Phylogenomically Guided Identification of Industrially Relevant GH1 β-Glucosidases through DNA Synthesis and Nanostructure-Initiator Mass Spectrometry

Abstract

Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 β-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different β-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) [C2mim][OAc] over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.

Biocatalysts for biomass deconstruction from environmental genomics

“…Targeted database searches combined with gene expression studies provide one effective route toward functional validation of known and novel CAZymes from individual genomes and metagenomes. For example, Heins and colleagues [14 ] selected and synthesized a set of 175 diverse GH1 genes from the CAZy database and a metagenomic study of the bovine rumen for characterization under biorefining relevant conditions (70 8C, 20% (v/v) ionic liquids) [15].…”

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

“…Codon optimization, including restriction site removal, and oligo design (150 mers) were performed using GeneDesign [70]. Oligonucleotides were pooled for synthesis using acoustic deposition (Labcyte Echo 550), and synthesis was performed at the Joint Genome Institute using a two-step polymerase chain assembly (PCA) approach in 2 μL final volume as previously described [71]. PCA products were purified by gel excision and cloned into a Pml I-digested pDRf1-4CL5-DsRed vector using Gibson assembly to generate the pDRf1-4CL5-BAHDs vectors.…”

A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

“…While the potential for high-throughput NIMS-based analysis of complex enzyme reaction mixtures has frequently been alluded to,sofar all experiments describing the analysis of complex samples using NIMS have been limited to small studies, [8,9,12] and all successful high-throughput NIMS experiments have been conducted with purified protein in low ionic strength buffer,avoiding the need for in situ fluorous affinity purification. [43,45,46] Thescreening campaign described here is the first demonstration of ah igh-throughput NIMS-based analysis of enzyme activity in ac omplex matrix.…”