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Mentions: Although Rut-C30 is a good cellulase-producer microorganism, some improvements are still possible . We sought to explore possibilities to engineer the cellulase production capacity of the overproducer Rut-C30 by alteration of a metabolic pathway. The idea was to eliminate phosphoglucose isomerase (PGI) activity in order to redirect the carbon flux through the pentose phosphate pathway (PPP). The two central pathways of carbon metabolism are glycolysis and the PPP (Figure 1). The PPP is the major source of NADPH, needed for the biosynthesis of many biomolecules, in particular fats . It also provides intermediates for the synthesis of amino acids: histidine is synthesised from ribose 5-phosphate (ribose-5-P), and erythrose 4-phosphate is one of the metabolic precursors needed for the synthesis of aromatic amino acids: phenylalanine, tyrosine and tryptophan . For this reason, it is obvious that the PPP plays an important role in the production of proteins and therefore this pathway is expected to be relevant especially in an organism with an efficient protein production system. The PPP intermediate ribose-5-P is also essential for the synthesis of nucleotides and nucleic acids.
The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30
Bottom Line: But, they did not increase the expression of gpdh.Morphological characteristics were the result of the pgi1 deletion.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.
Affiliation: VTT, P,O, Box 1000, (Tietotie 2, Espoo), FIN-02044 VTT, Finland. email@example.com
Background: 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 Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 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, Δpgi1 and cre1+Δpgi1 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. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose.
Conclusions: The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 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.
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