NPRONET - Natural products discovery and bioengineering network

Funded Proof of Concept Projects – Call 3

Understanding global transcription patterns of industrial Streptomyces strains in soil – Lorena T Fernández-Martínez

Project Partners Paul A Hoskisson – University of Strathclyde


Understanding the regulation of antibiotic gene clusters under soil conditions, particularly in industrially relevant strains, will help us understand how the production of these compounds is regulated in their natural environment. Expression of most of these antibiotic gene clusters appears dormant (cryptic) under laboratory conditions but the clusters are maintained in the genomes of these strains, therefore indicating that they must play important roles in adaptation and survival within their ecological niches. These cryptic pathways represent an untapped resource in terms of new metabolites and novel chemistry that could be very useful in the clinic. By growing industrial strains on iChips for the first time, we aim to understand the pathways which lead to the activation of transcription of these antimicrobial clusters in order to design new strategies to find novel metabolites. This will allow us to establish global regulatory pathways which lead to the production of these compounds. Moreover this knowledge can then be used to generate genetically modified industrial strains in order to increase the yield of both well characterised and unknown compounds under industrial fermentation conditions. This will ultimately increase the number of compounds which will enter the clinical trial pipeline.

The specific objectives we aim to achieve with this project are:

1. Optimisation of S. venezuelae and S. rimosus growth on iChips under different soil conditions
2. Optimisation of RNA extraction from iChip samples
3. RNA sequencing and analysis of global transcriptional patterns, particularly transcription of antibiotic gene clusters in these industrial strains.

The hunt for thioamide, a potentially revolutionary natural product tailoring. – John Heap – Imperial College London


Biologically active small molecules are of central importance to both medicine and industrialised agriculture. Despite great successes to-date, there remains a huge and growing unmet need due to the antibiotic crisis, the health and nutrition needs of the growing and ageing world population, and increasing biotic and abiotic pressures faced by agriculture, particularly climate change and decreasing water availability.

Secondary metabolite biosynthesis clusters, in particular the ‘assembly line’ systems (non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS) and their hybrids), now arguably represent the best hope for identifying bioactive compounds, for three main reasons: (1) NRPS/PKS clusters are modular, with largely predictable core module roles often preserved in new combinations, giving rise to new compounds. (2) The explosive growth of genome sequences has revealed that NRPS/PKS systems are very widespread and the vast majority are uncharacterised and unexploited. (3) In addition to the core modules, NRPS/PKS systems can include ‘tailoring’ enzymes, which add other moieties to natural product scaffolds, and which can also be ‘portable’ between systems, giving them the ability to act on non-native substrates.

Despite the advantages of biosynthesis, synthetic chemistry can incorporate a far greater variety of useful chemistries. The tailoring activities of NRPS/PKS systems may offer a route to improve the diversity of chemistries available to engineered biological systems for chemical synthesis. This project will study the secondary metabolite clusters of an organism which produces an interesting compound whose biosynthesis is not yet understood, and may involve a novel and useful tailoring activity. Identification of the relevant biosynthesis cluster is the first key step towards understanding and potentially exploiting this activity, which may allow the development of new biologically active compounds with medical and/or industrial uses.

Promiscuity is where it is AT: Exploiting a promiscuous acyltransferase for diversifying polyketide natural products – Ryan F. Seipke – University of Leeds


Microbial natural products underpin most new pharmaceutical leads. Chemical synthesis and semi-synthesis are typically used to generate a battery of analogues for testing which results in an optimised structure suitable for clinical trials. However, many natural products are too complex to be synthesised or to be modified by semi-synthesis. Thus, there is considerable interest in using bioengineering / synthetic biology to find biological solutions to chemical problems. We recently discovered the hybrid non-ribosomal peptide synthetase (NRPS) / polyketide synthase (PKS) biosynthetic pathway for antimycins, which comprise a very large family (>40) of anticancer molecules. The chemical diversity of antimycins is due in part to the catalytic promiscuity of the
acyltransferase (AT) domain of the unimodular AntD PKS. AntD-AT accepts a myriad of ‘atypical’ extender units produced by AntE, a crotonyl-CoA reductase. We predict that the functionalities of AntD-AT / AntE can exploited as a novel platform for analogue expansion of polyketides and we are well positioned to explore this opportunity. We recently cloned the biosynthetic machinery for a structurally similar compound called neoantimycin and developed a platform for its heterologous production. We propose to use our platform to evaluate whether AntD-AT / AntE can be utilised to extend polyketide natural products. Our strategy relies upon generating five strategic point mutations in two substrate selective motifs within the NatC-AT domain that we hypothesise will confer the promiscuity of AntD-AT. We will also define, the minimum set of motif alterations required for broadening substrate specificity, which will suggest obvious experimental strategies for adapting this strategy to other biosynthetic systems. At the conclusion of this project, we will be in an excellent position to apply for additional funds to further understand and develop our technology and translate our findings into the biotechnology sector.

