氨基香豆素抗生素：生物合成和生物技术
The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are produced in different Streptomyces strains and are potent inhibitors of DNA gyrase. Cloning and sequencing of the corresponding biosynthetic gene clusters allowed detailed investigations of their biosynthetic pathways as well as the generation of novel antibiotics by metabolic engineering, hemo-enzymatic synthesis and precursor-directed biosynthesis. On the other hand, only limited knowledge is available about the regulation of the biosynthesis of the aminocoumarin antibiotics.
The biosynthetic gene cluster of novobiocin, clorobiocin and coumermycin A1 each contains two putative regulatory genes with high similarity in between the clusters, i.e. novG/cloG/couG and novE/cloE/couE. The function of NovG as a DNA binding
protein and positive regulator of novobiocin biosynthesis has been established
previously. In the first part of this thesis, functional proof for the role of novE as a positive regulator of novobiocin biosynthesis is provided. Overexpression of novE, using a replicative shuttle vector in S. coelicolor strains carrying the intact novobiocin cluster has been shown to lead to almost two-fold overproduction of novobiocin, suggesting that novobiocin production is limited by the availability of NovE protein. In contrast, a novE-defective mutant, generated by an in-frame deletion in this study, produced only 0.7 % of the novobiocin amount formed by an S. coelicolor strain harboring the intact novobiocin cluster. Novobiocin production in this ΔnovE mutant could be restored by introduction of an intact copy of novE, but also by overexpression of the regulatory gene novG.
NovE was expressed in E. coli and purified. However, in contrast to NovG, no DNA binding properties could be shown for NovE. The following RT-PCR experiments showed that at least some novG transcription can occur in the absence of NovE, and that novE transcription can occur in the absence of NovG. Correspondingly, overexpression of novG under control of its own promoter stimulated novobiocin production even in a novE-defective mutant.
Another part of this thesis focuses on the determination of promoter regions within the novobiocin biosynthetic gene cluster. For this purpose Ω (omega) interposons, i.e. DNA fragments containing an antibiotic resistance marker flanked by short inverted repeats containing termination signals for transcription, were introduced into genes downstream of putative promoter regions, i.e. into novE, novF, novG, novH, novO, novP, novQ and novS, resulting in termination of mRNA synthesis at the place of insertion. Transcription is re-initiated at the next active promoter-sequence downstream of the Ω insertion. RT-PCR analysis of the generated mutants showed that the novobiocin biosynthetic gene cluster contains, in addition to previously identified promoter regions upstream of novE and gyrBR, six further promoter regions situated upstream of novF, novG, novH, novO, novP and novQ.
In order to confirm the importance of the promoter regions identified upstream of novO, novP and novQ, quantitative RT-PCR experiments were carried out to quantify transcription of novH, novP and novQ in the ΩnovH mutant in comparison to S.coelicolor strains containing the intact novobiocin cluster. The results of these investigations clearly showed that the Ω insertion into novH resulted not only in an almost complete loss of novH transcription (< 1 %), but additionally in a very strong reduction of transcription of novP and novQ (<3 %). This finding strongly suggests that transcription of novO, novP and novQ (and of the genes located downstream thereof) is mainly controlled by the novH promoter initiating a large transcript of at least 18 kb, i.e. from novH to novW. Furthermore, quantitative RT-PCR was used for investigations of the interplay of the two positive regulators novE and novG, as well as on their influence on the novobiocin biosynthetic genes. These investigations showed that both novE and novG act as transcriptional activators of the genes of novobiocin biosynthesis by initiating transcription from the novH-promoter. novE and novG act in a cascade-like reaction mechanism, i.e. novE positively regulates transcription of novG and NovG regulates transcription of all genes from the novH promoter by binding to a well-defined inverted repeat sequence in the intergenic region between novG and novH.
Based on the results presented above, the final part of this thesis deals with the uncoupling of novobiocin production from its natural regulation cascade by replacing the entire novEFG-region, including the promoter region upstream of novH, by a strong inducible promoter. For this purpose, the tetracycline-controllable promoter 830 (tcp830) was used. It has been shown previously, by using luxAB genes expressing luciferase as a reporter system, that induction factors of up to 270 could be obtained for tcp830 by induction with anhydrotetracycline. HPLC analysis of novobiocin production in the resulting mutants showed that an induced tcp830 promoter is sufficient to cause transcription of the genes from novH to novW, i.e. of a polycistronic mRNA of >18.000 nt, resulting in a two-fold overproduction of novobiocin in comparison to strains containing the unmodified novobiocin gene cluster. Therefore, regulation of novobiocin production by tcp830 has been confirmed as a strategy to uncouple novobiocin production from its natural regulation and notably as a further, newly discovered tool to enhance antibiotic production.
English
