(676a) Induced Pathway Optimization By Antibiotic Doses in Saccharomyces Cerevisiae
Saccharomyces cerevisiae has increasingly been engineered as a cell factory for efficient and economic production of fuels and chemicals from renewable resources. Here we report a new and simplest synthetic biology approach, named induced pathway optimization by antibiotic doses (iPAD), to improve the performance of multi-gene biosynthetic pathways in Saccharomyces cerevisiae. First, plasmids with step-wise increased copy numbers were constructed by engineering the expression level of selection marker proteins, including both auxotrophic and dominant markers. More importantly, the copy number of the plasmids with engineered dominant markers (5-100 copies per cell) showed a positive correlation with the concentration of antibiotics supplemented to the growth media. Based on this finding, iPAD was developed for modular optimization of multi-gene pathways by different combinations of antibiotic concentrations. To demonstrate this approach, iPAD was applied to optimize the lycopene and n-butanol biosynthetic pathways, with the production of lycopene and n-butanol increased by 10- and 100-fold, respectively. Finally, the pathways optimized by iPAD were integrated to the chromosome to increase the strain stability and eliminate the requirement of antibiotic supplementation, by taking advantage of the iPAD and CRISPR-Cas9 technologies for multiplex pathway integration.