The multipurpose cell factory Aspergillus niger can be engineered to produce hydroxylated collagen.

Advances in tissue printing and wound healing necessitate a continuous global supply of collagen. Microbial systems are highly desirable to meet these demands as recombinant collagenous proteins can be guaranteed as free from animal viruses. The filamentous cell factory Aspergillus niger has been instrumental for decades in the production of organic acids, enzymes and proteins, yet this fungus has not been explored for recombinant collagen production. In this study, we conducted extensive genetic engineering and fermentation optimization to provide proof of principle that A. niger can produce hydroxylated collagen. We used a modular cloning system to generate a suite of cassettes encoding numerous N-terminal secretion signals, native collagen genes and, additionally, various prolyl-4-hydroxylases (P4H) for protein hydroxylation. Collagen transcription was supported by both luciferase reporter and eGFP tagged approaches. Peptide sequencing from culture supernatant confirmed A. niger produced partially hydroxylated collagen. We then conducted a range of media optimizations and RNA sequencing to, respectively, increase collagen production and identify proteases which we hypothesized were detrimental to recombinant protein titers. Thus, we deleted an endopeptidase encoding gene, protA, which was likely responsible for degrading secreting collagen. Ultimately, we were able to generate an isolate capable of producing hydroxylated collagen at titers of 5 mgL in shake flask models of fermentation. This study thus proves A. niger is a promising heterologous system to address the demand for virus-free collagen.