Large scale expression for metabolic pathway engineering

代谢途径工程的大规模表达

• 批准号: 6740185
• 负责人: Thomas W. Jeffries
• 金额: $ 18.05万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6740185
• 关键词: Krebs' cycle; Saccharomyces cerevisiae; artificial chromosomes; biotechnology; carbohydrate metabolism; fungal genetics; gene expression; genetic manipulation; high throughput technology; liquid chromatography mass spectrometry; microorganism metabolism; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; polymerase chain reaction; technology /technique development; xylose

DESCRIPTION (provided by applicant): The long-term objective of this research is to enable coordinated, optimal expression of multiple genes for metabolic pathways in eukaryotes. The ultimate goal is to enable metabolic engineering for maximizing pathway flux. This would have application in the correction of metabolic imbalances or for the efficient formation of metabolites in microbes, plants and animals. The technology could eventually be used to engineer complex traits. Large-scale expression is the next step in metabolic engineering of eukaryotic cells. The specific objective of this work is to maximize the flux of metabolites from xylose to ethanol by manipulating the expression of 15 to 20 genes in the central pentose phosphate, glycolytic and TCA cycle pathways of Saccharomyces cerevisiae. The research will develop a technology for the systematic, large-scale expression of genes from synthetic artificial chromosomes (SACs) created from defined genetic elements. This technology will enable the engineering of large, complex metabolic pathways for the balanced synthesis of selected products. It will be highly applicable to various eukaryotic expression systems. Multiple genes will be introduced into DNA segments by extension overlap PCR, ligation and concatenation. A series of individual genes and defined genomic elements will be synthesized using appropriate promoters and linkers, ligated together into modules and expressed in S. cerevisiae. This work will test the hypothesis that over expression of genes for one part of a pathway simply results in the accumulation of intermediates in another part. If one gene is over expressed, another becomes limiting. Metabolic efficiency is impaired by imbalanced enzyme synthesis, and over expression of some genes can inhibit cell growth. This research will approach an optimum for gene expression in a complete pathway. By examining transformants for growth on and fermentation of xylose, which the parental cell cannot assimilate, it will determine effects of SACs on biochemistry metabolite flux, and cellular physiology. It will measure expression levels of the introduced genes using real-time PCR. The effects of SACs on the accumulation of transcripts for other genes will be evaluated in selected transformants by genome-wide expression studies. Finally, the effects of SACs on metabolite levels will be assessed by a combination of 31P NMR and LC/MS. It will employ statistical methods to interpret these results and approach an optimum level of metabolite flux through iterative trials.

描述（由申请人提供）：本研究的长期目标是使真核生物中代谢途径的多个基因能够协调、最佳表达。最终目标是使代谢工程最大化途径通量。这将应用于纠正代谢失衡或微生物、植物和动物中代谢物的有效形成。这项技术最终可以用来设计复杂的特征。大规模表达是真核细胞代谢工程的下一步。这项工作的具体目标是最大限度地提高从木糖到乙醇的代谢产物的流量，通过操纵的中央戊糖磷酸，糖酵解和TCA循环途径的酿酒酵母中的15至20个基因的表达。该研究将开发一种技术，用于从定义的遗传元件创建的合成人工染色体（SAC）中系统地大规模表达基因。这项技术将使大型，复杂的代谢途径的工程选择的产品的平衡合成。它将高度适用于各种真核表达系统。通过延伸重叠PCR、连接和串联将多个基因引入DNA片段中。一系列单独的基因和确定的基因组元件将使用适当的启动子和接头合成，连接在一起形成模块并在S.啤酒。这项工作将测试的假设，过度表达的基因的一部分，一个途径只是在另一个部分的中间产物的积累结果。如果一个基因过度表达，另一个基因就会受到限制。代谢效率因酶合成不平衡而受损，某些基因的过度表达可抑制细胞生长。这项研究将接近一个完整的途径中的基因表达的最佳状态。通过检查转化体在亲本细胞不能同化的木糖上的生长和发酵，将确定SAC对生物化学代谢物通量和细胞生理学的影响。它将使用实时PCR测量引入基因的表达水平。将通过全基因组表达研究在选定的转化体中评价SAC对其他基因转录物积累的影响。最后，将通过31 P NMR和LC/MS的组合评估SAC对代谢物水平的影响。将采用统计学方法解释这些结果，并通过迭代试验接近代谢物通量的最佳水平。