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.
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