Redesigning transcription factor Cre1 for alleviating carbon catabolite repression in Trichoderma reesei.

Carbon catabolite repression (CCR), which is mainly mediated by Cre1 and triggered by glucose, leads to a decrease in cellulase production in Trichoderma reesei. Many studies have focused on modifying Cre1 for alleviating CCR. Based on the homologous alignment of CreA from wild-type Penicillium oxalicum 114-2 (Po-0) and cellulase hyperproducer JUA10-1(Po-1), we constructed a C-terminus substitution strain-Po-2-with decreased transcriptional levels of cellulase and enhanced CCR. Results revealed that the C-terminal domain of CreAPo-1 plays an important role in alleviating CCR. Furthermore, we replaced the C-terminus of Cre1 with that of CreAPo-1 in T. reesei (Tr-0) and generated Tr-1. As a control, the C-terminus of Cre1 was truncated and Tr-2 was generated. The transcriptional profiles of these transformants revealed that the C-terminal chimera greatly improves cellulase transcription in the presence of glucose and thus upregulates cellulase in the presence of glucose and weakens CCR, consistent with truncating the C-terminus of Cre1 in Tr-0. Therefore, we propose constructing a C-terminal chimera as a new strategy to improve cellulase production and alleviate CCR in the presence of glucose.

A Tet-on and Cre-loxP Based Genetic Engineering System for Convenient Recycling of Selection Markers in Penicillium oxalicum.

The lack of selective markers has been a key problem preventing multistep genetic engineering in filamentous fungi, particularly for industrial species such as the lignocellulose degrading Penicillium oxalicum JUA10-1(formerly named as Penicillium decumbens). To resolve this problem, we constructed a genetic manipulation system taking advantage of two established genetic systems: the Cre-loxP system and Tet-on system in P. oxalicum JUA10-1. This system is efficient and convenient. The expression of Cre recombinase was activated by doxycycline since it was controlled by Tet-on system. Using this system, two genes, ligD and bglI, were sequentially disrupted by loxP flanked ptrA. The successful application of this procedure will provide a useful tool for genetic engineering in filamentous fungi. This system will also play an important role in improving the productivity of interesting products and minimizing by-product when fermented by filamentous fungi.