Metabolic engineering of an obligate methanotroph, Methylomonas sp. strain 16a, for the production of C₄₀ carotenoids from methane has been accomplished. In this presentation, I will describe the approaches used for the production of C₄₀ carotenoids, including a discussion of genetic tools developed to enable metabolic engineering of methanotrophs. Because long-term strain stability plus the lack of antibiotic resistance markers are essential for commercial production, genetic tools were developed to make markerless chromosomal deletions, as well as targeted or random chromosomal insertions. The genes necessary for the production of the C₄₀ carotenoids were absent in wild type Methylomonas and were isolated from nonpathogenic environmental isolates. Novel carotenoid gene clusters were introduced into Methylomonas and it was shown that carbon flux through the carotenoid pathway is greatly impacted by gene order. Additionally, the integration site of the gene clusters within the Methylomonas genome significantly impacts carotenoid gene expression. The stacking of additional copies of key carotenoid genes was shown to further increase carotenoid titers, and the re-direction of carbon flow to the isoprenoid pathway was accomplished by applying the metabolic understanding derived from the genome sequence analysis. The two-liter fermentation results revealed the strong effects of different carotenoid gene clusters, and their chromosomal integration sites, on growth, titer and carotenoid selectivity. This work show that it is possible to go from an environmental isolate, with no genetic system, to a metabolically engineered strain that meets production metrics in a short period of time via an interdisciplinary team of molecular biologist and engineers.