Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii.
Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii.
复制标题
DOI：
10.1002/bit.26846
复制
发表时间：
2018-12
影响因子：
3.8
通讯作者：
Oliver SG
中科院分区：
文献类型：
--
作者：
Cankorur-Cetinkaya A;Narraidoo N;Kasavi C;Slater NKH;Archer DB;Oliver SG
目前工业生物技术的趋势是从分批或补料分批发酵转向连续操作。这一转变的成功将需要开发遗传稳定的生产菌株，使用强大的构成启动子，并开发允许在细胞生长和产品形成之间实现适当平衡的新的培养基配方。我们发现了在不同的稳态条件下在Komagataella phaffii中高表达的基因，并探索了这些基因的启动子(ChR1-4_0586和FragB_0052)在优化两种不同的r蛋白-人溶菌酶(HuLy)和抗独特型抗体片段Fab-3H6的表达方面的作用，并与广泛使用的甘油醛-3-磷酸脱氢酶启动子进行了比较。我们的结果表明，启动子的强度高度依赖于培养条件，因此构建的载体应该在一系列条件下进行测试，以确定表达目的蛋白的最佳克隆和理想的启动子。连续生产的一个重要好处是，它促进了基因组规模代谢模型在菌株和培养基设计中的使用。在硅胶中，通量分布表明，任何一种蛋白质的产生都通过芳香氨基酸的生物合成增加了通量。添加酪氨酸提高了这两种蛋白的产量，而添加色氨酸没有引起任何显著变化，添加苯丙氨酸增加了HuLy的表达，但降低了Fab-3H6的表达。这些结果表明，基因组规模的代谢模型可以用来评估特定r-蛋白合成施加的代谢负担，然后这些信息可以用来定制培养基以提高产量。
The current trend in industrial biotechnology is to move from batch or fed‐batch fermentations to continuous operations. The success of this transition will require the development of genetically stable production strains, the use of strong constitutive promoters, and the development of new medium formulations that allow an appropriate balance between cell growth and product formation. We identified genes that showed high expression in Komagataella phaffii during different steady‐state conditions and explored the utility of promoters of these genes (Chr1–4_0586 and FragB_0052) in optimizing the expression of two different r‐proteins, human lysozyme (HuLy), and the anti‐idiotypic antibody fragment, Fab‐3H6, in comparison with the widely used glyceraldehyde‐3‐phosphate dehydrogenase promoter. Our results showed that the promoter strength was highly dependent on the cultivation conditions and thus constructs should be tested under a range of conditions to determine both the best performing clone and the ideal promoter for the expression of the protein of interest. An important benefit of continuous production is that it facilitates the use of the genome‐scale metabolic models in the design of strains and cultivation media. In silico flux distributions showed that production of either protein increased the flux through aromatic amino acid biosynthesis. Tyrosine supplementation increased the productivity for both proteins, whereas tryptophan addition did not cause any significant change and, phenylalanine addition increased the expression of HuLy but decreased that of Fab‐3H6. These results showed that a genome‐scale metabolic model can be used to assess the metabolic burden imposed by the synthesis of a specific r‐protein and then this information can be used to tailor a cultivation medium to increase production.
登录
查看更多内容
影响因子：
14.9
作者：
Hartner FS;Ruth C;Langenegger D;Johnson SN;Hyka P;Lin-Cereghino GP;Lin-Cereghino J;Kovar K;Cregg JM;Glieder A
通讯作者：
Glieder A
影响因子：
5
作者：
Ahn, Jungoh;Hong, Jiyeon;Lee, Hongweon
通讯作者：
Lee, Hongweon
影响因子：
5
作者：
Kunert R;Reinhart D
通讯作者：
Reinhart D
影响因子：
3.8
作者：
Cankorur-Cetinkaya A;Dikicioglu D;Oliver SG
通讯作者：
Oliver SG
影响因子：
6.4
作者：
Carnicer, Marc;ten Pierick, Angela;Ferrer, Pau
通讯作者：
Ferrer, Pau