title: Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.
abstract: 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.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology Toyama Prefectural University Imizu Toyama Japan.
authors.forename: Nobuya
authors.initials: N
authors.lastname: Itoh
authors.affiliation: Biotechnology Research Center and Department of Biotechnology Toyama Prefectural University Imizu Toyama Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Kazama
authors.affiliation: Biotechnology Research Center and Department of Biotechnology Toyama Prefectural University Imizu Toyama Japan.
authors.forename: Nami
authors.initials: N
authors.lastname: Takeuchi
authors.affiliation: Biotechnology Research Center and Department of Biotechnology Toyama Prefectural University Imizu Toyama Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Isotani
authors.affiliation: Biotechnology Research Center and Department of Biotechnology Toyama Prefectural University Imizu Toyama Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Kurokawa
keywords: bioprospecting; enzyme engineering; gene‐specific amplicons; metagenome; phenylacetaldehyde reductase