Engineering the cbh1 Promoter of Trichoderma reesei for Enhanced Protein Production by Replacing the Binding Sites of a Transcription Repressor ACE1 to Those of the Activators.

The strong and inducible cbh1 promoter is most widely used to express heterologous proteins, useful in food and feed industries, in Trichoderma reesei. Enhancing its ability to direct transcription provides a general strategy to improve protein production in T. reesei. The cbh1 promoter was engineered by replacing eight binding sites of the transcription repressor ACE1 to those of the activators ACE2, Hap2/3/5, and Xyr1. While changing ACE1 to Hap2/3/5-binding sites completely abolished the transcription ability, replacements with ACE2- and Xyr1-binding sites (designated cbh1pA and cbh1pX promoters, respectively) largely improved the promoter transcription efficiency, as reflected by expression of a reporter gene DsRed. The cbh1pA and cbh1pX promoters were applied to improve secretory expression of a codon-optimized mannanase from Aspergillus niger to 3.6- and 5.0-fold higher, respectively, which has high application potential in feed industry.