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Hopanoids and carotenoids are two of the major isoprenoid‐derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols. Methylobacterium extorquens contains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate the role of isoprenoid lipids in surface membrane function and cellular fitness. By genetically knocking out hpnE and crtB we disrupted the production of squalene and phytoene in M. extorquens PA1, which are the presumed precursors for hopanoids and carotenoids respectively. Deletion of hpnE revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in pigmentation with a C₃₀ backbone, rather than the previously predicted canonical C₄₀ phytoene‐derived pathway. Phylogenetic analysis suggested that M. extorquens may have acquired the C₃₀ pathway through lateral gene …