Glasgow Anum Azam, Wong Han Teng, Tullman-Ercek Danielle
UC Berkeley-UCSF Graduate Program in Bioengineering, University of California Berkeley , Berkeley, California 94720, United States.
Department of Plant and Microbial Biology, University of California Berkeley , Berkeley, California 94720, United States.
ACS Synth Biol. 2017 Jun 16;6(6):1006-1015. doi: 10.1021/acssynbio.6b00335. Epub 2017 Mar 16.
The bacterial type III secretion system (T3SS) is an important target for enabling high-titer production of proteins of biotechnological interest as well as for synthetic biology applications that rely on protein delivery to host cells. The T3SS forms a membrane-embedded needle complex that is capped by the translocon proteins and extends into the extracellular space. The needle tip complex in Salmonella enterica consists of three translocon proteins: SipB, SipC, and SipD. It is known that knocking out sipD disrupts T3SS regulation to cause constitutive secretion of native proteins. However, we discovered that complementation of SipD in trans via exogenous addition to T3SS-expressing cultures further improves heterologous protein secretion titers, suggesting a previously unknown but important role for this protein. Building on this knowledge, we have engineered a hyper-secreting strain of S. enterica for a greater than 100-fold improvement in the production of a variety of biotechnologically valuable heterologous proteins that are challenging to produce, such as toxic antimicrobial peptides and proteolysis-prone biopolymer proteins. We determined that transcription by several T3SS promoters is upregulated with the addition of SipD, that the N-terminal domain of SipD is sufficient to observe the increased secretion phenotype, and that the effect is post-transcriptional and post-translational. These results lend support to the use of bacterial secretion as a powerful protein production strategy, and the hypothesis that translocon proteins contribute to type III secretion regulation.
细菌III型分泌系统（T3SS）是实现高滴度生产具有生物技术意义的蛋白质以及依赖蛋白质递送至宿主细胞的合成生物学应用的重要靶点。T3SS形成一个膜嵌入的针状复合物，该复合物由转运蛋白封端并延伸至细胞外空间。肠炎沙门氏菌中的针尖复合物由三种转运蛋白组成：SipB、SipC和SipD。已知敲除sipD会破坏T3SS调控，导致天然蛋白质的组成型分泌。然而，我们发现通过向表达T3SS的培养物中外源添加SipD进行反式互补可进一步提高异源蛋白质的分泌滴度，这表明该蛋白具有此前未知但重要的作用。基于这一认识，我们构建了一种肠炎沙门氏菌的高分泌菌株，用于生产多种难以生产的具有生物技术价值的异源蛋白质，如有毒抗菌肽和易受蛋白水解的生物聚合物蛋白，产量提高了100倍以上。我们确定，添加SipD后，几个T3SS启动子的转录上调，SipD的N端结构域足以观察到分泌增加的表型，且这种效应是转录后和翻译后的。这些结果支持将细菌分泌作为一种强大的蛋白质生产策略，并支持转运蛋白有助于III型分泌调控的假说。
