DNA shuffling method for generating highly recombined genes and evolved enzymes

Coco, Wayne M.; Levinson, William E.; Crist, Michael; Hektor, Harm J.; Darzins, Al; Pienkos, Philip T.; Squires, Charles H.; Monticello, Daniel J.

doi:10.1038/86744

Cited by 240 publications

(108 citation statements)

References 29 publications

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“…are filamentous soil saprophytes but also include pathogenic agents that cause nocardiosis in humans and animals in the lung, central nervous system, brain, and skin (42), while another species, N. asteroides, is suspected to contribute to Parkinson's disease (189). Moreover, Nocardia species can produce industrially important bioactive molecules such as antibiotics and enzymes (78,393,422).…”

Section: General Featuresmentioning

confidence: 99%

Genomics ofActinobacteria: Tracing the Evolutionary History of an Ancient Phylum

Ventura

Canchaya

Tauch

et al. 2007

Microbiol Mol Biol Rev

933

638

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SUMMARY Actinobacteria constitute one of the largest phyla among Bacteria and represent gram-positive bacteria with a high G+C content in their DNA. This bacterial group includes microorganisms exhibiting a wide spectrum of morphologies, from coccoid to fragmenting hyphal forms, as well as possessing highly variable physiological and metabolic properties. Furthermore, Actinobacteria members have adopted different lifestyles, and can be pathogens (e.g., Corynebacterium, Mycobacterium, Nocardia, Tropheryma, and Propionibacterium), soil inhabitants (Streptomyces), plant commensals (Leifsonia), or gastrointestinal commensals (Bifidobacterium). The divergence of Actinobacteria from other bacteria is ancient, making it impossible to identify the phylogenetically closest bacterial group to Actinobacteria. Genome sequence analysis has revolutionized every aspect of bacterial biology by enhancing the understanding of the genetics, physiology, and evolutionary development of bacteria. Various actinobacterial genomes have been sequenced, revealing a wide genomic heterogeneity probably as a reflection of their biodiversity. This review provides an account of the recent explosion of actinobacterial genomics data and an attempt to place this in a biological and evolutionary context.

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Section: General Featuresmentioning

confidence: 99%

Genomics ofActinobacteria: Tracing the Evolutionary History of an Ancient Phylum

Ventura

Canchaya

Tauch

et al. 2007

Microbiol Mol Biol Rev

933

638

View full text Add to dashboard Cite

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“…Another feature of the nocardia is that many strains, even clinical isolates, also have the capability to produce bioactive molecules such as antibiotics (4,5) and enzymes that are industrially important (6). A monobactam antibiotic, nocardicin, was isolated from Nocardia sp.…”

mentioning

confidence: 99%

The complete genomic sequence of Nocardia farcinica IFM 10152

Ishikawa

Yamashita

Mikami

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

270

202

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We determined the genomic sequence of Nocardia farcinica IFM 10152, a clinical isolate, and revealed the molecular basis of its versatility. The genome consists of a single circular chromosome of 6,021,225 bp with an average G؉C content of 70.8% and two plasmids of 184,027 (pNF1) and 87,093 (pNF2) bp with average G؉C contents of 67.2% and 68.4%, respectively. The chromosome encoded 5,674 putative protein-coding sequences, including many candidate genes for virulence and multidrug resistance as well as secondary metabolism. Analyses of paralogous protein families suggest that gene duplications have resulted in a bacterium that can survive not only in soil environments but also in animal tissues, resulting in disease.N ocardia are filamentous-growing Gram-positive soil saprophytes that belong to the family Actinomycetales, which also includes clinically and industrially important genera such as Mycobacterium, Streptomyces, Corynebacterium, and Rhodococcus. Many species of Nocardia cause the disease nocardiosis in humans and animals on lung, central nervous system, brain, and cutaneous tissues (1), and a species Nocardia asteroides is suspected to be an etiological agent of Parkinson's disease (2). Nocardiosis is on the rise, with an estimated 109-136 new cases occurring annually in Japan (3) and 500-1,000 in the U.S. (www.cdc.gov͞ncidod͞dbmd͞diseaseinfo͞nocardiosis t.htm). However, there are only a few studies on the mechanisms of nocardial virulence. Nocardia species are resistant to many front-line antibiotics. Because treatment for nocardiosis relies heavily on chemotherapy, their intrinsic multiple drug resistance is a serious problem.Another feature of the nocardia is that many strains, even clinical isolates, also have the capability to produce bioactive molecules such as antibiotics (4, 5) and enzymes that are industrially important (6). A monobactam antibiotic, nocardicin, was isolated from Nocardia sp. (7), and a terpenoid brasilicardin with immunosuppressive activity was isolated from a clinical isolate (4).Nocardia farcinica IFM 10152 was isolated from the bronchus of a 68-year-old male Japanese patient. Despite the complicated taxonomy of the nocardia, the species N. farcinica was found to be nearly homogeneous (8). Subsequently, to elucidate the molecular basis of the versatility of the nocardia, we analyzed the genomic sequence of N. farcinica IFM 10152. MethodsGenome Sequencing and Assembly. The nucleotide sequence of the N. farcinica IFM 10152 genome was determined by a wholegenome shotgun strategy. We constructed small-insert (2 kb) and large-insert (10 kb) genomic libraries and generated 127,077 sequences (giving 9-fold coverage) from both ends of the genomic clones. Sequence assembly was carried out by using the PHRED͞PHRAP͞CONSED package (9). Remaining gaps were closed by transcriptional sequencing (Nippon Gene, Toyama, Japan) or by primer walking. There were no ambiguous nucleotides in the genomic sequence that we could determine. Consistency of the final assembly was confirmed in terms of res...

