使用优化的Yarrowia lipolytica表达系统生产Rhizopus oryzae脂酶
Lea Vidal1, Zehui Dong2,3, Kim Olofsson3
1INRAE, AgroParisTech, Micalis Institute, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
FEMS yeast research
|July 27, 2023
概括
研究人员开发了新的克隆载体和酵母菌株,以改善异质蛋白质的生产. 优化的系统将脂酶活性提高了三倍以上,展示了Yarrowia lipolytica.
科学领域:
- 生物技术是生物技术.
- 微生物工程 微生物工程
- 蛋白质生产 蛋白质生产
背景情况:
- Yarrowia lipolytica是一种用于异质蛋白质生产的多功能酵母平台.
- 有效的克隆策略和优化的表达系统对于最大限度地提高蛋白质产量至关重要.
研究的目的:
- 开发新的自克隆载体和改进的受体菌株,以提高Yarrowia lipolytica中的蛋白质生产.
- 为了确定最佳的信号序列和可诱导的促进器,用于高水平的Rhizopus oryzae ROL脂酶的分泌.
主要方法:
- 开发了18种具有可减税辅性标记物 (URA3ex,LYS5ex,LEU2ex) 和各种促进物 (pTEF,pHp4d,pEYK1,pEYL1衍生物) 的染色体克隆载体.
- 创建一个改进的受体菌株 (JMY8647),取消了丝和氨酸辅 (Lys-).
- 评估了八个针对ROL脂酶分泌的向序列,并比较了不同的促进剂强度和基因拷贝数量.
主要成果:
- 与野生类型的ROL信号序列相比,SP6信号序列提高了23%的脂酶活性.
- 混合红醇诱导性促进剂 (pHU8EYK,pEYL1-5AB) 的特异性脂酶活性比构成性pTEF促进剂高1.9倍和2.2倍.
- 双拷贝菌株的脂酶活性比pTEF单拷贝菌株增加了3.3倍 (266.7比79.7mU/mg).
结论:
- 开发的自克隆载体和受体菌株显著加速基因克隆,并增强Yarrowia lipolytica中异构蛋白质的产生.
- 信号序列和诱导性促进体的优化,以及基因拷贝数量的增加,导致脂酶分泌的实质性改善.
- 这一战略为最大限度地利用Yarrowia lipolytica生产有价值的蛋白质提供了一个强大的平台.
相关概念视频
01:22Bioreactor Controls-III
Strain improvement is a foundational strategy in industrial microbiology aimed at maximizing microbial productivity, particularly because natural isolates typically yield commercially valuable products in very low concentrations. Although optimizing the culture medium and environmental conditions can improve yields, these adjustments are inherently limited by the organism’s genetic potential. As a result, the focus shifts toward genetic modifications to enhance biosynthetic capacity. The...
01:27Upstream Processing
Upstream processing represents a critical phase in biomanufacturing, wherein biological systems such as microorganisms, mammalian cells, or insect cells are cultivated to produce therapeutic proteins, vaccines, enzymes, or other biologically derived products. This phase encompasses all steps from the selection and genetic manipulation of the production organism to the cultivation of cells in bioreactors under tightly controlled environmental conditions.Host Selection and Genetic OptimizationThe...
01:27Production of Alcohol
Continuous fermentation is a key strategy in industrial ethanol production, particularly when efficiency, scalability, and high yields are essential. This approach allows for uninterrupted operation and optimized resource utilization. The primary feedstock, corn starch, undergoes enzymatic hydrolysis facilitated by α-amylase and glucoamylase. These enzymes break down the starch into fermentable sugars such as glucose, which are readily assimilated by fermentative microorganisms.Fermentation...
01:25Production of Organic Acids
Lactic acid, an important organic acid extensively applied in food, pharmaceutical, and biodegradable polymer industries, is primarily produced via microbial fermentation. This method is favored over chemical synthesis due to its environmental sustainability and capacity for enantiomerically pure product formation. Among various microbial processes, the fermentation of starch-based substrates stands out due to the abundance and renewability of raw materials like corn and potatoes.Hydrolysis of...
01:27Production of Antibiotics
Penicillin, one of the earliest and most widely used antibiotics, is produced industrially by the filamentous fungus Penicillium chrysogenum. Large stirred-tank bioreactors ranging from tens to hundreds of thousands of liters maintain tightly controlled temperature, pH, and dissolved oxygen conditions to support fungal metabolism and maximize antibiotic yield. Penicillin is a secondary metabolite, synthesized primarily during the stationary growth phase, which requires a carefully managed...
01:30Production of Pharmaceuticals
Industrial insulin production uses genetically engineered E. coli expressing a proinsulin gene controlled by a tryptophan promoter and containing a methionine linker for later cleavage. The cells also carry ampicillin resistance for selective growth. Seed cultures are stored at −80 °C and production begins by thawing a small amount to inoculate starter cultures, which are progressively scaled to a 50,000-L bioreactor. In the bioreactor, E. coli grow in nutrient-rich media under sterile, tightly...