Phthalic acid esters (PAEs) have long been known as the most widely used plasticizer with a broad range of industrial application. PAEs are ubiquitous in different environments and our daily life due to their large and widespread application. Recent PAEs research mainly focused on their environmental fate (including leaching, migration, transformation) and toxicology and risk assessment. With the comprehensive recognition of their potential hazard, the elimination of PAEs has attracted worldwide concerns. Although many factors may contribute to the degradation of PAEs, the dominant role of biodegradation was widely reported. Many PAEs-degrading bacteria were isolated, metabolites and metabolic pathways were proposed, and enzymes involved in the degradation were identified. The current paper presents an overview of available reports about PAEs-degrading bacteria and related molecular mechanisms. The metabolic pathways deduced from the identified intermediates were presented. The upstream and downstream pathways of PAEs metabolism were summarized, including the aerobic and anaerobic pathways of phthalic acid (PA) degradation. Known enzymes involved in the hydrolysis of ester bonds were characterized according to their properties. Based on phylogenetic analysis, all these enzymes were distributed in four families of esterases and one unknown family. For these five families, conserved sequence motifs were identified and the biological properties of these motifs were characterized. Challenges and emerging opportunities are also discussed.
Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.
Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.
The rise of bacterial multi drug resistance becomes a global threat to the mankind. Therefore it is essential to find out alternate strategies to fight against these “super bugs.” Quorum sensing (QS) is a cell-to-cell communication mechanism by which many bacteria regulate their biofilm and virulence factors expression to execute their pathogenesis. Hence, interfering the quorum sensing is an effective alternate strategy against various pathogens. In this study, we aimed to find out potential CviR-mediated quorum sensing inhibitors (QSIs) against Chromobacterium violaceum. Virtual screening from a natural products database, in vitro biofilm and violacein inhibition assays have been performed. Biofilm formation was investigated using confocal microscopy and gene expression studies were carried out using qRT-PCR. Further, to study the biomolecular interaction of QSIs with purified CviR Protein (a LuxR homologue), microscale thermophoresis (MST) analysis was performed. Results suggested that phytochemicals SPL, BN1, BN2, and C7X have potential GScore when compared to cognate ligand and reduced the biofilm formation and violacein production significantly. Especially, 100 μM of BN1 drastically reduced the biofilm formation about 82.61%. qRT-PCR studies revealed that cviI, cviR, vioB, vioC, vioD genes were significantly down regulated by QSIs. MST analysis confirmed the molecular interactions between QSIs and purified CviR protein which cohere with the docking results. Interestingly, we found that BN2 has better interaction with CviR (Kd = 45.07 ±1.90 nm). Overall results suggested that QSIs can potentially interact with CviR and inhibit the QS in a dose dependent manner. Since, LuxR homologs present in more than 100 bacterial species, these QSIs may be developed as broad spectrum anti-infective drugs in future.
Natural products play vital roles against infectious diseases since ancient times and most drugs in use today are derived from natural sources. Worldwide, multi-drug resistance becomes a massive threat to the society with increasing mortality. Hence, it is very crucial to identify alternate strategies to control these ‘super bugs’. Pseudomonas aeruginosa is an opportunistic pathogen reported to be resistant to a large number of critically important antibiotics. Quorum sensing (QS) is a cell–cell communication mechanism, regulates the biofilm formation and virulence factors that endow pathogenesis in various bacteria including P. aeruginosa. In this study, we identified and evaluated quorum sensing inhibitors (QSIs) from plant-based natural products against P. aeruginosa. In silico studies revealed that catechin-7-xyloside (C7X), sappanol and butein were capable of interacting with LasR, a LuxR-type quorum sensing regulator of P. aeruginosa. In vitro assays suggested that these QSIs significantly reduced the biofilm formation, pyocyanin, elastase, and rhamnolipid without influencing the growth. Especially, butein reduced the biofilm formation up to 72.45% at 100 µM concentration while C7X and sappanol inhibited the biofilm up to 66% and 54.26% respectively. Microscale thermophoresis analysis revealed that C7X had potential interaction with LasR (KD = 933±369 nM) and thermal shift assay further confirmed the biomolecular interactions. These results suggested that QSIs are able to substantially obstruct the P. aeruginosa QS. Since LuxR-type transcriptional regulator homologues are present in numerous bacterial species, these QSIs may be developed as broad spectrum anti-infectives in the future.
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