Deletion of hxk1 gene results in derepression of xylose utilization in Scheffersomyces stipitis.

A major problem in fermenting xylose in lignocellulosic substrates is the presence of glucose and mannose which inhibit xylose utilization. Previous studies showed that catabolite repression in some yeasts is associated with hexokinases and that deletion of one of these gene(s) could result in derepressed mutant strain(s). In this study, the hxk1 encoding hexokinase 1 in Scheffersomyces stipitis was disrupted. The ∆hxk1 SS6 strain retained the ability to utilize the main hexoses and pentoses commonly found in lignocellulosic hydrolysates as efficiently as the wild-type (WT) strain. SS6 also fermented the dominant sugars to ethanol; however, on xylose, the ∆hxk1 strain produced more xylitol and less ethanol than the WT. On mixed sugars, as expected the WT utilized glucose ahead of xylose and xylose utilization did not commence until all the glucose was consumed. In contrast, the ∆hxk1 mutant showed derepression in that it started to utilize xylose even when considerable glucose (about 1.72%, w/v) remained in the medium. Similarly, mannose did not repress xylose utilization by the ∆hxk1 mutant and xylose and mannose were simultaneously utilized. The results are of interest in efforts to engineer yeast strains capable of efficiently utilizing glucose and xylose simultaneously for lignocellulosic biomass conversion.

Chemosensory mediated behaviors and gene transcription profiles in wild yellow perch (Perca flavescens) from metal contaminated lakes.

The olfactory system of fish is sensitive to the toxic effects of low concentrations of contaminants. To investigate the effects of long-term metal exposure on olfaction in wild yellow perch (Perca flavescens), fish from one clean (Geneva Lake) and two metal-contaminated lakes (Ramsey and Hannah lakes) were collected in and around the metal-mining district of Sudbury, ON. Two different techniques were used to measure the effects of exposure to environmental contamination: (i) behavioral responses were recorded in response to conspecific skin extract and (ii) gene transcription differences in olfactory rosettes were characterized using a novel, 1000-candidate gene yellow perch microarray. Behavioral assays performed on fish from the clean lake demonstrated avoidance of a conspecific skin extract, while fish from metal contaminated lakes showed no avoidance response. A total of 109 out of the 1000 genes were differentially transcribed among the lakes. Most of the differentially transcribed genes were between the two metal contaminated lakes relative to either of the contaminated lakes and the reference lake. No genes were differentially expressed between Geneva Lake (clean) and Hannah Lake (metal contaminated). These results demonstrated that even though the different populations of fish from both Hannah and Ramey lakes were affected at the behavioral level, the impairment of olfaction was not measurable using gene transcriptional changes in olfactory rosettes.

Separation of lignocelluloses from spent liquor of NSSC pulping process via adsorption.

Hemicelluloses and lignin present in the spent liquor (SL) of neutral sulfite semichemical (NSSC) pulping process can potentially be converted into value-added products such as furfural, hydroxymethylfurfural, levulinic acid, phenols and adhesives. However, the direct conversion of hemicelluloses and lignin of SL into value-added products is uneconomical due to the dilute nature of the SL. To have a feasible downstream process for utilizing lignocelluloses of SL, the lignocelluloses should initially be separated from the SL. In this study, an adsorption process (via applying activated carbon) was considered for isolating the dissolved lignin and hemicelluloses from the SL of an NSSC pulping process. Under the optimal conditions of pH, SL/AC weight ratio, time and temperature of 5.7, 30, 360 min and 30 °C, the maximum lignin and hemicellulose adsorptions were 0.33 and 0.25 g/g on AC. The chemical oxygen demand (COD) and turbidity of the SL were decreased by 11% and 39%, respectively, as a result of lignocellulose adsorption on AC. Also, the incineration behavior of the SL-treated AC was studied with a thermo-gravimetric analysis (TGA).

Overexpression of an exotic thermotolerant ß-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw.

BACKGROUND: Trichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of ß-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding ß-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient ß-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH. RESULTS: We successfully engineered a thermostable ß-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher ß-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg) at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant ß-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain. CONCLUSIONS: The recombinant T. reesei overexpressing Periconia sp. ß-glucosidase in this study showed higher ß-glucosidase and total cellulase activities within a shorter incubation time (24 h) as well as higher hydrolysis activity using biomass residues. These features suggest that the transformants can be used for ß-glucosidase production as well as improving the biomass conversion using cellulases.

Addressing Manufacturing Challenges for Commercialization of iPSC-Based Therapies.

