消除粘稠度挑战在连续培养酵母产生GLP-1样肽。

IF 4.9 2区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Ioannis Voulgaris, Anders Nygaard Nielsen, Tine Petersen, Sanne Jensen
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引用次数: 0

摘要

背景:用于治疗糖尿病、肥胖和其他疾病的glp -1的出现使得人们更加关注寻找有效的方法来生产足够数量的肽以满足不断增长的需求。尽管使用微生物宿主是大批量生产这些肽的最便宜、最简单和最安全的方法,但仍然存在降低生产能力的工艺挑战。主要的生产挑战之一是培养肉汤的高粘度,这会减少质量和氧气传递,从而产生底物和氧气梯度,可能导致不必要的二次代谢,最终损害产能。结果:介绍了在酿酒酵母连续培养中重组生产GLP-1前体过程中用于鉴定高粘性肉汤潜在因素的方法。发现了导致高粘稠肉汤的两个根本原因,并确定了解决方案。第一个是在肉汤的可溶性部分中发现的,与GLP-1前体分子的聚集有关,这种聚集导致了高粘性、剪切变薄的培养肉汤。讨论了发生聚合的培养条件以及对培养和产品回收的影响。粘度的第二个来源是在培养肉汤的不溶性部分中发现的,并且与由于Amn1p依赖的母细胞和子细胞的不完全分离而导致的细胞聚集有关。这种类型的细胞聚集导致细胞团块的形成,并导致具有轻度剪切增稠特性的高粘度培养肉汤。结论:为了消除GLP-1肽相关的黏度,使用了新一代酵母菌宿主菌株,该菌株能够耐受pH值升高的培养,高于导致GLP-1前体聚集的pH值。在细胞衍生黏度的情况下,采用了携带AMN1基因缺失或整合非结块AMN1D368V基因变体的酵母菌株。这些变化的实施导致了一个可扩展的培养过程,其特点是由于培养肉汤的低粘度和牛顿行为而显著改善了氧传质。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Eliminating viscosity challenges in continuous cultivation of yeast producing a GLP-1 like peptide.

Background: The emergence of GLP-1s for the treatment of diabetes, obesity and other diseases has led to increased focus on finding efficient ways to produce the peptides in sufficient amounts to satisfy the ever-increasing demand. Although the use of microbial hosts constitutes the cheapest, easiest and safest way to produce these peptides in high volumes, process challenges still exist that reduce the production capacity. One of the main production challenges is the high viscosity of cultivation broths, which reduces the mass and oxygen transfer, thereby creating substrate and oxygen gradients that potentially lead to unwanted secondary metabolism and eventually compromises capacity.

Results: The methodology used to identify the underlying factors of highly viscous broths during the recombinant production of GLP-1 precursors in S. cerevisiae in continuous cultivation is presented. Two root causes leading to highly viscous broths were uncovered and solutions identified. The first one is found in the soluble fraction of the broth and relates to the aggregation of GLP-1 precursor molecules that leads to highly viscous, shear thinning cultivation broths. The cultivation conditions under which the aggregation occurs and the consequences for both cultivation and product recovery are discussed. The second source of viscosity is found in the insoluble fraction of the cultivation broth and relates to cell aggregation due to Amn1p dependent incomplete separation of mother and daughter cells. This type of cell aggregation causes formation of cell clumps and leads to high viscosity cultivation broths with mild shear thickening properties.

Conclusions: To eliminate the GLP-1 peptide related viscosity, a new generation of yeast host strains that tolerates cultivation at increased pH values, above those that cause GLP-1 precursor aggregation, were utilized. In the case of the cell derived viscosity, yeast strains carrying either a deletion of the AMN1 gene or integration of the non-clumping AMN1D368V gene variant were employed. The implementation of these changes led to a scalable cultivation process characterized by a significant improved oxygen mass transfer attributed to the low viscosity and Newtonian behaviour of the cultivation broth.

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来源期刊
Microbial Cell Factories
Microbial Cell Factories 工程技术-生物工程与应用微生物
CiteScore
9.30
自引率
4.70%
发文量
235
审稿时长
2.3 months
期刊介绍: Microbial Cell Factories is an open access peer-reviewed journal that covers any topic related to the development, use and investigation of microbial cells as producers of recombinant proteins and natural products, or as catalyzers of biological transformations of industrial interest. Microbial Cell Factories is the world leading, primary research journal fully focusing on Applied Microbiology. The journal is divided into the following editorial sections: -Metabolic engineering -Synthetic biology -Whole-cell biocatalysis -Microbial regulations -Recombinant protein production/bioprocessing -Production of natural compounds -Systems biology of cell factories -Microbial production processes -Cell-free systems
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