Implementation of stable and complex biological systems through recombinase-assisted genome engineering.
Evaluating the performance of engineered biological systems with high accuracy and precision is nearly impossible with the use of plasmids due to phenotypic noise generated by genetic instability and natural population dynamics. Minimizing this uncertainty therefore requires a paradigm shift towards engineering at the genomic level. Here, we introduce an advanced design principle for the stable installment and implementation of complex biological systems through recombinase-assisted genome engineering (RAGE). We apply this concept to the development of a robust strain of Escherichia coli capable of producing ethanol directly from brown macroalgae. RAGE significantly expedites the optimal implementation of a 34 kb heterologous pathway for alginate metabolism based on genetic background, integration locus, copy number and compatibility with two other pathway modules (alginate degradation and ethanol production). The resulting strain achieves a ~40% higher titre than its plasmid-based counterpart and enables substantial improvements in titre (~330%) and productivity (~1,200%) after 50 generations.
1] Bio Architecture Lab, Inc., 604 Bancroft Way, Suite A, Berkeley, California 94710, USA [2].
Christine Nicole S
CN
Santos
Drew D
DD
Regitsky
Yasuo
Y
Yoshikuni
D000464:Alginates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D005818:Genetic Engineering; D016680:Genome, Bacterial; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D016254:Mutagenesis, Insertional; D000459:Phaeophyceae / Q000737:chemistry; D010957:Plasmids; D045522:Recombinases / Q000235:genetics* / Q000378:metabolism
D000464:Alginates; D029968:Escherichia coli Proteins; D006603:Hexuronic Acids; D045522:Recombinases; D000431:Ethanol; D020723:Glucuronic Acid