| ?-poly-L-lysine(?-PL), firstly discovered in the culture filtrates of Streptomyces albulus NBRC14147, is a natural homopolymer characterized by isopeptide linkages between ?-carboxyl groups and ?-amino groups of the L-lysine residues with diverse polymerization degrees. The often found polymerization degrees are 25~35. ?-PL containing more than nine residues exhibits remarkable antimicrobial activities against a broad spectrum of microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and virus. ?-PL is stable, soluble and biodegradable, and has been proven to be non-mutagenic and practically non-toxic to human beings. ?-PL is generally regarded as safe(GRAS) as a food preservative, and has been approved in several countries such as Japan and America. ?-PL has also been used as drug vectors, dietary agents, and biomaterials. The utilization of ?-PL is dependent on its polymerization degrees. However, the mechanism controlling the ?-PL polymerization degree has not been fully elucidated.The ?-PL synthesizing enzyme(Pls) is responsible for ?-PL biosynthesis and polymerization degree. It has been shown that the genes in the context of the pls gene such as pldII are not responsible for the production nor have effect on the polymerization degree of ?-PL. Pls is a nonribosomal peptide-synthetase(NRPS) and a membrane protein as well. The sequence structure of Pls has been well characterized, containing an adenylation domain(A domain), a thiolation domain(T domain), three condensation domains(C1, C2 and C3 domains) that all are essential for the biosynthesis of ?-PL, six transmembrane(TM) domains flanking the condensation domains and the C-terminal of the T domain, and three linkers connecting the TM domains. To explore the distribution and sequence features of Pls. We developed a bioinformatics method to predict putative Pls-encoding genes in the completely sequenced genomes, via identification of the substrate-recognition and condensation domains and amino acid residues that determine the substrate specificity. A total of 113 Pls were identified from 110 genomes, mostly distributed in Actinobacteria, with two identified in Gram-negative bacteria.Interestingly, a Streptomyces cattleya strain DSM 46488 was found to carry two pls genes, with one on the chromosome and one on the plasmid, whose products display the closest phylogenetic distances among the predicted Pls proteins. Here we proved that S. cattleya DSM 46488 is a ?-PL producer, consistent with the prediction result. The produced ?-PL contains 14~29 L-lysine residues. To clarify which pls gene controls the ?-PL synthesis in this strain, the two genes were deleted separately. Results showed that the plasmid-borne pls gene controls the ?-PL production and polymerization degree, rather than the one on the chromosome. Complementation was not performed as corresponding mutants could not be obtained, since successful genetic manipulation on S. cattleya DSM 46488 is currently hard to achieve. Our previous study has constructed a pls-deleted non-?-PL-producing mutant derived from a reported ?-Pl producer Streptomyces diastatochromogenes CGMCC3145. Here we introduced the two pls gene into the mutant and the outcome further supported the above conclusion. In addition, heterologous expression of the two pls genes in Streptomyces lividans TK24 was also performed but in vain. The plasmid-borne pls was not found to transcript in this heterologous host.Furthermore, to explore the effect of modules in Pls on polymerization degree, recombinant Pls containing domains from different Pls enzymes that control different polymerization degrees, were constructed. However, no recombinant Pls was found functional so far. In addition, a very recent research has demonstrated that the amino acid substitutions incertain positions could result in shift in polymerization degree. Therefore we systematically compared the Pls sequences, known and predicted, and uncovered the amino acid distribution pattern in each module, which will aid future mechanism study regarding Pls. This study may shed light on the biological engineering strategies that enable ?-PL polymerization with specific degrees. |
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