Influence of prefoldin subunit 4 on the tolerance of Kluyveromyces marxianus to lignocellulosic biomass-derived inhibitors.

BACKGROUND: Kluyveromyces marxianus is a potentially excellent host for microbial cell factories using lignocellulosic biomass, due to its thermotolerance, high growth rate, and wide substrate spectrum. However, its tolerance to inhibitors derived from lignocellulosic biomass pretreatment needs to be improved. The prefoldin complex assists the folding of cytoskeleton which relates to the stress tolerance, moreover, several subunits of prefoldin have been verified to be involved in gene expression regulation. With the presence of inhibitors, the expression of a gene coding the subunit 4 of prefoldin (KmPFD4), a possible transcription factor, was significantly changed. Therefore, KmPFD4 was selected to evaluate its functions in inhibitors tolerance. RESULTS: In this study, the disruption of the prefoldin subunit 4 gene (KmPFD4) led to increased concentration of intracellular reactive oxygen species (ROS) and disturbed the assembly of actin and tubulin in the presence of inhibitors, resulting in reduced inhibitor tolerance. Nuclear localization of KmPFD4 indicated that it could regulate gene expression. Transcriptomic analysis showed that upregulated gene expression related to ROS elimination, ATP production, and NAD+ synthesis, which is a response to the presence of inhibitors, disappeared in KmPFD4-disrupted cells. Thus, KmPFD4 impacts inhibitor tolerance by maintaining integration of the cytoskeleton and directly or indirectly affecting the expression of genes in response to inhibitors. Finally, overexpression of KmPFD4 enhanced ethanol fermentation with a 46.27% improvement in productivity in presence of the inhibitors. CONCLUSION: This study demonstrated that KmPFD4 plays a positive role in the inhibitor tolerance and can be applied for the development of inhibitor-tolerant platform strains.

Analysis of the response of the cell membrane of Saccharomyces cerevisiae during the detoxification of common lignocellulosic inhibitors.

Gaining an in-depth understanding of the response of Saccharomyces cerevisiae to the different inhibitors generated during the pretreatment of lignocellulosic material is driving the development of new strains with higher inhibitor tolerances. The objective of this study is to assess, using flow cytometry, how three common inhibitors (vanillin, furfural, and acetic acid) affect the membrane potential, the membrane permeability and the concentration of reactive oxygen species (ROS) during the different fermentations. The membrane potential decreased during the detoxification phase and reflected on the different mechanisms of the toxicity of the inhibitors. While vanillin and furfural caused a metabolic inhibition and a gradual depolarization, acetic acid toxicity was related to fast acidification of the cytosol, causing an immediate depolarization. In the absence of acetic acid, ethanol increased membrane permeability, indicating a possible acquired tolerance to ethanol due to an adaptive response to acetic acid. The intracellular ROS concentration also increased in the presence of the inhibitors, indicating oxidative stress. Measuring these features with flow cytometry allows a real-time assessment of the stress of a cell culture, which can be used in the development of new yeast strains and to design new propagation strategies to pre-adapt the cell cultures to the inhibitors.

Impact of stress-response related transcription factor overexpression on lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae environmental isolates.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5-hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis-folded protein. Additional stress-related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains.

Effects of shading on lignin biosynthesis in the leaf of tea plant (Camellia sinensis (L.) O. Kuntze).

Shading can effectively reduce photoinhibition and improve the quality of tea. Lignin is one of the most important secondary metabolites that play vital functions in plant growth and development. However, little is known about the relationship between shading and xylogenesis in tea plant. To investigate the effects of shading on lignin accumulation in tea plants, 'Longjing 43' was treated with no shading (S0), 40% (S1) and 80% (S2) shading treatments, respectively. The leaf area and lignin content of tea plant leaves decreased under shading treatments (especially S2). The anatomical characteristics showed that lignin is mainly distributed in the xylem of tea leaves. Promoter analysis indicated that the genes involved in lignin pathway contain several light recognition elements. The transcript abundances of 12 lignin-associated genes were altered under shading treatments. Correlation analysis indicated that most genes showed strong positive correlation with lignin content, and CsPAL, Cs4CL, CsF5H, and CsLAC exhibited significant positively correlation under 40% and 80% shading treatments. The results showed that shading may have an important effect on lignin accumulation in tea leaves. This work will potentially helpful to understand the regulation mechanism of lignin pathway under shading treatment, and provide reference for reducing lignin content and improving tea quality through shading treatment in field operation.

