Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars

Abstract

It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in Escherichia coli or tobacco chloroplasts. A PCR-based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10, 751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3100-fold, and pectate lyase is 1057 or 1480-fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coli extracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails.

Figure 1

Regeneration and analysis of transplastomic lines (a) Schematic representation of the chloroplast 16S trnI/trnA region. Transgenes were inserted at the trnI/trnA spacer region in the tobacco chloroplast genome. (b) Schematic representation of the chloroplast transformation vectors. The gene of interest (GOI) is celD, celO, pelA, pelB, pelD, cutinase, lipY, egI, swo1, xyn2, axe1 or bgl1. Prrn, rRNA operon promoter; aadA, aminoglycoside 3′-adenylytransferase gene; 5′ UTR, promoter and 5′ untranslated region of psbA gene; 3′ UTR, 3′ untranslated region of psbA gene. (c) Evaluation of transgene integration and homoplasmy by Southern blot of pelB, (d) pelD and (e) celD transplastomic Petite Havana lines hybridized with the flanking sequence probe (1, untransformed; 2 to 4, transplastomic lines). (f) Phenotypes of untransformed (UT) and transplastomic lines (Petite Havana) grown in green house showing normal growth.

Figure 2

Western blot analysis and quantitation of transplastomic lines. Western blot of transplastomic lines expressing (a) PelB or (b) PelD. UT: untransformed, mature leaves harvested at 10 AM, 2 PM, 6 PM and 10 PM; 5ng, 10ng and 25ng: PelA purified protein, young, mature and old leaves. (c) Western blot analysis of PelB and PelD with His-tag antibody. Lane 1, protein marker, lane 2, untransformed plant extract; lane 3, PelD plant extract; lanes 4 & 5, PelD E. coli; lane 6, untransformed E. coli; lane 7, PelB plant extract; lane 8, blank; lanes 9 & 10, PelB E. coli. Enzyme units of PelB and PelD (d) or CelD (e) from one g or 100 mg leaf of different age or harvesting time.

Figure 3

Effect of substrate, pH, temperature and cofactors on cpPelB, rPelB, cpPelD, rPelD, rCelD and cpCelD enzyme activity. (a) Effect of increasing PGA concentration on pectate lyases activity. (b) Effect of pH on pectate lyases activity in the absence of CaCl₂ and (c) in the presence of CaCl₂ (d) Effect of temperature (30 to 70°C) on enzyme activity at pH 8.0 in the absence of CaCl₂ and (e) in the presence of CaCl₂. (f) Optimization of pH and (g) effect of increasing temperature for cpCelD and rCelD enzyme activity (h) Enhancement of cpCelD (25 μg TSP/ml reaction) activity using 10 mM CaCl₂ and 20 μg/ml BSA individually or in combination with 50 mM sodium acetate during the prolonged enzymatic hydrolysis. The hydrolysis was carried out up to 36 hours at 60°C, pH 6.0 in the presence of CMC (2%). Untransformed E. coli and leaf crude extracts did not yield any detectable level of unsaturated galacturonic acid or reducing sugar under these assay conditions.

Figure 4

E. coli vs. chloroplast derived enzymes at different protein concentrations of crude extracts. (a) Enzyme kinetics of cpCelD and rCelD using carboxymethyl cellulose (2%) substrate. The reaction mixture contained increasing concentration of cpCelD and rCelD TSP (μg/ml) with 10 mM CaCl₂ and 50 mM sodium acetate buffer, pH 6.0. Enzyme hydrolysis was carried out for 30 minutes at 60°C. Figure inset shows enzyme kinetics saturation point for cpCelD TSP amount (μg/ml) towards CMC (2%). Eppendorf tubes with reaction mixture shown in inset represents, 1 untransformed plant, 2 and 3 rCelD and cpCelD 10μg TSP. (b) Effect of cpPelB, cpPelD, rPelB, and rPelD on hydrolysis of 5.0 mg/ml sodium polygalacturonate substrate. The reaction mixture contained increasing concentration of cpPelB, cpPelD, rPelB, and rPelD (μg/ml) in 20 mM Tris-HCl buffer (pH 8.0). Enzyme hydrolysis was carried out for 2 hour at 40°C on rotary shaker at 150 rpm.

Figure 5

In planta bioassays. Five- to 7-mm areas of untransformed, PelB and PelD transplastomic tobacco cv Petit Havana leaves were scraped with fine-grain sandpaper. Twenty microliters of 10⁸, 10⁶, 10⁴ and 10²_cells from an overnight culture of E. carotovora were inoculated to each prepared area. Photos were taken 5 d after inoculation.

Figure 6

Enzyme cocktails for filter paper, pine wood and citrus peel. (a) Enzyme cocktail activity on Whatman No1 filter paper (50 mg/ml). (b) Hydrolysis of pine wood sample (200 mg/5 ml reaction). (c) Hydrolysis of Valencia orange peel and albedo portion (200 mg/5 ml reaction).