Related Experiment Videos
Growth-phase dependent expression of the mevalonate pathway in a terpenoid antibiotic-producing Streptomyces strain
Yoshimitsu Hamano1, Tohru Dairi, Masahiro Yamamoto
1Biotechnology Research Center, Toyama Prefectural University, Japan.
Abstract:
Streptomyces griseolosporeus MF730-N6, a terpenoid antibiotic-terpentecin (Tp) producer, has both the nonmevalonate and mevalonate pathways for the formation of IPP. The Tp biosynthetic gene (ter) and the mevalonate pathway gene cluster (mev) including an HMG-CoA reductase gene (hmgr) had previously been cloned from strain MF730-N6. In this study, two distinct dxs genes (dxs 1 and dxs 2) and a dxr gene, which encode DXP synthases and DXP reductoisomerase, and participate in the initial and the second step of the nonmevalonate pathway, respectively, were cloned. These gene products were expressed in E. coli and confirmed to have the expected activities. The dxs 1, dxs 2, dxr, mev, and ter genes were used for Northern blot and primer extension analyses to examine temporal expression of these genes together with a gap gene coding for GAP dehydrogenase, which was also cloned in this study and used as an internal control. Transcripts of the dxs 1, dxs 2, dxr, and gap genes were detected throughout the cultivation. On the other hand, messages of the mev and ter genes were not detected at early growth phase but appeared when Tp production started. These results suggested that the nonmevalonate pathway and the mevalonate pathway were mainly used for the primary metabolism and the secondary metabolism, respectively, and that both of the two dxs genes were actually transcribed in this strain.
Insights
Streptomyces griseolosporeus uses two pathways for IPP production. The nonmevalonate pathway supports primary metabolism, while the mevalonate pathway is crucial for terpentecin antibiotic production during secondary metabolism.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Streptomyces griseolosporeus MF730-N6 produces the terpenoid antibiotic terpentecin (Tp).
- This strain possesses both nonmevalonate and mevalonate pathways for isopentenyl pyrophosphate (IPP) synthesis.
- Previous work cloned the Tp biosynthetic gene (ter) and the mevalonate pathway gene cluster (mev).
Purpose of the Study:
- To clone and characterize genes involved in the nonmevalonate pathway (dxs 1, dxs 2, dxr) in S. griseolosporeus.
- To investigate the temporal expression of nonmevalonate and mevalonate pathway genes in relation to Tp production.
- To elucidate the roles of these pathways in primary and secondary metabolism.
Main Methods:
- Cloning of dxs 1, dxs 2, dxr, and gap genes.
- Expression of cloned genes in E. coli to confirm enzymatic activity.
- Northern blot and primer extension analyses to determine gene expression patterns.
- Quantitative analysis of gene transcripts throughout cultivation.
Main Results:
- Two distinct dxs genes (dxs 1, dxs 2) and a dxr gene from the nonmevalonate pathway were successfully cloned and their activities confirmed.
- Transcripts for dxs 1, dxs 2, dxr, and the housekeeping gap gene were detected throughout the cultivation period.
- Mevalonate (mev) and terpentecin (ter) pathway genes were not expressed during the early growth phase but were upregulated upon initiation of Tp production.
Conclusions:
- The nonmevalonate pathway is primarily involved in primary metabolism, while the mevalonate pathway is predominantly utilized for secondary metabolism (Tp production).
- Both dxs genes are actively transcribed in Streptomyces griseolosporeus.
- This study provides insights into the coordinated regulation of primary and secondary metabolic pathways in antibiotic production.