Abstract
Trichoderma reesei is an established protein production host with high natural capacity to secrete enzymes. The lack of efficient genome engineering approaches and absence of robust constitutive gene expression systems limits exploitation of this organism in some protein production applications. Here we report engineering of T. reesei for high-level production of highly enriched lipase B of Candida antarctica (calB) using glucose as a carbon source. Multiplexed CRISPR/Cas9 in combination with the use of our recently established synthetic expression system (SES) enabled accelerated construction of strains, which produced high amounts of highly pure calB. Using SES, calB production levels in cellulase-inducing medium were comparable to the levels obtained by using the commonly employed inducible cbh1 promoter, where a wide spectrum of native enzymes were co-produced. Due to highly constitutive expression provided by the SES, it was possible to carry out the production in cellulase-repressing glucose medium leading to around 4 grams per liter of fully functional calB and simultaneous elimination of unwanted background enzymes.
Highlights
The use of recombinant proteins in various industries is steadily increasing
We tested simultaneous deletion of three major cellulase genes: cellobiohydrolase II, endoglucanase I, and endoglucanase II. The deletion of these genes would lead to a strain with significantly reduced production of background proteins in the culture supernatant
Development of industrial protein production strains of T. reesei often requires several simultaneous genetic modifications. These include introducing the protein product-encoding expression cassette and deletion of selected native genes, such as proteases or cellulases. These actions are taken to maximize the productivity during extended cultivations, to decrease proteolytic activity, or to avoid secretion of unwanted proteins
Summary
The use of recombinant proteins in various industries is steadily increasing. For instance, the chemical industry aims at novel practices, where use of enzymes would ensure improved process performance, increased safety, and lower impact on the environment[1]. Protein purification from the typical production culture supernatant containing a wide mixture of enzymes may require extensive downstream processing and significantly affects the overall economic and technical feasibility of the bioprocess, especially considering the industrial scale of a typical cultivation[19] Due to these reasons, T. reesei has not been extensively used as a host in applications in which purity of the end-product www.nature.com/scientificreports/. To minimize co-production of unwanted proteins, deletion of the major cellulase genes or the main transcriptional regulator xyr[121] can provide a partial solution This may not be desired and straightforward, because the lack of cellulase activities limits conversion of carbon source substrate into smaller inducing molecules and may decrease the production output of T. reesei, in cellulose-containing conditions[14]
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