Antelo-Varela Minia, Aguilar Suárez Rocío, Bartel Jürgen, Bernal-Cabas Margarita, Stobernack Tim, Sura Thomas, van Dijl Jan Maarten, Maaß Sandra, Becher Dörte
Centre of Functional Genomics of Microbes, Department of Microbial Proteomics, Institute of Microbiology, University of Greifswald, Greifswald, Germany.
Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.
Front Bioeng Biotechnol. 2020 Feb 28;8:143. doi: 10.3389/fbioe.2020.00143. eCollection 2020.
has been extensively used as a microbial cell factory for industrial enzymes due to its excellent capacities for protein secretion and large-scale fermentation. This bacterium is also an attractive host for biopharmaceutical production. However, the secretion potential of this organism is not fully utilized yet, mostly due to a limited understanding of critical rearrangements in the membrane proteome upon high-level protein secretion. Recently, it was shown that bottlenecks in heterologous protein secretion can be resolved by genome minimization. Here, we present for the first time absolute membrane protein concentrations of a genome-reduced strain ("midi") expressing the immunodominant antigen A (IsaA). We quantitatively characterize the membrane proteome adaptation of midi during production stress on the level of molecules per cell for more than 400 membrane proteins, including determination of protein concentrations for ∼61% of the predicted transporters. We demonstrate that ∼30% of proteins with unknown functions display a significant increase in abundance, confirming the crucial role of membrane proteins in vital biological processes. In addition, our results show an increase of proteins dedicated to translational processes in response to IsaA induction. For the first time reported, we provide accumulation rates of a heterologous protein, demonstrating that midi secretes 2.41 molecules of IsaA per minute. Despite the successful secretion of this protein, it was found that there is still some IsaA accumulation occurring in the cytosol and membrane fraction, leading to a severe secretion stress response, and a clear adjustment of the cell's array of transporters. This quantitative dataset offers unprecedented insights into bioproduction stress responses in a synthetic microbial cell.
由于其出色的蛋白质分泌能力和大规模发酵能力，已被广泛用作生产工业酶的微生物细胞工厂。这种细菌也是生物制药生产的理想宿主。然而，该生物体的分泌潜力尚未得到充分利用，主要是因为对高水平蛋白质分泌时膜蛋白质组中的关键重排了解有限。最近，研究表明通过基因组最小化可以解决异源蛋白质分泌的瓶颈问题。在此，我们首次展示了表达免疫显性抗原A（IsaA）的基因组简化菌株（“midi”）的绝对膜蛋白浓度。我们在每个细胞分子水平上对midi在生产压力下的膜蛋白质组适应性进行了定量表征，涉及400多种膜蛋白，包括对约61%预测转运蛋白的蛋白质浓度测定。我们证明，约30%功能未知的蛋白质丰度显著增加，证实了膜蛋白在重要生物过程中的关键作用。此外，我们的结果表明，响应IsaA诱导，参与翻译过程的蛋白质有所增加。我们首次报告了异源蛋白质的积累速率，表明midi每分钟分泌2.41个IsaA分子。尽管该蛋白质成功分泌，但发现仍有一些IsaA在细胞质和膜部分积累，导致严重的分泌应激反应，以及细胞转运蛋白阵列的明显调整。这个定量数据集为合成微生物细胞中的生物生产应激反应提供了前所未有的见解。
