Large scale expression for metabolic pathway engineering

描述(申请人提供)：这项研究的长期目标是在真核生物中实现代谢途径的多个基因的协调、最佳表达。最终的目标是使代谢工程能够最大化途径的流量。这将在纠正代谢失衡或在微生物、植物和动物中有效形成代谢物方面有应用。这项技术最终可能被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通过调控15到20个基因在酿酒酵母的中心戊糖磷酸、糖酵解和TCA循环途径中的表达，最大化从木糖到乙醇的代谢产物的通量。这项研究将开发一种系统、大规模表达人工合成染色体(SACs)基因的技术，这些人造染色体是由特定的遗传元素创造出来的。这项技术将使设计大型、复杂的代谢途径成为可能，以平衡合成选定的产品。它将高度适用于各种真核表达系统。通过延伸重叠聚合酶链式反应、连接和连接将多个基因引入到DNA片段中。将使用适当的启动子和连接子合成一系列单独的基因和定义的基因组元件，连接在一起形成模块并在酿酒酵母中表达。这项工作将检验这样一种假设，即途径的一个部分的基因过度表达只会导致中间产物在另一部分积累。如果一个基因过度表达，另一个基因就会受到限制。代谢效率受到酶合成不平衡的影响，某些基因的过度表达会抑制细胞的生长。这项研究将接近在一个完整的途径中基因表达的最佳水平。通过检测转化子对木糖的生长和发酵，亲本细胞不能同化，它将确定SACS对生化代谢产物通量和细胞生理学的影响。它将使用实时聚合酶链式反应(Real-time PCR)测量导入基因的表达水平。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，将结合31P核磁共振和LC/MS来评估SACS对代谢物水平的影响。它将使用统计学方法来解释这些结果，并通过迭代试验接近代谢物通量的最佳水平。

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.