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.

Ultrahigh‐Throughput Detection of Enzymatic Alcohol Dehydrogenase Activity in Microfluidic Droplets with a Direct Fluorogenic Assay

“…Despite the importance of ADHs in biocatalysis for synthetic chemistry, [13b,c] combinatorial exploration of libraries has thus far either relied on in vivo selections (based on growth advantages through release of a carbon source by the enzymatic reaction [20] ) or relatively low throughput assays using colorimetric detection on agar or microtiter plates. [20b] As the chances of finding ADHs in metagenomic samples are likely to be low and high throughput assays are as yet unavailable, [21] sequence-based approaches [22] have naturally dominated enzyme discovery over functional metagenomic strategies in the past, despite promising examples for the latter at ultrahigh throughput. [4b] In this work, we have demonstrated enzyme screens suitable for discovery of novel ADHs in functional metagenomics or in directed evolution [23] experiments.…”

Construction and characterization of a functional chimeric laccase from metagenomes suitable as a biocatalyst

“…S-GAM is defined by steps 1 and 3, which enable omission of time-consuming subcloning and expression optimization procedures. To date, at least three enzyme genes including short-chain alcohol dehydrogenase (Itoh et al 2014 ), zinc-dependent medium-chain alcohol dehydrogenase (Itoh et al 2016b ), and styrene monooxygenase genes have been isolated by this method and characterized. In this study, we applied this approach to bacterial MCO genes in the soil environment because bacterial MCOs represented by Bacillus sp.…”

“…The screening of gene-specific amplicons from metagenomes (S-GAM) approach we have developed is a powerful technique for the efficient isolation of target genes from metagenomes (Itoh et al 2014 , 2016b ; Itoh 2017 ). This approach can overcome a major disadvantage of previous techniques, namely, low efficiency in obtaining target genes from metagenomes.…”