Stachelhaus code residues as “D G E S I S V V N K” which matches that from Methylomarinum sp showed biosynthesis of an unusual polymer ε-Poly β-lysine synthetase 10 . Codon-optimized synthesized DNA for adenylation domains of the two genes were cloned and expressed in E. coli BL21 (DE3) strain.…”

“…Building on these early studies with εPLs, homologous enzymes that produce polymers of 2,3-diaminopropionate (DAP) 8 and both D- and L-isomers of 2,4-diaminobutyrate (DAB) 9 have been reported. A recent addition to the cationic homopolymeric amino acids (CHPA) family aided by the discovery of a gene encoding a lysine-2,3-aminomutase adjacent to a εPLs homolog has further extended the family of natural CPHAs to include ε-poly-β-lysine 10 . Finally, an unusual branched polymer, poly[β-(L-diaminopropionyl-L-diaminopropionic acid)] has also been identified 11 .…”

First direct evidence for direct cell-membrane penetrations of polycationic homopoly(amino acid)s produced by bacteria

“…1a ). To date, six polymers, ε-poly- l -α-lysine (ε-PαL, 1 ) 1 , 2 , ε-oligo- l -β-lysine (ε-OβL, 2 ) 3 , γ-poly- l / d -diaminobutyric acid 4 , 5 , β-poly- l -diaminopropionic acid 6 , ε-poly- l -β-lysine (our recent finding) 7 , and γ-poly- l / d -glutamic acid 1 , 8 have been identified. In addition to these linear polymers characterized by the isopeptide backbones, two branched polymers consisting of repeated dipeptide chains have also been found in bacteria: multi- l -arginyl-poly- l -aspartic acid (also referred to as cyanophycin) 9 , 10 and multi- l -diaminopropionyl-poly- l -diaminopropionic acid 11 .…”

“…Given the proof of these cell-penetrating activities, we propose the name polycationic isopeptides entering cells (PIECEs) for 1 and 2 . Although they are the first examples of bacterial PIECEs, further investigations of the cell-penetrating activities in bacterial polycationic isopeptides, such as γ-poly- l / d -diaminobutyric acid 4 , 5 , β-poly- l -diaminopropionic acid 6 , ε-poly- l -β-lysine 7 , cyanophycin 9 , 10 , and multi- l -diaminopropionyl-poly- l -diaminopropionic acid (Supplementary Fig. 1a ) 11 , would expand the potential applications of the CPP-driven technology.…”