Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases

Abstract

The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

Efficient and Supplementary Enzyme Cocktail from Actinobacteria and Plant Biomass Induction

“…In the proposed study, therefore, 850 isolates of Actinobacteria were evaluated via a three‐step screening process for their utility in a possible biomass‐degrading enzyme cocktail. Among the previous researches for biomass‐degrading enzymes from Actinobacteria , C. fimi , T. fusca , and C. flavigena have been studied extensively. In the proposed study, the halo size of each strain was ≈14.5 mm for C. fimi (NBRC15513), ≈0 mm for T. fusca (NBRC14071), and ≈15.47 mm for C. flavigena (NBRC3775) (Table ).…”

“…One indicator of this role is the smell of fertile soil from metabolites such as geosmin, and another is the pest control of rice blast via metabolites such as kasugamycin . These metabolisms have been focal points in biological engineering, but the biomass degrading titer has only been partially evaluated using lower isolates of Actinobacteria such as Thermobifida fusca ( T. fusca ), Cellulomonas fimi ( C. fimi ), Cellulomonas flavigena ( C. flavigena ), and Streptomyces lividans . Recently, progress in genomic sequencing technology enabled the genome identification of plant biomass‐degrading enzymes .…”

Green Biotechnology: A Brief Update on Plastid Genome Engineering

“…However, transplastomic lines exhibited pigment-deficient phenotypes (Petersen and Bock 2011). Klinger et al (2015) proposed that enzymes of prokaryotic origin are efficiently expressed in plants than in a bacterial production platform. Espinoza-Sánchez et al (2015) expressed pectin lyase and manganese peroxidase in tobacco plastids and recombinant enzymes exhibited improved enzyme activity.…”

The Effects of Carbon Source and Growth Temperature on the Fatty Acid Profiles of Thermobifida fusca

“…The readily available T. fusca genome sequence reveals that it has the capacity to express many enzymes useful for hydrolyzing biomass, including numerous cellulases, xylanases, and carbohydrate transporters for sugar uptake ( Lykidis et al, 2007 ). Many of these thermally stable enzymes have been heterologously expressed in alternative microbial hosts and analyzed for their applicability to biomass conversion ( Ghangas and Wilson, 1987 ; Ali et al, 2015 ; Klinger et al, 2015 ; Saini et al, 2015 ; Zhao et al, 2015 ; Setter-Lamed et al, 2017 ; Yan and Fong, 2018 ; Ali et al, 2020 ).…”