Fungal arabinan and l-arabinose metabolism

l-Arabinose is the second most abundant pentose beside d-xylose and is found in the plant polysaccharides, hemicellulose and pectin. The need to find renewable carbon and energy sources has accelerated research to investigate the potential of l-arabinose for the development and production of biofuels and other bioproducts. Fungi produce a number of extracellular arabinanases, including α-l-arabinofuranosidases and endo-arabinanases, to specifically release l-arabinose from the plant polymers. Following uptake of l-arabinose, its intracellular catabolism follows a four-step alternating reduction and oxidation path, which is concluded by a phosphorylation, resulting in d-xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. The genes and encoding enzymes l-arabinose reductase, l-arabinitol dehydrogenase, l-xylulose reductase, xylitol dehydrogenase, and xylulokinase of this pathway were mainly characterized in the two biotechnological important fungi Aspergillus niger and Trichoderma reesei. Analysis of the components of the l-arabinose pathway revealed a number of specific adaptations in the enzymatic and regulatory machinery towards the utilization of l-arabinose. Further genetic and biochemical analysis provided evidence that l-arabinose and the interconnected d-xylose pathway are also involved in the oxidoreductive degradation of the hexose d-galactose

The Promoter Toolbox for Recombinant Gene Expression in Trichoderma reesei

The ascomycete Trichoderma reesei is one of the main fungal producers of cellulases and xylanases based on its high production capacity. Its enzymes are applied in food, feed, and textile industry or in lignocellulose hydrolysis in biofuel and biorefinery industry. Over the last years, the demand to expand the molecular toolbox for T. reesei to facilitate genetic engineering and improve the production of heterologous proteins grew. An important instrument to modify the expression of key genes are promoters to initiate and control their transcription. To date, the most commonly used promoter for T. reesei is the strong inducible promoter of the main cellobiohydrolase cel7a. Beside this one, there is a number of alternative inducible promoters derived from other cellulase- and xylanase encoding genes and a few constitutive promoters. With the advances in genomics and transcriptomics the identification of new constitutive and tunable promoters with different expression strength was simplified. In this review, we will discuss new developments in the field of promoters and compare their advantages and disadvantages. Synthetic expression systems constitute a new option to control gene expression and build up complex gene circuits. Therefore, we will address common structural features of promoters and describe options for promoter engineering and synthetic design of promoters. The availability of well-characterized gene expression control tools is essential for the analysis of gene function, detection of bottlenecks in gene networks and yield increase for biotechnology applications

The Hypocrea jecorina (Trichoderma reesei) hypercellulolytic mutant RUT C30 lacks a 85 kb (29 gene-encoding) region of the wild-type genome

<p>Abstract</p> <p>Background</p> <p>The hypercellulolytic mutant <it>Hypocrea jecorina </it>(anamorph <it>Trichoderma reesei</it>) RUT C30 is the <it>H. jecorina </it>strain most frequently used for cellulase fermentations and has also often been employed for basic research on cellulase regulation. This strain has been reported to contain a truncated carbon catabolite repressor gene <it>cre1 </it>and is consequently carbon catabolite derepressed. To date this and an additional frame-shift mutation in the glycoprotein-processing β-glucosidase II encoding gene are the only known genetic differences in strain RUT C30.</p> <p>Results</p> <p>In the present paper we show that <it>H. jecorina </it>RUT C30 lacks an 85 kb genomic fragment, and consequently misses additional 29 genes comprising transcription factors, enzymes of the primary metabolism and transport proteins. This loss is already present in the ancestor of RUT C30 – NG 14 – and seems to have occurred in a palindromic AT-rich repeat (PATRR) typically inducing chromosomal translocations, and is not linked to the <it>cre1 </it>locus. The mutation of the <it>cre1 </it>locus has specifically occurred in RUT C30. Some of the genes that are lacking in RUT C30 could be correlated with pronounced alterations in its phenotype, such as poor growth on α-linked oligo- and polyglucosides (loss of maltose permease), or disturbance of osmotic homeostasis.</p> <p>Conclusion</p> <p>Our data place a general caveat on the use of <it>H. jecorina </it>RUT C30 for further basic research.</p

A versatile toolkit for high throughput functional genomics with Trichoderma reesei

<p>Abstract</p> <p>Background</p> <p>The ascomycete fungus, <it>Trichoderma reesei </it>(anamorph of <it>Hypocrea jecorina</it>), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies.</p> <p>Results</p> <p>Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in <it>T. reesei</it>. We provide a primer database for gene deletion using the <it>pyr4, amdS </it>and <it>hph </it>selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a <it>T. reesei </it>strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.</p> <p>Conclusions</p> <p>Using this strategy and the materials provided, high throughput gene deletion in <it>T. reesei </it>becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of <it>T. reesei </it>for cellulase expression and hence second generation biofuel production.</p

Metabolic engineering strategies for the improvement of cellulase production by Hypocrea jecorina

Hypocrea jecorina (= Trichoderma reesei) is the main industrial source of cellulases and hemicellulases used to depolymerise plant biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. Cellulases are formed adaptively, and several positive (XYR1, ACE2, HAP2/3/5) and negative (ACE1, CRE1) components involved in this regulation are now known. In addition, its complete genome sequence has been recently published, thus making the organism susceptible to targeted improvement by metabolic engineering. In this review, we summarise current knowledge about how cellulase biosynthesis is regulated, and outline recent approaches and suitable strategies for facilitating the targeted improvement of cellulase production by genetic engineering

Kinetic transcriptome analysis reveals an essentially intact induction system in a cellulase hyper-producer Trichoderma reesei strain

International audienceBackground: The filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG 14 and RUT C30. In this follow-up work, we examine how these mutations affect phenotype as regards the transcriptome and cultivation behaviour. Results: We performed kinetic transcriptome analysis of the NG 14 and RUT C30 strains of early enzyme production induced by lactose using bioreactor cultivations close to an industrial cultivation regime. RUT C30 exhibited both earlier onset of protein production (3 h) and higher steady-state productivity. A rather small number of genes compared to previous studies were regulated (568), most of them being specific to the NG 14 strain (319). Clustering analysis highlighted similar behaviour for some functional categories and allowed us to distinguish between induction-related genes and productivity-related genes. Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting that these clusters contain genes playing a role in strain performance. Conclusions: This is the first kinetic analysis of a transcriptomic study carried out under conditions approaching industrial ones with two related strains of T. reesei showing distinctive cultivation behaviour. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei. Genes from two specific clusters might be potential targets for such genetic engineering