Dip to Ingegneria Chimica e Alimentare, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy.
Microb Cell Fact. 2009 Dec 30;8:70. doi: 10.1186/1475-2859-8-70.
Saccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1beta (IL1beta), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1beta] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Deltayca1 [PIR4-IL1beta], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Deltayca1 mutant strain.
Our results show that the concentrations of ACA in the feeding solution, corresponding to those routinely used in the literature, are limiting for the growth of S. cerevisiae BY4741 [PIR4-IL1beta] in fed-batch reactor. Even in the presence of a proper ACA supplementation, S. cerevisiae BY4741 [PIR4-IL1beta] did not achieve a high cell density. The Deltayca1 deletion did not have a beneficial effect on the overall performance of the strain, but it had a clear effect on its viability, which was not impaired during fed-batch operations, as shown by the kd value (0.0045 h-1), negligible if compared to that of the parental strain (0.028 h-1). However, independently of their robustness, both the parental and the Deltayca1 mutant ceased to grow early during fed-batch runs, both strains using most of the available carbon source for maintenance, rather than for further proliferation. The mathematical model used evidenced that the energy demand for maintenance was even higher in the case of the Deltayca1 mutant, accounting for the growth arrest observed despite the fact that cell viability remained comparatively high.
The paper points out the relevance of a proper ACA formulation for the outcome of a fed-batch reactor growth carried out with S. cerevisiae BY4741 [PIR4-IL1beta] strain and shows the sensitivity of this commonly used auxotrophic strain to aerated fed-batch operations. A Deltayca1 disruption was able to reduce the loss of viability, but not to improve the overall performance of the process. A mathematical model has been developed that is able to describe the behaviour of both the parental and mutant producer strain during fed-batch runs, and evidence the role played by the energy demand for maintenance in the outcome of the process.
酿酒酵母 BY4741 是一种常用的营养缺陷型菌株。在这项工作中，它被用作人白细胞介素-1β（IL1β）表达和分泌的宿主，使用细胞壁蛋白 Pir4 作为融合伴侣。为了实现高密度细胞和高产物产量，使用补充有氨基酸的定义矿物质培养基，通过通气分批补料培养，用 Pir4-IL1β 对 BY4741 [PIR4-IL1beta] 进行了培养。氨基酸（ACA）作为补充物。还对携带 caspase 样蛋白基因（参与凋亡反应）缺失的酿酒酵母突变株 BY4741 Deltayca1 [PIR4-IL1beta]进行了通气分批补料培养，并与亲本菌株进行了比较，以测试该突变对菌株健壮性的影响。在运行过程中检查了生产菌株的存活率，并开发了一个数学模型，该模型考虑了反应器中存在的活生物质和葡萄糖消耗，用于描述和解释过程的时间进程。为两种情况，即亲本 BY4741 株和 BY4741 Deltayca1 突变株。
我们的结果表明，在补料分批培养中，补料溶液中 ACA 的浓度对应于文献中常规使用的浓度，对酿酒酵母 BY4741 [PIR4-IL1beta]的生长有限制。即使进行了适当的 ACA 补充，酿酒酵母 BY4741 [PIR4-IL1beta]也无法达到高细胞密度。Deltayca1 缺失对菌株的整体性能没有有益影响，但对其存活率有明显影响，这在补料分批操作过程中没有受到损害，如 kd 值（0.0045 h-1）所示，如果与亲本菌株（0.028 h-1）相比，可以忽略不计。然而，无论是在稳健性方面，无论是亲本还是 Deltayca1 突变体，在补料分批运行过程中都早早停止生长，两种菌株都将大部分可用碳源用于维持，而不是进一步增殖。使用的数学模型表明，对于 Deltayca1 突变体，维持的能量需求甚至更高，这解释了尽管细胞活力仍然相对较高，但观察到的生长停滞现象。
本文指出了对于用酿酒酵母 BY4741 [PIR4-IL1beta]菌株进行通气分批补料培养，适当的 ACA 配方对结果的重要性，并显示了这种常用的营养缺陷型菌株对通气分批操作的敏感性。Deltayca1 缺失能够降低存活率损失，但不能改善整个过程的性能。已经开发了一种数学模型，能够描述亲本和突变体生产菌株在补料分批运行期间的行为，并证明维持能量需求在过程结果中所起的作用。
