High-level Expression Of ?-mannanase In Trichoderma Reesei

Mannan is a main component of the plant cell wall,and is widely present in plant tissues.?-mannanase is the main mannan-degrading enzyme,whose applications have been established in different industries,such as animal feed,food,bio-refinery,textiles,detergents and papermaking.Therefore,increasing the production level and reducing the production cost of ?-mannanase have great application value and practical significance.Filamentous fungi(e.g.Trichoderma reesei)have the ability to efficiently synthesize and secrete cellulase and hemicellulase,and have great application prospects in the production of ?-mannanase.Promoters play an important role in the expression of proteins.Screening and modifying efficient promoters is a key strategy to achieve high-efficiency expression of target proteins.In this study,we selected the ?-mannanase Man5A derived from Penicillium oxalicum,and studied its high-efficiency expression strategy in T.reesei.The main results are as follows.(1)The constitutive promoter Pcdnal was used to realize the expression of Man5A in glucose medium.The man5A expression cassette driven by Pcdnal was constructed and randomly inserted into the genome of T.reesei strain QP4.The obtained Qman strains could produce relatively pure Man5A.A preliminary study on the properties of the obtained Man5A was carried out,which found that the enzyme can maintain high activity in the range of pH 2.5-5.5.The optimal temperature of Man5A is 80?,and its melting temperature was 80? as determined by differential scanning calorimetry.The key sequences affecting the efficiency of Pcdna1 were identified.When the Pcdna1 sequence was shortened from the origina1 1160 bp to about 831 bp,the efficiency was reduced to 81%.When it was shortened to 505 bp,the efficiency was significantly reduced to 10%.The performance of mixtures of Man5A-containing crude enzyme produced by T.reesei and ?-galactosidase-rich crude enzyme produced by P.oxalicum in the degradation of soybean meal was explored.The degradation efficiency was the highest when the two crude enzymes were mixed in equal proportion.(2)The inducible promoter Pcbh1 was used to realize the high-efficiency expression of Man5A in cellulose medium,and the expression level was further improved through the modifications of the promoter sequence and a transcription activator.By sequence truncation and engineering the transcription factor binding sites in the Pcbh1 sequence,three different Pcbh1 mutants were constructed.The efficiency of Pcbh1-IR was increased to twice that of the original promoter.Based on a high copy number strain that efficiently expresses ?-mannanase,overexpression of constitutive transcriptional activator XYR1A824V enabled high expression of ?-mannanase under both glucose and cellulose cultivation conditions.