Characterization of modification enzyme NukM and engineering of a novel thioether bridge in lantibiotic nukacin ISK-1.
ABSTRACT The lantibiotic nukacin ISK-1 is an antimicrobial peptide containing unusual amino acids such as lanthionine and dehydrobutyrine. The nukacin ISK-1 prepeptide (NukA) undergoes posttranslational modifications, such as the dehydration and cyclization reactions required to form the unusual amino acids by the modification enzyme NukM. We have previously constructed a system for the introduction of unusual amino acids into NukA by coexpression of NukM in Escherichia coli. Using this system, we describe the substrate specificity of NukM by the coexpression of a series of NukA mutants. Our results revealed the following characteristics of NukM: (1) its dehydration activity is not coupled to its cyclization activity; (2) its dehydration activity is site-specific; (3) the length of the substrate is important for its dehydration activity. Furthermore, we succeeded in introducing a novel thioether bridge in NukA by replacing an unmodified Ser at position 27 with a Cys residue.
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ABSTRACT: With the advent of next-generation DNA sequencing technologies, the number of microbial genome sequences has increased dramatically, revealing a vast array of new biosynthetic gene clusters. Genomics data provide a tremendous opportunity to discover new natural products, and also to guide the bioengineering of new and existing natural product scaffolds for therapeutic applications. Notably, it is apparent that the vast majority of biosynthetic gene clusters are either silent or produce very low quantities of the corresponding natural products. It is imperative therefore to devise methods for activating unproductive biosynthetic pathways to provide the quantities of natural products needed for further development. Moreover, on the basis of our expanding mechanistic and structural knowledge of biosynthetic assembly-line enzymes, new strategies for re-programming biosynthetic pathways have emerged, resulting in focused libraries of modified products with potentially improved biological properties. In this review we will focus on the latest bioengineering approaches that have been utilised to optimise yields and increase the structural diversity of natural product scaffolds for future clinical applications.Current opinion in biotechnology 04/2012; · 7.82 Impact Factor
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ABSTRACT: Lacticin Q (LnqQ) produced by Lactococcus lactis QU 5 is an unmodified linear bacteriocin, which is synthesized without an N-terminal leader peptide. In vitro synthesis and in vivo expression of LnqQ have revealed the intracellular toxicity of this leaderless peptide as well as the necessity of a dedicated secretion and self-immunity system of producer cells. Further DNA sequencing and analysis has discovered 11 putative orfs in LnqQ locus. None of the orfs showed similarities to any of the bacteriocin biosynthetic genes characterized to date; however, 6 orfs (orfs2q-7q) other than the structure gene (lnqQ) were highly conserved in the locus of lacticin Z (orfs2z-7z), which is a LnqQ-homologue produced by L. lactis QU 14. The ORF2q (ORF2z), located upstream of the structure gene, is a putative transcriptional regulator, whereas the ORF6q and ORF7q (ORF6z and ORF7z) compose a putative ATP-binding cassette (ABC) transporter. The ORFs3q-5q (ORFs3z-5z) are all predicted as membrane proteins with no clear functions. Co-expression of LnqQ and ORFs3q-7q in a heterologous host allowed the extracellular production of LnqQ; additionally, the expression of ORFs3q-7q rendered the host cells immune to LnqQ. This self-immunity was facilitated possibly by two means; firstly by secreting the active LnqQ peptides thus reducing the intracellular toxicity, and secondly by protecting the host cells from extracellularly released LnqQ. This is the first report describing an intracellular toxicity of a leaderless bacteriocin, and providing a rare example of biosynthetic genes that are required for bacteriocin secretion and immunity.Microbiology 10/2012; · 3.06 Impact Factor
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ABSTRACT: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with a high degree of structural diversity and a wide variety of bioactivities. Understanding the biosynthetic machinery of these RiPPs will benefit the discovery and development of new molecules with potential pharmaceutical applications. In this Concept article, we discuss the features of the biosynthetic pathways to different RiPP classes, and propose mechanisms regarding recognition of the precursor peptide by the post-translational modification enzymes. We propose that the leader peptides function as allosteric regulators that bind the active form of the biosynthetic enzymes in a conformational selection process. We also speculate how enzymes that generate polycyclic products of defined topologies may have been selected for during evolution.Chemistry 05/2013; · 5.93 Impact Factor