A large and growing number of complete genomes from diverse species open tremendous opportunities for getting deep insights into cell metabolism. This increased understanding strongly supports engineering of cell metabolism for microbial production. In spite of the recent progress, a large fraction of genes in most of the available genomes remain incorrectly or imprecisely annotated. In this paper we review some of the new comparative genomics techniques used to reconstruct regulatory and metabolic networks from genomic data, reveal gaps in current knowledge, and identify previously uncharacterized genes. The application will be discussed by using a recent example–reconstruction of xylose utilization pathway in Clostridium acetobutylicum.