Coupling Biosensor and Multi-Chromosomal SCRaMbLE for High-Throughput Screening of Isobutanol Strains

Abstract

Improvement of BCHA production through metabolic engineering has been a goal and challenge of the industry for many years. Novel poly-synthetic yeast cells that incorporate the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a method for generating genotype diversity. Here, we utilize single copy genome integration of the mitochondrial metabolic pathway into both synthetic and non-synthetic chromosomes for the biosynthesis of isobutanol. Single copy integration combined with the integration of an isobutanol biosensor aids in the high-throughput screening of mutant strains. Following the SCRaMbLEing of haploid strains the yield of isobutanol is increased to 4.5-fold after 3 cycles of Fluorescence Activated single Cell Sorting (FACS) in strains where a single copy was integrated into a synthetic chromosome. This strain provides a potentially powerful tool for increasing the production of bio-based BCHAs and for faster identification of beneficial mutant cells.