Trehalolipid biosynthesis in marine Rhodococcus sp.: gene expression and bioactivity assessment – Steven L. Kelly – Swansea University

Project partners – Charlotte Cook – Plymouth Marine Laboratory


Synthetically derived surfactants are currently utilised in a diverse array of applications covering a wide range of global industries and have a significant global market value. In 2009, the world surfactant market reached $24.33 billion. The potential to in-part replace these compounds with natural, biologically derived surface active compounds (biosurfactants) is commercially attractive owing to increasing consumer demand for natural alternatives and the obvious potential market share. Biosurfactants have advantageous characteristics over synthetic counterparts that include novel bioactivity (including antimicrobial activity); they are non-toxic and are biodegradable; they are more stable to extremes in temperature, pH and ionic concentration, and they can be produced from renewable feedstocks. In comparison to other biosurfactants, trehalolipids remain largely unexplored for their biotechnological potential (including bioactivity assessment) despite them demonstrating a range of functionalities and bioactivities for a wide range of applications.
The first objective of this project is to produce and interpret novel genetic information (transcriptomic studies) regarding the biosynthesis of unique trehalolipid compounds produced by a recently discovered marine bacterium. This approach will, for the first time, help to elucidate the genes responsible for trehalolipid biosynthesis in bacterium, since a fundamental understanding of the genetic control of trehalolipid biosynthesis in microorganisms, which is required for strain development and improvement, is distinctly lacking. The second objective of this project is to test the antimicrobial activity of and surface active properties of trehalolipid products produced by the novel marine bacterium, and to comprehensively identify the different trehalolipid analogues contained within extracts using analytical approaches. When combined, this work will provide foundation data (proof of concept) for further research and development of an innovative commercially scalable biosurfactant (trehalolipid) biotechnology platform. The proposed collaboration between the academic and industrial partners will ensure the commercially focussed approach of the research with a view to product development that will have applications and benefits in a number of industries benefitting a wide range of potential stakeholders.

Identifying the Biosynthetic Origins of Nybomycin, a Reverse Antibiotic – Barrie Wilkinson – John Innes Centre

Project partners – Tony Maxwell – John Innes Centre


This project will investigate the biosynthesis of nybomycin, a Streptomyces natural product. Nybomycin is a molecule of unique chemical structure and highly unusual biological activity. It was first reported in 1955 and while it was found to have useful antibacterial potency, it was also shown to be extremely insoluble in water and did not find clinical utility. It was rediscovered in 2012 during a screen for substances active against multidrug-resistant Staphylococcus aureus M50 (MRSA) and it was found to have a curious activity profile: nybomycin was inactive against strains carrying intact gyrase genes (quinolone sensitive) and active against those with mutated gyrase genes (quinolone resistant). Unexpectedly, rather than select for further additional mutations to generate dual resistance, quinolone resistant strains treated with nybomycin reverted to quinolone sensitivity by reversion of the relevant mutation. This means that any bacteria that develop resistance to nybomycin will revert to being sensitive to quinolone antibiotics, potentially bypassing the usual cycle of endless accumulation of antibiotic resistances. Theoretically, the efficacy of quinolone antibiotics can be preserved by sequential application of nybomycin and quinolone antibiotics. Due to these phenomena nybomycin was deemed to be the first of a novel group termed ‘reverse antibiotics’.
The aim of this research project is to identify the nybomycin biosynthetic genes using comparative transcriptional analysis and/or transposon mutagenesis. Inspection of the chemical structure in conjunction with preliminary stable isotope feeding data from the 1970s suggest that nybomycin biosynthesis does not conform to any general, known biosynthetic pathway and likely comprises novel chemical and biochemical mechanisms. In our hands, typical genome mining techniques (antiSMASH 3.0 and MultiGeneBlast) have failed to identify the biosynthetic gene cluster (BGC), providing further support to the notion that the biosynthetic pathway is unique. Thus, we will be applying novel methodology towards the identification of an unprecedented BGC thereby enabling the understanding of a novel biosynthetic pathway that will involve new chemistry and enzymology.