1. Conclusion Abstract Marine microorganisms are rich source of active compounds for drug discovery. In our previous studies, marine actinomycetes were isolated from the marine sediment samples collected from Dalian sea area, and the potential producers of type I polyketides were identified by gene-based screening. In this study, degenerate primers that target the FADH2-dependent halogenase genes were designed using CODEHOP, and PCR products of about 1 kb were obtained from some marine isolates, which were further confirmed as putative halogenase gene fragments by DNA sequencing. Alignment of the putative halogenase sequence from a newly characterized species Streptomyces xinghaiensis revealed that this fragment shows high identity with the halogenases that modify glycopeptide antibiotics. Bioassay analysis showed that this strain harbors activities against many pathogenic bacteria, including drug-resistant isolates of Pseudomonas aeruginosa and Staphyloccocus aureus MRSA strains. A possible novel compound was identified from this isolate and the structure elucidation is now under investigation. Antibiotic biosynthesis potential of a marine actinomycete Streptomyces xinghaiensis Xinqing Zhao *, Wence Jiao, Tianhong Yang, Shanshan Zhang School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China *Corresponding author, Tel: +86-411-84706308, email: xqzhao@dlut.edu.cn Introduction Acknowledgement Fig.1. Culture characteristics of S. xinghaiensis on Bennett’s agar and its antibacterial activities. Streptomyces xinghaiensis During the studies on the marine actinomycetes isolated in Dalian sea area in China, we identified a new Streptomyces species, for which we named as S. xinghaiensis [1, 2]. The culture characteristics of this species and its antibacterial activities were shown in Fig.1. S. xinghaiensis has strong inhibitory activities against many gram positive and gram negative test strains, including E. coli, S. aureus, B. subtilis as well as P. aeruginosa. It also has the potential of producing lipopeptide antibiotics. Therefore, it is of great interest to further explore its chemical diversity. The availability of large amount of genomic sequences and information on the biosynthetic enzymes and pathways have provided basis for the genome mining of the microbial producers for natural compound discovery [3]. To explore the biosynthetic capability of marine isolates, a gene based screening was established using FADH2-dependent halogenase as probe [4], which has been successful in the gene-based screening of natural compounds using various streptomycetes. And S. xinghaiensis showed positive results in the screening. Results References 1.Design of new halogenase primers and analysis of the PCR products To facilitate further functional analysis, a long pair of halogenase primers was designed using CODEHOP, and touchdown PCR was performed. 2. Identification of a possible novel compound from S. xinghaiensis. Fig. 3 Identification of a novel compound from S. xinghaiensis based on gene-mining. a-c, HPLC analysis of the culture broth (a), empty medium (b), ethyl acetate extract of the culture broth (c) at 330 nm; d, UV spectrum of the novel compound. e, extraction procedure of the compound; f, Fragmentation patterns in the mass spectra of the compound. Fig. 2 Isolation and identifation of a halogenase from S. xinghaiensis. a, Amplification of halogenase by PCR; b, phylogenetic analysis of the sequence of halogenase; c, homology analysis of the identified halogenase with other known halogenases. 1, Zhao XQ, Li WJ, Jiao WC, et al., Streptomyces xinghaiensis sp. nov., from marine sediment. International Journal of System and Evolutionary Microbiology, 2009, 59: 2870-2874. 2, Zhao XQ, Jiao WC, Yuan WJ, et al, Screening and identification of actinobacteria from marine sediments: Investigation of potential producers for antimicrobial agents and type I polyketides. World Journal of Microbiology Biotechnology, 2009, 25: 859- 866. 3, Malek Z, Gregory L, Challis. Strategies for the discovery of new natural products by genome mining. ChemBioChem 2009,10: 625-633. 4, Hornung A, Bertazzo M, Dziarnowski A, et al. A genomic screening approach to the structure-guided identification of drug candidates from natural sources. ChemBioChem, 2007, 8: 757-766. This study was supported by the grant from the open project program of the key laboratory to the State Key Laboratory of Fine Chemicals from the State Key Laboratory of Bioreactor Engineering (SKLBE) in ECUST, Shanghai to XQ Zhao. The authors appreciate the help of Prof. Dr. Hans-Peter Fiedler in University of Tuebingen, Germany for sample analysis and thought-probing discussions. 1.A long PCR primer pair was designed and halogenase fragement of about 1kb was amplified from a new streptomyces species S. xinghaiensis. The sequence of the fragment showed high homology with those involved in glycopeptide biosynthesis. 2. A possible new compound was isolated from the culture extract of S. xinghaiensis, and the structure of this compound is now under investigation. a b Gene-based analysis indicated that S. xinghaiensis has the potential as a glycopeptide producer, therefore, a selective purification procedure was performed to identify its active compound(s). A compound with maximum absorption at 330 nm was identified in the acidified ethyl acetate extract, and the same compound was not detected in the phylogenetically closed relatives of S. xinghaiensis. The structure of this compound is now under investigation. Streptomyces lavendulae (AAK81830.1) Streptomyces chartreusis (ABF82429.1) Streptomyces bingchenggensis (ADI03797.1) Frankia sp. CcI3 (YP 481561.1) Streptomyces sp. L133(2010) (ADC55283.1) Streptomyces sp. CB2312 (ABF82428.1) Streptomyces sp. CB2023 (ABF82427.1) Amycolatopsis orientalis (CAA11780.1) Actinoplanes teichomyceticus (CAG15020.1) Nonomuraea sp. ATCC 39727 (CAD91205.1) Streptomyces toyocaensis StaI (AAM80532.1) Streptomyces toyocaensis StaK (AAM80530.1) Streptomyces globisporus (AAL06656.1) Myxobacterial Chondrochloren Cndh (3E1T) Streptomyces sviceus ATCC 29083 (ZP 05023122.1) Streptomyces antibioticus (AAZ77674.1) 100 48 97 63 60 95 93 96 92 68 93 53 45 0.1 Marine actinomycetes and natural products discovery Gene-based mining of S. xinghaiensis for natural products discovery Atinobacteria from marine environments have aroused the keen interests of researchers due to the species diversity and the special metabolites they produce during recent years. Metabolites with useful properties such as antimicrobial agents, enzyme inhibitors and antitumor compounds have been discovered in certain marine isolates. The novel antibiotics identified in marine actinobacteria provide new hope for combating the emergence of antibiotic-resistant bacteria. c a b c d f e Culture broth Adjust pH to 3.0 Ethyl acetate extraction (1:1) Ethyl acetate phase collection