在大肠杆菌中生产重组丝素蛋白和弹性蛋白样肽的构建毒性、菌株优化和一级序列设计的新见解

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Alexander Connor , Caleb Wigham , Yang Bai , Manish Rai , Sebastian Nassif , Mattheos Koffas , R. Helen Zha
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引用次数: 3

摘要

蜘蛛丝蛋白(spidroins)是一类引人注目的生物材料,表现出高价值属性的独特组合,可以加工成多种形态,用于不同领域的靶向应用。蜘蛛蛋白的重组生产代表了建立该材料工业生产的最有前途的途径,然而,重组蜘蛛丝的生产存在根本困难,包括低滴度、质粒不稳定和翻译效率低下。在这项工作中,我们试图通过在多个大肠杆菌菌株中生产一组系统变化的蜘蛛蛋白序列,来更深入地了解该领域存在的上游瓶颈。对基础表达的限制和与应激反应相关的特异性遗传突变被确定为促进重组丝构建体更高滴度的主要因素。利用这些发现,产生了一种新的大肠杆菌菌株,其产生的重组丝构建体的水平是标准BL21(DE3)的4-33倍。然而,这些发现并没有延伸到类似的重组蛋白,一种弹性蛋白样肽。研究发现,重组丝蛋白,而不是弹性蛋白样肽,可能通过其高度的内在紊乱对大肠杆菌宿主系统产生毒性。与菌株工程一起,发现利用较长培养时间和减弱诱导的生物工艺设计可以将重组丝滴度提高7倍并减轻毒性。还发现对重组丝构建体的一级序列的靶向改变可以减轻毒性。这些发现为未来寻求进一步优化丝蛋白重组生产的工作确定了多个重点,也是第一项将某些蜘蛛蛋白构建体的内在紊乱和随后的毒性确定为与生产困难相关的主要因素的工作。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Novel insights into construct toxicity, strain optimization, and primary sequence design for producing recombinant silk fibroin and elastin-like peptide in E. coli

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 E. coli 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 E. coli 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 E. coli 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.

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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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