Raschmanová Hana, Zamora Iwo, Borčinová Martina, Meier Patrick, Weninger Astrid, Mächler Dominik, Glieder Anton, Melzoch Karel, Knejzlík Zdeněk, Kovar Karin
Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czechia.
Institute of Chemistry and Biotechnology, School of Life Sciences and Facility Management, Zurich University of Applied Sciences ZHAW, Wädenswil, Switzerland.
Front Microbiol. 2019 Feb 27;10:335. doi: 10.3389/fmicb.2019.00335. eCollection 2019.
(.) is broadly used for the production of secreted recombinant proteins. Due to the high rate of protein production, incorrectly folded proteins may accumulate in the endoplasmic reticulum (ER). To restore their proper folding, the cell triggers the unfolded protein response (UPR); however, if the proteins cannot be repaired, they are degraded, which impairs process productivity. Moreover, a non-producing/non-secreting subpopulation of cells might occur, which also decreases overall productivity. Therefore, an in depth understanding of intracellular protein fluxes and population heterogeneity is needed to improve productivity. Under industrially relevant cultivation conditions in bioreactors, we cultured strains producing three different recombinant proteins: penicillin G acylase from (PGA), lipase B from (LB) and xylanase A from (XynA). Extracellular and intracellular product concentrations were determined, along with flow cytometry-based single-cell measurements of cell viability and the up-regulation of UPR. The cell population was distributed into four clusters, two of which were viable cells with no UPR up-regulation, differing in cell size and complexity. The other two clusters were cells with impaired viability, and cells with up-regulated UPR. Over the time course of cultivation, the distribution of the population into these four clusters changed. After 30 h of production, 60% of the cells producing PGA, which accumulated in the cells (50-70% of the product), had up-regulated UPR, but only 13% of the cells had impaired viability. A higher proportion of cells with decreased viability was observed in strains producing LB (20%) and XynA (27%). The proportion of cells with up-regulated UPR in LB-producing (35%) and XynA-producing (30%) strains was lower in comparison to the PGA-producing strain, and a smaller proportion of LB and XynA (<10%) accumulated in the cells. These data provide an insight into the development of heterogeneity in a recombinant population during a biotechnological process. A deeper understanding of the relationship between protein production/secretion and the regulation of the UPR might be utilized in bioprocess control and optimization with respect to secretion and population heterogeneity.
（某系统）被广泛用于分泌型重组蛋白的生产。由于蛋白质生产速率高，错误折叠的蛋白质可能会在内质网（ER）中积累。为了恢复其正确折叠，细胞会触发未折叠蛋白反应（UPR）；然而，如果蛋白质无法修复，它们就会被降解，这会损害生产过程的生产率。此外，可能会出现不产生/不分泌的细胞亚群，这也会降低总体生产率。因此，需要深入了解细胞内蛋白质通量和群体异质性以提高生产率。在生物反应器中与工业相关的培养条件下，我们培养了生产三种不同重组蛋白的菌株：来自（某菌）的青霉素G酰化酶（PGA）、来自（某菌）的脂肪酶B（LB）和来自（某菌）的木聚糖酶A（XynA）。测定了细胞外和细胞内产物浓度，以及基于流式细胞术的细胞活力单细胞测量和UPR的上调情况。细胞群体被分为四个簇，其中两个是没有UPR上调的活细胞，在细胞大小和复杂性方面有所不同。另外两个簇是活力受损的细胞和UPR上调的细胞。在培养过程中，群体在这四个簇中的分布发生了变化。生产30小时后，积累在细胞中（占产物的50 - 70%）的生产PGA的细胞中有60%的细胞UPR上调，但只有13%的细胞活力受损。在生产LB（20%）和XynA（27%）的菌株中观察到活力下降的细胞比例更高。与生产PGA的菌株相比,生产LB（35%）和XynA（30%）的菌株中UPR上调的细胞比例更低，并且积累在细胞中的LB和XynA比例更小（<10%）。这些数据为生物技术过程中重组（某生物）群体异质性的发展提供了见解。对蛋白质生产/分泌与UPR调节之间关系的更深入理解可用于生物过程中关于分泌和群体异质性的控制和优化。
