The mega-enzymes non-ribosomal peptide synthetases / polyketide synthases encoded in Lactic Acid Bacteria

            The secondary metabolites also known as natural products exhibit a variety of important bioactivities and excellent pharmaceutical properties (as antibiotics, anti-cancers and immune-suppressants) that have successfully been used in the pharmaceutical industry. The secondary metabolites are synthesized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) mega-enzymes, which consist of multiple functional domains that cluster in one or more modules. The presence of these mega-enzymes in Lactic Acid Bacteria (LAB) has so far been studied only to a limited extent.

            The acquisition of a series of functional genes, enabled the strain Lactobacillus plantarum WCFS1 to be highly adapted to a wide variety of environmental niches such as dairy products and the human gastrointestinal (GI) tract. The lactis-specific genes, on the other hand enabled Lactococcus lactis KF147 to adapt to the plant niche. Researches have been conducted to study the role of both LAB in human food and health. As the first examples in LAB, a novel non-ribosomal peptide synthetase (NRPS) and a hybrid non-ribosomal peptide synthetase-polyketide synthase (hybrid NRPS-PKS) systems were discovered in the genomes of these bacteria. In this PhD work, homology searches revealed novel homologous NRPS systems similar in module and domain organization to the NRPS of Lactobacillus plantarum WCFS1 in nine other strains of Lactobacillus plantarum, namely RI-515, PS128, B21, K35, P86, P67, P26, TMW 1.708 and strain 19.1, two strains of Clostridium (Clostridium sp. DMHC 10 and Clostridium akagii DSM 12554), Carnobacterium inhibens subsp. inhibens DSM 13024 K1, six strains of Enterococcus (Enterococcus devriesei DSM 22802, Enterococcus sp. KPPR-6, Enterococcus thailandicus DSM 21767, Enterococcus faecium KACC16100,  Enterococcus faecium E2039 and Enterococcus faecium KACC16106) and four strains of Streptococcus (Streptococcus sp. HMSC034F02, Streptococcus sp. HMSC034B04, Streptococcus oralis subsp. tigurinus AZ_3a and Streptococcus anginosus isolate BVI). On the other hand, homologous hybrid systems having similar gene, module and domain organization were also found in six other Lactococcus lactis subsp. Lactis strains (KF134, NCDO 2118, KF196, Li-1, KF146 and YF11), and twentyfive strains of the dental pathogen Streptococcus mutans.

            In this work we aimed to characterize the two mega-enzymes that are encoded in the two LAB. For this purpose a novel comprehensive bioinformatics approach was used to characterize the orphan NRPS and the hybrid NRPS-PKS metabolic pathways in Lactobacillus plantarum WCFS1 and Lactococcus lactis KF147 and their homologous systems. The approach included the analysis of 140 sequenced microbial genomes using Hidden Markov Model profiles of all core domains of NRPS/PKS systems, which led to the identification of numerous systems. Several genomes were found to contain three or more systems including hybrids, these included mainly microorganisms with genomes larger than 4 Mb isolated from soil or aquatic environments.

            As a main step in the characterization strategy, the ability to identify substrate specificity of the adenylation (A) and acyltransferase (AT) domains of the NRPS and PKS systems, respectively, which  recognize the building blocks, can aid in the rapid characterization, discovery and/or engineering of novel products of these systems. In this direction we developed an efficient generic tool to recognize the substrate specificity of the A and AT domains, using Ensembles of Substrate Specific Hidden Markov Models (ESSHMM). The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

            Using the recent most accurate tools to predict the substrate specificity of the six adenylation domains of the NRPS system of Lactobacillus plantarum WCFS1 and the homologues, only three out six were predicted, confirming the novelty of these systems. The predicted substrate specificity of the six A domains and the iterative transAT domain of the hybrid NRPS-PKS system encoded by Lactococcus lactis KF147 and the homologues, was used to predict the primary structure of the putative hybrid natural product to be (Leu-DLeu-Asp-DAsn-Gly-MC-MC-MC-DAsp).

            Regulation of the two systems and their homologues was studied to help in the characterizing approach. The findings suggest that the NRPS of Lactobacillus plantarum WCFS1 and its homologous systems in the other strains are most likely regulated similarly as a whole gene operon with their conserved upstream PTS systems mediated by σ54 factors. With respect to the regulation of the hybrid NRPS-PKS of the studied Lactococcus lactis strains and the homologous systems in the Streptococcus mutans strains, based on the inspected regulatory features we assume that they are regulated by the highly conserved two-component systems present upstream of the NRPS-PKS gene operons. Furthermore the findings led to the hypothesis that the two-component system transduces the signals for biological cell responses to trigger a hybrid metabolite (NRP-PK) production and biofilm formation as well as oxidative stress resistance. The new specificity recognition techniques and the computational strategy can be used to characterize other newly discovered orphan NRPS/PKS systems that have significantly increased as the result of large scale sequencing efforts in metagenomics.