In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

Formic Acid Regenerated Mori, Tussah, Eri, Thai, and Muga Silk Materials: Mechanism of Self-Assembly.

Flexible and water-insoluble regenerated silk materials have caught considerable interest due to their mechanical properties and numerous potential applications in medical fields. In this study, regenerated Mori (China), Thai, Eri, Muga, and Tussah silk films were prepared by a formic acid-calcium chloride (FA) method, and their structures, morphologies, and other physical properties were comparatively studied through Fourier transform infrared spectroscopy (FTIR), wide-angle X-ray scattering (WAXS), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), and thermogravimetric analysis (TGA). FTIR results demonstrated that the secondary structures of those five types of silk films are different from those of their respective natural silk fibers, whose structures are dominated by stacked rigid intermolecular ß-sheet crystals. Instead, intramolecular ß-sheet structures were found to dominate these silk films made by FA method, as confirmed by WAXS. We propose that silk I-like structures with intramolecular ß-sheets lead to water insolubility and mechanical flexibility. This comparative study offers a new pathway to understanding the tunable properties of silk-based biomaterials.

Protein-Based Bioelectronics.

The desire for flexible electronics is booming, and development of bioelectronics for health monitoring, internal body procedures, and other biomedical applications is heavily responsible for the growing market. Most current fabrication techniques for flexible bioelectronics, however, do not use materials that optimize both biocompatibility and mechanical properties. This Review explores flexible electronic technologies, fabrication methods, and protein materials for biomedical applications. With favorable sustainability and biocompatibility, naturally derived proteins are an exceptional alternative to synthetic materials currently used. Many proteins can take on various forms, such as fibers, films, and scaffolds. The fabrication of resistors and organic solar cells on silk has already been proven, and optoelectronics made of collagen and keratin have also been explored. The flexibility and biocompatibility of these materials along with their proven performance in electronics make them ideal materials in the advancement of biomedical devices.