Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

Rabausch, Ulrich; Jüergensen, Julia; Ilmberger, Nele; Böhnke, Stefanie; Fischer, Simon; Schubach, B; Schulte, Michael; Streit, Wolfgang R.

doi:10.1128/aem.01077-13

Cited by 70 publications

(25 citation statements)

References 73 publications

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“…To date, no thermolabile APs from metagenomes have been isolated or characterized. This might be due to the limited availability of sensitive and reliable screening systems, which enable the screening of large libraries of metagenome clones [ 17 , 18 ]. Recently, we developed a genetic circuit termed the Genetic Enzyme Screening System (GESS) that allows for the measurement of enzyme activity as in vivo fluorescence intensity and is highly useful for high-throughput metagenomic screening of enzymes [ 19 ].…”

Section: Introductionmentioning

confidence: 99%

A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments

et al. 2015

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BackgroundAlkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Here, we report on the first isolation and biochemical characterization of a thermolabile AP from a metagenome.ResultsThe gene encoding a novel AP was isolated from a metagenomic library constructed with ocean-tidal flat sediments from the west coast of Korea. The metagenome-derived AP (mAP) gene composed of 1,824 nucleotides encodes a polypeptide with a calculated molecular mass of 64 kDa. The deduced amino acid sequence of mAP showed a high degree of similarity to other members of the AP family. Phylogenetic analysis revealed that the mAP is shown to be a member of a recently identified family of PhoX that is distinct from the well-studied classical PhoA family. When the open reading frame encoding mAP was cloned and expressed in recombinant Escherichia coli, the mature mAP was secreted to the periplasm and lacks an 81-amino-acid N-terminal Tat signal peptide. Mature mAP was purified to homogeneity as a monomeric enzyme with a molecular mass of 56 kDa. The purified mAP displayed typical features of a psychrophilic enzyme: high catalytic activity at low temperature and a remarkable thermal instability. The optimal temperature for the enzymatic activity of mAP was 37°C and complete thermal inactivation of the enzyme was observed at 65°C within 15 min. mAP was activated by Ca2+ and exhibited maximal activity at pH 9.0. Except for phytic acid and glucose 1-phosphate, mAP showed phosphatase activity against various phosphorylated substrates indicating that it had low substrate specificity. In addition, the mAP was able to remove terminal phosphates from cohesive and blunt ends of linearized plasmid DNA, exhibiting comparable efficiency to commercially available APs that have been used in molecular biology.ConclusionsThe presented mAP enzyme is the first thermolabile AP found in cold-adapted marine metagenomes and may be useful for efficient dephosphorylation of linearized DNA.

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Section: Introductionmentioning

confidence: 99%

A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments

et al. 2015

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“…For more than a decade, metagenome research has demonstrated that it is a powerful tool for the discovery of novel biocatalysts and other valuable biomolecules by using either function or sequence-based screening technologies, addition to its impact on bacterial biodiversity. Sequence-based approaches allow the identification of candidate genes [24]. In the last years were studies initiated to investigate the metagenome [13].…”

Section: Discussionmentioning

confidence: 99%

Strategies for the Extraction, Purification and Amplification of Metagenomic DNA from Soil Growing Sugarcane

Gutiérrez-Lucas¹,

Montor-Antonio²,

Cortés-López³

et al. 2014

ABC

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Recently, studies were initiated to investigate the metagenome, which represents the genomes of cultured and uncultured microbes, as a rich source for isolation of many novel genes. The metagenomic approach originated from the molecular analysis of microbial communities, which revealed that the majority of microorganisms in nature were not cultivable by standard culturing techniques. Therefore, most microorganisms in nature have not been characterized. Although numerous methods have been reported for direct DNA isolation and purification from microorganisms in soil, the sample preparation procedures and experimental conditions used in different studies vary widely. Soils are therefore one of the most challenging environmental matrices from which to obtain microbial DNA that will support PCR. The Papaloapan River is the second largest river basin in México. For the climatic conditions of this region, there is great diversity in plants, animals and microorganisms. In the Papaloapan region different fruits are grown, however, the main crops are sugarcane and pineapple. In this work the extraction of DNA from soils of sugarcane cultivation was performed. We used PCR tests to assess the quality of DNA extracted from soil by amplifying the 16S rDNA gene. Changes in both protocols were performed; satisfactory results were obtained as to the quality of DNA and gene amplification. These results will allow continuing the metagenomic studies, such as sequencing, library construction and identification of enzymes cellulase and amylase activity. It is the first time these studies were performed in the Papaloapan region.

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“…Other approaches and techniques that have been successfully utilized for fine-tuning gene expression for the needs of metabolic engineering were thoroughly reviewed in [192]. Furthermore, a new screening technique based on high-performance thin-layer chromatography (HPTLC) has been developed for the discovery of flavonoid-modifying enzymes [193]. The authors claim that this metagenome extract thin-layer chromatography analysis (META) allows rapid detection of glycosyltransferases and other flavonoid-decorating enzymes, as well as that the system is highly sensitive, being able to detect of as little as 4 ng of a modified molecule.…”

Section: Strategies For Improving Production Of Polyphenolic Compoundmentioning

confidence: 99%

Engineering of Microbial Cell Factories for the Production of Plant Polyphenols with Health-Beneficial Properties

Dudnik

Gaspar

Neves

et al. 2018

CPD

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Polyphenols form a group of important natural bioactive compounds with numerous ascribed healthbeneficial attributes (e.g. antioxidant, anti-inflammatory, anti-microbial and tumor-suppressing properties). Some polyphenols can also be used as natural dyes or plastic precursors. Notwithstanding their relevance, production of most of these compounds still relies on extraction from plant material, which for most of it is a costly and an inefficient procedure. The use of microbial cell factories for this purpose is an emerging alternative that could allow a more efficient and sustainable production. The most recent advances in molecular biology and genetic engineering, combined with the ever-growing understanding of microbial physiology have led to multiple success stories. Production of multiple polyphenolic compounds or their direct precursors has been achieved not only in the common production hosts, such as Escherichia coli and Saccharomyces cerevisiae, but also in Corynebacterium glutamicum and Lactococcus lactis. However, boosting production of native compounds or introduction of heterologous biosynthetic pathways also brings certain challenges, such as the need to express, balance and maintain efficient precursor supply. This review will discuss the most recent advances in the field of metabolic engineering of microorganisms for polyphenol biosynthesis and its future perspectives, as well as outlines their potential health benefits and current production methods.

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Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

Cited by 70 publications

References 73 publications

A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments

A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments

Strategies for the Extraction, Purification and Amplification of Metagenomic DNA from Soil Growing Sugarcane

Engineering of Microbial Cell Factories for the Production of Plant Polyphenols with Health-Beneficial Properties

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