The invention provides methods and compositions for assembling a modular replacement
genome in a host
microorganism. After such
assembly, the
host organism's
genome is inactivated or ablated to permit full control of host
cellular functions by the replacement
genome. A modular replacement genome comprises an
assembly of
nucleic acid fragments, or segments, derived from one or more natural organisms or from synthetic polynucleotides or from a combination of both. Such an
assembly, or set, of segments making up a replacement genome comprises a substantially complete set of genes and regulatory elements for carrying out minimal life functions under predefined culture conditions. The invention provides modular genomes having modules that are amenable to facile replacement, deletion, and / or additions. Such modules may be synthetic polynucleotides and may be designed for controlling
gene content, excluding of genes that
encode inhibitors or otherwise undesirable competing enzymes that divert a host
cell from desired metabolic / synthetic processes; modifying codon usage to maximize or minimize
protein production; modifying regulatory elements, including promoters, enhancers, repressors, activator, or the like, to modulate
gene expression; balancing enzymatic and transport activities to optimize fluxes of substrates, intermediates, and products in metabolic pathways, and like objectives.