Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250-320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications.metabolic engineering | glycyl-tRNA | silk fiber | Nephila clavipes | spinning S pider dragline silk, used by spiders as the safety line and the web frame, is exceptionally strong and elastic; it is five times stronger by weight than steel, three times tougher than the top quality man-made fiber Kevlar (1, 2). The dragline silk is primarily composed of two proteins, the major ampullate spidroins 1 (MaSp1) and 2 (MaSp2) (3, 4). These spidroins are highly modular, each with a long repetitive sequence that is flanked on both sides by nonrepetitive amino-and carboxy-termini of approximately 100 amino acids (5). The repetitive sequence is rich in glycine and alanine, and characterized by stretches of alanine that are interrupted by glycine-rich repeats (4). The poly alanine regions form hydrophobic crystalline domains that are responsible for the high tensile strength, whereas the glycine-rich regions are hydrophilic and responsible for the links between crystalline domains as well as the elasticity of dragline fiber (6).Due to the unique mechanical properties, spider dragline silk has received much attention as a promising material for numerous industrial applications such as parachute cords, protective clothing, and composite materials in aircrafts. Also, many biomedical applications are envisioned due to its biocompatibility and biodegradability. For example, silk-based materials have been developed as sutures for wounds, coatings for biomedical implants, drug carriers for drug delivery, and scaffolds for cell culture and organ replacement (7-9). Unfortunately, natural spider dragline silk cannot be conveniently obtained by farming spiders because they are highly territorial and aggressive. Thus, many attempts have been made to produce recombinant dragline silk proteins (10-13) followed ...

Silk-elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Due to the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk to elastin blocks in a SELP’s repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering.

The interactions of C2C12 myoblasts and human bone marrow stem cells (hMSCs) with silk-tropoelastin biomaterials, and the capacity of each to promote attachment, proliferation, and either myogenic- or osteogenic-differentiation were investigated. Temperature-controlled water vapor annealing was used to control beta-sheet crystal formation to generate insoluble silk-tropoelastin biomaterial matrices at defined ratios of the two proteins. These ratios controlled surface roughness and micro/nano-scale topological patterns, and elastic modulus, stiffness, yield stress, and tensile strength. A combination of low surface roughness and high stiffness in the silk-tropoelastin materials promoted proliferation and myogenic-differentiation of C2C12 cells. In contrast, high surface roughness with micro/nano-scale surface patterns was favored by hMSCs. Increasing the content of human tropoelastin in the silk-tropoelastin materials enhanced the proliferation and osteogenic-differentiation of hMSCs. We conclude that the silk-tropoelastin composition facilitates fine tuning of the growth and differentiation of these cells.

A four carbon linear chain diamine, putrescine (1,4-diaminobutane), is an important platform chemical having a wide range of applications in chemical industry. Biotechnological production of putrescine from renewable feedstock is a promising alternative to the chemical synthesis that originates from non-renewable petroleum. Here we report development of a metabolically engineered strain of Escherichia coli that produces putrescine at high titer in glucose mineral salts medium. First, a base strain was constructed by inactivating the putrescine degradation and utilization pathways, and deleting the ornithine carbamoyltransferase chain I gene argI to make more precursors available for putrescine synthesis. Next, ornithine decarboxylase, which converts ornithine to putrescine, was amplified by a combination of plasmid-based and chromosome-based overexpression of the coding genes under the strong tac or trc promoter. Furthermore, the ornithine biosynthetic genes (argC-E) were overexpressed from the trc promoter, which replaced the native promoter in the genome, to increase the ornithine pool. Finally, strain performance was further improved by the deletion of the stress responsive RNA polymerase sigma factor RpoS, a well-known global transcription regulator that controls the expression of ca. 10% of the E. coli genes. The final engineered E. coli strain was able to produce 1.68 g L À1 of putrescine with a yield of 0.168 g g À1 glucose. Furthermore, high cell density cultivation allowed production of 24.2 g L À1 of putrescine with a productivity of 0.75 g L À1 h À1 . The strategy reported here should be useful for the bio-based production of putrescine from renewable resources, and also for the development of strains capable of producing other diamines, which are important as nitrogen-containing platform chemicals.