Production of hyperthermostable GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants enables complete hydrolysis of methylglucuronoxylan to fermentable sugars for biofuel production

Abstract

Overcoming the recalcitrance in lignocellulosic biomass for efficient hydrolysis of the polysaccharides cellulose and hemicellulose to fermentable sugars is a research priority for the transition from a fossilfuel-based economy to a renewable carbohydrate economy. Methylglucuronoxylans (MeGXn) are the major components of hemicellulose in woody biofuel crops. Here, we describe efficient production of the GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants and demonstrate exceptional stability and catalytic activities of the in planta produced enzyme. Fully expanded leaves from homotransplastomic plants contained enzymatically active Xyl10B at a level of 11-15% of their total soluble protein. Transplastomic plants and their seed progeny were morphologically indistinguishable from non-transgenic plants. Catalytic activity of in planta produced Xyl10B was detected with poplar, sweetgum and birchwood xylan substrates following incubation between 40 and 90 °C and was also stable in dry and stored leaves. Optimal yields of Xyl10B were obtained from dry leaves if crude protein extraction was performed at 85 °C. The transplastomic plant derived Xyl10B showed exceptional catalytic activity and enabled the complete hydrolysis of MeGXn to fermentable sugars with the help of a single accessory enzyme (α-glucuronidase) as revealed by the sugar release assay. Even without this accessory enzyme, the majority of MeGXn was hydrolyzed by the transplastomic plant-derived Xyl10B to fermentable xylose and xylobiose.

Identification and Functional Analysis of S-AdenosylMethionine Synthetase (HvSAMS) genes in Early Maturing Barley (Hordeum vulgare subsp. vulgare)

“…Following amplification, the coding sequence was used as a probe for Southern blot analysis. Probe labeling, hybridization, and signal detection were performed as described by Kim et al (2011).…”

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

“…Although in planta expression of thermophilic endoglucanases has been achieved in the range of 10-26% TSP without any detrimental effects on transgenic plants [37][38][39][40][41][42][43][44][45][46][47][48], yields of 0.1-5% TSP are more typical, and several studies have described negative phenotypes associated with in planta expression of recombinant mesophilic cellulolytic enzymes [32,49].…”

“…N-terminal or C-terminal transit peptides generally possess the necessary information for the correct targeting of proteins to subcellular organelles. Subcellular targeting of heterologous proteins generally yields higher accumulation levels in transgenic plants than expression in the cytosol [32][33][34][35][36][37][38][39][40][41][42]47,51]. When expressed in subcellular compartments, heterologous proteins typically display correct folding and activity, appropriate glycosylation, reduced degradation, and increased stability, as compared to expression and accumulation in the cytosol [8,44].…”

“…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.…”

“…In general, the most successful attempts to express hyperthermophilic enzymes in plant tissues (Table S1 in the supplementary material online) have come from targeted expression in the chloroplast [47,48]. For example, a hyperthermophilic endoglucanase (Cel5A) gene was targeted to Arabidopsis chloroplasts using the N-terminal transit peptides of nuclear-encoded plastid proteins, the lightharvesting chlorophyll a/b-binding protein (CAB), Rubisco small subunit (RS), and Rubisco activase (RA).…”