Biology subjects

Seipke, R. F.

Publications and source records attributed to Seipke, R. F..

4 recordsLinked to original sources

bioRxiv2019

A chromatogram-simplified Streptomyces albus host for heterologous production of natural products

Cloning natural product biosynthetic gene clusters from cultured or uncultured sources and their subsequent expression by genetically tractable heterologous hosts is an essential strategy for the elucidation and characterisation of novel microbial natural products. The availability of suitable expression hosts is a critical aspect of this workflow. In this work, we mutagenised five endogenous biosynthetic gene clusters from Streptomyces albus S4, which reduced the complexity of chemical extracts generated from the strain and eliminated antifungal and antibacterial bioactivity. We showed that the resulting quintuple mutant can express foreign BGCs by heterologously producing actinorhodin, cinnamycin and prunustatin. We envisage that our strain will be a useful addition to the growing suite of heterologous expression hosts available for exploring microbial secondary metabolism.

microbiology↗

bioRxiv2019

Direct proteolytic control of an extracytoplasmic function RNA polymerase sigma factor

The survival of any microbe relies upon its ability to respond to environmental change. Use of Extra Cytoplasmic Function (ECF) RNA polymerase sigma ({sigma}) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF {sigma} factors; nearly all of which regulate genes required for morphological differentiation and/or response to environmental stress, except for {sigma}AntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF {sigma} factor, whose activity is regulated by a cognate anti-{sigma} factor, {sigma}AntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstitute in vitro ClpXP proteolysis of {sigma}AntA, but not a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of {sigma}AntA in vivo is enhanced by removal of the ClpXP recognition sequence, and that levels of the protein rise when cellular ClpXP protease activity is abolished. These data establish direct proteolysis as an alternative and thus far unique control strategy for an ECF RNA polymerase {sigma} factor and expands the paradigmatic understanding of microbial signal transduction regulation. ImportanceNatural products produced by Streptomyces species underpin many industrially- and medically-important compounds. However, the majority of the ~30 biosynthetic pathways harboured by an average species are not expressed in the laboratory. This undiscovered biochemical diversity is believed to comprise an untapped resource for natural products drug discovery. A major roadblock preventing the exploitation of unexpressed biosynthetic pathways is a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of {sigma}AntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

microbiology↗

bioRxiv2017

A phylogenetic and evolutionary analysis of antimycin biosynthesis

Streptomyces species and other Actinobacteria are ubiquitous in diverse environments worldwide and are the source of, or inspiration for, the majority of pharmaceuticals. The genomic era has enhanced biosynthetic understanding of these valuable chemical entities and has also provided a window into the diversity and distribution of natural product biosynthetic gene clusters. Antimycin is an inhibitor of mitochondrial cytochrome c reductase and more recently was shown to inhibit Bcl-2/Bcl-XLsrelated anti-apoptotic proteins commonly overproduced by cancerous cells. Here we identify 65 putative antimycin biosynthetic gene clusters (BGCs) in publicly available genome sequences of Actinobacteria and classify them based on the presence or absence of cluster-situated genes antP and antQ, which encode a kynureninase and phosphopantetheinyl transferase (PPTase), respectively. The majority of BGCs possess either both antP and antQ (L-form) or neither (S-form), while a minority of them lack either antP or antQ (IQ or IPform, respectively). We also evaluate the biogeographical distribution and phylogenetic relationships of antimycin producers and BGCs. We show that antimycin BGCs occur on five of the seven continents and are frequently isolated from plants and other higher organisms. We also provide evidence for two distinct phylogenetic clades of antimycin producers and gene clusters, which delineate S-form from L- and I-form BGCs. Finally, our findings suggest that the ancestral antimycin producer harboured an L-form gene cluster which was primarily propagated by vertical transmission and subsequently diversified into S-, IQ and IPform biosynthetic pathways.

microbiology↗

bioRxiv2016

MtrA is an essential regulator that coordinates antibiotic production and development in Streptomyces species

Streptomyces bacteria make numerous secondary metabolites, including half of all known antibiotics. Understanding the global regulation of secondary metabolism is important because most Streptomyces natural products are not made under laboratory conditions and unlocking cryptic biosynthetic gene clusters (BGCs) is a major focus for natural product discovery. Production is coordinated with sporulation but the regulators that coordinate development with antibiotic biosynthesis are largely unknown. Here we characterise a highly conserved actinobacterial response regulator called MtrA in antibiotic-producing Streptomyces species. We show that MtrA is an essential global regulator of secondary metabolism that directly activates antibiotic production in in S. coelicolor and S. venezuelae. MtrA also controls key developmental genes required for DNA replication and cell division and we propose that MtrA is the missing link that coordinates secondary metabolism with development in Streptomyces species.

microbiology↗