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

Abstract

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^-1 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.

Keywords: Aspergillus niger; Collagen; Heterologous expression system; Transcriptomics.

Fig. 1

Modular cloning for the construction of a suite of collagen expressing cassettes. The modular cloning system allows interchangeable transcriptional units (TU) to be ligated together into a single plasmid backbone. TU-1 contains the collagen III sequence fused to the glaA secretion signal. Constructs utilized polycistronic expression of luciferase using a P2A sequence or an eGFP tagged collagen III. TU-4 contains the prolyl-4-hydroxylase variant in bold. Transcriptional units may also be replaced by „dummy “ units, 8 bp flanked by the 4 bp sticky ends for ligation that confer no biological function. All plasmids in this study were constructed with transcriptional units in this order. P: promoter; T: terminator

Fig. 2

A Luciferase luminescence as a reporter for collagen expression: The P2A sequence between the collagen constructs and luciferase reporter gene causes ribosomal skipping during gene translation, resulting in no peptide bond formation between the glycine and proline residues within the P2A region. This produces equimolar translation of each peptide, collagen III and luciferase, under the Tet-on promoter. Strains containing Tet-on inducible collagen cassette were inoculated (5.0x10⁶ spores/mL) to a 96 well plate into CM (200μl) containing beetle luciferin (0.4 mM) and doxycycline (20 μg/mL). Luminescence was detected over 24 h using a Perkin Elmer 2030 Multilabel Reader VictorTM X3 with the average of experimental triplicates normalised to OD at 595 nm depicted. Negative control (NC) was uninoculated media, positive control (PC) was VG8.27 expressing luciferase under the Tet-on promoter. Reduction in luciferase expression at ~20 h was consistent with sporulation of these isolates causing subsequent blocking of luciferase detection. B Fluorescence microscopy confirms confirms translation of eGFP tagged collagen III: TM17.1 expressing a collagen III::eGFP cassette. Spores (1.0x10⁶) were cultivated for 24 h at room temperature on solid minimal medium containing 20 μg/mL doxycycline and fluorescence was measured with excitation at 450-490 nm and emission at 500-550 nm with a differential interference microscope (Leica)

Fig. 3

Hydroxylated collagen III is detectable in the supernatant of the prtT ⁻ mutant TM4.2.1: Total proteins from culture filtrate (20 µl 5 × concentrated) from GalUA medium shake flask cultures inoculated with multiple collagen expressing strains were separated using SDS-PAGE (12%). Highlighted bands were excised and analysed by LC/MS for identification. Peptide fragments (F1–F11) from the 17-kDa band spanning the expected collagen III amino acid sequence were detected. The fragments indicated that a number of proline residues were hydroxylated (hyp, grey square) in the Y position of the Gly–X–Y tripartite repeat

Fig. 4

Preferred nitrogen sources decrease supernatant degradation of hydrolysed collagen: Supernatant hydroxyproline concentration as an indicator of the utilisation of collagen as a nitrogen source. Progenitor isolate FGE2.1 was inoculated in 50 mL shake-flask cultures containing 70 mM of various nitrogen sources and 1.5 mgL⁻¹ Peptan^® (hydrolysed collagen, Rousselot, Belgium). Culture filtrate was sampled every 24 h up to 72 h. Percentage loss of collagen calculated as the average loss in hydroxyproline relative to concentration at t=0 of biological and experimental duplicates.

Fig. 5

A. niger strains expressing collagen III under the tef1 promoter secrete the protein into the supernatant A. Dot Blot HiBiT Assay: Isolates inoculated to 50 mL CM (1% Glucose) and grown for 96 hours at 30°C and 200 rpm with protease inhibitors (Pierce, Roche). 100 μl supernatant was mixed 1:1 with 1xPBS and 200 μl was added to the 0.2 μM nitrocellulose membrane via vacuum filtration before use in the HiBiT detection assay (Promega). Membrane was imaged using the ChemiDoc imager (BioRad) with cumulative image acquirement, 30 s intervals for 5 min. S – supernatant; P – promoter; SS – secretion signal

Fig. 6

A proline specific endopeptidase, protA, is highly upregulated in the collagen-secreting isolate TM30.4. Comparative transcriptomic analyses for differential gene expression between TM30.4 and the protease deficient progenitor FGE2.1 were performed. Isolates were cultivated in 50 mL CM shake flask and total RNA samples extracted at t = 15, 18 and 21 h after inoculation, based on previous supernatant signal observed (Supplementary Fig. 1). High stringency differential gene expression was performed with a mean raw count > 200, a log₂ fold-change > 1 (upregulated) or log₂ fold-change < − 1 (downregulated) and a multiple hypothesis corrected p-value < 0.05

Fig. 7

∆protA isolate TM44.2 expressing a viral P4H secretes partially hydroxylated collagen with increased supernatant stability. A Total unmodified culture filtrate proteins (15 μL) from shake-flask cultures of GalUA medium supplemented with 5% glucose were separated by SDS-PAGE (4-15%, Biorad) and either transferred to nitrocellulose for HiBiT blotting (right) or stained for 30 min in Coomassie G in 30 mM HCl (left). Arrows indicate bands extracted for analysis by LC-MS. B Lower protein band marked in A was excised and sent for protein identification using LC-MS against the MASCOT database and A. niger secretome, allowing for mass changes corresponding to hydroxylation of proline residues. The peptide fragments (F1-F3) identified are marked in red in the expected amino acid sequence of the HiBiT tagged collagen III. Oxidation (hydroxylation) present on methionine (M) or proline (P) residues is underlined (right). Hydroxylated proline residues (hyp) marked in grey (left)