Screening And Identification Of Marine Microbial Type Ⅰ PKS Gene Resources

The biosphere is dominated by microorganisms, which produce numerous secondary metabolites with various biological activities. The efforts to discover new bioactive molecules from microbes have lasted one century. However, the number of novel compounds discovered in recent years has not increased in proportion to the progress in culture-based screening methods, because only a small fraction of all microbes can be cultured by traditional methods. The knowledge about the"underexplored majority"is still poor.To avoid culture limit, modern biological technology approaches have provide direct access to explore genes or gene clusters that are responsible for the synthesis of microbial secondary metabolites. Polyketide synthase (PKS) gene clusters are suitable to be the screening target. PKSs synthesize polyketides, a large family of secondary metabolites that include many clinically important drugs such as erythromycin (antibacterial), epothilone (antitumor), soraphen (antifungal), rapamycin (immunosuppressant) and lovastatin (anti-hypercholesterolemic).In this study, a set of degenerate oligonucleotide primers, designed for amplification KSs domains, had been employed to identify KS gene fragments from soil and seawater DNA samples. Our purpose was to develop a culture-independent method to directly access PKSⅠgene diversity in Chinese soil and seawater, because the related knowledge was incomplete. This would open up the possibilities of using the results for DNA fingerprints of secondary metabolites and as the basis for a search for attractive antibiotic biosynthesis genes that could be used in the heterogenous expression and combinatorial biosynthesis. Furthermore, the amplified KS gene fragments can also be used as homologous hybridization probes to detect the clones harbored PKS gene clusters in the recombinant metagenomic libraries that would be constructed in the following researches.Part 1. DNA extractionsDNA extractions were carried out by using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc.). Compared with an adaptation of the procedure described by Zhou et al, the MO BIO kit produced DNA with a higher level of purity that can be used for the following PCR directly.DNA extractions of the soil and seawater samples were performed following the manufacturers'instructions. The DNA yield were calculated from the A260. The DNA were loaded on a 1.0% agarose gels and ethidium bromide staining to determine size and concentration. All extracted DNA were stored at -20℃until use.Part 2. Design of the degenerate PCR primersWith the help of The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) and manually correction, a set of degenerate PCR primers were designed from conserved regions of KS domains of bacterial PKSⅠgenes.The forward primers KSF (5'-CGC TCC ATG GAY CCS CAR CA-3') were based on the conserved motif SDPQQR. The reverse primers KSR (5'-GTC CCG GTG CCR TGS SHY TCS A -3') were based on the conserved motif HGTGT.The specificity of the primer set had been confirmed by testing with a collection of polyketide-producing strain (Streptomyces rimosus 8229, Streptomyces coelicolor ATCC101478, Streptomyces avermitlis, ATCC 31271) prior to the following PCR reactions with the environmental samples. The specific fragments amplified with KSF-KSR were about 700 bp in length.Part 3. Isolation of 23 ketosynthase fragments from soil and sea water In this study, the degenerate PCR primers were employed to amplified the soil and seawater DNA samples.The 25μl PCR mixture consisting of 2.5μl 10×Ex Taq Buffer (Mg2+ Plus), 2μl dNTP Mixture( 2.5 mmol/L each), 0.125μl TaKaRa Ex Taq (5U/μl), 1.5μl DMSO(dimethyl sulfoxide), 0.25μl each primer (20μmol/L), 1μl template DNA (0.03 g/L), and 17.375μl ddH2O.The initial denaturation step at 94℃for 5 minutes was followed by 35 cycles of DNA denaturation at 94℃for 1 minute, primer annealing at 65℃for 1 minute, and DNA strand extension at 72℃for 1 minute, and a final extension step at 72℃for 10 minutes.The result PCR products about 700 bp were purified on agarose gels (mini-DNA rapid purification kit, BioDev-Tech) and then cloned into PMD18-T Vector according to the manufacturer's instructions (TaKaRa). positive recombinants were then submitted for sequencing using an ABI3730 DNA Sequencer (USA) with M13 primer at the Invitrogen Biotechnology Company.DNA sequences obtained from the PCR product had been translated into amino acid sequences using the Primer Premier 5 software after cutting off the vector and primer regions. Sequence analyses were performed using the BLASTP programs provided by the National Center for Biotechnology Information (NCBI).After excluding the identical clones, 23 unique nucleotide fragments (ranged from 630 bp to 690 bp after cutting off the primer and vector sequences) were obtained. Among them 19 clones were amplified from soil (DQ640993, DQ640997, DQ641926, DQ641927, and DQ673137～DQ673151) and 4 from the seawater (DQ673151 and EF554859～EF554861).The GC content of the resulting 23 KS gene fragments ranged from 49% to 73% with an average value 66.7%. The alignment of the predicted protein sequences revealed that all the nucleotide sequences encoded the highly conserved region corresponding to the active site of the beta-ketoacyl synthetase consensus region of PKS I genes, these KS gene fragments showed 45% to 85% identities to the known PKS I amino acid sequences in the GenBank database.Among the 23 KS sequences obtained in present study, 9 sequences displayed a unique pattern N(DE)KD, 22 amino acids upstream from the cysteine active site in the KS domain and the conserved pattern VDTACSSS was replaced by VQTACSTS.These two patterns were shown to identify KS domains belonging to hybrid NRPS/PKS systems, which require specialized KS domains capable of using the peptidyl substrate of the NRPS donor site. On the other hand, the remained 15 KS sequences (the 4 KS sequences derived from the sea water included) showed typical conserved patterns of KS domains. No KSQ like fragment was found from all the 23 KS fragments.Part 4. Isolation and functional analysis of the PKS genes in the marine bacteria X-2 which produce MacrolactinsMacrolactins are 24-membered macrolides produced by unidentified marine bacterium, Actinomadura sp. and Bacillus sp., which exhibit both antibacterial and antitumor activities in vitro. In addition, macrolactins were shown to inhibit mammalian herpes simplex virus and HIV replication. Although cloning of the macrolactins biosynthetic genes has not been reported, its chemical structure suggests that it is assembled by a modular PKS system.The environmental strain X-2 which was isolated from the sediment of the East China Sea producing Macrolatin A, B and O. KS domains cloned from the X-2 showed 98% identities to the known pks2 gene cluster in environmental strain Bacillus amyloliquefaciens FZB42. The result showed that the pks2 gene cluster may be play a important role in the biosynthesis of Macrolatins.Data presented in this study showed that the PCR method using degenerate primer to isolate the secondary metabolites biosynthesis gene fragments from the environmental samples and strains were practically effective. This study will provide the foundation for the biosynthesis and heterogeneous expression of polyketides and contribute to the exploitation of microbial genetic resources belong to the"underexplored majority".