| Laccases are multicopper oxidases which can catalyze the oxidation of phenols and aromatic amine subtrates. These enzymes have been widely used in industry and biotechnology, such as the pulp lignin biodegradation, detoxification of wastewater, biological sensors and so on.The white rot fungus Trametes sp. AH28-2can secrete three laccases including laccase A (LacA), LacB and LacC. LacA is the main enzyme component, which has been applied to the degradation of straw stalk lignin and the reduction of oxygen consumption in pulp wastewater treatment. The lacA gene has been cloned and expressed in Pichia and Trichoderma reesei. However, the thermal stability of LacA expressed in Pichia is poor and the optimal pH is reduced. Nevertheless, the enzymatic properties of LacA expressed in Trichoderma reesei are closer to those from the original strain Trametes sp. AH28-2.Since Trichoderma reesei can produce large amounts of cellulases, it’s one of the important fungal strains used for cellulose degradation. Meanwhile, it’s also the potential eukaryotic host for heterologous protein expression. The major cellulase cellobiohydrolase Ⅰ (CBHI or Cel7a) accounts for about60%of the total protein secreted outside the cell, and its coding gene cbhl is single copy, so the cbhl promoter Pcbh1is a widely used strong promoter in heterologous protein expression. Furthermore, the transcription of eukaryotic genes could be affected by their location, so the cbhl locus may also be a potential site for efficient transcription and expression. In this study, on the one hand, the lacA gene under the control of the strong cbhl promoter was randomly integrated into the Trichoderma reesei chromosome for heterologous expression; on the other hand, the cbhl gene locus was used as the target locus for heterologous expression of lacA gene. This study used these two strategies to explore the potential of heterologous protein expression in Trichoderma reesei and at the same time analyzed the enzymatic properties of the recombinant LacA.Firstly, the lac A gene expression vector under the control of Pcbh1called pCGTLacA was constructed by Double-joint PCR and molecular cloning techniques. The vector consisted of the lacA gene expression cassette Pcbh1-lacA under the control of the cbhl promoter and the cbhl signal peptide from Trichoderma reesei, the orotidine-5-monophosphate decarboxylase gene expression cassette pyrG (as a selection marker) and the downstream sequence of the cbhl gene Tcbh1. The fragment containing the lacA and pyrG expression cassette Pcbh1-lacA-pyrG was amplified using the pCGTLacA as template and transformed into Trichoderma reesei protoplasts for random integration.126transformants were chosen in the uracil-free plate, and finally7laccase-expressing strains containing1or2copies of the lacA gene were obtained by ABTS plate analysis, PCR analysis and Southern blot. Different culture media were applied to the fermention experiments, but all the fungal strains could not grow happily, secreted very low level of extracellular proteins, and no laccase activity was detected. Subsequently, we detected the transcription level of the lacA gene and the genes related to the unfoled protein stress response (UPR) as well as the endoplasmic reticulum associated degradation pathway (ERAD) in the transfromant Tulac118under the lactose induction condition. The results shew that the lacA gene was successfully transcribed and reached the highest mRNA level at about12h. In addition, the UPR and ERAD related genes were up-regulated in different degrees, which may be the reason why laccase couldn’t be secreted outside the cell in the liquid fermentation conditions.Using pCGTLacA as template, we amplified the lacA expression cassette called Pcbh1-lacA-pyrG-Tcbhl to replace the cbhl gene locus. In this expression cassette, the Pcbh1not only acted as the promoter of lacA gene, but also as the upstream sequence of cbhl together with downstream sequence called Tcbhl for homologous recombination (gene replacement) in the Trichoderma reesei chromosome. This gene replacement fragment was transformed into Trichoderma reesei protoplasts, and transformants were selected on the uracil-free plate, followed by PCR validation and ABTS identification. Finally, the TLac3tranformtant whose cbhl gene locus was successfully replaced by the lacA gene was obtained. The laccase activity was detected in the fermentation medium, and the highest activity was168.32U/L. the optimum temperature of the recombinant laccase was35℃, the optimum pH was4.0, and the pH stability was between2.4to8.0. TLac3was cultivated in the induction condition to test gene expression level, and the lacA transcription level peaked at44h. Comparative analysis of lacA and cbhl expression levels shew that, the highest expression levels of lacA and cbhl were comparable, but the lacA was about10h lagging behind. The above results indicated that the lacA gene was successfully heterologously expressed by replacing the cbhl locus in Trichoderma reesei, the recombinant laccase was secreted to extracellular matrix, and its enzymatic properties were stable. |
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