Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

Chen, I-Chien; Lin, Wei‐De; Hsu, S.K.; Venkatesan, T.; Hsu, Wen-Hwei

doi:10.1128/aem.00074-09

Cited by 34 publications

(10 citation statements)

References 46 publications

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“…The racemization of chiral lysine by Lyr was analyzed by high-performance liquid chromatography using Crown CR (?) column (Daicel Chemical Industries Ltd, Japan) at an absorbance of 200 nm (Chen et al 2009). …”

Section: Lyr Activity Assaymentioning

confidence: 99%

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Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

et al. 2009

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A non-antibiotic based selection system using L-lysine as selection agent and the lysine racemase (lyr) as selectable marker gene for plant transformation was established in this study. L-lysine was toxic to plants, and converted by Lyr into D-lysine which would subsequently be used by the transgenic plants as nitrogen source. Transgenic tobacco and Arabidopsis plants were successfully recovered on L-lysine medium at efficiencies of 23 and 2.4%, respectively. Phenotypic characterization of transgenic plants clearly revealed the expression of normal growth and developmental characteristics as that of wild-type plants, suggesting no pleiotropic effects associated with the lyr gene. The specific activity of Lyr in transgenic tobacco plants selected on L: -lysine ranged from 0.77 to 1.06 mU/mg protein, whereas no activity was virtually detectable in the wild-type plants. In addition, the composition of the free amino acids, except aspartic acid, was not affected by the expression of the lyr gene in the transgenic tobacco plants suggesting very limited interference with endogenous amino acid metabolism. Interestingly, our findings also suggested that the plant aspartate kinases may possess an ability to distinguish the enantiomers of lysine for feedback regulation. To our knowledge, this is the first report to demonstrate that the lysine racemase selectable marker system is novel, less controversial and inexpensive than the traditional selection systems.

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Section: Lyr Activity Assaymentioning

confidence: 99%

“…The lyr gene (NCBI Accession Number FJ405258) was isolated from a soil metagenome as described previously (Chen et al 2009) and cloned into the TA cloning vector pGEM-T (Promega). The PCR primers 5 0 -CGCGGATCC ATGGCGCATACTGGCCG-3 0 (forward) and 5 0 -CCAAG CTTGGCTTACGGTTGCAGGC-3 0 (reverse) were used to amplify the lyr fragment.…”

Section: Vector Constructionmentioning

confidence: 99%

Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

et al. 2009

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“…Lysine racemization is performed by two different enzymes: those acting preferentially on Lys and Arg (namely Lys racemases) and broad spectrum enzymes that are also active against other amino acids (Wu et al ., ; Espaillat et al ., ). Although Lys racemase activity was reported almost 50 years ago (Huang et al ., ), isolation and characterization of a Lys racemase was only accomplished recently from soil metagenomic studies (Chen et al ., ). This study has been followed by the characterization of additional Lys racemases, from Oenococcus oeni (Kato et al ., ) and Proteus mirabilis (Kuan et al ., ), whose activity, by contrast, appears to be PLP dependent.…”

Section: Lysine Racemasesmentioning

confidence: 99%

Environmental roles of microbial amino acid racemases

Hernández

Cava

2015

Environmental Microbiology

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SummaryEnzymes catalysing the stereo-chemical interconversion of amino acids are known as amino acid racemases. In bacteria, these enzymes are fundamental to synthesize the D-Ala and D-Glu that are critical components of the peptidoglycan. In addition to this structural function in cell wall assembly, D-amino acids produced by microbial amino acid racemases have been described as relevant constituents in other prokaryotic structures (e.g. capsule, non-ribosomal peptides) and have been associated to growth fitness and to processes such as biofilm development, spore germination and signalling. The recent discovery of broad spectrum racemases able to produce and release several D-amino acids to the environment suggests that these enzymes might have a great impact in microbial ecology. Consequently, new data on the biochemistry and regulation of racemases is key to understand the biological significance of D-enantiomers in nature, in particular their effect on microbial social networks. This review summarizes current knowledge on the environmental roles of bacterial racemases with an emphasis on the potential roles of the new broad spectrum enzymes in natural environments.

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“…Growth is exclusively observed in the case of recombinant clones that possess the gene of interest and produce an active product. This strategy has been applied for the detection of enzymes involved in poly-3-hydroxybutyrate metabolism (Wang et al, 2006), DNA polymerase I (Simon et al, 2009), operons for biotin biosynthesis (Entcheva et al, 2001), lysine racemases (Chen et al, 2009), glycerol dehydratases (Knietsch et al, 2003) and naphthalene dioxygenase (Ono et al, 2007). …”

Section: Metagenomic Library Screeningmentioning

confidence: 99%

Size does matter: Application-driven approaches for soil metagenomics

Kakirde

Parsley

Liles

2010

Soil Biology and Biochemistry

106

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Metagenomic analyses can provide extensive information on the structure, composition, and predicted gene functions of diverse environmental microbial assemblages. Each environment presents its own unique challenges to metagenomic investigation and requires a specifically designed approach to accommodate physicochemical and biotic factors unique to each environment that can pose technical hurdles and/or bias the metagenomic analyses. In particular, soils harbor an exceptional diversity of prokaryotes that are largely undescribed beyond the level of ribotype and are a potentially vast resource for natural product discovery. The successful application of a soil metagenomic approach depends on selecting the appropriate DNA extraction, purification, and if necessary, cloning methods for the intended downstream analyses. The most important technical considerations in a metagenomic study include obtaining a sufficient yield of high-purity DNA representing the targeted microorganisms within an environmental sample or enrichment and (if required) constructing a metagenomic library in a suitable vector and host. Size does matter in the context of the average insert size within a clone library or the sequence read length for a highthroughput sequencing approach. It is also imperative to select the appropriate metagenomic screening strategy to address the specific question(s) of interest, which should drive the selection of methods used in the earlier stages of a metagenomic project (e.g., DNA size, to clone or not to clone). Here, we present both the promising and problematic nature of soil metagenomics and discuss the factors that should be considered when selecting soil sampling, DNA extraction, purification, and cloning methods to implement based on the ultimate study objectives.

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Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

Cited by 34 publications

References 46 publications

Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

Environmental roles of microbial amino acid racemases

Size does matter: Application-driven approaches for soil metagenomics

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