(67f) Optimization Driven Top-Down Synthesis of Genome Minimized Strains for Bioproduction

Genome minimized strains are obtained by removing genome segments associated with genes or processes either detrimental or unneeded under bioproduction conditions. They offer advantages as production chassis [1, 2] by reducing transcriptional cost, eliminating competing functions and limiting unwanted regulatory interactions. Genetically minimized strains have been experimentally explored before, demonstrating beneficial characteristics [3]. Existing approaches for identifying stretches of DNA to remove are largely ad hoc based on information on presumably dispensable regions through experimentally determined non-essential genes and comparative genomics. As more sophisticated genome editing tools (e.g., CRISPR) are becoming commonplace, the need for a computational aid that will help sequentially minimize genomes consistent with a set of performance criteria is becoming more pressing. Here we introduce a versatile genome reduction algorithm that implements a mixed integer linear program (MILP) to iteratively identify the largest contiguous sequences within the genome that can be deleted without affecting the organism’s growth rate or other desired attributes. Known essential genes or genes that cause significant fitness or performance loss can be flagged and their deletion is thus prohibited. This optimization-based procedure also accounts for promoter regions ensuring that retained genes will be properly transcribed. The algorithm avoids the deletion of synthetic lethal pairs by maintaining biomass production even upon the simultaneous deletion of multiple stretches. In addition, we assess the possibility of removing even larger stretches of DNA if only one or two essential genes are within the deleted sequence. These deleted essential genes can then be re-inserted into the genome in a different location. We applied the algorithm to design minimized E. coli strains and found that we were able to recapitulate the long deletions identified in previous experimental studies [3]. Moreover, we discovered alternative combinations of deletions which have not been explored in vivo. Identification of large DNA stretches allows us to significantly reduce the time needed to perform deletions during each cycle of the genome reduction process.

[1] Lian, Jiazhang, et al. "Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains." Metabolic engineering 24 (2014): 139-149.

[2] Vickers, Claudia E., et al. "Grand challenge commentary: Chassis cells for industrial biochemical production." Nature chemical biology 6.12 (2010): 875.

[3] Juhas, Mario, et al. "Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering." Microbiology 160.11 (2014): 2341-2351.