The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reeseiRut-C30

Limón, Pakula Tiina, Saloheimo Markku, Penttilä, Penttilä Merja

Description

Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1 . It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1 , encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δ pgi1 mutant. Both Δ pgi1 and cre1 + Δ pgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δ pgi1 and cre1 + Δ pgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1 . Δ pgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δ pgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δ pgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh . Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1 .

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 24
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production inTrichoderma reeseiRutC30 Limónet al.

Limónet al.Microbial Cell Factories2011,10:40 http://www.microbialcellfactories.com/content/10/1/40 (24 May 2011)

Limónet al.Microbial Cell Factories2011,10:40 http://www.microbialcellfactories.com/content/10/1/40

R E S E A R C HOpen Access The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production inTrichoderma reeseiRutC30 1,2* 11 1 M Carmen Limón, Tiina Pakula , Markku Saloheimoand Merja Penttilä

Abstract Background:Cellulase and hemicellulase genes in the fungusTrichoderma reeseiare repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reeseistrain RutC30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene,cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose6P from glucose6P. Results:We deleted the genepgi1, encoding PGI, in theT. reeseistrain RutC30 and we introduced thecre1gene + in aΔpgi1mutant. BothΔpgi1andcre1 Δpgi1mutants showed a pelletlike and growth as well as morphological alterations compared with RutC30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose,Δpgi1andcre1 + Δpgi1mutants showed higher cellulase activity than RutC30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene,xyr1.Δpgi1mutants were also affected in the extracellularbgalactosidase activity. Levels of mRNA of the glucose 6phosphate dehydrogenase did not increase inΔpgi1during growth on glucose. Conclusions:The ability to grow in media with glucose as the sole carbon source indicated thatTrichoderma Δpgi1mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of thepgi1deletion. Deletion ofpgi1in RutC30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with thecre11mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression ofxyr1.

Background Trichoderma reesei, an anamorph of the speciesHypo crea jecorina, stands out for its ability to degrade cellu lose and hemicellulose. Cellulose is a long polymer ofb 1,4linked Dglucospyranose units. Degradation of cellu lose to glucose requires the synergistic action of differ ent types of enzymes. InT. reesei, at least several endoglucanases (EGI/Cel7B, EGII/Cel5A, EGIII/Cel12A,

* Correspondence: [e-mail] 1 VTT, P.O. Box 1000, (Tietotie 2, Espoo), FIN02044 VTT, Finland Full list of author information is available at the end of the article

EGIV/Cel61A, and EGV/Cel45A), exoglucanases (the cellobiohydrolases CBHI/Cel7A and CBHII/Cel6A) and bglucosidases (BGLI/Cel3A and BGLII/Cel1A) contri bute to the total cellulase activity [1,2]. An additional protein, swollenin, disrupts crystalline cellulose struc tures, making polysaccharides more accessible [3]. Cellulase induction inT. reeseiis modulated by several physiological and environmental conditions, such as growth rate [4], sulphur [5] and light [6,7]. Cellulose and disaccharides such as lactose, cellobiose and sophor ose (glycosylb1,2glucose) [8,9] can act as inducers

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