In vivo synergistic activity of a CAZyme cassette from Acidothermus cellulolyticus significantly improves the cellulolytic activity of the C. bescii exoproteome

Abstract

The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of Caldicellulosiruptor bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In this work, co-expression of the A. cellulolyticus Acel_0615 β-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the β-glucanase alone. Our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP. Biotechnol. Bioeng. 2017;114: 2474-2480. © 2017 Wiley Periodicals, Inc.

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

“…Initially, modular Caldicellulosiruptor β-glucosidases were introduced in H. thermocellum to relieve cellobiose inhibition of cellulases [103][104][105], and in turn, two different enzymes, a β-glucosidase from A. cellulolyticus [135] and cellobiose phosphorylase [136], were produced in C. bescii, which increased the efficiency of cellulose hydrolysis. An important first step in increasing cellulolytic activity in C. bescii was demonstrated by chromosomal expression of a β-glucanase and/or endoglucanse from A. cellulolyticus, which resulted in synergistic increases in cellulose hydrolysis, and over a 17-fold improvement of C. bescii growth on cellulose [137]. Heterologous expression of a cellobiose phosphorylase from Thermotoga maritima to relieve cellobiose inhibition improved the performance of the C. bescii endoglucanase knock-in strain [91], and the best gains in cellulolytic capacity were observed when all three enzymes (β-glucanase, endoglucanse, and cellobiose phosphorylase) were co-expressed in C. bescii [138].…”

Innovations in CAZyme gene diversity and its modification for biorefinery applications

“…To refine the efficiency of Caldicellulosiruptor bescii as a plant degrader, CAZyme from Acidothermus cellulolyticus was introduced to act synergistically with the C. bescii exproteome. The study demonstrated that E1 endo-1,4-β- d -glucanase (GH5) of Acidothermus cellulolyticus acting in concert with CelA , increased the exproteome activity of Caldicellulosiruptor bescii on plant biomass [ 175 ].…”

Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus

“…Additionally, some of these enzymes show synergy with the more classical CAZymes with simpler architectures [ 7 ] in vitro and in vivo. For example, heterologous expression of the multifunctional GuxA (GH6/GH12) from A. cellulolyticus, in Caldicellulosiruptor bescii resulted in enhanced growth and cellulolytic activity of its exoproteome, but only in the presence of the endoglucanase, E1, from A. cellulolyticus [ 12 , 13 ]. E1 is a highly active and well characterized GH5 endoglucanase that was shown to exhibit high levels of synergy with several exoglucanases [ 12 , 13 ].…”

“…For example, heterologous expression of the multifunctional GuxA (GH6/GH12) from A. cellulolyticus, in Caldicellulosiruptor bescii resulted in enhanced growth and cellulolytic activity of its exoproteome, but only in the presence of the endoglucanase, E1, from A. cellulolyticus [ 12 , 13 ]. E1 is a highly active and well characterized GH5 endoglucanase that was shown to exhibit high levels of synergy with several exoglucanases [ 12 , 13 ]. GuxA on the other hand contains an N-terminal GH6 domain linked to a CBM3 followed by a GH12 catalytic domain linked to a CBM2 on the C-terminal end.…”