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

今天的大部分S.酿酒酵母采取了一种简单且通常是幼稚的方法，其中几种蛋白质，有时来自广泛不同的来源，如植物，真菌，细菌或人类，在细胞中共同表达以重建生物化学途径。为了评估工程化途径的功效，测量底物、产物和有时中间体。通常，这些工程菌株的第一代显示出非常差的产量和滴度，并且必须优化菌株以具有任何商业价值。传统上，这些菌株中的每一种的开发都需要很长的时间，通常每个开发周期消耗超过5-10年。随着路径复杂性的增加，开发时间也在增加。不足的异源蛋白表达或活性可能是重建途径性能差的主要原因。其他限制包括前体供应不足、导致通量降低的不平衡途径、毒性中间体的积累、中间体对竞争途径或分泌的损失、重组蛋白的异常或次优空间分布。在本申请中，我们提出解决后者。

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.