Multidimensional engineering strategies for strengthening exogenous synthesis of collagens and elastin monomers

Collagens and elastin (ELN), the most abundant proteins in the human body, prefer specific amino acids and post-translational modification (PTM), making their exogenous synthesis challenging. Here we developed multidimensional strategies to improve collagen exogenous synthesis. In the transcriptional layer, we optimized the phage-assisted noncontinuous evolution method to enhance T7 RNA polymerase processivity on the collagen III (CO3A1) gene, obtaining a mutant (K345E and P474L) with over twofold higher transcription performance. In the PTM layer, we screened the Moumouvirus prolyl 4-hydroxylase mutants using structure-based protein clustering and fluorescence-activated droplet sorting method, obtaining a mutant (Δ2-23, S109P, L122F, V169I, and P181Y) with about threefold increased activity to hydroxylate prolines in CO3A1 and ELN. Moreover, in the RNA stability and translation layer, we improved a cell-free protein synthesis system, significantly boosting CO3A1 and ELN yields to 0.31 and 2.58 g/L, respectively. These findings are of valuable implications for industrial collagens and ELN production.