Full-length title: NRPPUR database search and in vitro analysis identify an NRPS-PKS biosynthetic gene cluster with a potential antibiotic effect

Bioinformatics

Antibiotics have achieved major advances in medicine and surgery, saving patients’ lives and extending the expected human lifespan [1]. Following the golden era when natural antibiotics were discovered and prescribed in 1925–1950, the chemistry era followed in the 1970s, with synthetic tweaking to improve activity. However, in 2000, the resistance era, largely due to the overuse and misuse of these medications, began [2, 3]. This coincided with the development of new technologies such as the manipulation of recombinant DNA and the high-throughput synthesis of chemicals that has given rise to hopes of drug discoveries other than antibiotics [4]. Although the high-throughput biochemical screening of large collections of syntheses has provided some interesting leads, the complexity and diversity of these molecules has been insufficient to provide the same level of bioactivity as found in naturally occurring antibiotics. It has been suggested that the coexistence of microbes with other microbes and fungi in the environment leads to selection of the most potent targets so that the best source of new antibiotics are compounds naturally produced by microorganisms [5]. It has therefore been recommended that natural products are revisited as an alternative to synthetic collections following the methods in the “golden age of the discovery of antibiotics” that screened microbial cell extracts from soil to find new antibiotic scaffolds. It has been also recommended that new technologies are embraced to overcome problems of compound discovery. Thus, the exploration of genome sequences of microorganisms and data from metagenomics of the microbial dark matter- microorganisms that have resisted to easy cultivation in the laboratory [6] has revealed a very large spectrum of potential for secondary metabolites with potential antibiotic functions [7].

Microbial secondary metabolites are organic compounds that are not directly involved in primary growth and development, but rather have auxiliary functions including defense and communication [8]. Natural antimicrobial products consist mainly of two groups i) bacteriocins [9] where biosynthesis is carried out conventionally via ribosome, and ii) polyketides (PKS) and non-ribosomal peptides (NRPS) where biosynthesis is ribosome independent. The atypical biosynthesis of NRPS and PKS known as “thiotempling” is supported by a multi-enzymatic, multi-domain synthases NRPSs and PKSs, respectively that add amino-acid monomers for NRPS and acyl Coenzyme A for PKS products. The primary sequence of the peptide product is determined by the sequential arrangement of active sites called modules within NRPSs and modular PKSs. These modules contain multiple functional domains that are necessary for catalyzing each condensation and chain elongation or modification reaction [1012]. Genes encoding biosynthetic enzymes for the synthesis of these secondary metabolites are typically co-localized on the chromosome and are referred to as “biosynthetic gene clusters” (BGCs). Since the first elucidation of the PKS gene cluster for erythromycin in the early 1990s [13, 14], many gene clusters responsible for the biosynthesis of NRPS and PKS have been reported and deposited in International Nucleotide Sequence Database Collection (INSDC) entries (DDBJ/GenBank/EMBL) [15]. In addition, the community-driven website developed many specialized pieces of software such as Antismash [1618] and Streptome DB [19] that enabled the detection of NRPS and PKS [2024] in a wide range of microorganisms such as Bacteria, Fungi, Archaea and Eukarya. The general principle behind in silico mining consists of using a library of enzymes/protein domains commonly observed in secondary metabolite biosynthetic pathways to identify homologues in the genome sequences of the organisms of interest. For this task, sequence based comparison software, such as BLAST [25] or DIAMOND [26], or profile-based tools such as HMMer [27] are usually used. Together, the stunning advances in genome sequencing and informatics tools are creating the conditions necessary to support the discovery of narrow-spectrum potent antibiotics. However, large-scale gene dispensability studies using microbial gene cloning, protein expression and high-throughput screening revealed that these databases contain numerous targets that were not always bioactive when tested in vitro [28, 29].

In this paper, we present an in silico/in vitro combined strategy for identifying NRPS and PKS in the human gut microbiota. With this aim, we built an updated database, named NRPPUR (Non-Ribosomal Peptide and Polyketide Urmite), containing gene sequences for NRPS/PKS clusters, which products and corresponding extracts have demonstrated an interesting activity using antimicrobial testing methods during in vitro investigation. NRPPUR was queried to make the functional annotation using RPS-BLAST (Reverse Position-Specific Blast) in order to decipher NRPS-PKS BGCs on 49 bacterial genomes first isolated from human gut microbiota using the “culturomics” approach [30]. The antimicrobial activity of the identified producers has been tested in vitro using the cross-streak method. The combined strategy using the “culturomics- genomic-bioinformatic-antibiogram” platform has significant potential to discover new candidate antibiotic producers.

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