Superior cellulolytic activity of Trichoderma guizhouense on raw wheat straw.

Lignocellulosic plant biomass is the world's most abundant carbon source and has consequently attracted attention as a renewable resource for production of biofuels and commodity chemicals that could replace fossil resources. Due to its recalcitrant nature, it must be pretreated by chemical, physical or biological means prior to hydrolysis, introducing additional costs. In this paper, we tested the hypothesis that fungi which thrive on lignocellulosic material (straw, bark or soil) would be efficient in degrading untreated lignocellulose. Wheat straw was used as a model. We developed a fast and simple screening method for cellulase producers and tested one hundred Trichoderma strains isolated from wheat straw. The most potent strain-UB483FTG2/ TUCIM 4455, was isolated from substrate used for mushroom cultivation and was identified as T. guizhouense. After optimization of growth medium, high cellulase activity was already achieved after 72 h of fermentation on raw wheat straw, while the model cellulase overproducing strain T. reesei QM 9414 took 170 h and reached only 45% of the cellulase activity secreted by T. guizhouense. Maximum production levels were 1.1 U/mL (measured with CMC as cellulase substrate) and 0.7 U/mL (ß-glucosidase assay). The T. guizhouense cellulase cocktail hydrolyzed raw wheat straw within 35 h. Our study shows that screening for fungi that successfully compete for special substrates in nature will lead to the isolation of strains with qualitatively and quantitatively superior enzymes needed for their digestion which could be used for industrial purposes.

Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei.

The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1. IMPORTANCE: Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism.

Xpp1 regulates the expression of xylanases, but not of cellulases in Trichoderma reesei.

BACKGROUND: The ascomycete Trichoderma reesei is industrially used for the production of cellulases. During the production process xylanases are co-secreted, which uses energy and nutrients. Cellulases and xylanases share the same main regulators, which makes a knowledge-based strain design difficult. However, previously a cis-element in the promoter of the main xylanase-encoding gene was identified as binding site for a putative repressor. Subsequently, three candidate repressors were identified in a pull-down approach. The expression of the most promising candidate, Xpp1 (Xylanase promoter-binding protein 1), was reported to be up-regulated on the repressing carbon source d-glucose and to bind the cis-element in vitro. RESULTS: In this study, Xpp1 was deleted and over-expressed in T. reesei. An in vivo DNA-footprint assay indicated that Xpp1 binds a palindromic sequence in the xyn2 promoter. Comparison of the deletion, the over-expression, and the parent strain demonstrated that Xpp1 regulates gene expression of xylanolytic enzymes at later cultivation stages. Xpp1 expression was found to be up-regulated, additionally to d-glucose, by high d-xylose availability. These findings together with the observed xyn2 transcript levels during growth on xylan suggest that Xpp1 is the mediator of a feedback mechanism. Notably, Xpp1 has neither influence on the d-xylose metabolism nor on the expression of cellulases. CONCLUSIONS: Xpp1 as regulator acting on the expression of xylanases, but not cellulases, is a highly promising candidate for knowledge-based strain design to improve the cellulases-to-xylanases ratio during industrial cellulase production.

Biocatalyzed approach for the surface functionalization of poly(L-lactic acid) films using hydrolytic enzymes.

Poly(lactic acid) as a biodegradable thermoplastic polyester has received increasing attention. This renewable polyester has found applications in a wide range of products such as food packaging, textiles and biomedical devices. Its major drawbacks are poor toughness, slow degradation rate and lack of reactive side-chain groups. An enzymatic process for the grafting of carboxylic acids onto the surface of poly(L-lactic acid) (PLLA) films was developed using Candida antarctica lipase B as a catalyst. Enzymatic hydrolysis of the PLLA film using Humicola insolens cutinase in order to increase the number of hydroxyl and carboxylic groups on the outer polymer chains for grafting was also assessed and showed a change of water contact angle from 74.6 to 33.1° while the roughness and waviness were an order of magnitude higher in comparison to the blank. Surface functionalization was demonstrated using two different techniques, (14) C-radiochemical analysis and X-ray photoelectron spectroscopy (XPS) using (14) C-butyric acid sodium salt and 4,4,4-trifluorobutyric acid as model molecules, respectively. XPS analysis showed that 4,4,4-trifluorobutyric acid was enzymatically coupled based on an increase of the fluor content from 0.19 to 0.40%. The presented (14) C-radiochemical analyses are consistent with the XPS data indicating the potential of enzymatic functionalization in different reaction conditions.

Chlorination of lignin by ubiquitous fungi has a likely role in global organochlorine production.

Soils and decayed plant litter contain significant quantities of chlorinated aromatic polymers that have a natural but largely unknown origin. We used cupric oxide ligninolysis coupled with gas chromatography/mass spectrometry to show that Curvularia inaequalis, a widely distributed litter ascomycete, chlorinated the aromatic rings of lignin in wood that it was degrading. In aspen wood decayed for 24 weeks, two chlorolignin fragments, 5-chlorovanillin and 2-chlorosyringaldehyde, were each found at approximately 10 mug/g of wood (dry weight). These levels resemble those of similar structures generally found in unpolluted environmental samples. Fractionation of the extractable proteins followed by tandem mass spectrometric analysis showed that the colonized wood contained a previously described C. inaequalis chloroperoxidase that very likely catalyzed lignin chlorination. Chlorolignin produced by this route and humus derived from it are probably significant components of the global chlorine cycle because chloroperoxidase-producing fungi are ubiquitous in decaying lignocellulose and lignin is the earth's most abundant aromatic substance.

Chlorination and cleavage of lignin structures by fungal chloroperoxidases.

Two fungal chloroperoxidases (CPOs), the heme enzyme from Caldariomyces fumago and the vanadium enzyme from Curvularia inaequalis, chlorinated 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane, a dimeric model compound that represents the major nonphenolic structure in lignin. Both enzymes also cleaved this dimer to give 1-chloro-4-ethoxy-3-methoxybenzene and 1,2-dichloro-4-ethoxy-5-methoxybenzene, and they depolymerized a synthetic guaiacyl lignin. Since fungal CPOs occur in soils and the fungi that produce them are common inhabitants of plant debris, CPOs may have roles in the natural production of high-molecular-weight chloroaromatics and in lignin breakdown.