Background

Youth and beauty in the human body are synonymous with health and vitality, which always run through people's life to become an important concern of society (Goyal et al., 2022). Cell activity and state are fundamental factors in maintaining beauty, which can be regulated by proteins such as collagens and elastin, ribonucleic acids, fatty acids and active small molecules (e.g., peptides, ectoine) (Avila Rodriguez et al., 2018).

As the most abundant protein in the human body containing 28 subtypes with 44 members, collagens act widely as connectors in conjunctival and connective tissue, such as skin, joints, and bones (Holmes et al., 2018). It has been verified that the thickness and strength of collagen fibers are destroyed with age increasing, which strongly relate with skin aging phenomena, such as wrinkles, rarefaction of bone, and muscle weakness (Shoulders and Raines, 2009). Collagens are star molecules in the beauty and medical fields for their cytology function and weak immunogenicity. In medical treatment, collagens can interact with platelets and act as scaffold to produce clotting phenomena and accelerate wound healing (Avila Rodriguez et al., 2018). Cosmetically, collagen backfilling therapy is a clinically proven safe treatment for facial wrinkles, scars or facial skin defects. In the diet and spreads, supplementing with collagen lysate will maintain the youthful state of the skin (Shoulders and Raines, 2009). Therefore, collagen-related products have a huge market demand, which has reached more than $15 billion in 2022 (You et al., 2023).

There are two main types of collagens on the market. One is animal collagen oligopeptides (from pig, cattle and fish), which mainly industrialized by acid and enzymatic lysis to reduce immune rejection and allergic reactions. This method has the advantages of lower cost, large yield and mature technology, but there is a risk of difficult purity control and infection of animal-derived diseases (Avila Rodriguez et al., 2018; Martinez-Puig et al., 2023). The other is recombinant human collagens produced in genetic engineering cells, such as E. coli, yeast, insect cells and genetically modified crops (Brodsky and Ramshaw, 2017; Fang et al., 2023). This method has the advantages of high protein purity and low immunogenicity, and has greater application prospects in the advanced market. However, due to the large molecular weight and structural repeatability of collagens, the ability of natural strains is too poor to synthesize collagen. Taking the human collagen III as example, one of the most widely used collagens, which is trihelix fibrous protein composed of three homologous CO3A1 polypeptide chains. CO3A1 consists of 1466 amino acids, which contain 413 glycine residues and 281 proline residues. What’s more, about 80% of CO3A1 polypeptide are repetitive and disordered region, in which over half of prolines were hydroxylated verified by mass spectrometry (Seyer and Kang, 1981). These bring great difficulty to the synthesis of full-length natural collagen. Such problems also exist in the synthesis elastin. Therefore, the efficient synthesis of collagen and elastin in its natural state has important application value in the fields of medical treatment, skin care and nutrition.

In this study, we systematically analyzed the function of collagens and elastin using bioinformatic methods, and improved the yield and PTM efficiency of recombinant full-length collagens in engineered E. coli and CFPS system using multi-dimensional directed evolution techniques.