Hyperthermophilic endoglucanase for in planta lignocellulose conversion

Abstract

BackgroundThe enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass.ResultsHere we show that a cellulase gene (sso1354) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade α-cellulose or even complex cell wall preparations under those pretreatment conditions.ConclusionThe hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.

Co-expression of Cellulases in the Chloroplasts of Nicotiana tabacum

“…An alternative method to prevent damage to the plant host is the use of cellulolytic enzymes derived from thermophilic hosts, such as Thermobifida fusca or Sulfolobus solfataricus, which show minimal or no activity when expressed at the mesophilic temperatures at which the transformed plants grow, with the cellulases activated by a rise in temperature [37,59]. Another possible method is to use inducible promoters for target gene expression.…”

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

“…Recombinant T inact values 10-158C > T opt . Details can be found in [63,64,67,68]. Abbreviations: T inact , temperature at which enzyme are 50% inactive; T opt , optimum temperature for activity.…”

“…Enzyme activities are effectively switched on as processing temperatures rise, and the integrated activity (a function of both the longevity of enzyme survival and the specific activities achieved) should, theoretically, be much greater for hyperthermophilic enzymes than for mesophilic enzymes [35,47,67]. This effect could be further enhanced by modifying the heating profile so as to optimize the activity of the particular recombinant enzyme.…”

“…The expression of a hyperthermophilic endoglucanase (SSO1354) has been successfully targeted to the apoplast using the plant codon optimized murine antibody mAb24 (LPH) signal peptide driven by the widely used CaMV double 35S promoter with a polyadenylation signal (pA35S) at the C terminus and a 5 0 -untranslated region (UTR) translational enhancer from the Petroselinum hortense chalcone synthase gene combined with an endoplasmic reticulum retention sequence (KDEL) [67]. However, yields of 1.1% TSP are lower than have been reported for other cellulolytic enzymes in plant expression systems (Table S1 in the supplementary material online), which can be improved by codon optimization of the gene corresponding to this enzyme.…”

The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

“…However, the use of plant-expressed CBH as an additive to a commercial cellulase mixture may offer the best option to substantially reduce the costs of enzymes for lignocellulosic saccharification [24][25][26]. There are few studies describing the ability of plant-expressed cellulase to hydrolyse cellulose in pretreated biomass, either alone [27][28][29][30] or in combination with sub-optimal doses of commercial cellulase mixtures [31]. The present study complements these studies and is an extension of our previous work [23], wherein we examined saccharification of bagasse pretreated using dilute NaOH, H 2 SO 4 , HCl, and acidified glycerol at the pilot scale.…”