(596b) Silk Fibroin Production in Escherichia coli is Limited By a Positive Feedback Loop between Metabolic Burden and Toxicity Stress

To investigate the metabolic elasticity and production bottlenecks for recombinant silk proteins in Escherichia coli, we performed a comprehensive characterization of one elastin-like peptide strain (ELP) and two silk protein strains (A5 4mer, A5 16mer). Our approach included ¹³C metabolic flux analysis, genome-scale modeling, transcription analysis, and ¹³C-assisted media optimization experiments. Three engineered strains maintained their central flux network during growth, while measurable metabolic flux redistributions (such as the Entner–Doudoroff pathway) were detected. Under metabolic burdens, the reduced TCA fluxes forced the engineered strain to rely more on substrate-level phosphorylation for ATP production, which increased acetate overflow. Acetate (as low as 10 mM) in the media was highly toxic to silk-producing strains, which reduced 4mer production by 43% and 16mer by 84%, respectively. Due to the high toxicity of large-size silk proteins, 16mer's productivity was limited, particularly in the minimal medium. Therefore, metabolic burden, overflow acetate, and toxicity of silk proteins may form a vicious positive feedback loop that fractures the metabolic network. Three solutions could be applied: 1) addition of building block supplements (i.e., eight key amino acids: His, Ile, Phe, Pro, Tyr, Lys, Met, Glu) to reduce metabolic burden; 2) disengagement of growth and production; and 3) use of non-glucose based substrate to reduce acetate overflow. Other reported strategies were also discussed in light of decoupling this positive feedback loop.