Discovering novel hydrolases from hot environments

Abstract

Novel hydrolases from hot and other extreme environments showing appropriate performance and/or novel functionalities, and new approaches for their systematic screening are of great interest for developing new processes, for improving safety, health and environment issues. Existing processes could benefit as well from their properties. The workflow, based on the HotZyme project, describes a multitude of technologies and their integration from discovery to application, providing new tools for discovering, identifying and characterizing more novel thermostable hydrolases with desired functions from hot terrestrial and marine environments. To this end, hot springs worldwide were mined, resulting in hundreds of environmental samples and thousands of enrichment cultures growing on polymeric substrates of industrial interest. Using high-throughput sequencing and bioinformatics, 15 hot spring metagenomes, as well as several sequenced isolate genomes and transcriptomes were obtained. To facilitate the discovery of novel hydrolases, the annotation platform Anastasia and a whole-cell bioreporter-based functional screening method were developed. Sequence-based screening and functional screening together resulted in about 100 potentially new hydrolases of which more than a dozen have been characterized comprehensively from a biochemical and structural perspective. The characterized hydrolases include thermostable carboxylesterases, enol lactonases, quorum sensing lactonases, gluconolactonases, epoxide hydrolases, and cellulases. Apart from these novel thermostable hydrolases, the project generated an enormous amount of samples and data, thereby allowing the future discovery of even more novel enzymes.

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

“…Indeed, no HSDHs have been characterized so far from extremophilic microorganisms, e. g., thermophiles and halophiles. However, enzymes from these sources may have practical interest exceeding the scope of the present work, since, thanks to their higher robustness, they could be more suitable than mesophilic enzymes to industrial applications …”

“…The search of novel HSDH sequences was carried out by using either the Blastp tool (https://blast.ncbi.nlm.nih.gov/) for mining into the NCBI database (https://www.ncbi.nlm.nih.gov/) or the program LAST (http://last.cbrc.jp/) for the analysis of metagenomes of samples collected in hot terrestrial environments . In both cases, only sequences of functionally characterized HSDHs (Table S1 in the Supporting Information) were used as queries during the multiple sequence alignments.…”

XynDZ5: A New Thermostable GH10 Xylanase

“…Such organisms are encountered in habitats with temperatures above 60 • C, where they degrade plant biomass through the action of an extensive repertoire of enzymes, mainly GHs. Environmental sampling followed by culture-free or culture-dependent DNA extraction and functional in vivo screening or bioinformatic analysis, have proven powerful strategies toward the discovery of industrially relevant biocatalysts (Zarafeta et al, 2016b,c;Wohlgemuth et al, 2018).…”

Engineering acyl-homoserine lactone-interfering enzymes toward bacterial control

“…Indeed, this strain is impaired (by transposon insertions) in AHL synthesis (40), but its endogenous QS system is functional, and QS activation leads to the production of the purple pigment violacein, which acts as an antibiotic at high cell density. Therefore, it can be used for screening purposes and responds best to exogenous AHLs with chain lengths ranging from C4-HSL to C8-HSL, including OC6-HSL and OC8-HSL (41)(42)(43). In particular, C. violaceum CV026 was used to screen a metagenomic library of 250,000 clones from a hypersaline soil located in Spain with a pool strategy of 50 clones/well, which resulted in the identification of a single lactonase, called HqiA, which had no homology with any known lactonase or acylase.…”