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

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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.