Efficient Overproduction of Membrane Proteins in Lactococcus lactis Requires the Cell Envelope Stress Sensor/Regulator Couple CesSR

BackgroundMembrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein. Methods and FindingsWe describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold. ConclusionsThe results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins.背景：膜蛋白是一类至关重要的分子类群，其研究长期受限于多项固有局限，例如自身的疏水性，以及正确折叠所需的额外严苛条件。此外，将膜蛋白功能性整合至膜中并监测其折叠状态所需的细胞机制极为复杂，这使得研究者既难以预判膜蛋白的可获得产量，也无法精准预测某一特定蛋白的最优生产宿主。 方法与结果：我们提出了一种理性设计策略，以优化乳酸菌乳酸乳球菌（Lactococcus lactis）作为膜蛋白生产宿主的性能。转录组（transcriptome）数据显示，当乳酸乳球菌被迫过量表达内源性膜蛋白BcaP——支链氨基酸通透酶（branched-chain amino acid permease）——时，感知不同来源细胞包膜应激的双组分系统CesSR是主要响应通路之一。当CesSR系统本身或其调控子（regulon）的特定成员（尤其是ftsH、oxaA2、llmg_2163及rmaB基因）被敲除（knocked out）时，表达BcaP的乳酸乳球菌菌株的生长能力与膜蛋白生产水平均会受到严重抑制。过表达（overexpressing）cesSR可有效缓解生长缺陷，从而直接提升BcaP的生产产量。将该策略推广应用于真核蛋白时，针对其中部分生产难度公认极高的蛋白（例如具有重要医学价值的早老素复合物（presenilin complex）），我们成功大幅减轻了野生型菌株中出现的生长缺陷，并将早老素变体PS1Δ9-H6的生产产量提升了4倍以上。 结论：本研究结果揭示了乳酸乳球菌中一种关键且或许处于核心地位的膜蛋白质量控制机制。调控CesSR的表达可显著提升不同来源膜蛋白的生产产量。上述发现进一步证实，乳酸乳球菌可作为生产膜蛋白的可靠替代宿主。