The Impact Of Kynurenine Pathway On Daptomycin Biosynthesis

Daptomycin is an acid lipopeptide which is derived from fermentation by a nonribosomal peptide synthetase mechanism of Streptomyces roseosporus. FDA approved daptomycin for the treatment of complicated skin and skin structure infections caused by Gram-positive pathogens in 2003 and for the treatment of bacteremia and right-sided endocarditis caused by Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in 2006. Its mode of action is destroy bacterial membrane function so that rapidly kills Gram-positive bacteria without cross-resistance with other approved antibiotics. Its potency against Gram-positive pathogens make it an attractive choice for first-line treatment in these types of infections. Studys on daptomycin was focused on two aspects:1. To increase production of daptomycin in S. roseosporus. With the traditional natural isolation, mutation and selection, rational screening and fermentation technology optimization, daptomycin production have been made a great progress. These researchs provide an important foundation for the industrialized production of daptomycin.2. To find better efficacy daptomycin analogue by combinatorial biosynthesis. These studies reveal the important function of amino acid which locate in the peptide core of daptomycin on its antibacterial activity, and shed light on development of new daptomycin analogue.Amino acids, locate in the peptide core of daptomycin, have important functions on the antibacterial activity of daptomycin and the biosynthetic pathway of these amino acids aslo have significant impact on daptomycin biosynthesis in S. roseosporus. As the unique amino acid which is observed exclusively in the peptide core of daptomycin, kynurenine has a crucial role on its mode of action. But little known about the biosynthesis of kynurenine in S. roseosporus. Kynurenine is generated via kynurenine pathway that is a main path of tryptophan metabolism in prokaryotes and eukaryotes. We speculate that S. roseosporus NRRL 11379 (v4) may have the kynurenine pathway and this pathway supply kynurenine for the biosynthesis of daptomycin.Objective:To confirm the biosynthesis pathway of kynurenine and its impact on daptomycin biosynthesis in S. roseosporus NRRL 11379 (v4).Methods:1. We established a genetic transformation system by using Escherichia coli-mediated conjugal transfer and optimized the condition of conjugal transfer.2. According to the S. roseosporus genome, we designed primers for cloning genes of kynurenine pathway and analyzed these genes by bioinformation analysis.3. In order to investigate this pathway’s impact on daptomycin biosynthesis in S. roseosporus NRRL 11379 (v4), key genes in this pathway were disrupted. The tdo deletion mutant (DM), dptJ DM and kyn DM were obtained after disrupting the gene of tdo, dptJ and kyn respectively. At the same time, tdo overexpression mutant (OM) and dptJ OM were gain after tdo or dptJ interted into the attB site of genome. Then, the daptomycin production of wild type (WT), DM, complemention strain (DMC) and OM were measured through bioassay after cultured in MM liquid mediun.Results:1. In this study, we successfully established a genetic transformation system by using Escherichia coli-mediated conjugal transfer. The best spore thermal shock condition is 50℃for 10 min; the best antibiotic cover time is 16-18 h; the best concentration of apramycin is 10μg/ml and nalidixic acid is 25μg/ml.2. tdo, kynf, kyn and dptJ were successfully cloned from S. roseosporus NRRL 11379 (v4). Bioinformation analysis revealed that tdo code for tryptophan 2,3-dioxygenase (TDO), as well as dptJ. kynf code for kynurenine formamidase (KYNF) and kyn code for kynureninase (KYN). These genes are conservation in prokaryotes and eukaryotes. The recombination protein KYN successfully converted kynurenine to anthranilate. Results showed that the kynurenine pathway which convert the tryptophane to anthranilate through TDO, KYNF and KYN indeed exist in Streptomyces roseosporus NRRL 11379 (v4).3. Deletion of tdo or dptJ, the daptomycin production was was dramatically decreased by 55% or 58% in DM compared to wild type. After complemention, strain restored production partly. Daptomycin production was increased 7% and 112% compared to the control after overexpression of tdo and dptJ respectively. The production of kyn DM was 32.75% higer than WT.Conclusion:All of the results demonstrated kynurenine pathway exist in Streptomyces roseosporus NRRL 11379 (v4). This pathway is required for daptomycin production as the provider of kynurenine molecule and our data illustrating that kynureine pathway have an important role on daptomycin biosynthesis. Meanwhile, this study will provide useful information for further understanding the biosynthesis of daptomycin and shed light on strain breeding and strategies to find new daptomycin analogue.