Scale up of fermentation of recombinant Escherichia coli for efficient production of spider drag silk protein MaSp1s and its dimers

Background: Spider dragline silk exhibits ultrahigh tensile strength and excellent ductility, making it one of the best-performing natural biomaterials. The major ampullate spidroin (MaSp1) has promising applications in the biomedical, chemical, and military industries owing to its good biocompatibility, biodegradability, and low immunogenicity. The generation of recombinant spidroin can significantly facilitate its scaled production but has several challenges, including the high cost of the downstream spidroin solubilization process and the resulting toxicity due to the use of organic solvents. Unlike common MaSp, MaSp short (MaSp1s) from Cyrtophoramoluccensis is a low-molecular-weight spidroin, lacking the typical repetitive sequences and long poly(A) motif. These features enable the heterologous production of soluble spidroin. Results: In this study, rMaSp1 and its dimer rMaSp1s-2Core were expressed in soluble form by introducing the SUMO fusion tag and the self-shearing peptide intein. To improve the yield of recombinant spidroin using shake-flask fermentation, response surface analysis was used to optimize the conditions. The yields of rMaSp1 and rMaSp1s-2Core were 218.9 and 95.76 mg/L, respectively. Subsequently, fermentation was scaled up in a 5 L fermenter after adding metal ions and other growth factors to the medium. The optimal inoculation amount, induction temperature, loaded liquid, and feeding strategy were explored. Finally, the yields of rMaSp1 and rMaSp1s-2Core reached 1,112.2 and 297.8 mg/L, respectively. Furthermore, the dimerization of rMaSp1 monomers was achieved by introducing disulfide bonds via exogenous cysteine residues in the C-terminal domain. The secondary structure and self-assembly of rMaSp1 were also analyzed. Conclusion: This study successfully addressed key challenges in recombinant spidroin production by employing fusion tags (SUMO and self-shearing peptide intein) to enable the soluble expression of rMaSp1 and its dimer rMaSp1s-2Core. The secondary structure and self-assembly analyses further contributed to our understanding of recombinant spidroin. These findings enable the large-scale production of spidroin and its potential applications in the biomedical, chemical, and military industries, overcoming previous hurdles related to the solubility and toxicity associated with downstream processing.