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“…With respect to carbohydrate enzymology, recent applications include the tagatose-1,6-bisphosphate aldolase modified by in vitro evolution toward an unnatural stereoselectivity (25), an evolved N-acetylneuraminic acid aldolase for L-sialic acid synthesis (26), or a 2-deoxy-D-ribose-5-phosphate aldolase with an expanded substrate range after site-directed mutagenesis (27). Usually, in vitro evolution strategies include error-prone PCR for gene diversification (28,29) and͞or locating critical amino acid residues for saturation mutagenesis (30) or, more prominently, shuffling of fragmented diversified genes or gene families according to a number of different protocols (31)(32)(33)(34)(35)(36). Subsequently, the diversified proteins are subjected to a screen.…”

mentioning

confidence: 99%

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Hoffmeister

Yang²,

Liu³

et al. 2003

Proceedings of the National Academy of Sciences

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Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK. From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as D-galacturonic acid, D-talose, L-altrose, and L-glucose, all of which failed as wild-type GalK substrates. Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme. Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme. In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.galactokinase ͉ glycorandomization ͉ in vitro evolution ͉ enzyme M any clinically important medicines are derived from glycosylated natural products, the D-or L-sugar substituents of which often dictate their overall biological activity. This paradigm is found throughout the anticancer and antiinfective arenas with representative clinical examples (Fig. 1a), including enediynes (calicheamicin, 1), polyketides (doxorubicin, 2; erythromycin, 3), indolocarbazoles (staurosporine, 4), nonribosomal peptides (vancomycin, 5), polyenes (nystatin, 6), coumarins (novobiocin, 7), and cardiac glycosides (digitoxin, 8) (1-7). Given the importance of the sugars attached to these and other biologically significant metabolites, extensive effort has been directed in recent years toward altering sugars as a means to enhance or alter natural product-based therapeutics by both in vivo and in vitro approaches (ref. 8 and references therein). Among these, in vitro glycorandomization (IVG) makes use of the inherent or engineered substrate promiscuity of nucleotidylyltransferases and glycosyltransferases to activate and attach chemically synthesized sugar precursors to various natural product scaffolds (6,7,(9)(10)(11)(12)(13)(14)(15), the advantage of which is the ability to efficiently incorporate highly functionalized ''unnatural'' sugar substitutions into the corresponding natural product scaffold (Fig. 1b). In a recent demonstration of IVG, Ͼ50 analogs of 5 were generated, some of which displayed enhanced and distinct antibacterial profiles from the parent natural product (13).As starting materials, sugar phosphates play a key role in the entire IVG process. Thus, the ability to rapidly construct sugar phosphate ...

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DNA shuffling method for generating highly recombined genes and evolved enzymes

Cited by 240 publications

References 29 publications

Genomics ofActinobacteria: Tracing the Evolutionary History of an Ancient Phylum

Genomics ofActinobacteria: Tracing the Evolutionary History of an Ancient Phylum

The complete genomic sequence of Nocardia farcinica IFM 10152

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

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