Identification And Biosynthesis Of Phenazine Compounds From Lysobacter Antibioticus

The genus Lysobacter was established in 1978 by Christensen and Cook. Lysobacter belongs to the Xanthomonadaceae family which is within the Gamma-proteobacteria. They are ubiquitous bacteria, existing in such diverse habitats as marine, tar pits, compost sludge and volcanic ash, as well as soil and aquatic environments. Lysobacter species display a number of unique features that distinguish them from other related microbes, such as high G+C content (65 to 72%), gliding motility but without flagella, and prolific production of extracellular lytic enzymes and bioactive secondary metabolites. These Gram-negative gliding bacteria have attracted increased attention because of their proficiency in producing bioactive secondary metabolites. L.antibioticus OH 13 was initially isolated from the rhizosphere of rice and exhibited potent biocontrol activity against bacterial and fungal pathogens of plants.We isolated six compounds from L. antibioticus strain OH13 using sephadex chromatography column and HPLC. The six compounds were identified as (1)6-methoxy-1 -phenazinol 5,10-dioxide, (2) 6-methoxy-1 -phenazinol-10-oxide. (3)1 -phenazinol-5,10-dioxide, (4) 1,6-dimethoxy-phenazinol-5-oxide, (5)1,6-dimethoxyphenazine, (6) 1 -hydroxy-6-methoxyphenazine by NMR and MS.Compounds 1-4 are N-oxide phenazines, and compound 1, which was previously reported as myxin, shows effective antibiotic activities against a variety of microorganisms.Compound 3 is known as iodinin which was first isolated from Brevibacterium iodinum.Iodinin exhibits potent antibiotic activity against Gram-positive bacteria, several actinomycetes, and some fungi. To our knowledge, compounds 4 and 5 have not been reported as natural products, although they had been chemically synthesized.Phenazine natural products have been extensively studied because of their antibacterial,antifungal, antiviral and antitumor activities, especially phenazine-1-carboxylic acid (PCA)and phenazine-1, 6-dicarboxylic acid (PDC), which are derived from the shikimic acid pathway. A conserved seven-gene cluster, phzABCDEFG, was shown to be responsible for the biosynthesis of PCA or PDC in microorganisms. PCA and PDC are considered as precursors for more complex phenazine metabolites. However, little is known about the biosynthesis of phenazine N-oxides, such as myxin (1) and iodinin (3).Besides the phenazine N-oxides, there are only a handful of natural products that contain aromatic N-oxide functional groups, but often with potent biological activities.They are an interesting class of antitumor agents exhibiting bioreductively activated,hypoxia-selective DNA-damaging properties.However, the mechanism for N-oxidation of these N-oxides is unclear. Here, we study on mechanism of N-oxidation of phenazine N-oxides from L.antibioticus OH13.Subsequently we sequenced the OH 13 genome and identified a 10-gene cluster (LaPhz).The LaPhz cluster contains six putative core genes, LaPhzB-G, and four putative modification genes, LaPhzN01, LaPhzS, LaPhzX, and LaPhzM. To verify the LaPhz gene cluster responsible for biosynthesis of phenazines in L.antibioticus OH13, we first mutated three core genes, LaPhzC, LaPhzD and LaPhzB. HPLC analysis showed that phenazines were abolished from mutants LaPhzC and LaPhzB, and mutant LaPhzD produced a trace amount of compound 2. The antibacterial activity of the mutants was consistent with the HPLC results. LaPhzC and LaPhzB mutants lost the antibacterial activity and mutant LaPhzD exhibited trace activity. These results demonstrated that the core genes are required for phenazine biosynthesis in OH13.LaPhzNO1 in the cluster was homologous to genes encoding cyclohexanone monooxygenases, flavoproteins catalyzing the Baeyer-Villiger type oxidation. Mutation of LaPhzNO1 abolished all N-oxides, while two non-oxide phenazines were markedly increased. We then expressed LaPhzNO1 in E. coli and demosntrated the enzyme as an NADPH-dependent, flavin N-monooxygenase. Together with LaPhzS, another flavin monooxygenase, LaPhzNO1 converted phenazine 1,6-dicarboxylic acid to 1,6-dihydroxy phenazine N5,N10-dioxide (iodinin, 3). Moreover, LaPhzNO1 could catalyze nitrogen oxidation of 8-hydroxyquinoline besides phenazines, which indicated a potential use of this enzyme in chemoenzymatic synthesis of aromatic N-oxides. LaPhzNO1 represents the first experimentally characterized N-monooxygenase in the biosynthesis of heterocyclic aromatic natural products.