TY  - THES
T1  - Studies on the biosynthesis and heterologous expression of tubulysin
A1  - Chai,Yi
Y1  - 2011/12/06
N2  - The tubulysins are a family of nine complex peptides with promising cytotoxic activity against multi drug-resistant tumors. This thesis reports a third tubulysin producing strain, Cystobacter sp. SBCb004. Although the tubulysin biosynthetic gene clusters appear to be incomplete in both SBCb004 and An d48, comparison of the clusters reveals a conserved 11-gene architecture, which allowed the assignment of cluster boundaries. Knockout mutagenesis and in vitro assay of genes located at both ends of the conserved cluster revealed their involvement and possible functions in the tubulysin pathway. Pretubulysin was identified from both natural producer strains, confirming its intermediacy in the pathway. Using a combination of feeding studies and high-resolution tandem mass spectrometry, An d48 and SBCb004 were found to biosynthesize 32 additional tubulysin derivatives. Using Red/ET recombineering, the entire tubulysin core gene cluster from strain SBCb004 was reconstituted and successfully expressed in heterologous host strains Pseudomonas putida and Myxococcus xanthus. Finally, BLAST analysis of the SBCb004 genome data was carried out to identify candidate monooxygenases, whose involvements in tubulysin assembly were evaluated using a combination of knockout mutagenesis and heterologous expression. 
KW  - Biosynthese,  Heterologe Genexpression
KW  - Sekundärmetabolit
CY  - Saarbrücken
PB  - Universitäts- und Landesbibliothek
AD  - Postfach 151141, 66041 Saarbrücken
UR  - http://scidok.sulb.uni-saarland.de/volltexte/2011/4519
ER  -