(136b) Learning New Tricks for an Old Problem: Spider Silk Production in Recombinant Microorganisms

Spider silk proteins, or spidroins, possess a unique combination of desirable properties, including exceptional toughness, biocompatibility, biodegradability, self-assembly capability, thermostability, solvent resistance, and the ability to function as optical waveguides. The development of recombinant proteins that mimic spider silk is particularly appealing due to their tunable properties and potential for large-scale production. However, challenges such as low production titers, limited understanding of how construct design influences expression outcomes, and a lack of research on host systems beyond E. coli hinder progress in this field.

In our study, we investigated the expression of systematically varied spidroin-mimetic genes in different E. coli strains. Our findings reveal that a novel strain designed with both a reduction in basal expression and specific genetic mutations can enhance the titer of recombinant spidroins to levels twelve times greater than baseline. However, these benefits did not extend to a similar elastin-like peptide. Metabolic Flux Analysis (MFA) provided critical insights into the metabolic alterations induced by the introduction of spider silk genes, revealing that the high intrinsic disorder of the silk constructs may generate strong toxicity, contributing to atypical flux profiles. Notably, MFA highlighted that adding seven amino acids to M9 minimal media can significantly boost spider silk protein production.

We also explored secretion as a strategy to address the challenges of industrial silk production. Our research establishes the first successful secretion of recombinant silk proteins in Bacillus megaterium, utilizing the Sec secretion pathway along with N-terminal signal peptides, which strongly depend on the specific sequence of the signal peptide. Various parameters, such as inducer concentration, fermentation temperature, and cell growth, were examined to enhance the titers of secreted proteins and to assess whether secretion mitigates construct toxicity.

Moreover, we have developed innovative strains of the Pseudomonas genus capable of producing spider silk. We optimized these strains by testing different expression levels through chromosomal integration and refining media formulations. Notably, we successfully employed linear alkanes as carbon sources for spider silk production, and we demonstrated the ability to produce spider silk from depolymerized polyethylene (PE) mixtures. This groundbreaking work paves the way for upcycling plastic waste into renewable biomaterials, contributing to sustainability efforts in the future.