NPRONET - Natural products discovery and bioengineering network

Funded Proof of Concept Projects – Call 4

A Sustainable Route to Scytonemin Production in Cyanobacteria – Samantha Bryan – University of Nottingham

Project Partners – Steve Skill – Greenskill Ltd

Microbial cell factories offer extensive opportunities for the industrial production of complex biomolecules for cost effective biological synthesis. Microbial fermentation often reduces the need for energy intensive reaction conditions, toxic organic solvents, heavy metal catalysts and strong acids/bases, widely used in chemical synthesis routes.

We are developing new ways of producing key complex biomolecules from waste industrial CO2, a process which is environmentally restorative and requires no fossil reserves as carbon feedstock. Cyanobacteria are robust, metabolically diverse cell factories capable of generating a vast treasure trove of natural products. We anticipate that our research will be at the forefront of generating a metabolically engineered strain capable of generating industrially relevant biomolecules, which will have a significant impact on the next range of skincare products, as well as developing a new class of pharmaceuticals.

Exploring, exploiting and evolving ketomemicin biosynthesis for production of pseudopeptides. – Prof. Dominic Campopiano – University of Edinburgh

Project Partners – Dr. Yasushi Ogasawara – Hokkaido University, Japan

Peptide therapeutics are of great interest to the pharmaceutical industry due to their efficacy and selectivity but they are limited by their biological instability. In contrast, peptidomimetics, or pseudopeptides, are gaining increased popularity since they overcome the inherent bioavailability and stability problems associated with peptides. In a pseudopeptide the typical amide backbone is replaced with a non-hydrolysable alternative. Pseudopeptides are typically manufactured via multi-step synthetic procedures but the overarching goals of the natural product biosynthesis and biocatalysis fields is to discover and exploit new enzymes to catalyse the synthesis of high value products in a greener, environmentally-friendly way. One of the NPRONET aims is “to develop more efficient and diverse routes for the production of “non natural” products, fine and commodity chemicals, APIs, and food products”. A valuable source of new biocatalysts are the enzymes from natural product biosynthetic pathways, but few natural products have a pseudopeptide core. However, the exciting recent discovery of the ketomemicins, isolated from the microbes Streptomyces mobaraensis, Salinispora tropica, and Micromonospora sp. led to the identification of the six enzymes involved in building the complex ketomemicin structure from simple building blocks. A key enzyme, KtmB, is crucial for the biosynthetic pathway since it catalyses the formation of the pseudopeptide core. KtmB is also key for introducing variability into the ketomemicin pseudopeptides since different versions of KtmB display selectivities for different amino acid building blocks. We propose that bioengineering of KtmB will lead to the development of a range of novel pseudopeptides via the use of alternative amino acids, which would have far reaching implications for the development of novel therapeutics. The aim of our project is to explore and exploit the substrate promiscuity of KtmB enzymes and to investigate their use in introducing structural diversity into ketomemicins. This will be accomplished by carrying out a detailed biochemical analysis of the KtmB enzymes, in which we will use our chemical knowledge to explore the substrate scope of KtmB. This will be complemented by structural characterisation of the KtmB enzymes in the presence of their substrates. This PoC study will provide a springboard for the bioengineering of KtmB which will lead to the production of novel ketomemicin pseudopeptide derivatives with enhanced properties.

Can epistasis be exploited to understand industrial antibiotic production in Streptomyces? – Paul Hoskisson – University of Strathclyde

Project Partners – Ryan F Seipke – University of Leeds

There is an urgent need to develop new antimicrobial drugs to treat drug resistant infections and the Actinobacteria, especially Streptomyces, still remain one of the best sources of these drugs. A key to exploiting these organisms is an understanding of how these organisms produce such a diverse array of bioactive metabolites. To achieve industrial scale production of natural products from Streptomyces iterative rounds of random mutagenesis, followed by selection for high-producing strains is normally performed. The nature and role played by many of these mutations is not known or how these mutations interact with each other. It is well known from natural evolutionary processes that interaction between unconnected genetic loci (epistatic effects) can have profound effects on biological fitness. This process has not been studied in Streptomyces. This proposal will ‘evolve’ parallel lineages of an antibiotic production mutant of S. coelicolor and at specified points reintroduce the ability to produce the polyketide antibiotic, actinorhodin. This will enable the identification of genome wide mutations that alter production within a specific lineage. Using genome sequencing and bioinformatics we will map these mutations to identify mutations that ultimately succeed or fail in the context of natural product production during strain evolution and use these data to identify common mutations that occur across lineages. To examine epistasis in Streptomyces in an industrially relevant context we will investigate lineages of another polyketide producing strain, the oxytetracycline producer S. rimosus to identify the key mutagenic innovations acquired by the strains over 40 years of strain improvement to enhance their antibiotic production capabilities. Combining these two approaches will allow the formulation of hypotheses for experimental work in a larger application to BBSRC.
This proposal is novel and innovative because this process has not been studied in these organisms before and the nature of epistasis and its impact on antibiotic production is a ‘blackbox’ to industrial scientists. Detailed information about key mutations that enhance production that lay outside of biosynthetic clusters can bring about a step-change in how we exploit these organisms.

Hijacking nutrient sensing to improve the yield of natural products made by Actinobacteria – Helen O’Hare – University of Leicester

Project Partners – Matt Gregory – Isomerase Therapeutics Ltd

We have identified a novel nutrient sensor that is conserved in most Actinobacteria and regulates central metabolism. We propose to manipulate this sensory/regulatory system as a novel route to enhance the yield of valuable natural products. The aim of this proposal is to carry out a proof of concept experiment to genetically manipulate this pathway and determine the effects on primary metabolism and antibiotic yield.


