An engineered GH1 β-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 β-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 β-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis

Revealing the interaction mode of the highly flexible Sorghum bicolor Hsp70/Hsp90 organizing protein (Hop) : a conserved carboxylate clamp confers high affinity binding to Hsp90

Proteostasis is dependent on the Hsp70/Hsp90 system (the two chaperones and their co-chaperones). Of these, Hop (Hsp70/Hsp90 organizing protein), also known as Sti1, forms an important scaffold to simultaneously binding to both Hsp70 and Hsp90. Hop/Sti1 has been implicated in several disease states, for instance cancer and transmissible spongiform encephalopathies. Therefore, human and yeast homologous have been better studied and information on plant homologous is still limited, even though plants are continuously exposed to environmental stress. Particularly important is the study of crops that are relevant for agriculture, such as Sorghum bicolor, a C4 grass that is among the five most important cereals and is considered as a bioenergy feedstock. To increase the knowledge on plant chaperones, the hop putative gene for Sorghum bicolor was cloned and the biophysical and structural characterization of the protein was done by cross-linking coupled to mass spectroscopy, small angle X-ray scattering and structural modeling. Additionally, the binding to a peptide EEVD motif, which is present in both Hsp70 and Hsp90, was studied by isothermal titration calorimetry and hydrogen/deuterium exchange and the interaction pattern structurally modeled. The results indicate SbHop as a highly flexible, mainly alpha-helical monomer consisting of nine tetratricopeptide repeat domains, of which one confers high affinity binding to Hsp90 through a conserved carboxylate clamp. Moreover, the present insights into the conserved interactions formed between Hop and Hsp90 can help to design strategies for potential therapeutic approaches for the diseases in which Hop has been implicated191191201CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP305018/2015-9; 306943/2015-888887.125517/2016-002012/50161-8; 2014/17264-3; 2015/15822-1This study was funded by Fundação de Amparo do Estado de São Paulo FAPESP (2012/50161-8, 2014/17264-3 and 2015/15822-1), CNPq (305018/2015-9 and 306943/2015-8) and CAPES (88887.125517/2016-00). We thank the National Laboratory of Synchrotron Light (Campinas, SP, Brazil) and its staff for the use of SAXS beam line facilities. We acknowledge the Protein Analysis Facility (Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Switzerland) for cross-linking MS dat

A rationally identified marine GH1 β‐glucosidase has distinguishing functional features for simultaneous saccharification and fermentation

The classical route for second‐generation ethanol from lignocellulosic biomass is hampered by high process costs, fostering the development of alternative strategies such as simultaneous saccharification and fermentation (SSF). However, the lack of compatible enzyme cocktails poses a challenge. In this study, the enzyme EmBgl from the marine bacterium Exiguobacterium marinum was rationally identified based on structural and phylogenetic analyses, known desirable properties of close orthologs, and the ecological niche of its organism source. EmBgl is a multifunctional and glucose‐tolerant enzyme that efficiently hydrolyzes cello‐oligosaccharides due to a positive‐subsite region that can accommodate long cello‐oligosaccharides without imposing steric impediments. The efficacy of EmBgl in an SSF process was demonstrated using pretreated sugarcane bagasse as feedstock, yielding 28 g L−1 of ethanol in 30 h. The distinguishing functional properties of EmBgl and its successful utilization in an SSF process highlight its potential in biotechnological applications in which lignocellulose deconstruction is desirable under milder temperatures14611631179FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP17/14253‐9; 16/19995‐0; 16/06509‐0; 19/08855‐1; 15/26982‐0; 18/02865‐2; 16/06509‐0; 16/19995‐0; 17/14253‐9; 18/02865‐2; 15/26982‐0; 19/08855‐