New insights into two yeast BDHs from the PDH subfamily as aldehyde reductases in context of detoxification of lignocellulosic aldehyde inhibitors.

At least 24 aldehyde reductases from Saccharomyces cerevisiae have been characterized and most function in in situ detoxification of lignocellulosic aldehyde inhibitors, but none is classified into the polyol dehydrogenase (PDH) subfamily of the medium-chain dehydrogenase/reductase (MDR) superfamily. This study confirmed that two (2R,3R)-2,3-butanediol dehydrogenases (BDHs) from industrial (denoted Y)/laboratory (denoted B) strains of S. cerevisiae, Bdh1p(Y)/Bdh1p(B) and Bdh2p(Y)/Bdh2p(B), were members of the PDH subfamily with an NAD(P)H binding domain and a catalytic zinc binding domain, and exhibited reductive activities towards lignocellulosic aldehyde inhibitors, such as acetaldehyde, glycolaldehyde, and furfural. Especially, the highest enzyme activity towards acetaldehyde by Bdh2p(Y) was 117.95 U/mg with cofactor nicotinamide adenine dinucleotide reduced (NADH). Based on the comparative kinetic property analysis, Bdh2p(Y)/Bdh2p(B) possessed higher specific activity, substrate affinity, and catalytic efficiency towards glycolaldehyde than Bdh1p(Y)/Bdh1p(B). This was speculated to be related to their 49% sequence differences and five nonsynonymous substitutions (Ser41Thr, Glu173Gln, Ile270Leu, Ile316Met, and Gly317Cys) occurred in their conserved NAD(P)H binding domains. Compared with BDHs from a laboratory strain, Bdh1p(Y) and Bdh2p(Y) from an industrial strain displayed five nonsynonymous mutations (Thr12, Asn61, Glu168, Val222, and Ala235) and three nonsynonymous mutations (Ala34, Ile96, and Ala369), respectively. From a first analysis with selected aldehydes, their reductase activities were different from BDHs of laboratory strain, and their catalytic efficiency was higher towards glycolaldehyde and lower towards acetaldehyde. Comparative investigation of kinetic properties of BDHs from S. cerevisiae as aldehyde reductases provides a guideline for their practical applications in in situ detoxification of aldehyde inhibitors during lignocellulose bioconversion.Key Points• Two yeast BDHs have enzyme activities for reduction of aldehydes.• Overexpression of BDHs slightly improves yeast tolerance to acetaldehyde and glycolaldehyde.• Bdh1p and Bdh2p differ in enzyme kinetic properties.• BDHs from strains with different genetic backgrounds differ in enzyme kinetic properties.

Recombinant Penicillium oxalicum 16 ß-Glucosidase 1 Displays Comprehensive Inhibitory Resistance to Several Lignocellulose Pretreatment Products, Ethanol, and Salt.

ß-Glucosidase (BGL) is a rate-limiting enzyme of lignocellulose hydrolysis for second-generation bioethanol production, but its inhibition by lignocellulose pretreatment products, ethanol, and salt is apparent. Here, the recombinant Penicillium oxalicum 16 BGL 1 (rPO16BGL1) from Pichia pastoris GS115 kept complete activity at 0.2-1.4 mg/mL furan derivatives and phenolic compounds, 50 mg/mL sodium chloride (potassium chloride), or 100 mg/mL ethanol at 40 °C. rPO16BGL1 retained above 50% residual activity at 30 mg/mL organic acid sodium, and 60% residual activity at 40 °C with 300 mg/mL ethanol. Sodium chloride and potassium chloride had a complicated effect on rPO16BGL1, which resulted in activation or inhibition. The inhibition kinetics of the enzyme reaction demonstrated that organic acids and organic acid sodium were non-competitive inhibitors and that ethanol was a competitive inhibitor at < 1.5 mg/mL salicin. Moreover, substrate inhibition of the enzyme was found at > 2 mg/mL salicin, and the Km/KI and Km/KSI average values revealed that the inhibitory strength was ranked as salicin-organic acids > organic acids > salicin-organic acid sodium salt > organic acid sodium salt > salicin > salicin-KCl > salicin-NaCl > salicin-ethanol > ethanol.

