Comparison of intracellular and secretion-based strategies for production of human α-galactosidase A in the filamentous fungus Trichoderma reesei

Abstract

BackgroundTrichoderma reesei is known as a good producer of industrial proteins but has hitherto been less successful in the production of therapeutic proteins. In order to elucidate the bottlenecks of heterologous protein production, human α-galactosidase A (GLA) was chosen as a model therapeutic protein. Fusion partners were designed to compare the effects of secretion using a cellobiohydrolase I (CBHI) carrier and intracellular production using a gamma zein peptide from maize (ZERA) which accumulates inside the endoplasmic reticulum (ER). The two strategies were compared on the basis of expression levels, purification performance, enzymatic activity, bioreactor cultivations, and transcriptional profiling.ResultsConstructs were cloned into the cbh1 locus of the T. reesei strain Rut-C30. The secretion and intracellular strains produced 20 mg/l and 636 mg/l of GLA respectively. Purifications of secreted product were accomplished using Step-Tactin affinity columns and for intracellular product, a method was developed for gravity-based density separation and protein body solubilisation. The secreted protein had similar specific activity to that of the commercially available mammalian form. The intracellular version had 5-10-fold lower activity due to the enzymes incompatibility with alkaline pH. The secretion strain achieved 10% lower total biomass than either the parental or the intracellular strain. The patterns of gene induction for intracellular and parental strains were similar, whereas the secretion strain had a broader spectrum of gene expression level changes. Identification of the genes involved indicated strong secretion stress in the secretion strain and to a lesser extent also in intracellular production. Genes involved in the unfolded protein response (UPR) and ER-associated degradation were induced by GLA production, including; hac1, pdi1, prp1, cnx1, der1, and bap31.ConclusionsActive human α-galactosidase could most effectively be produced intracellularly in Trichoderma reesei at >0.5 g/l by avoidance of the extracellular environment, although purification was challenging due to specific activity losses. Strain analysis revealed that in addition to the issues with secreted proteases, the processes of secretion stress including UPR and ER degradation remain as bottlenecks for heterologous protein production. Genetic engineering to eliminate these bottlenecks is the logical path towards establishing a strain capable of producing sensitive heterologous proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0091-y) contains supplementary material, which is available to authorized users.

Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei

“…A further strategy to target proteins to the ER uses the γ-zein peptide (ZERA) derived from the maize storage protein. Analogously, these self-assembling fusion proteins form protein bodies surrounded by ER membrane which protect them from proteolysis [ 102 ]. Developing fungi as efficient production hosts for mammalian proteins also requires the inactivation of the frequently encountered proteases in the fermentation broth.…”

Building a Versatile Protein Production Platform Using Engineered Trichoderma reesei

“…T. reesei has been applied to express a variety of heterologous proteins. − For example, up to approximately 1 g/L of β-glucosidase from Aspergillus terreus was expressed by T. reesei in a shake-flask culture …”

Novel genetic tools that enable highly pure protein production in Trichoderma reesei

“…The calB coding region was modified by replacing native N-terminal secretion signal with CBHI carrier protein and Kex2 cleavage site encoding sequences 10 . The N-terminal part of CBHI is commonly used as a fusion carrier protein for facilitating heterologous protein secretion into culture medium 10 , 35 – 37 . The high natural capacity of T .…”