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

Abstract

The strong and inducible <i>cbh1</i> promoter is most widely used to express heterologous proteins, useful in food and feed industries, in <i>Trichoderma reesei</i>. Enhancing its ability to direct transcription provides a general strategy to improve protein production in <i>T. reesei</i>. The <i>cbh1</i> 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 <i>DsRed.</i> The cbh1pA and cbh1pX promoters were applied to improve secretory expression of a codon-optimized mannanase from <i>Aspergillus niger</i> to 3.6- and 5.0-fold higher, respectively, which has high application potential in feed industry.