Collagen molecules are fiber-forming proteins that are composed of three amino acids—glycine, proline, and hydroxyproline. They arrange themselves into a tightly wound triple helix. The amino acid compositions of collagen types vary considerably between species, and these variations affect chemical and physical properties, thermal stability, solution viscosity, and crosslinking density. The mechanical and chemical variation of human-derived collagen, coupled with the potential for infection, immunogenicity, and batch-to-batch variability in animal-derived collagen, have led to an increased interest in alternatives such as recombinant collagen. Recombinant collagen biosynthesis offers the potential for consistent, biochemically-identical collagen production at scale.
Creative Biogene's successful collagen production platform based on microbial fermentation is setting new benchmarks for quality. We have developed a wide and growing spectrum of microbial expression systems that are becoming available. Our platform can already produce several different types of collagen.
Recombinant human collagen expression models have been successfully demonstrated in both prokaryotic and eukaryotic cells, including Escherichia coli and fungus. This method eliminates the risk of interspecies variance and pathogenic infection, while ensures homogeneity across batches. P. pastoris is the preferred production system for recombinant human collagen in medical and cosmetic applications. Engineered P. pastoris can secrete high levels of recombinant human collagen fragments with defined length, composition, and physiochemical properties. Recombinant human collagen results in products with improved safety, traceability, reproducibility, and quality. S. cerevisiae has also been used to produce recombinant human collagen. Engineered S. cerevisiae containing prolyl hydroxylases can produce human type III collagen, respectively.
Bacterial collagens represent a biosynthetic ground up approach, where a triple-helical non-animal collagen molecule with no specific bioactivity can be designed to include desired interactions and regulated degradation. More than 100 putative collagen-like proteins have been identified in bacterial genomes, of which eight have been recombinantly expressed in Escherichia coli. The gram-positive bacterium Streptococcus pyogenes contains two collagen-like proteins, Scl1 and Scl2, which have been well characterized in terms of structure and functional properties. The Scl2 protein has been possible to generate constructs in a recombinant E. coli system with various sequence modifications of Scl2 and to establish large scale production methods. The Scl2 recombinant bacterial collagen system has advantages compared to recombinant human collagen strategies for large scale production and biomedical applications, and may serve as a prototype for engineering novel collagen-based biomaterials.
Figure 1 Incorporation of hydroxyproline in bacterial collagen (Yong Y. Peng, et al. 2018)
|Expression System||Transduced Gene||Expressed Collagen||Descriptions|
|Prokaryote||Escherichia coli||COL1A1||Type I||Different amino acid expression when compared to natural collagen|
|Escherichia coli||COL3A1; L230, L593 (APMV)||Type III||Expression collagen III and mimivirus propyl and lysyl hydroxylases yielded hydroxylation levels similar to those expressed in humans|
|Yeast||Pichia pastoris||COL1A1, PH4A/B||Type I||-|
|Pichia pastoris||COL3A1, PH4A/B||Type III||Recombinant hydroxylated collagen III exhibited hemostatic properties in vivo|
|Saccharomyces cerevisiae||COL3A1, PH4A/B||Type III||Computational algorithm determined optimal oligonucleotide sequence|
|Addition of non-native cysteine residues created crosslinking and anchoring sites; increased melting point compared to other RHC|
Table 1 Summary of recombinant expression systems (Davison-Kotler E, et al. 2019)
Several collagen-like polymers have been recombinantly expressed by E. coli and exhibit thermostability, despite the lack of post-translational hydroxylation. Given the current issues surrounding hydroxylation and amino acid sequence parity, collagen produced by E. coli and similar bacterial systems is best suited for mechanical applications, rather than those relating to physiological interactions.
The persistence of yeast as a model recombinant system is due to the characteristic rapid growth rates and ease of genetic modifications, coupled with the ability to synthesize enzymes capable of post-translational modification and protein folding. Although yeast lacks native propyl 4-hydroxylase (P4H), the transduction of P4H α and β subunits together with a collagen-coding gene results in the production of hydroxylated collagen fibrils. Recombinant human collagen synthesized by yeast is more similar to native collagen than that produced by E. coli systems. Advances in genetic manipulation have vastly improved the yields and quality of collagen produced by yeast expression systems, leading to the industrial scale production.
Creative Biogene is also conducting in vitro studies in order to investigate the biofunctionality of the new fermentative collagen. We have the commercial experience and technological flexibility to scale manufacturing on a need-based or rolling basis.
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