Novel Hyperthermophilic Crenarchaeon Thermofilum adornatum sp. nov. Uses GH1, GH3, and Two Novel Glycosidases for Cellulose Hydrolysis

Abstract

A novel hyperthermophilic, anaerobic filamentous archaeon, Thermofilum adornatum strain 1910b T , is capable of growing with cellulose as its sole carbon and energy source. This strain was isolated from a terrestrial hot spring in Kamchatka, Russia. The isolate 1910b T grew optimally at a temperature of 80 • C and a pH of 5.5-6.0, producing cell-bound inducible cellulases. During genome analysis, genes, encoding various glycosidases (GHs) involved in oligo-and polysaccharide hydrolysis and genes for the fermentation of sugars were identified. No homologs of currently known cellulase families were found among the GHs encoded by the 1910b T genome, suggesting that novel proteins are involved. To figure this out, a proteomic analysis of cells grown on cellulose or pyruvate (as a control) was performed. Both in-depth genomic and proteomic analyses revealed four proteins (Cel25, Cel30, Cel40, and Cel45) that were the most likely to be involved in the cellulose hydrolysis in this archaeon. Two of these proteins (Cel30 and Cel45) were hypothetical according to genome analysis, while the other two (Cel25 and Cel40) have GH3 and GH1 domains, respectively. The respective genes were heterologously expressed in Escherichia coli BL21 (DE3), and enzymatic activities of recombinant proteins were measured with carboxymethyl cellulose (CMC), Avicel and cellobiose as substrates. It was revealed that the Cel30 and Cel25 proteins were likely exoglucanases with side beta-glucosidase and endoglucanase activities, that Cel40 was a multifunctional glucanase capable of hydrolyzing beta-1,4-glucosides of various lengths, and that Cel45 was an endoglucanase with side exoglucanase activity. Taking into account that the cellulolytic activity of T. adornatum 1910b T surface protein fractions was inducible, that recombinant Cel25 and Cel30 were much less active than Cel40 and Cel45, and that their gene expressions were (almost) non-induced by CMC, we suggest that Cel40 and Cel45 play a major role in the degradation of cellulose, while Cel25 and Cel30 act only as accessory enzymes.

High Potential for Biomass-Degrading Enzymes Revealed by Hot Spring Metagenomics

“…The majority of archaea capable of cellulose degradation are affiliated to Crenarchaeota and Euryarchaeota , which also contain the majority of archaeal isolates, and have optimal temperatures ranging from 80 to 115°C when grown on cellulose ( Suleiman et al, 2020 ). Isolates and enriched communities of Crenarchaeota have been recovered from several hyperthermophilic sources, including terrestrial hot springs ( Perevalova et al, 2010 ; Graham et al, 2011 ; Zayulina et al, 2020 ). While we did not recover CAZyme containing contigs affiliated with Euryarchaeota , crenarchaeotal contigs were detected in LCB-024 and SJ3, with the latter hot spring containing more such contigs.…”

“…Lignocellulolytic enzymes and their activities have been studied in detail ( Zarafeta et al, 2016 ; Zayulina et al, 2020 ), but the discovery of new enzymes has mainly been limited to isolated microorganisms or enrichment cultures. Most cellulolytic isolates come from only three classes of bacteria, Actinobacteria , Bacilli , and Clostridia ( Bayer et al, 2013 ).…”

Degradation of switchgrass by Bacillus subtilis 1AJ3 and expression of a beta-glycoside hydrolase

“…Raw material pretreatment has always been a major bottleneck in the degradation process of lignocellulose due to its hightemperature conditions (Chen and Qiu, 2010;Mishra and Ghosh, 2017). Studies on thermophilic microorganisms have shown that their biomass degradation ability requires their optimal temperature conditions (Strang et al, 2017;Bibra et al, 2018), which can reach 80 • C (Zayulina et al, 2019) and strain 1AJ3 also can stand these high temperatures. It promised that an acidic environment might promote straw biomass degradation, highlighting the potential value of acidheat resistant strains in industrial applications.…”

Biomass-degrading glycoside hydrolases of archaeal origin

“…Improved combinatorial approaches, such as metatranscriptomic- and proteomic-based screening coupled with prior high-temperature cultivation on plant biomass, will probably have a high impact on prospective identification of novel hyperthermozymes for application in various biotechnological processes including biorefineries. The successful application of such a combinatorial approach was recently described by Zayulina and colleagues, who coupled cultivation of a novel archaeon Thermofilum adornatum with proteomic analyses to identify four novel cellulolytic enzymes [ 117 ].…”