Metabolic engineering tools for Saccharomyces cerevisiae: Engineering the spatial organization of metabolic pathways

今天酿酒酵母的代谢工程大多采用了一种简单且往往幼稚的方法，即在一个细胞中集体表达几种蛋白质，有时来自不同的来源，如植物、真菌、细菌或人类，以重建一条生化途径。为了评估工程途径的有效性，我们测量了底物、产物(S)，有时还测量了中间体。通常情况下，这些工程菌株的第一代表现出非常低的产量和效价，必须进行优化，才能使菌株具有商业价值。传统上，每一种毒株的发育都需要很长的时间，每个发育周期通常需要5-10年以上的时间。随着途径复杂性的增加，开发的时间也会增加。不充分的异源蛋白表达或活性可能是重组途径表现不佳的主要原因。其他限制包括前体供应不足，导致通量减少的途径不平衡，有毒中间体(S)积累，中间体(S)损失到竞争途径(S)或分泌，重组蛋白的空间分布异常或次优。在本申请中，我们建议解决后者。

Much of today's metabolic engineering of S. cerevisiae assumes a simplistic and often naïve approach whereby several proteins, sometimes from widely different origins such as plants, fungi, bacteria or human are collectively expressed in a cell to reconstitute a biochemical pathway. To evaluate the efficacy of the engineered pathways, substrate, product(s) and sometimes intermediates are measured. More often than not, the first generations of these engineered strains show very poor yields and titers and must be optimized for the strains to have any commercial value. Traditionally, the development of each of these strains requires lengthy periods, often consuming over 5-10 years per development cycle. As the pathway complexity goes up so does the time for development. Less than adequate heterologous protein expression or activity is probably the leading cause of poor performance by reconstituted pathways. Other limitations include insufficient supply of precursors, imbalanced pathway leading to reduced flux, accumulation of toxic intermediate(s), loss of intermediate(s) to competing pathway(s) or secretion, aberrant or sub-optimal spatial distribution of recombinant proteins. In this current application, we propose to address the latter.