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

BACKGROUND: The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes. RESULTS: Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts were able to enhance the performance of Cellic CTec2 (a commercial cellulase mixture) up to fourfold on a range of residues from sugar cane bagasse pretreated at the laboratory (using acidified ethylene carbonate/ethylene glycol, 1-butyl-3-methylimidazolium chloride, and ball-milling) and pilot (dilute sodium hydroxide and glycerol/hydrochloric acid steam explosion) scales. We have demonstrated using tap water as a solvent (under conditions that mimic an industrial process) extraction of about 90% recombinant cellobiohydrolase from senescent, transgenic corn stover leaf that had minimal tissue disruption. CONCLUSIONS: The accumulation of recombinant cellobiohydrolase in senescent, transgenic corn stover leaf is a viable strategy to reduce the saccharification cost associated with the production of fermentable sugars from pretreated biomass. We envisage an industrial-scale process in which transgenic plants provide both fibre and biomass-degrading enzymes for pretreatment and enzymatic hydrolysis, respectively.

Stability of endoglucanases from mesophilic fungus and thermophilic bacterium in acidified polyols.

Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solvents (e.g., glycerol and ethylene glycol) at temperatures less than 100°C may allow the effective use of thermostable and non-thermostable cellulases in situ during the saccharification process. The potential of biomass saccharifying enzymes, endoglucanases (EG) from a thermophilic bacterium (Thermotoga maritima) and a mesophilic fungus (Trichoderma longibrachiatum), to retain their activity in aqueous buffer, acidified glycerol, and acidified ethylene glycol used as co-solvents at pretreatment temperatures at or below 100°C were examined. The results show that despite its origin, T. longibrachiatum EG (Tl-EG) retained 75% of its activity after exposure to 100°C for 5 min in aqueous buffer while T. maritima EG (Tm-EG) retained only 5% activity. However, at 90°C both enzymes retained over 87% of their activity. In acidified (0.1% (w/w) H2SO4) glycerol, Tl-EG retained similar activity (80%) to that obtained in glycerol alone, while Tm-EG retained only 35%. With acidified ethylene glycol under these conditions, both Tl-EG and Tm-EG retained 36% of their activity. The results therefore show that Tl-EG is more stable in both acidified glycerol and ethylene glycol than Tm-EG. A preliminary kinetic study showed that pure glycerol improved the thermal stability of Tl-EG but destabilized Tm-EG, relative to the buffer solution. The half-lives of both Tl-EG and Tm-EG are 4.5 min in acidified glycerol, indicating that the effectiveness of these enzymes under typical pretreatment times of greater than 15 min will be considerably diminished. Attempts have been made to explain the differences in the results obtained between the two enzymes.

Advancing energy cane cell wall digestibility screening by near-infrared spectroscopy.

Breeding energy cane for cellulosic biofuel production involves manipulating various traits. An important trait to optimize is cell wall degradability as defined by enzymatic hydrolysis. We investigated the feasibility of using near-infrared spectroscopy (NIRS) combined with multivariate calibration to predict energy cane cell wall digestibility based upon fiber samples from a range of sugarcane genotypes and related species. These samples produced digestibility values ranging between 6 and 31%. To preserve the practicality of the technique, spectra obtained from crudely prepared samples were used. Various spectral pre-processing methods were tested, with the best NIRS calibration obtained from second derivative, orthogonal signal-corrected spectra. Model performance was evaluated by cross-validation and independent validation. Large differences between the performance results from the two validation approaches indicated that the model was sensitive to the choice of test data. This may be remedied by using a larger calibration training set containing diverse sample types. The best result was obtained through independent validation which produced a R(2) value of 0.86, a root mean squared error of prediction (RMSEP) of 1.59, and a ratio of prediction to deviation (RPD) of 2.7. This study has demonstrated that it is feasible and practical to use NIRS to predict energy cane cell wall digestibility.

Co-ordinated synthesis of gentiobiitol and sorbitol, evidence of sorbitol glycosylation in transgenic sugarcane.

Sugarcane (a Saccharum spp. interspecific hybrid) was previously engineered to synthesize sorbitol (designated as sorbitolcane). Motivated by the atypical development of the leaves in some sorbitolcane, the polar metabolite profiles in the leaves of those plants were compared against a group of control sugarcane plants. Eighty-six polar metabolites were detected in leaf extracts by GC-MS. Principal component analysis of the metabolites indicated that three compounds were strongly associated with sorbitolcane. Two were identified as sorbitol and gentiobiose and the third was unknown. Gentiobiose and the unknown compound were positively correlated with sorbitol accumulation. The unknown compound was only abundant in sorbitolcane. This compound was structurally characterized and found to be a sorbitol-glucose conjugate. (13)C NMR analysis indicated that the glucopyranose and glucitol moieties were 1,6-linked. Ligand exchange chromatography confirmed that the compound was a beta-anomer, thus identifying the compound as 6-O-beta-d-glucopyranosyl-D-glucitol, or gentiobiitol.

Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink.

