Title: Recombinant Spidroins Fully Replicate Primary Mechanical Properties

24 Dragline spider silk is among the strongest and toughest bio-based materials, capable of 25 outperforming most synthetic polymers and even some metal alloys.1,2,3,4 These properties 26 have gained spider silk a growing list of potential applications that, coupled with the 27 impracticalities of spider farming, have driven a decades-long effort to produce 28 recombinant spider silk proteins (spidroins) in engineered heterologous hosts.2 However, 29 these efforts have so far been unable to yield synthetic silk fibers with mechanical 30 properties equivalent to natural spider silk, largely due to an inability to stably produce 31 highly repetitive, high molecular weight (MW) spidroins in heterologous hosts.1,5 Here we 32 address these issues by combining synthetic biology techniques with split intein (SI)33 mediated ligation for the bioproduction of spidroins with unprecedented MW (556 kDa), 34 containing 192 repeat motifs of the Nephila clavipes MaSp1 dragline spidroin. Fibers spun 35 from these synthetic spidroins display ultimate tensile strength (σ), modulus (E), 36 extensibility (ε), and toughness (UT) of 1.03 ± 0.11 GPa, 13.7 ± 3.0 GPa, 18 ± 6%, and 114 ± 37 51 MJ/m3, respectively—equivalent to the performance of natural N. clavipes dragline silk.6 38 This work demonstrates for the first time that microbially produced synthetic silk fibers 39 can match the performance of natural silk fibers by all common metrics (σ, E, ε, UT), 40 providing a more dependable source of high-strength fibers to replace natural spider silks 41 for mechanically demanding applications. Furthermore, our biosynthetic platform can be 42 potentially expanded for the assembly and production of other protein-based materials 43 with high MW and repetitive sequences that have so far been impossible to synthesize by 44 genetic means alone. 45