Iterative SCRaMbLE for engineering synthetic genome modules and chromosomes.

Area of Science:

• Synthetic biology
• Genomics
• Molecular and cell biology

Background:

• Saccharomyces cerevisiae is nearing completion of the first synthetic eukaryotic genome.
• Genome redesign includes LoxPsym sites for inducible rearrangements via Cre recombinase using SCRaMbLE.
• SCRaMbLE diversifies gene content and arrangement for phenotype enhancement.

Purpose of the Study:

• To demonstrate iterative SCRaMbLE for reorganizing synthetic genome modules and improving functions.
• To introduce SCOUT, a reporter system for sorting SCRaMbLEd cells into high-diversity pools.
• To map genotype abundance and genotype-phenotype relationships using SCOUT and long-read sequencing.

Main Methods:

• Utilizing SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxPsym-mediated Evolution) for genome diversification.
• Implementing the SCOUT (SCRaMbLE Continuous Output and Universal Tracker) reporter system for cell sorting.
• Applying iterative SCRaMbLE to yeast strains with synthetic chromosomes and histidine biosynthesis modules.

Main Results:

• Iterative SCRaMbLE successfully reorganizes synthetic genome modules.
• SCOUT facilitates the creation of high-diversity cell pools for analysis.
• Genotype-phenotype relationships were mapped using SCOUT and long-read sequencing.
• Optimization of a histidine biosynthesis module was achieved through iterative SCRaMbLE.

Conclusions:

• Iterative SCRaMbLE is a powerful tool for modular genome design.
• The SCOUT system enables efficient tracking and selection of modified yeast cells.
• This approach supports data-driven optimization of synthetic genomes for improved functions.