Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends

Abstract

SummaryRecent reports have suggested that the establishment of industrially relevant enzyme collections from environmental genomes has become a routine procedure. Across the studies assessed, a mean number of approximately 44 active clones were obtained in an average size of approximately 53 000 clones tested using naïve screening protocols. This number could be significantly increased in shorter times when novel metagenome enzyme sequences obtained by direct sequencing are selected and subjected to high‐throughput expression for subsequent production and characterization. The pre‐screening of clone libraries by naïve screens followed by the pyrosequencing of the inserts allowed for a 106‐fold increase in the success rate of identifying genes encoding enzymes of interest. However, a much longer time, usually on the order of years, is needed from the time of enzyme identification to the establishment of an industrial process. If the hit frequency for the identification of enzymes performing at high turnover rates under real application conditions could be increased while still covering a high natural diversity, the very expensive and time‐consuming enzyme optimization phase would likely be significantly shortened. At this point, it is important to review the current knowledge about the success of fine‐tuned naïve‐ and sequence‐based screening protocols for enzyme selection and to describe the environments worldwide that have already been subjected to enzyme screen programmes through metagenomic tools. Here, we provide such estimations and suggest the current challenges and future actions needed before environmental enzymes can be successfully introduced into the market.

Relationships between Substrate Promiscuity and Chiral Selectivity of Esterases from Phylogenetically and Environmentally Diverse Microorganisms

“…Promiscuous esterases acting against multiple substrates, while at the same time being enantio-selective, appear to be rare in nature, or at least in the habitats from where the esterases herein described were isolated [5]. As these enzymes are of interest for application purposes [1][2][3][4][5][6]32], protein engineering and rational design may be needed to obtain esterases being promiscuous and selective for industrial applications. We anticipate that the possibility to transform a promiscuous but not selective esterase into an efficient enantio-selective biocatalyst would require less engineering effort because increasing the selectivity for an enantiomer may involve a reduced number of contacts close to the active sites (for a recent example see reference [33]).…”

“…Along with requirements of a technical nature, such as process development and optimization, it is however widely recognized that the establishment of enzymatic processes is mainly a problem of finding, optimizing, or designing new and/or better performing enzymes. Nature is a rich reservoir from where enzymes can be isolated [3,4], because they are continuously evolving as a consequence of natural selection. Promiscuous enzymes are effective for converting multiple substrates, thus, they are industrially relevant [4][5][6].…”

“…Nature is a rich reservoir from where enzymes can be isolated [3,4], because they are continuously evolving as a consequence of natural selection. Promiscuous enzymes are effective for converting multiple substrates, thus, they are industrially relevant [4][5][6]. Enzymes need to also be robust and, preferably, chiral selective to reduce raw material costs in the synthesis of pure chiral compounds [1,2,4,7].…”

“…Promiscuous enzymes are effective for converting multiple substrates, thus, they are industrially relevant [4][5][6]. Enzymes need to also be robust and, preferably, chiral selective to reduce raw material costs in the synthesis of pure chiral compounds [1,2,4,7]. That is, they need to be able to cleave preferentially only one chiral ester when offered a racemic mixture.…”

“…The activity of these esterases relies mainly on a catalytic triad usually formed by Ser, Asp/Glu, and His [8]. This enzyme class was selected for a number of reasons: it is widely distributed in the environment, it has important physiological functions, it includes hydrolases that are among the most important industrial biocatalysts, and extensive biochemical knowledge has been accumulated [4,5,7].…”