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Gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases

Screening gene‐specific amplicons from metagenomes (S‐GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the prod...

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Autores principales: Itoh, Nobuya, Kazama, Miki, Takeuchi, Nami, Isotani, Kentaro, Kurokawa, Junji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887972/
https://www.ncbi.nlm.nih.gov/pubmed/27419059
http://dx.doi.org/10.1002/2211-5463.12067
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author Itoh, Nobuya
Kazama, Miki
Takeuchi, Nami
Isotani, Kentaro
Kurokawa, Junji
author_facet Itoh, Nobuya
Kazama, Miki
Takeuchi, Nami
Isotani, Kentaro
Kurokawa, Junji
author_sort Itoh, Nobuya
collection PubMed
description Screening gene‐specific amplicons from metagenomes (S‐GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full‐length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence‐ and activity‐based screening of these libraries identified different homologous par genes, Hpar‐001 to ‐036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error‐prone PCR to exhibit increased activity in the presence of concentrated 2‐propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM‐based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM‐based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies.
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spelling pubmed-48879722016-07-14 Gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases Itoh, Nobuya Kazama, Miki Takeuchi, Nami Isotani, Kentaro Kurokawa, Junji FEBS Open Bio Research Articles Screening gene‐specific amplicons from metagenomes (S‐GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full‐length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence‐ and activity‐based screening of these libraries identified different homologous par genes, Hpar‐001 to ‐036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error‐prone PCR to exhibit increased activity in the presence of concentrated 2‐propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM‐based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM‐based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies. John Wiley and Sons Inc. 2016-05-13 /pmc/articles/PMC4887972/ /pubmed/27419059 http://dx.doi.org/10.1002/2211-5463.12067 Text en © 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Itoh, Nobuya
Kazama, Miki
Takeuchi, Nami
Isotani, Kentaro
Kurokawa, Junji
Gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases
title Gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases
title_full Gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases
title_fullStr Gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases
title_full_unstemmed Gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases
title_short Gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases
title_sort gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887972/
https://www.ncbi.nlm.nih.gov/pubmed/27419059
http://dx.doi.org/10.1002/2211-5463.12067
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