The development of reprogramming technology to generate human induced pluripotent stem cells (iPSCs) has tremendously influenced the field of regenerative medicine and clinical therapeutics where curative cell replacement therapies can be used in the treatment of devastating diseases such as Parkinson's disease (PD) and diabetes. In order to commercialize these therapies to treat a large number of individuals, it is important to demonstrate the safety and efficacy of these therapies and ensure that the manufacturing process for iPSC-derived functional cells can be industrialized at an affordable cost. However, there are a number of manufacturing obstacles that need to be addressed in order to meet this vision. It is important to note that the manufacturing process for generation of iPSC-derived specialized cells is relatively long and fairly complex and requires differentiation of high-quality iPSCs into specialized cells in a controlled manner. In this chapter, we have summarized our efforts to address the main challenges present in the industrialization of iPSC-derived cell therapy products with focus on the development of a current Good Manufacturing Practice (cGMP)-compliant iPSC manufacturing process, a comprehensive iPSC characterization platform, long-term stability of cGMP compliant iPSCs, and innovative technologies to address some of the scale-up challenges in establishment of iPSC processing in 3D computer-controlled bioreactors.

Cellulase activities in biomass conversion: measurement methods and comparison.

Cellulose, the major constituent of all plant materials and the most abundant organic molecule on the Earth, is a linear biopolymer of glucose molecules, connected by ß-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires mixtures of hydrolytic enzymes including endoglucanases, exoglucanases (cellobiohydrolases), and ß-glucosidases acting in a synergistic manner. In biopolymer hydrolysis studies, enzyme assay is an indispensable part. The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article. In addition, some novel approaches recently used for enzyme assays are summarized.

Transfer of plasmid into the pentose-fermenting yeast Pachysolen tannophilus.

The pentose-fermenting yeast Pachysolen tannophilus can convert glucose and xylose in lignocellulosic hydrolysates to ethanol. However, it performs poorly in industrially relevant lignocellulosic hydrolysates containing mixed sugars and inhibitors. Efforts have been directed at improving the performance of this yeast to enable efficient lignocellulosic biomass conversion. While some successes have been reported using random mutagenesis and/or hybridization-based approaches, further genetic improvement of this yeast is hampered by the lack of efficient gene transfer methods as well as limited genetic information to guide further construction of robust strains of P. tannophilus. In this study, we aimed to address this short-coming by establishing the optimal conditions needed for efficient gene transfer into P. tannophilus. We ascertained that plasmids can be transferred into P. tannophilus through trans-kingdom conjugation or lithium acetate (LiAc) transformation. The efficiency of plasmid YEp13 (2-micron, LEU2) transferred into a P. tannophilus leucine auxotroph (Leu-) reached as high as 1.93 × 10-2 transconjugants per input recipient and 3.25 × 104 transformants per µg plasmid DNA through trans-kingdom conjugation and transformation, respectively. In trans-kingdom conjugation, the number of recipient P. tannophilus cells played an important role, while the ratio of donor (Escherichia coli) to recipient cells was less important. For efficient transformation in P. tannophilus, the use of PEG 3350 was essential, as no transformants were obtained in its absence. The transformation efficiency increased with the addition of single-stranded carrier DNA and incubation at 30 °C for >60 min. Plasmids with different replication origins or 2-micron plasmids with different CUG codon-optimized antibiotic resistance markers were unable to transform P. tannophilus under our experimental conditions. The results are of interest in the genetic manipulation and improvement of P. tannophilus.

Acidification of prehydrolysis liquor and spent liquor of neutral sulfite semichemical pulping process.

Acidification has been commercialized for producing kraft lignin from black liquor of kraft pulping process. This work intended to evaluate the effectiveness of acidification in extracting lignocelluloses from the spent liquor of neutral sulfite semichemical pulping (NSSC) process and from prehydrolysis liquor (PHL) of kraft-based dissolving pulp production process. The results showed that the NSSC and PHL spent liquors had some lignin-carbohydrate complexes (LCC), and that the square weighted counts of particles with a chord length of 50-150µm in the spent liquors were significantly increased as pH dropped to 1.5. Interestingly, the acidification reduced the lignosulfonate/lignin content of NSSC and PHL by 13% or 20%, while dropped their oligosugars content by 75% and 38%, respectively. On a dry basis, the precipitates had more carbon, hydrogen and a high heating value of 18-22MJ/kg, but less oxygen, than spent liquors. The precipitates of PHL could be used as fuel.

A combined adsorption and flocculation process for producing lignocellulosic complexes from spent liquors of neutral sulfite semichemical pulping process.