QTL analysis of natural Saccharomyces cerevisiae isolates reveals unique alleles involved in lignocellulosic inhibitor tolerance.

Decoding the genetic basis of lignocellulosic inhibitor tolerance in Saccharomyces cerevisiae is crucial for rational engineering of bioethanol strains with enhanced robustness. The genetic diversity of natural strains present an invaluable resource for the exploration of complex traits of industrial importance from a pan-genomic perspective to complement the limited range of specialised, tolerant industrial strains. Natural S. cerevisiae isolates have lately garnered interest as a promising toolbox for engineering novel, genetically encoded tolerance phenotypes into commercial strains. To this end, we investigated the genetic basis for lignocellulosic inhibitor tolerance of natural S. cerevisiae isolates. A total of 12 quantitative trait loci underpinning tolerance were identified by next-generation sequencing linked bulk-segregant analysis of superior interbred pools. Our findings corroborate the current perspective of lignocellulosic inhibitor tolerance as a multigenic, complex trait. Apart from a core set of genetic variants required for inhibitor tolerance, an additional genetic background-specific response was observed. Functional analyses of the identified genetic loci revealed the uncharacterised ORF, YGL176C and the bud-site selection XRN1/BUD13 as potentially beneficial alleles contributing to tolerance to a complex lignocellulosic inhibitor mixture. We present evidence for the consideration of both regulatory and coding sequence variants for strain improvement.

Quantitative proteomic analysis of xylose fermentation strain Pichia stipitis CBS 5776 to lignocellulosic inhibitors acetic acid, vanillin and 5-hydroxymethylfurfural.

To obtain a global insight into the dynamic protein expression pattern in Pichia stipitis during xylose fermentation in the presence of three representative inhibitors (acetic acid, vanillin and 5-hydroxymethylfurfural), proteins were extracted for quantitative proteomic analysis using 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) on a liquid chromatography-mass/mass spectrometry instrument. Interestingly, aconitase (Aco1p) and NAD-isocitrate dehydrogenase (Idh1p) were upregulated during the middle exponential phase in the presence of the three inhibitors during tricarboxylic acid cycle. We speculated that yeast cells adaptively increased the expression of the tricarboxylic acid cycle proteins to compensate for low NADH derived from glycolysis in the presence of the three inhibitors. Proteins related to amino acid metabolism, aminoacyl tRNA synthesis and stress response were also significantly affected in the presence of the three inhibitors. Taken together, quantitative proteomic analysis is capable of monitoring P. stipitis xylose fermentation under inhibitor conditions and identifying physiological changes, such as stress response.

Feasibility studies with lignin blocking additives in enhancing saccharification and cellulase recovery: Mutant UV-8 of T. verruculosus IIPC 324 a case study.

The process economics of fermentable sugar production is dependent on the performance of cellulase cocktail on realistic lignocellulosic biomass and their capability to be recovered and recycled. Feasibility studies were conducted to enhance the digestibility of acid pretreated sugarcane bagasse using novel cellulase cocktail obtained from stable mutant UV-8 of Talaromyces verruculosus IIPC 324 in presence of lignin blocking additives. PEG 6000 was shortlisted as the best additive as it could simultaneously enhance saccharification and overall cellulase recoveries namely cellobiohydrolase, endoglucanase and cellobiase. Addition of 0.3 g PEG 6000/g acid-insoluble lignin content, resulted in 55% and 49.2% saccharification yields in terms of reducing sugars and glucose respectively using this cellulase cocktail (25 mg protein/g cellulose content) after 72 h from acid pretreated sugarcane bagasse loaded at 7.5%. The study also suggested that the endoglucanase of this mutant was unique with high desorption capability as 85% activity was observed in the saccharified broth devoid of any lignin blocking additive. At its optimum concentration, PEG 6000 was able to retain 94 ± 0.79% cellobiohydrolase I and 97.97 ± 1.16% cellobiase enzyme in the saccharified broth which were otherwise lost in residual biomass by ∼80%, in the absence of this polymeric additive. These results suggest that PEG 6000 was the most promising facilitator for recycling of cellulases obtained from mutant UV-8 of Talaromyces verruculosus IIPC 324 in particular. It paved a way towards the production of cheaper fermentable sugars which serve as a starting raw material for the production of green chemicals and fuels.

