Dataset Information

Biocatalyst Screening with a Twist: Application of Oxygen Sensors Integrated in Microchannels for Screening Whole Cell Biocatalyst Variants.

ABSTRACT: Selective oxidative functionalization of molecules is a highly relevant and often demanding reaction in organic chemistry. The use of biocatalysts allows the stereo- and regioselective introduction of oxygen molecules in organic compounds at milder conditions and avoids the use of complex group-protection schemes and toxic compounds usually applied in conventional organic chemistry. The identification of enzymes with the adequate properties for the target reaction and/or substrate requires better and faster screening strategies. In this manuscript, a microchannel with integrated oxygen sensors was applied to the screening of wild-type and site-directed mutated variants of naphthalene dioxygenase (NDO) from Pseudomonas sp. NICB 9816-4. The oxygen sensors were used to measure the oxygen consumption rate of several variants during the conversion of styrene to 1-phenylethanediol. The oxygen consumption rate allowed the distinguishing of endogenous respiration of the cell host from the oxygen consumed in the reaction. Furthermore, it was possible to identify the higher activity and different reaction rate of two variants, relative to the wild-type NDO. The meander microchannel with integrated oxygen sensors can therefore be used as a simple and fast screening platform for the selection of dioxygenase mutants, in terms of their ability to convert styrene, and potentially in terms of substrate specificity.

SUBMITTER: Fernandes AC

PROVIDER: S-EPMC6027248 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Biocatalyst Screening with a Twist: Application of Oxygen Sensors Integrated in Microchannels for Screening Whole Cell Biocatalyst Variants.

Fernandes Ana C AC Halder Julia M JM Nestl Bettina M BM Hauer Bernhard B Gernaey Krist V KV Krühne Ulrich U

Bioengineering (Basel, Switzerland) 20180409 2

Selective oxidative functionalization of molecules is a highly relevant and often demanding reaction in organic chemistry. The use of biocatalysts allows the stereo- and regioselective introduction of oxygen molecules in organic compounds at milder conditions and avoids the use of complex group-protection schemes and toxic compounds usually applied in conventional organic chemistry. The identification of enzymes with the adequate properties for the target reaction and/or substrate requires bette ...[more]

PMID: 29642515

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Project description:BackgroundStallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions.MethodsGene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human und mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologous genes were searched in whole genome sequence data of seven stallions and four mares and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen 2 software. All genetic variants with the missing homozygous mutant genotype were genotyped on 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis was employed for an association analysis with de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT).ResultsWe screened next generation sequenced data of whole genomes from 11 horses for equine genetic variants in 1194 human and mice genes involved in male fertility and linked through common gene ontology (GO) with male reproductive processes. Variants were filtered for high-impact on protein structure and validated through SIFT and Polyphen 2. Only those genetic variants were followed up when the homozygote mutant genotype was missing in the detection sample comprising 11 horses. After this filtering process, 17 single nucleotide polymorphism (SNPs) were left. These SNPs were genotyped in 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. An association analysis in 216 Hanoverian stallions revealed a significant association of the splice-site disruption variant g.37455302G>A in NOTCH1 with the de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). For 9 high-impact variants within the genes CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C) absence of the homozygous mutant genotype in the validation sample of all 337 fertile stallions was obvious. Therefore, these variants were considered as potentially deleterious factors for stallion fertility.ConclusionsIn conclusion, this study revealed 17 genetic variants with a predicted high damaging effect on protein structure and missing homozygous mutant genotype. The g.37455302G>A NOTCH1 variant was identified as a significant stallion fertility locus in Hanoverian stallions and further 9 candidate fertility loci with missing homozygous mutant genotypes were validated in a panel including 19 horse breeds. To our knowledge this is the first study in horses using next generation sequencing data to uncover strong candidate factors for stallion fertility.

Dataset Information

Biocatalyst Screening with a Twist: Application of Oxygen Sensors Integrated in Microchannels for Screening Whole Cell Biocatalyst Variants.

Publications

Biocatalyst Screening with a Twist: Application of Oxygen Sensors Integrated in Microchannels for Screening Whole Cell Biocatalyst Variants.

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