Exploitation of underused Streptomyces through a combined metabolomics-genomics workflow to enhance natural product diversity.

Burns, Joshua

Abstract

The genus Streptomyces is the source of approximately two-thirds of all clinically-used antibiotics. Despite being the source of so many specialised metabolites, genomic analysis indicates that most Streptomyces strains have the potential to produce around twenty-five bioactive metabolites, some of which may be the basis of novel therapies. This makes culture collections of Streptomyces spp. an easily accessible (but under-used) resource to mine for genomic and metabolomic variety. Therefore, the main aim of this project was to initiate exploitation of the culture collection at NCIMB Ltd., by expanding the available chemical space from under-utilised Streptomyces for the production of novel antibiotics. This primarily used a mixture of metabolomic and genomic methods. A high-throughput culture parameter screen was designed around multiple carbon sources, nitrogen sources and extraction sample times. This was tested on the model species S. coelicolor A3(2) to compare differences in the production of known specialised metabolites, using UPLC-MS to analyse crude extracts from growth on agar. Data was analysed using MZmine and putative metabolites were identified using freely-available MS/MS databases - primarily GNPS. This showed clear variation in production of nine identified metabolites - including deferoxamines, germicidins, undecylprodigiosin and coelichelin - as a result of different culture parameters. Therefore, the screen successfully expanded the available chemical space, so was applied to non-model Streptomyces strains. The screen was used to compare the total metabolomic variety produced by three Streptomyces, isolated from different environments, in order to select a strain for further investigation. Comparing metabolomic features using principal component analysis showed the Costa Rican soil isolate S. costaricanus to produce the most variety versus the other two Streptomyces strains. The metabolite family most responsible for principal component separation was identified as the actinomycins. Scale-up of both agar and broth culture was used for metabolite dereplication and bioassays against multidrug resistant Acinetobacter baumannii, which is one of the bacteria on the World Health Organisation's list of pathogens that most urgently require new therapies. Fractions were derived from broth culture supernatant and agar crude extract by flash chromatography, resulting in semi-purified fractions. The predominant metabolite families in fractions were actinomycins and deferoxamines, which were further split by polarity into separate fractions. This resulted in rapid purification of metabolites, with one fraction comprising 80% deferoxamine B by weight. Fractions were tested against A. baumannii using the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay, which showed partial inhibition of growth at 50 µg/ml. Examining the bioactive fractions showed potentially novel minor peaks that could be responsible for bioactivity. A high-quality full genome of S. costaricanus was obtained using a combination of MiSeq and MinION sequences. This was analysed with RAST and antiSMASH to determine the specialised metabolite potential of S. costaricanus. AntiSMASH detected thirty-three biosynthetic gene clusters (BGCs), above the mean for Streptomyces. Thus, the confirmed genomic potential also suggested a wider metabolite variety, as indicated by the metabolomic screen. Some of the thirty-three BGC products had been previously detected by UPLC-MS, like actinomycin D and deferoxamine B. Other BGCs had 0% homology to known BGCs, including a terpene BGC which only showed core gene homology to two other Streptomyces. One of these strains shared all of the BGCs with S. costaricanus, including their sequential order and closely approximated genomic locations. Comparison of marker genes with autoMLST gave preliminary evidence for the taxonomic reclassification of S. costaricanus as a strain of S. griseofuscus. Starting from a large collection of unexploited Streptomyces, this project catalogued the metabolomic and genomic diversity of a single strain and its bioactive potential. Together, the project stages formed a workflow for further exploitation of NCIMB Streptomyces and other microbes.

Citation

BURNS, J. 2020. Exploitation of underused streptomyces through a combined metabolomics-genomics workflow to enhance natural product diversity. Robert Gordon University [online], PhD thesis. Available from: https://openair.rgu.ac.uk

• Thesis Type: Thesis
• Deposit Date: Jul 20, 2020
• Publicly Available Date: Jul 20, 2020
• Keywords: Streptomyces; Antibiotics; Bacterial culture screening; Microbes
• Public URL: https://rgu-repository.worktribe.com/output/950245
• Award Date: May 31, 2020