Profiling of Saccharomyces cerevisiae transcription factors for engineering the resistance of yeast to lignocellulose-derived inhibitors in biomass conversion.

BACKGROUND: Yeast transcription factors (TFs) involved in the regulation of multidrug resistance (MDR) were investigated in experiments with deletion mutants, transformants overexpressing synthetic genes encoding TFs, and toxic concentrations of lignocellulose-derived substances added to cultures as complex mixtures or as specific compounds, viz. coniferyl aldehyde, 5-hydroxymethylfurfural, and furfural. RESULTS: In the presence of complex mixtures of toxic substances from spruce wood, transformants overexpressing YAP1 and STB5, TFs involved in oxidative stress response, exhibited enhanced relative growth rates amounting to 4.589 ± 0.261 and 1.455 ± 0.185, respectively. Other TFs identified as important for resistance included DAL81, GZF3, LEU3, PUT3, and WAR1. Potential overlapping functions of YAP1 and STB5 were investigated in experiments with permutations of deletions and overexpression of the two genes. YAP1 complemented STB5 with respect to resistance to 5-hydroxymethylfurfural, but had a distinct role with regard to resistance to coniferyl aldehyde as deletion of YAP1 rendered the cell incapable of resisting coniferyl aldehyde even if STB5 was overexpressed. CONCLUSIONS: We have investigated 30 deletion mutants and eight transformants overexpressing MDR transcription factors with regard to the roles the transcription factors play in the resistance to toxic concentrations of lignocellulose-derived substances. This work provides an overview of the involvement of thirty transcription factors in the resistance to lignocellulose-derived substances, shows distinct and complementary roles played by YAP1 and STB5, and offers directions for the engineering of robust yeast strains for fermentation processes based on lignocellulosic feedstocks.

Clostridium species strain BOH3 tolerates and transforms inhibitors from horticulture waste hydrolysates.

Conversion of lignocellulosic hydrolysate to biofuels is impeded by the toxic effects of inhibitors that are generated during pretreatment and hydrolysis processes. Here we describe a wild-type Clostridium sp. strain BOH3 with high tolerance to the lignocellulose-derived inhibitors and its capability to transform these inhibitors. Strain BOH3 is capable of tolerating over 60 mM furfural, 60 mM hydroxymethylfurfural, and 6.6 mM vanillin, respectively, and is able to convert 53.74 ± 0.37 mM furfural into furfuryl alcohol within 90 h. The high furfural tolerance and its biotransformation by strain BOH3, which is correlated to the high transcription levels of two short-chain dehydrogenase/reductases, enable strain BOH3 to produce 5.15 ± 0.52 g/L butanol from dilute sulfuric acid pretreated horticultural waste hydrolysate (HWH) that bypassed the detoxification step. The capability of strain BOH3 to produce butanol from un-detoxified HWH lays the foundation of cost-effective biofuel production from lignocellulosic materials.

GH11 xylanase from Emericella nidulans with low sensitivity to inhibition by ethanol and lignocellulose-derived phenolic compounds.

