An engineered Pichia pastoris platform for the biosynthesis of silk-based nanomaterials with therapeutic potential.

Silk fibroin (SF) from the cocoon of silkworm has exceptional mechanical properties and biocompatibility and is used as a biomaterial in a variety of fields. Sustainable, affordable, and scalable manufacturing of SF would enable its large-scale use. We report for the first time the high-level secretory production of recombinant SF peptides in engineered Pichia pastoris cell factories and the processing thereof to nanomaterials. Two SF peptides (BmSPR3 and BmSPR4) were synthesized and secreted by P. pastoris using signal peptides and appropriate spacing between hydrophilic sequences. By strain engineering to reduce protein degradation, increase glycyl-tRNA supply, and improve protein secretion, we created the optimized P. pastoris chassis PPGSP-8 to produce BmSPR3 and BmSPR4. The SF fed-batch fermentation titers of the resulting two P. pastoris cell factories were 11.39 and 9.48 g/L, respectively. Protein self-assembly was inhibited by adding Tween 80 to the medium. Recombinant SF peptides were processed to nanoparticles (NPs) and nanofibrils. The physicochemical properties of nanoparticles R3NPs and R4NPs from the recombinant SFs synthesized in P. pastoris cell factories were similar or superior to those of RSFNPs (Regenerated Silk Fibroin NanoParticles) originating from commercially available SF. Our work will facilitate the production by microbial fermentation of functional SF for use as a biomaterial.

Building a Versatile Protein Production Platform Using Engineered Trichoderma reesei.

Trichoderma reesei has an extremely high capacity for synthesizing and secreting proteins, thus exhibiting promise as an expression platform for heterologous proteins. However, T. reesei secretes large amounts of native proteins, which hinders its widespread application for heterologous protein production. Here, we designed and built a series of T. reesei chassis using an iterative gene deletion approach based on an efficient genome editing system. Donor DNAs with specially designed construct facilitated screening of positive deletion strains without ectopic insertion. Finally, marker-free T. reesei chassis with lower rates of native protein secretion and low levels of extracellular protease activity were constructed after 11 consecutive rounds of gene deletion. Higher production levels of three heterologous proteins─a bacterial xylanase XYL7, a fungal immunomodulatory protein LZ8, and the human serum albumin HSA─were achieved with these chassis using the cbh1 promoter. It is possible that diverse high-value proteins might be produced at a high yield using this engineered platform.