Yushchuk O. Genetic control of teicoplanin biosynthesis in Actinoplanes teichomyceticus

Some aspects of genetic control of teicoplanin biosynthesis in Actinoplanes teichomyceticus were studied in this thesis. The post-assembly line tailoring processes in teicoplanin biosynthesis were characterized. We have knocked out genes of glycosyltransferases tei3* and tei10*, as well as genes putatively involved in aliphatic side chain attachment: tei13*, tei30* and tei11*. HPLC-MS analysis of teicoplanin derivatives produced by these mutants revealed the putative sequence of teicoplanin aglycon (AGT) decoration reactions. First, glycosyltransferase Tei10* attaches GlcNAc to the fourth amino acid of AGT, giving GlcNAc-AGT. The latter is then deacetylated with Tei2*. This reaction is followed with the attachment of aliphatic side chain involving Tei11* and the attachment of GlcNAc to sixth amino acid of AGT involving Tei1. Finally, Tei3* attaches mannose residue to the seventh amino acid of AGT. Teicoplanin biosynthesis cluster (TBC) includes three genes encoding pathway-specific regulators, particularly tei15*, tei16* (both shown to be crucial for teicoplanin biosynthesis) and tei31* (with uninvestigated functions). Knock-out as well as overexpression of tei31* did not affected the production of teicoplanin. In addition, transcriptional analysis revealed that tei31* was not expressed in teicoplanin-producing culture of A. teichomyceticus. We have also studied transcriptional organization of TBC and found eighteen operons and monogenic cistrons. Expression of the majority of these transcriptional units was revealed to be dependent on Tei15* and Tei16*. Finally, expression of tei15* was found to be absolutely dependent on Tei16*. The role of tei14* and tei24* genes from TBC was investigated in our work. Both genes encode enzymes of tyrosine metabolism and were shown to be important for teicoplanin biosynthesis: overexpression of these genes led to teicoplanin production increase. Finally, we have also studied global regulatory mechanisms of teicoplanin biosynthesis and A. teichomyceticus morphogenesis. In vitro and in vivo studies have shown that closest AdpA-homologues from A. teichomyceticus are neither able to reconstitute bld-phenotype in S. griseus ?adpA nor to bind classical AdpA operator. This data along with in silico analysis of A. teichomyceticus genome allowed us to suggest the absence of AdpA-mediated regulation in A. teichomyceticus. However, overexpression of some adpA homologues in A. teichomyceticus increased the teicoplanin production level. We revealed that overexpression of two global negative regulators, BldDAT and AbsBAT, blocked the development of A. teichomyceticus on the stages of sporangiferous hyphae germination and sporangia development respectively. BldDAT protein was shown to bind its own operator region, as well as operator regions of some other putative morphogens, including whiGAT and sigHAT. BldD-mediated regulation in A. teichomyceticus appeared to share many features with the BldD-mediated regulation studied for streptomycetes.