An endo-ß-1,4-xylanase (X22) was purified from crude extract of Emericella nidulans when cultivated on submerged fermentation using sugarcane bagasse as the carbon source. The purified protein was identified by mass spectrometry and was most active at pH and temperature intervals of 5.0-6.5 and 50-60°C, respectively. The enzyme showed half-lives of 40, 10 and 7 min at 28, 50 and 55°C, respectively, and pH 5.0. Apparent Km and Vmax values on soluble oat spelt xylan were 3.39 mg/mL and 230.8 IU/mg, respectively, while Kcat and Kcat/Km were 84.6 s(-1) and 25.0 s(-1) mg(-1) mL. Incubation with phenolic compounds showed that tannic acid and cinnamic acid had an inhibitory effect on X22 but no time-dependent deactivation. On the other hand, ferulic acid, 4-hydroxybenzoic acid, vanillin and p-coumaric acid did not show any inhibitory effect on X22 activity, although they changed X22 apparent kinetic parameters. Ethanol remarkably increased enzyme thermostability and apparent Vmax and Kcat values, even though the affinity and catalytic efficiency for xylan were lowered.

Nitrite promotes the growth and decreases the lignin content of indica rice calli: a comprehensive transcriptome analysis of nitrite-responsive genes during in vitro culture of rice.

As both major macronutrients and signal molecules, nitrogen metabolites, such as nitrate and nitrite, play an important role in plant growth and development. In this study, the callus growth of indica rice cv. 9311 was significantly enhanced by nitrite, whereas the soluble protein content remained unchanged. The deep RNA sequencing technology (RNA-seq) showed that the transcriptional profiles of cv. 9311 calli were significantly changed after adding nitrite to the nitrate-free medium, and these nitrite-responsive genes were involved in a wide range of plant processes, particularly in the secondary metabolite pathways. Interestingly, most of the genes involved in phenylpropanoid-related pathways were coordinately down-regulated by nitrite, such as four cinnamoyl-CoA reductase, and these in turn resulted in the decrease of lignin content of indica calli. Furthermore, several candidate genes related to cell growth or stress responses were identified, such as genes coding for expansins, SMALL AUXIN UP RNA (SAUR) and HSP20s, and these suggested that nitrite could probably serve as a transcriptome signal to enhance the indica calli growth by regulation of various downstream genes expression. This study contributes to a better understanding of the function of nitrite during the process of plant tissue culture and could aid in the application of this technology to improved indica genetic transformation efficiency.

Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa.

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ∼40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome-based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.

Global regulator engineering significantly improved Escherichia coli tolerances toward inhibitors of lignocellulosic hydrolysates.

Lignocellulosic biomass is regarded as the most viable source of feedstock for industrial biorefinery, but the harmful inhibitors generated from the indispensable pretreatments prior to fermentation remain a daunting technical hurdle. Using an exogenous regulator, irrE, from the radiation-resistant Deinococcus radiodurans, we previously showed that a novel global regulator engineering (GRE) approach significantly enhanced tolerances of Escherichia coli to alcohol and acetate stresses. In this work, an irrE library was subjected to selection under various stresses of furfural, a typical hydrolysate inhibitor. Three furfural tolerant irrE mutants including F1-37 and F2-1 were successfully obtained. The cells containing these mutants reached OD(600) levels of 4- to 16-fold of that for the pMD18T cells in growth assay under 0.2% (v/v) furfural stress. The cells containing irrE F1-37 and F2-1 also showed considerably reduced intracellular oxygen species (ROS) levels under furfural stress. Moreover, these two irrE mutants were subsequently found to confer significant cross tolerances to two other most common inhibitors, 5-hydroxymethyl-2-furaldehyde (HMF), vanillin, as well as real lignocellulosic hydrolysates. When evaluated in Luria-Bertani (LB) medium supplemented with corn stover cellulosic hydrolysate (prepared with a solid loading of 30%), the cells containing the mutants exhibited lag phases markedly shortened by 24-44 h in comparison with the control cells. This work thus presents a promising step forward to resolve the inhibitor problem for E. coli. From the view of synthetic biology, irrE can be considered as an evolvable "part" for various stresses. Furthermore, this GRE approach can be extended to exploit other exogenous global regulators from extremophiles, and the native counterparts in E. coli, for eliciting industrially useful phenotypes.

Effect of lignocellulosic inhibitory compounds on growth and ethanol fermentation of newly-isolated thermotolerant Issatchenkia orientalis.

