Microbial production of megadalton titin yields fibers with advantageous mechanical properties.

Manmade high-performance polymers are typically non-biodegradable and derived from petroleum feedstock through energy intensive processes involving toxic solvents and byproducts. While engineered microbes have been used for renewable production of many small molecules, direct microbial synthesis of high-performance polymeric materials remains a major challenge. Here we engineer microbial production of megadalton muscle titin polymers yielding high-performance fibers that not only recapture highly desirable properties of natural titin (i.e., high damping capacity and mechanical recovery) but also exhibit high strength, toughness, and damping energy - outperforming many synthetic and natural polymers. Structural analyses and molecular modeling suggest these properties derive from unique inter-chain crystallization of folded immunoglobulin-like domains that resists inter-chain slippage while permitting intra-chain unfolding. These fibers have potential applications in areas from biomedicine to textiles, and the developed approach, coupled with the structure-function insights, promises to accelerate further innovation in microbial production of high-performance materials.

Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk.

Despite significant efforts to engineer their heterologous production, recombinant spider silk proteins (spidroins) have yet to replicate the unparalleled combination of high strength and toughness exhibited by natural spider silks, preventing their use in numerous mechanically demanding applications. To overcome this long-standing challenge, we have developed a synthetic biology approach combining standardized DNA part assembly and split intein-mediated ligation to produce recombinant spidroins of previously unobtainable size (556 kDa), containing 192 repeat motifs of the Nephila clavipes dragline spidroin. Fibers spun from our synthetic spidroins are the first to fully replicate the mechanical performance of their natural counterparts by all common metrics, i.e., tensile strength (1.03 ± 0.11 GPa), modulus (13.7 ± 3.0 GPa), extensibility (18 ± 6%), and toughness (114 ± 51 MJ/m3). The developed process reveals a path to more dependable production of high-performance silks for mechanically demanding applications while also providing a platform to facilitate production of other high-performance natural materials.

Exploiting nongenetic cell-to-cell variation for enhanced biosynthesis.

Biosynthesis enables renewable production of manifold compounds, yet often biosynthetic performance must be improved for it to be economically feasible. Nongenetic, cell-to-cell variations in protein and metabolite concentrations are naturally inherent, suggesting the existence of both high- and low-performance variants in all cultures. Although having an intrinsic source of low performers might cause suboptimal ensemble biosynthesis, the existence of high performers suggests an avenue for performance enhancement. Here we develop in vivo population quality control (PopQC) to continuously select for high-performing, nongenetic variants. We apply PopQC to two biosynthetic pathways using two alternative design principles and demonstrate threefold enhanced production of both free fatty acid (FFA) and tyrosine. We confirm that PopQC improves ensemble biosynthesis by selecting for nongenetic high performers. Additionally, we use PopQC in fed-batch FFA production and achieve 21.5 g l(-1) titer and 0.5 g l(-1) h(-1) productivity. Given the ubiquity of nongenetic variation, PopQC should be applicable to a variety of metabolic pathways for enhanced biosynthesis.

Single-Walled Carbon Nanotubes in Highly Viscous Media: A Comparison between the Dispersive Agents [BMIM][BF4 ], L121, and Triton X-100.

Dispersions of single-walled carbon nanotubes (SWNTs) have been prepared by using the room-temperature ionic liquid [BMIM][BF4 ] (1-butyl-3-methylimidazolium tetrafluoroborate), the triblock copolymer Pluronic L121 [poly(ethylene oxide)5 -poly(propylene oxide)68 -poly(ethylene oxide)5 ] and the non-ionic surfactant Triton X-100 (TX100) in the pure state. The size of the SWNTs aggregates and the dispersion degree in the three viscous systems depend on the sonication time, as highlighted by UV/Vis/NIR spectroscopy and optical microscopy analysis. A nonlinear increase in conductivity can be observed as a function of the SWNTs loading, as suggested by electrochemical impedance spectroscopy. The generation of a three-dimensional network of SWNTs showing a viscoelastic gel-like behavior above a critical percolation concentration has been found at 25 °C in all the investigated systems by oscillatory rheology measurements.

Engineering Escherichia coli for Conversion of Glucose to Medium-Chain ω-Hydroxy Fatty Acids and α,ω-Dicarboxylic Acids.

In search of sustainable approaches to plastics production, many efforts have been made to engineer microbial conversions of renewable feedstock to short-chain (C2-C8) bifunctional polymer precursors (e.g., succinic acid, cadaverine, 1,4-butanediol). Less attention has been given to medium-chain (C12-C14) monomers such as ω-hydroxy fatty acids (ω-OHFAs) and α,ω-dicarboxylic acids (α,ω-DCAs), which are precursors to high performance polyesters and polyamides. Here we engineer a complete microbial conversion of glucose to C12 and C14 ω-OHFAs and α,ω-DCAs, with precise control of product chain length. Using an expanded bioinformatics approach, we screen a wide range of enzymes across phyla to identify combinations that yield complete conversion of intermediates to product α,ω-DCAs. Finally, through optimization of culture conditions, we enhance production titer of C12 α,ω-DCA to nearly 600 mg/L. Our results indicate potential for this microbial factory to enable commercially relevant, renewable production of C12 α,ω-DCA-a valuable precursor to the high-performance plastic, nylon-6,12.

Interferon-inducible transmembrane protein 3 (IFITM3) restricts reovirus cell entry.

Reoviruses are double-stranded RNA viruses that infect the mammalian respiratory and gastrointestinal tract. Reovirus infection elicits production of type I interferons (IFNs), which trigger antiviral pathways through the induction of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified, the functions of many of these genes are unknown. The interferon-inducible transmembrane (IFITM) proteins are one class of ISGs that restrict the cell entry of some enveloped viruses, including influenza A virus. One family member, IFITM3, localizes to late endosomes, where reoviruses undergo proteolytic disassembly; therefore, we sought to determine whether IFITM3 also restricts reovirus entry. IFITM3-expressing cell lines were less susceptible to infection by reovirus, as they exhibited significantly lower percentages of infected cells in comparison to control cells. Reovirus replication was also significantly reduced in IFITM3-expressing cells. Additionally, cells expressing an shRNA targeting IFITM3 exhibited a smaller decrease in infection after IFN treatment than the control cells, indicating that endogenous IFITM3 restricts reovirus infection. However, IFITM3 did not restrict entry of reovirus infectious subvirion particles (ISVPs), which do not require endosomal proteolysis, indicating that restriction occurs in the endocytic pathway. Proteolysis of outer capsid protein µ1 was delayed in IFITM3-expressing cells in comparison to control cells, suggesting that IFITM3 modulates the function of late endosomal compartments either by reducing the activity of endosomal proteases or delaying the proteolytic processing of virions. These data provide the first evidence that IFITM3 restricts infection by a nonenveloped virus and suggest that IFITM3 targets an increasing number of viruses through a shared requirement for endosomes during cell entry.