Large scale expression for metabolic pathway engineering

描述（由申请人提供）：本研究的长期目标是实现真核生物代谢途径中多个基因的协调、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、植物和动物体内代谢物的有效形成。这项技术最终可以用于设计复杂的性状。大规模表达是真核细胞代谢工程的下一步。这项工作的具体目的是通过控制酿酒酵母中心戊糖磷酸、糖酵解和TCA循环途径中15至20个基因的表达，最大限度地提高木糖到乙醇的代谢物通量。该研究将开发一种技术，用于系统地、大规模地表达由确定的遗传元素产生的合成人工染色体（SACs）的基因。这项技术将使工程大，复杂的代谢途径的平衡合成选定的产品。它将高度适用于各种真核表达系统。将多个基因通过延伸重叠PCR、结扎和串联等方法引入DNA片段。一系列单独的基因和定义的基因组元件将通过合适的启动子和连接子合成，连接在一起形成模块并在酿酒酵母中表达。这项工作将验证一种假设，即通路中某一部分基因的过度表达只会导致另一部分中间产物的积累。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会抑制细胞生长。本研究将在一个完整的途径中接近基因表达的最佳状态。通过检测转化体在木糖上的生长和发酵，确定SACs对生物化学代谢物通量和细胞生理学的影响，而木糖是亲本细胞不能吸收的。它将使用实时聚合酶链反应（real-time PCR）测量引入基因的表达水平。SACs对其他基因转录本积累的影响将通过全基因组表达研究在选定的转化子中进行评估。最后，SACs对代谢物水平的影响将通过31P NMR和LC/MS的组合来评估。它将采用统计方法来解释这些结果，并通过反复试验接近代谢物通量的最佳水平。

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.