A newly isolated thermotolerant ethanologenic yeast strain, Issatchenkia orientalis IPE 100, was able to produce ethanol with a theoretical yield of 85% per g of glucose at 42°C. Ethanol production was inhibited by furfural, hydroxymethylfurfural and vanillin concentrations above 5.56 gL(-1), 7.81 gL(-1), and 3.17 gL(-1), respectively, but the strain was able to produce ethanol from enzymatically hydrolyzed steam-exploded cornstalk with 93.8% of theoretical yield and 0.91 gL(-1)h(-1) of productivity at 42°C. Therefore, I. orientalis IPE 100 is a potential candidate for commercial lignocelluloses-to-ethanol production.

Down-regulation of lignin biosynthesis in transgenic Leucaena leucocephala harboring O-methyltransferase gene.

In the present study, a 0.47 kb OMT gene construct from aspen, encoding for an enzyme O-methyltransferase (OMT, EC 2.1.1.6), in antisense orientation was used to down-regulate lignin biosynthesis in Leucaena leucocephala. The plants were transformed with Agrobacterium tumefaciens strain harboring the antisense gene, and the transformation was confirmed by PCR amplification of the npt II gene. The integration of a heterologous antisense OMT gene construct in transformed plants led to a maximum of 60% reduction in OMT activity relative to control. The evaluation of total lignin content by the Klason method revealed a maximum of 28% reduction. Histochemical analyses of stem sections depicted a reduction in lignin content and normal xylem development. The results also suggested a probable increase in aldehyde levels and a decrease in syringyl units. Lignin down-regulation was accompanied by an increase in methanol soluble phenolics to an extent that had no impact on wood discoloration, and the plants displayed a normal phenotype. Concomitantly, an increase of up to 9% in cellulose content was also observed. Upon alkali extraction, modified lignin was more extractable as evident from reduced Klason lignin in saponified residue and increased alkali soluble phenolics. The results together suggested that the extent of down-regulation of OMT activity achieved may lead to quality amelioration of Leucaena with respect to its applicability in pulp and paper manufacture as well as nutritive and easily digestible forage production.

Induction of neurite outgrowth by (-)-(7R, 8S)-dihydrodehydrodiconiferyl alcohol from PC12 cells.

A lignan derivative, (-)-(7R, 8S)-dihydrodehydrodiconiferyl alcohol (DHDA), was isolated from Kalopanax septemlobus L. and was observed to have neuritogenic activity. DHDA at 50 microM caused a marked induction of neurite outgrowth and an enhancement of nerve growth factor (NGF)-mediated neurite outgrowth from PC12 cells. However, it did not exhibit any neurotrophic action. At 50 microM, DHDA enhanced NGF-induced neurite-bearing activity. This activity was partially blocked by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and by GF109203X, a protein kinase C (PKC) inhibitor. These results suggest that DHDA can induce neurite outgrowth and enhance NGF-induced neurite outgrowth from PC12 cells by amplifying up-stream steps such as MAPK and PKC.

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei.

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.

beta-fluoro-coniferyl alcohol does not inhibit lignin biosynthesis in suspension cultures of Picea abies (L.) Karst.

A fluorinated analogue of coniferyl alcohol has been reported to be a specific inhibitor of oxidases involved in the biosynthesis of lignin. The Z isomer of beta-fluoro-coniferyl alcohol was synthesized and used for the preparation of dehydrogenation polymers (DHPs) and was also tested on lignin producing suspension cultures of spruce (Picea abies (L.) Karst.). The growth of the cells or the production of lignin by the suspension cultures was not significantly affected by the addition of fluoroconiferyl alcohol. This analogue did not form polymers quite as easily as did coniferyl alcohol in oxidation with hydrogen peroxide and horseradish peroxidase. In both cases the beta-fluoroconiferyl alcohol became incorporated in the polymeric product. We were unable to detect any specific inhibition of peroxidase activity, which is at variance with earlier reports of pronounced inhibition of lignin biosynthesis in poplar plantlets by fluoroconiferin, a potential inhibitor of oxidases involved in lignin biosynthesis.