title: Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli.
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.
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA, United States of America.|Biological Systems and Engineering, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, United States of America.
authors.forename: Pouya
authors.initials: P
authors.lastname: Javidpour
authors.affiliation: Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA, United States of America.
authors.forename: Samuel
authors.initials: S
authors.lastname: Deutsch
authors.affiliation: Environmental Genomics and Systems Biology, LBNL, Berkeley, CA, United States of America.
authors.forename: Vivek K
authors.initials: VK
authors.lastname: Mutalik
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA, United States of America.|Biological Systems and Engineering, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, United States of America.|Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA, United States of America.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Hillson
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA, United States of America.|Biological Systems and Engineering, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, United States of America.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA, United States of America.|Biological Systems and Engineering, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, United States of America.|Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, United States of America.|Department of Bioengineering, University of California, Berkeley, CA, United States of America.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA, United States of America.|Earth and Environmental Sciences, LBNL, Berkeley, CA, United States of America.
authors.forename: Harry R
authors.initials: HR
authors.lastname: Beller
mesh_terms: D001421:Bacteria, Anaerobic / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003503:Cyclobutanes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D009876:Operon; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003503:Cyclobutanes; D011994:Recombinant Proteins