Efficient Extracellular Production of Insulin Glargine in Trichoderma reesei by an Integrative Secretion‐Based Strategy

Insulin glargine, a long-acting insulin analog, is essential for diabetes treatment. However, its industrial production remains challenging due to limitations in conventional expression systems. Here, we employed the industrial filamentous fungus Trichoderma reesei as an alternative expression host for insulin glargine production, leveraging its superior protein secretion capacity. Initially, expression constructs containing the constitutive Pcdna1 promoter and CBH1 signal peptide (SP1) showed successful transcription but failed to achieve extracellular secretion, presumably due to induced endoplasmic reticulum (ER) stress. Consequently, we implemented a fusion protein strategy utilising three distinct carrier proteins (CBH1, CBH2, and LA-20) to enhance glargine secretion. Notably, the CBH1 fusion not only enabled detectable glargine secretion but also significantly alleviated the ER stress. Furthermore, replacement of SP1 with the Aspergillus niger β-glucoamylase signal peptide achieved a fourfold enhancement in glargine secretion and further reduced cellular stress responses. Following these systematic optimizations, a final yield of 58.95 mIU/L glargine was achieved in shake-flask cultures. Thus, the combined strategy described here could achieve extracellular production of glargine in T. reesei, suggesting that it is a promising host for secretory production of therapeutic recombinant proteins, particularly complex analogs like glargine.