Abstract
Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and experimental results developed in this study to elucidate the biosynthetic pathway that produces unique and intriguing ladderane lipids.
Highlights
IntroductionLadderanes (e.g., Fig 1) are hydrocarbon chains with three or five fused cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria [1,2,3]
Ladderanes (e.g., Fig 1) are hydrocarbon chains with three or five fused cyclobutane rings that are uniquely produced as membrane lipid components by anammox bacteria [1,2,3]
Microbially produced fuels derived from conventional fatty acids, such as fatty acid ethyl esters or medium-chain methyl ketones, have recently been developed and have favorable properties as diesel fuel blending agents [6,7,8,9,10], it is plausible that fuels derived from ladderane fatty acids would have excellent jet fuel properties, in particular, high volumetric energy density
Summary
Ladderanes (e.g., Fig 1) are hydrocarbon chains with three or five fused cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria [1,2,3]. Microbially produced fuels derived from conventional fatty acids, such as fatty acid ethyl esters or medium-chain methyl ketones, have recently been developed and have favorable properties as diesel fuel blending agents [6,7,8,9,10], it is plausible that fuels derived from ladderane fatty acids would have excellent jet fuel properties, in particular, high volumetric energy density. Estimations of volumetric energy densities of ladderane structures suggest that a [5]-ladderane could have ~46% greater volumetric energy density than conventional jet fuel used in the U.S (S1 Table), which would lead to greater energy efficiency. The primary challenge in producing ladderane-derived fuels in a renewable fashion, for example, using engineered microbes to make them from cellulosic sugars, is that the underlying biochemistry and genetics of ladderane biosynthesis are unknown. An elegant chemical synthesis of [5]-ladderane fatty acid (pentacycloanammoxic acid) was accomplished [12], the synthesis was laborious, low yielding (~2%), and doi:10.1371/journal.pone.0151087.g001
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