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.

Bioproduction of biomacromolecules for antiviral applications.

The societal damage brought on by viral epidemics indicates that next-generation antiviral treatments must be developed and deployed. Biomacromolecules are a diverse class of compounds that can potentially exhibit potent antiviral activity. Their efficacy and mechanisms of action are dependent upon multiple structural factors, including molecular weight, degree and position of sulfation, and backbone stereochemistry. Extracting biomacromolecules from animals and plants for healthcare applications is undesirable, as these methods are unable to yield products with well-defined chemical structures. Modern advances utilizing recombinant microbes and metabolic pathway engineering can be a key step towards large-scale bioproduction of tailored biomacromolecules for targeted antiviral applications.

Chemical Synthesis of Silk-Mimetic Polymers.

Silk is a naturally occurring high-performance material that can surpass man-made polymers in toughness and strength. The remarkable mechanical properties of silk result from the primary sequence of silk fibroin, which bears semblance to a linear segmented copolymer with alternating rigid ("crystalline") and flexible ("amorphous") blocks. Silk-mimetic polymers are therefore of great emerging interest, as they can potentially exhibit the advantageous features of natural silk while possessing synthetic flexibility as well as non-natural compositions. This review describes the relationships between primary sequence and material properties in natural silk fibroin and furthermore discusses chemical approaches towards the synthesis of silk-mimetic polymers. In particular, step-growth polymerization, controlled radical polymerization, and copolymerization with naturally derived silk fibroin are presented as strategies for synthesizing silk-mimetic polymers with varying molecular weights and degrees of sequence control. Strategies for improving macromolecular solubility during polymerization are also highlighted. Lastly, the relationships between synthetic approach, supramolecular structure, and bulk material properties are explored in this review, with the aim of providing an informative perspective on the challenges facing chemical synthesis of silk-mimetic polymers with desirable properties.