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Nonribosomal peptide synthetases (NRPSs) are giant multi-enzymes that carry out sequencial assembly line couplings of amino acids to generate linear or cyclic peptides. NRPSs are composed of repeating enzyme domains with modular organization to activate and couple specific amino acids in a particular order. From a synthetic biology perspective, they can be considered as peptide assembly machines composed of devices to couple fatty acids to L-amino acids, L-amino acids to L-amino acids, and D-amino acids to L-amino acids. The coupling devices are composed of specific parts that contain two or more enzyme domains that can be exchanged combinatorially to generate novel peptide assembly machines to produce novel peptides. The potent lipopeptide antibiotics daptomycin and A54145E have identical cyclic depsipeptide ring structures and stereochemistry but have divergent amino acid sequences. As their biosynthetic gene clusters are derived from an ancient ancestral lipopetide pathway, these lipopeptides provided an attractive model to develop combinatorial biosynthesis to generate antibiotics superior to daptomycin. These studies on combinatorial biosynthesis have helped generate guidelines for the successful assembly of NRPS parts and devices that can be used to generate novel lipopeptide structures and have established a basis for future synthetic biology studies to further develop combinatorial biosynthesis as a robust approach to natural product drug discovery. (Figure Presented). © 2012 American Chemical Society. Source


Baltz R.H.,CognoGen Biotechnology Consulting
Journal of Industrial Microbiology and Biotechnology | Year: 2016

Actinomycetes continue to be important sources for the discovery of secondary metabolites for applications in human medicine, animal health, and crop protection. With the maturation of actinomycete genome mining as a robust approach to identify new and novel cryptic secondary metabolite gene clusters, it is critical to continue developing methods to activate and enhance secondary metabolite biosynthesis for discovery, development, and large-scale manufacturing. This review covers recent reports on promising new approaches and further validations or technical improvements of existing approaches to strain improvement applicable to a wide range of Streptomyces species and other actinomycetes. © 2015, Society for Industrial Microbiology and Biotechnology. Source


Chemical mutagenesis continues to be an important foundational methodology for the generation of highly productive actinomycete strains for the commercial production of antibiotics and other secondary metabolites. In the past, the determination of frequencies of chemically induced resistance to rifampicin (RifR), spectinomycin (SpcR) and streptomycin (StrR) have served as surrogate markers to monitor the efficiencies and robustness of mutagenic protocols. Recent studies indicate that high level RifR, SpcR and StrR phenotypes map to specific regions of the rpoB, rpsE and rpsL genes, respectively, in actinomycetes. Moreover, mutagenesis to RifR can occur spontaneously at many different sites in rpoB, and all six types of base-pair substitutions, as well as in-frame deletions and insertions, have been observed. The RifR/rpoB system provides a robust method to rank mutagenic protocols, to evaluate mutagen specificity and to study spontaneous mutagenesis mechanisms involved in the maintenance of high G+C content in Streptomyces species and other actinomycetes. © 2014 Japan Antibiotics Research Association All rights reserved 0021-8820/14. Source


Baltz R.H.,CognoGen Biotechnology Consulting
Journal of Industrial Microbiology and Biotechnology | Year: 2012

øC31, øBT1, R4, and TG1 are temperate bacteriophages with broad host speciWcity for species of the genus Streptomyces. They form lysogens by integrating site-speciWcally into diverse attB sites located within individual structural genes that map to the conserved core region of streptomycete linear chromosomes. The target genes containing the øC31, øBT1, R4, and TG1 attB sites encode a pirin-like protein, an integral membrane protein, an acyl-CoA synthetase, and an aminotransferase, respectively. These genes are highly conserved within the genus Streptomyces, and somewhat conserved within other actinomycetes. In each case, integration is mediated by a large serine recombinase that catalyzes unidirectional recombination between the bacteriophage attP and chromosomal attB sites. The unidirectional nature of the integration mechanism has been exploited in genetic engineering to produce stable recombinants of streptomycetes, other actinomycetes, eucaryotes, and archaea. The øC31 attachment/ integration (Att/Int) system has been the most widely used, and it has been coupled with the øBT1 Att/Int system to facilitate combinatorial biosynthesis of novel lipopeptide antibiotics in Streptomyces fradiae. © Society for Industrial Microbiology and Biotechnology 2011. Source


Baltz R.H.,CognoGen Biotechnology Consulting
Journal of Industrial Microbiology and Biotechnology | Year: 2014

Advances in DNA sequencing technologies have made it possible to sequence large numbers of microbial genomes rapidly and inexpensively. In recent years, genome sequencing initiatives have demonstrated that actinomycetes with large genomes generally have the genetic potential to produce many secondary metabolites, most of which remain cryptic. Since the numbers of new and novel pathways vary considerably among actinomycetes, and the correct assembly of secondary metabolite pathways containing type I polyketide synthase or nonribosomal peptide synthetase (NRPS) genes is costly and time consuming, it would be advantageous to have simple genetic predictors for the number and potential novelty of secondary metabolite pathways in targeted microorganisms. For secondary metabolite pathways that utilize NRPS mechanisms, the small chaperone-like proteins related to MbtH encoded by Mycobacterium tuberculosis offer unique probes or beacons to identify gifted microbes encoding large numbers of diverse NRPS pathways because of their unique function(s) and small size. The small size of the mbtH-homolog genes makes surveying large numbers of genomes straight-forward with less than ten-fold sequencing coverage. Multiple MbtH orthologs and paralogs have been coupled to generate a 24-mer multiprobe to assign numerical codes to individual MbtH homologs by BLASTp analysis. This multiprobe can be used to identify gifted microbes encoding new and novel secondary metabolites for further focused exploration by extensive DNA sequencing, pathway assembly and annotation, and expression studies in homologous or heterologous hosts. © 2013 Society for Industrial Microbiology and Biotechnology. Source

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