Elucidating novel regulatory mechanisms of antimycin-type depsipeptide biosynthesis

Soil bacteria, namely streptomycetes, produce more than half of all drugs used in human medicine, ranging from antibiotics to cure infectious disease as well as drugs to treat cancer and enable organ transplantation. Only about one-tenth of the bacterial genes that enable the production of these compounds are expressed in the laboratory. In other words, the global pharmaceutical industry is based on ~10% of the biochemical diversity it could be. A lack of knowledge as to how bacteria control the production of these potentially useful chemicals is one of the most significant challenges faced by the field of drug discovery and fundamental advances in this area could result in the discovery and development of a multitude of new therapeutics for a wide range of diseases. We have identified a novel regulatory protein (called a 'transcription factor') that controls the expression of a subset of genes in the pathway that makes compounds with promising activity as anticancer agents and treatments for the neurodegenerative conditions, Alzheimer's and Parkinson's diseases. Treating these age-related diseases with a small molecule is very attractive and has the potential to decrease the mortality and improve the quality of life of patients. Our ultimate goal is elucidate other regulators of this pathway and understand in detail how production of these molecules is controlled so that we may engineer their overproduction. Doing so will result in knowledge-based improvements in yield and the approaches we take and tools we make could be used in the same with for other potential drugs, possibly making them less expensive and more widely available.

The deliverables of our proposal will be achieved through a multidisciplinary programme of work incorporating microbiology, molecular genetics, synthetic biology and biochemistry.

Section 1 (Leeds): To identify the unknown regulators controlling antimycin biosynthesis. Methodology in genomic library construction, reporter probe analysis and CRISPR/Cas9 genome editing will be used in order to identify these regulators. The role of these regulators will be further elucidated and characterised using methods in ultra performance liquid chromatography mass spectrometry (UPLC/MS) and in vivo DNA footprinting using ChIP-exonuclease.

Section 2 (Leeds/MIT): To determine if the activity of the ECF sigma factor AntA is modulated by the ClpXP protease. Techniques in site-directed mutagenesis, quantitative RT-PCR, recombinant protein purification, proteolytic activity assays and immunoblotting will be used in order to determine if AntA is directly targeted by ClpXP, the latter will be achieved by the PDRA during a secondment to Collaborator Tania Baker's lab at MIT.

Section 3 (Leeds): To construct an optimised Streptomyces albus expression host and overproduce antimycins and neoantimycins. Mutiplexed CRISPR/Cas9 genome editing combined with UPLC/MS will be used to construct a genome-minimsed S. albus strain, which will be an ideal expression platform for this project and beyond. Promoter engineering using CRISPR/Cas9/homology-direct repair will be used to construct antimycin and neoantimycin overproduction strains and small-scale laboratory fermentation, chemical extractions and comparative metabolic profiling using UPLC/MS will be used to assess the level of overproduction.

WHO WILL BENEFIT FROM THIS RESEARCH?
Longer term, the outputs of this research should benefit both the pharmaceutical industry and society as a whole. Shorter term, this work will be of value to fundamental and applied scientists in academia and industry as well as research clinicians.

HOW WILL THEY BENEIFT FROM THIS RESEARCH?
The value of the global pharmaceutical industry exceeds £200 billion per year. More than half of all drugs critical for human health and wellbeing are derived from or inspired by natural products produced by bacteria and in particular by Streptomyces species. These bacteria only express about 10% of their chemistry under normal laboratory growth conditions. Fundamental studies of understanding how the production of natural products is controlled, like that proposed here, not only have the potential to unearth new chemistry from silent biosynthetic pathways but lead to knowledge-based improvements in yield, which can circumvent the frequent stumbling block of not having enough material for clinical trials. Alternative RNA polymerase sigma factors are emerging as a mechanism for controlling natural product biosynthesis. We have exciting preliminary data suggesting that the alternative sigma factor that regulates biosynthesis of antimycin-type depsipeptides (potential anticancer agents treatments for Alzheimer's and Parkinson's diseases) is controlled directly by the protease, ClpXP and not an anti-sigma factor, overturning longstanding models. Despite this being an unprecedented method of controlling RNA polymerase sigma factor activity, it is unlikely to be unique. Thus, our proposed work offers a valuable opportunity to understand this strategy for controlling production of natural products early on. Our ultimate goal is to develop Streptomyces albus as an optimised heterologous expression platform and use our novel insight into regulation of natural products to engineer the overproduction of antimycins and neoantimycins using synthetic biology. The World looks to the UK as the leader in actinomycete genetics and the approaches used throughout this project will ensure this remains true as we move into the synthetic biology era.

WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT FROM THIS RESEARCH?
We will disseminate the results of this project to the scientific community through publications and presentations at conferences and workshops. We will publish our data in open access journals when possible in order to increase their availability. The commercial potential of our work will be identified during regular self-assessments of progress and appropriate discoveries will be discussed (with a view to patenting) with Commercialisation Services at the U of Leeds and partner company IP Group Plc. The purpose of IP Group Plc is to bring scientific results from Leeds-based scientists into public use for public benefit. This is an established route within the University, which currently boasts 37 spin-out companies. The potential for future financial links with the industry sector will be explored through the U of Leeds Pharmaceutical and Biopharmaceutical Hub and through the BBSRC Natural Products Network (NPRONET). Ryan Seipke will work closely with the University of Leeds Media Relations Communication Team and the Faculty Marketing Team to maximise publicity and press coverage for the high impact papers we expect to publish from this work to audiences outside academia, for example in print (e.g. London Evening Standard, Yorkshire Evening Post, New Scientist), online (e.g. BBC, Daily Telegraph). Seipke will ensure the wider public benefit from this work by becoming involved in initiatives to inspire school children to study science including workshops and science fairs coordinated through National Science and Engineering Week, such as Discovery Zone and Social Mobility Foundation, a charity that sponsors children from low-income families to gain work experience and mentoring for entry into university.