The spent liquor (SL) of a neutral sulfite semichemical pulping process contains lignocelluloses that are currently treated in a waste water system. In this work, an adsorption process using activated carbon (AC) was considered for isolating the lignin and hemicelluloses from SL. The maximum adsorptions of 0.9 g/g lignin and 0.43 g/g of hemicelluloses on AC were achieved under the conditions of 30°C, pH 7 and 3h with SL/AC weight ratio of 90. The addition of polydiallyldimethylammonium chloride (PDADMAC) to the SL/AC system significantly improved the adsorption of lignin to 2.5 g/g on AC. The molecular weight of PDADMAC considerably affected the results in that the higher MW PDADMAC led to less lignin, but more hemicelluloses, turbidity and chemical oxygen demand removals from the SL. The thermal analysis also revealed that the higher MW PDADMAC generated precipitates with a lower incineration temperature and heating value.

Overexpression of D-xylose reductase (xyl1) gene and antisense inhibition of D-xylulokinase (xyiH) gene increase xylitol production in Trichoderma reesei.

T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.

Xylitol production by genetically engineered Trichoderma reesei strains using barley straw as feedstock.

Xylitol, a naturally occurring five-carbon sugar alcohol derived from D-xylose, is currently in high demand by industries. Trichoderma reesei, a prolific industrial cellulase and hemicellulase producing fungus, is able to selectively use D-xylose from hemicelluloses for xylitol production. The xylitol production by T. reesei can be enhanced by genetic engineering of blocking further xylitol metabolism in the D-xylose pathway. We have used two different T. reesei strains which are impaired in the further metabolism of xylitol including a single mutant in which the xylitol dehydrogenase gene was deleted (∆xdh1) and a double mutant where additionally L-arabinitol-4-dehydrogenase, an enzyme which can partially compensate for xylitol dehydrogenase function, was deleted (∆lad1∆xdh1). Barely straw was first pretreated using NaOH and Organosolv pretreatment methods. The highest xylitol production of 6.1 and 13.22 g/L was obtained using medium supplemented with 2 % Organosolv-pretreated barley straw and 2 % D-xylose by the ∆xdh1 and ∆lad1∆xdh1 strains, respectively.

Effect of different carbon sources on cellulase production by Hypocrea jecorina (Trichoderma reesei) strains.

The ascomycete Hypocrea jecorina, an industrial (hemi)cellulase producer, can efficiently degrade plant polysaccharides. At present, the biology underlying cellulase hyperproduction of T. reesei, and the conditions for the enzyme induction, are not completely understood. In the current study, three different strains of T. reesei, including QM6a (wild-type), and mutants QM9414 and RUT-C30, were grown on 7 soluble and 7 insoluble carbon sources, with the later group including 4 pure polysaccharides and 3 lignocelluloses. Time course experiments showed that maximum cellulase activity of QM6a and QM9414 strains, for the majority of tested carbon sources, occurred at 120 hrs, while RUT-C30 had the greatest cellulase activity around 72 hrs. Maximum cellulase production was observed to be 0.035, 0.42 and 0.33 µmol glucose equivalents using microcrystalline celluloses for QM6a, QM9414, and RUTC-30, respectively. Increased cellulase production was positively correlated in QM9414 and negatively correlated in RUT-C30 with ability to grow on microcrystalline cellulose.

Fungal biodegradation and enzymatic modification of lignin.

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H(2)O(2)-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed.

Fungal bioconversion of lignocellulosic residues; opportunities & perspectives.

The development of alternative energy technology is critically important because of the rising prices of crude oil, security issues regarding the oil supply, and environmental issues such as global warming and air pollution. Bioconversion of biomass has significant advantages over other alternative energy strategies because biomass is the most abundant and also the most renewable biomaterial on our planet. Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi such as Trichoderma reesei and Aspergillus niger produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and a few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex called cellulosome, which is associated with the cell wall. In filamentous fungi, cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and beta-glucosidases work efficiently on cellulolytic residues in a synergistic manner. In addition to cellulolytic/hemicellulolytic activities, higher fungi such as basidiomycetes (e.g. Phanerochaete chrysosporium) have unique oxidative systems which together with ligninolytic enzymes are responsible for lignocellulose degradation. This review gives an overview of different fungal lignocellulolytic enzymatic systems including extracellular and cellulosome-associated in aerobic and anaerobic fungi, respectively. In addition, oxidative lignocellulose-degradation mechanisms of higher fungi are discussed. Moreover, this paper reviews the current status of the technology for bioconversion of biomass by fungi, with focus on mutagenesis, co-culturing and heterologous gene expression attempts to improve fungal lignocellulolytic activities to create robust fungal strains.