1. To validate our approach we will mutate the regulator in an erythromycin over-producer strain to determine the effect on primary metabolism and on the yield of erythromycin (additional industrially relevant strains will also be tested).
2. Using the fundamental knowledge arising from (1), we aim to build a tool box of genetic and chemical manipulations that can be applied broadly to Actinobacteria in order to improve the yield of natural products of commercial interest.


The direct link between primary metabolism and control of antibiotic production in Streptomyces is firmly established, although incompletely understood (reviewed by Urem et al Mol Micro 2016). Genetic engineering of carbon metabolism (enzymes and regulators) has been used to increase actinorhodin production (Borodina et al JBC 2008), while carbon catabolite repression reduces production of many antibiotics: chloramphenicol, cephamycin, erythromycin and streptomycin.


We have identified a nutrient sensor that is unique to Actinobacteria and highly conserved within the Actinobacteria. Engineering genes in this pathway significantly alters the balance between carbon and nitrogen metabolism of pathogenic Mycobacterium tuberculosis and amino-acid producing Corynebacterium glutamicum. The functions and applications have not been investigated in antibiotic producers previously because of their recent discovery (the sensor is still unpublished). Engineering the equivalent genes in streptomycetes is highly likely to shift the balance between C and N metabolism, which, according to published work, is likely to influence natural product yield. This pathway could be a broad target to improve the yield of many natural products.

Towards a second Golden Age of antibiotic discovery: a platform for identifying small molecule elicitors of secondary metabolism in Streptomyces – Alex O’Neill – University of Leeds

Project Partners – Ryan Seipke – University of Leeds

New antibiotics are urgently needed to treat infections caused by bacteria resistant to our existing drugs. Historically, the most prolific source of clinically useful antibiotics has been the genus Streptomyces; the existence of a substantial untapped reservoir of secondary metabolites in this genus likewise suggests its potential to provide a pre-eminent source of antibiotics in the future. Unfortunately, the default setting for most secondary metabolite production in Streptomyces spp. is ‘off’ under laboratory growth conditions, thereby hampering the classical approach of screening for new antibiotics by testing these strains or extracts thereof for inhibition of an indicator bacterium. Whilst the concept that silent biosynthetic gene clusters (BGCs) can be activated using small molecule ‘elicitors’ is well-established, the variable and incomplete nature of the data regarding the ability of these compounds to stimulate secondary metabolism has to date precluded their routine deployment as an effective means to rejuvenate natural product antibiotic discovery.

This study seeks to build towards a more systematic and comprehensive analysis of elicitors. In preliminary work, we have begun to engineer a suite of reporter strains in the model antibiotic producer organism, Streptomyces coelicolor, in which activation of individual BGCs can be monitored via expression of the reporter gene, gusA. In the present proposal, we aim to grow this suite of strains to encompass all of the predicted BGCs in this organism, thereby creating a powerful new tool for the field; a first standardised platform for interrogating activation of secondary metabolism in Streptomyces. This platform will subsequently be employed both to gain a more detailed ‘activation profile’ for previously-reported elicitor molecules, and to conduct a screen of several thousand small molecules to identify novel chemical elicitors with broad applicability for awakening antibiotic production. Collectively, these studies will make a valuable contribution to developing methods for activating unproductive BGCs, and will underpin the longer-term aim of establishing a cipher for unlocking cryptic secondary metabolism.

Using antimicrobials to discover antibiotics; determining the efficiency of using antimicrobials as elicitor molecules to activate silent biosynthetic gene clusters in environmental bacteria. – Adam Roberts – Liverpool School of Tropical Medicine

The need for new antimicrobials to be used in human and animal medicine is extremely urgent due to the development and spread of antimicrobial resistance. Most antimicrobials we currently use are natural products (NPs) which are produced by actinomycete bacteria from soil; predominantly Streptomyces spp. Two major obstacles in the discovery of novel antimicrobials NPs are avoiding rediscovery of known compounds and turning on silent biosynthetic gene clusters (BGCs) responsible for NP biosynthesis. To de-replicate the discovery of NPs we are screening environmental isolates which are not isolated from soil, to determine if they can produce antimicrobials. We have screened thousands of isolates in this ongoing project, which are isolated from swabs that are returned by members of the public after they have swabbed their chosen environment.

We screen them against a range of Gram positive and negative bacterial isolates and the fungi Candida albicans. We are currently investigating approximately 20 Bacillus spp., and one Klebsiella sp which can inhibit the growth of multi-drug resistant Escherichia coli and / or C. albicans. We have also determined the genome sequence of these strains and are currently analysing interesting BGCs. In order to maximise the numbers of inhibitory strains we detect; and therefore, the number of antimicrobials we can isolate, we need to address the second challenge; ie. activating silent BGCs. This proposal aims to address this by determining of the effects of including sub-inhibitory concentrations of antibiotics as elicitor molecules in the growth media on which the inhibition assays are carried out. The elicitor molecules which we will investigate are antibiotics and antiseptics commonly used in medicine and may activate expression of silent BGCs. NPRONET funding will allow us to investigate the effect of sub-inhibitory concentrations of antimicrobials on the recovery of NPs during antimicrobial assays. We will use standard microbiological assays coupled with detailed molecular investigations to determine the exact mechanisms of activation of BGCs in bacteria other than Streptomyces spp. from a wide range of environments that have not been investigated previously.