Connor Alexander, Wigham Caleb, Bai Yang, Rai Manish, Nassif Sebastian, Koffas Mattheos, Zha R Helen
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Metab Eng Commun. 2023 Feb 3;16:e00219. doi: 10.1016/j.mec.2023.e00219. eCollection 2023 Jun.
Spider silk proteins (spidroins) are a remarkable class of biomaterials that exhibit a unique combination of high-value attributes and can be processed into numerous morphologies for targeted applications in diverse fields. Recombinant production of spidroins represents the most promising route towards establishing the industrial production of the material, however, recombinant spider silk production suffers from fundamental difficulties that includes low titers, plasmid instability, and translational inefficiencies. In this work, we sought to gain a deeper understanding of upstream bottlenecks that exist in the field through the production of a panel of systematically varied spidroin sequences in multiple strains. A restriction on basal expression and specific genetic mutations related to stress responses were identified as primary factors that facilitated higher titers of the recombinant silk constructs. Using these findings, a novel strain of was created that produces recombinant silk constructs at levels 4-33 times higher than standard BL21(DE3). However, these findings did not extend to a similar recombinant protein, an elastin-like peptide. It was found that the recombinant silk proteins, but not the elastin-like peptide, exert toxicity on the host system, possibly through their high degree of intrinsic disorder. Along with strain engineering, a bioprocess design that utilizes longer culturing times and attenuated induction was found to raise recombinant silk titers by seven-fold and mitigate toxicity. Targeted alteration to the primary sequence of the recombinant silk constructs was also found to mitigate toxicity. These findings identify multiple points of focus for future work seeking to further optimize the recombinant production of silk proteins and is the first work to identify the intrinsic disorder and subsequent toxicity of certain spidroin constructs as a primary factor related to the difficulties of production.
蜘蛛丝蛋白是一类非凡的生物材料，具有高价值属性的独特组合，并且可以加工成多种形态，用于不同领域的靶向应用。蜘蛛丝蛋白的重组生产是实现该材料工业化生产最具前景的途径，然而，重组蜘蛛丝生产存在一些基本困难，包括产量低、质粒不稳定和翻译效率低。在这项工作中，我们试图通过在多个菌株中生产一组系统变化的蜘蛛丝蛋白序列，更深入地了解该领域存在的上游瓶颈。确定基础表达的限制和与应激反应相关的特定基因突变是促进重组丝构建体更高产量的主要因素。利用这些发现，创建了一种新型菌株，其生产重组丝构建体的水平比标准BL21(DE3)高4至33倍。然而，这些发现并没有扩展到一种类似的重组蛋白——弹性蛋白样肽。研究发现，重组丝蛋白而非弹性蛋白样肽可能通过其高度的内在无序性对宿主系统产生毒性。除了菌株工程，一种利用更长培养时间和减弱诱导的生物工艺设计被发现可以将重组丝产量提高7倍并减轻毒性。对重组丝构建体一级序列的靶向改变也被发现可以减轻毒性。这些发现确定了未来工作中多个需要关注的点，旨在进一步优化丝蛋白的重组生产，并且是第一项确定某些蜘蛛丝蛋白构建体的内在无序性及随后的毒性是与生产困难相关的主要因素的工作。
