Expression of Trichoderma reesei β-mannanase in tobacco chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis

Abstract

Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost-effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, the man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds in the selection medium showed inheritance of transgenes into the progeny without any Mendelian segregation. Expression of endo-β-mannanase for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-β-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-β-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40 °C to 70 °C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6-7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different plant-derived enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the biofuel, paper, oil, pharmaceutical, coffee and detergent industries.

Figure 1. Characterization of transplastomic plants.

A & B, Schematic representation of chloroplast flanking sequences used for homologous recombination, probe DNA sequence (0.81 kb), primer annealing sites (3P/3M and 5P/2M) and expected products of untransformed and transgenic lines when digested with ApaI. Prrn, rRNA operon promoter; aadA, aminoglycoside 3′ adenylyltransferase gene; PpsbA, promoter and 5′ untranslated region of the psbA gene; tpsbA, 3′ untranslated region of the psbA gene. C, PCR analysis using primer pairs 3P/3M and D, 5P/2M. Lanes 1–3, transplastomic lines; UT, Untransformed (−ve C); +C, positive control for 3P/3M confirmed established transplastomic line, for 5P/2M pLD man1; 1 kb, 1 kb plus DNA ladder. E, Southern blot hybridized with the flanking sequence probe. Lanes 1–2, transplastomic lines; UT, Untransformed. F, Southern blot hybridized with man1 probe. Lanes 1–2, transplastomic lines; UT, Untransformed.

Figure 2. Phenotype of transplastomic mannanase plants.

A, Mannanase transplastomic plant growing autotrophically in the green house. Mannanase plants were fertile and set seeds. B, Untransformed (UT) plant C, Transplastomic (man1) seeds and Untransformed (UT) seeds germinated on MSO medium containing spectinomycin (500 mg/l) showing lack of Mendelian segregation.

Figure 3. Gel diffusion assay for mannanase activity.

Agar plate with 0.1% locust bean gum substrate stained with Congo red dye to evaluate mannanase activity. rMan, 100 µg of E.coli-derived mannanase crude extract; cpMan 100 µg of leaf extract from different transplastomic plant lines (Plant 1, 2 & 3); UT E.coli, Untransformed E.coli extract; UT plant, Untransformed plant extract; A.niger man, purified Aspergillus niger mannanase (Megazyme).

Figure 4. Characterization of chloroplast-derived mannanase.

A, Effect of increasing locust bean gum concentration on cpMan activity. B, Substrate locust bean gum (0.5%) incubated with crude enzyme extracts in increasing concentrations of total soluble protein at 70°C, pH 5.0 in a reaction for 16 hours. C, Effect of incubation time on cpMan activity. D, Effect of pH on cpMan and rMan activity. E, Effect of temperature on cpMan and rMan activity. 30 µg of total soluble protein was incubated with 0.5% of locust bean gum at indicated reaction parameters for 2 hrs. rMan, E.coli-derived mannanase crude extract; cpMan, leaf extract from transplastomic plants; UT E.coli, Untransformed E.coli extract; UT plant, untransformed plant extract. (Error bars indicates the standard deviation; n = 3).

Figure 5. Enzyme cocktail for pinewood hydrolysis.

Pinewood (200 mg/5 ml) hydrolysis using different formulations of crude enzyme cocktails. Glucose equivalents released were quantified using DNS method. 200 ug TSP of crude chloroplast derived enzyme extracts were used. Man, Mannanase; Xyn, Xylanase; Axe, Acetyl xylan esterase; CelD, Endoglucanase; CelO, Exoglucanase; Bgl, β glucosidase; Eg1, Endoglucanase; Swo, Swollenin; Pel A, B, D, Pectate lyase. (Error bar indicates standard deviation among triplicates, * p value = 0.038, ** p value = 0.013, p value were calculated using t- test).