Construction And Application Of Ordered Genomic Library Of Actinomycetes And Studies On Genetic Elements

The actinomycetes, filamentous bacteria belong to high G+C content gram-positive group, have a complex morphological development and differentiation life cycle with unique genetics characters. And produce more than 8700 kinds of antibiotics and physiological activity chemical. So actinomycetes were taken as important materials in fundermental and application research on microorganism. In this thesis, investigation on functional genome, antibiotic biosynthesis and DNA replication were carried out on Streptomyces avermililis and Actinomadura yumaensis (with high commercial values), Streptomyces coelicolor (the model actinomycetes), Streptomyces. sp.44414 and Nocardia sp.107. The results are following:1 The construction of an ordered genomic library of S. avermitilis and obtainment the genetic-engineering strain to produce Doramectin. A genomic library of S. avermitilis was constructed with the cosmid vector SuperCosl. After sequenced the. both ends of 1000 cosmid clones, an ordered cosmids library covered more than 91% chromosome of S. avermitilis was obtained on the basis of its genome sequence. Base on the cosmids library, a genetic manipulation system were established through theλRED mediated recombination and Escherichia coli-Streptomyces intergeneric conjugative transfer technique. Efficient and precised gene or gene cluster knockout and sequential gene knockout can be carried on in S. avermitilis with this system. An unprecedented genetic modified strain can produced Doramectin was obtained through secondary metabolism engineering and metabolic flux improvement with this system. In addition, a previous overlooked gene between aveD and aveF genes was discovered, aveC might involve in the biosynthesis of Avermectin.2 Genetic modification of industrial strains of Avermectin-and Doramectin-producers by using positive regulatory genes. Here we reported that several known regulatory genes, afsR, aveR, orfX, afsB, cprB and metK, were cloned into an integrative vector and introduced by conjugal transfer from E. coli to the Streptomyces avermitilis industrial stains that produced Avermectins or Doramectin. The Avermectin Bla yield in strain MMR630 increased about one fold after introduced individually the regulatory genes aveR, afsR or orfX. But strain G11 with the introduced individual 6 genes, including the former three genes, decreased the yield of Doramectin except the afsB gene increased 13%. Expression of afsB gene under the constitutive promoter permE~* decreased the productivity of Doramectin in strain G11. These results indicate that the regulatory genes have differential effects on the antibiotic production of different industrial strains, and suggest that regulatory of antibiotic biosynthesis indeed involves in a complicated network in Streptomyces.3. Establishment large chromosome fragment deletion on the basis of genomic library constructed with novel cosmid vector. Cosmid vector pHAQ31 was constructed to carry out large DNA fragment deletion research. Ordered cosmids library of S. coelicolor and S. avermtilis constructed with the conjugative cosmids vector pHAQ31 covered each genome 73% and 62% respectively. Both libraries can carry out large fragment deletion besides theλRED mediated gene PCR-targeting system. For example, 30kb fragment no-marker deletion efficiency is more than 10% with this system in S. avermitilis. Large chromosome fragment longer than 80kb deletion were also test in S. coelicolor. Nine vectors were constructed to delete 9 ploketide biosynthetic gene clusters except that of Avermectin used both librarys of S. avermitilis constructed with SuperCos1 and pHAQ31. Now complete the deletion of seven PKS clusters.4 Cloned DNA fragments related with the biosynthetic gene cluster of Maduramicin. A cosmids genomic library of A. yumaensis NRRL12515 constructed with cosmid vector pHZ1358. Nine positive clones were obtained through screened the library by in situ colony hybridization with the endogenious (3-keto-synthase gene fragment probe,which was amplified with designed degenerate primers on conserved motif in this strain. After both ends seuqnecing, the ends of four clone showed high homologous with PKS genes. It canbe concluded that these 4 clones probably related with the biosynthesis of Maduramicin.5 Replication of a temperature-sensitive linear plasmid pRL4. Streptomyces sp. 44414 harbored three linear plasmids, and one of them was lost during culturing at 40℃(temperature-sensitive). By partially digested plasmid DNA, cloned into an E. coli plasmid pQC156 and introduced by transformation into Streptomyces lividans ZX7, a replication origin of pRL4, consisting of a linear plasmid SAP1-like gene and a non-coding sequence, was identified. As expected, the SAP1 gene and adjacent non-coding sequence also contained activity of replication.6 Studies on the Nocardia circular plasmid pXT107. We report here a new identified plasmid pXT107 of Nocardia sp.107, one of the smallest circular plasmid found in Nocardia. The complete nucleotide sequence of pXT107 consisted of 4335-bp with 65% G+C content, encoding one Rep (replication protein) and six proteins of unknown functions. The Rep, double strand origin (dso) and single strand origin (sso) of pXT107 resembled these of typical rolling-circle-replication plasmids, such as pNI100 of Nocardia, pRE8424 of Rhodococcus and pIJ101 of Streptomyces. An E. coli-Nocardia shutter plasmid pHAQ22, containing the rep gene, propagated in Nocardia but not in Streptomyces.7 Cloning and functional studies of the chromosomal replication origin(oriC) of Actinomadura yumaensis. A 1.3kb fragment amplified in A. yumaensis with degenerate primers designed on the conserved motifs of dnaA and dnaN. Result showed that the oriC was located between the conserved genes dnaA and dnaN of chromosome and consisted of 919 bp. Unlike previous published actinomycetes oriC, the oriC of Actinomadura yumaensis contained 14 DnaA-boxes with conserved 9-bp sequence (T/C)(T/C)GTCC(A/C)CA and 2 AT-rich sequences. An E. coli plasmid carrying the oriC fragment was able to propagate in Streptomyces coelicolor in a low copy number. Phylogenetic trees based on the oriC sequences of four genera actinomycetes and of 16S rDNA were similar, supporting the previous hypothesis.