An efficient in planta sugarcane-based production system may be realized by coupling the synthesis of alternative products to the metabolic intermediates of sucrose metabolism, thus taking advantage of the sucrose-producing capability of the plant. This was evaluated by synthesizing sorbitol in sugarcane (Saccharum hybrids) using the Malus domestica sorbitol-6-phosphate dehydrogenase gene (mds6pdh). Mature transgenic sugarcane plants were compared with untransformed sugarcane variety Q117 by evaluation of the growth, metabolite levels and extractable activity of relevant enzymes. The average amounts of sorbitol detected in the most productive line were 120 mg/g dry weight (equivalent to 61% of the soluble sugars) in the leaf lamina and 10 mg/g dry weight in the stalk pith. The levels of enzymes involved in sucrose synthesis and cleavage were elevated in the leaves of plants accumulating sorbitol, but this did not affect sucrose accumulation in the culm. The activity of oxidative reactions in the pentose phosphate pathway and the non-reversible glyceraldehyde-3-phosphate dehydrogenase reaction were elevated to replenish the reducing power consumed by sorbitol synthesis. Sorbitol-producing sugarcane generated 30%-40% less aerial biomass and was 10%-30% shorter than control lines. Leaves developed necrosis in a pattern characteristic of early senescence, and the severity was related to the relative quantity of sorbitol accumulated. When the Zymomonas mobilis glucokinase (zmglk) gene was co-expressed with mds6pdh to increase the production of glucose-6-phosphate, the plants were again smaller, indicating that glucose-6-phosphate deficiency was not responsible for the reduced growth. In summary, sorbitol hyperaccumulation affected sugarcane growth and metabolism, but the outcome was not lethal for the plant. This work also demonstrated that impressive yields of alternative products can be generated from the intermediates of sucrose metabolism in Saccharum spp.

Initial evaluation of sugarcane as a production platform for p-hydroxybenzoic acid.

Sugarcane (Saccharum hybrids) was evaluated as a production platform for p-hydroxybenzoic acid using two different bacterial proteins (a chloroplast-targeted version of Escherichia coli chorismate pyruvate-lyase and 4-hydroxycinnamoyl-CoA hydratase/lyase from Pseudomonas fluorescens) that both provide a one-enzyme pathway from a naturally occurring plant intermediate. The substrates for these enzymes are chorismate (a shikimate pathway intermediate that is synthesized in plastids) and 4-hydroxycinnamoyl-CoA (a cytosolic phenylpropanoid intermediate). Although both proteins have previously been shown to elevate p-hydroxybenzoic acid levels in plants, they have never been evaluated concurrently in the same laboratory. Nor are there any reports on their efficacy in stem tissue. After surveying two large populations of transgenic plants, it was concluded that the hydratase/lyase is the superior catalyst for leaf and stem tissue, and further studies focused on this pathway. p-Hydroxybenzoic acid was quantitatively converted to glucose conjugates by endogenous uridine diphosphate (UDP)-glucosyltransferases and presumably stored in the vacuole. The largest amounts detected in leaf and stem tissue were 7.3% and 1.5% dry weight (DW), respectively, yet there were no discernible phenotypic abnormalities. However, as a result of diverting carbon away from the phenylpropanoid pathway, there was a severe reduction in leaf chlorogenic acid, subtle changes in lignin composition, as revealed by phloroglucinol staining, and an apparent compensatory up-regulation of phenylalanine ammonia-lyase. Although product accumulation in the leaves at the highest level of gene expression obtained in the present study was clearly substrate-limited, additional experiments are necessary before this conclusion can be extended to the stalk.

Microbial hyaluronic acid production.

Hyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.

Increased exopolysaccharide production in Lactococcus lactis due to increased levels of expression of the NIZO B40 eps gene cluster.

Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented food products. This is the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactococcus lactis leads to increased EPS production levels. A ninefold-elevated EPS plasmid copy number led to an almost threefold increase in the eps expression level, resulting in an almost fourfold increase in the NIZO B40 EPS production level. It was previously reported that increased EPS precursor levels did not influence NIZO B40 EPS production levels. However, the present results indicate that the maximal NIZO B40 EPS production level is limited by the activity level of the expression products of the eps gene cluster rather than by the level of EPS precursors.

Amplifying the cellular reduction potential of Streptococcus zooepidemicus.

The valuable pharmaceutical polymer, hyaluronic acid, is produced industrially using the gram-positive bacterium Streptococcus zooepidemicus. Synthesis of this polymer is a significant energetic burden upon the microorganism hence the native NADH oxidase gene was cloned and overexpressed to increase the energy yield of catabolism during aerobic cultivation on glucose. Elevated NADH oxidase levels led to a decline in lactic acid generation and prevented ethanol formation, leaving acetate as the main fermentation product. Biomass yield increased due to the energy gained from the formation of acetate. Evaluation of the acetate flux control coefficient over a range of NADH oxidase expression levels revealed that acetate production was sensitive to the NADH oxidase level. However, at high NADH oxidase levels, the acetate flux was mainly influenced by another factor. The concomitant excretion of pyruvate at high NADH oxidase levels suggested that the flux through the pyruvate dehydrogenase enzyme complex was limiting the conversion of pyruvate to acetate.