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,more »
- Authors:
-
- Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
- Joint BioEnergy Institute, Emeryville, CA (United States)
- Imperial College, London (United Kingdom)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1379122
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Accepted Manuscript
- Journal Name:
- PLoS ONE
- Additional Journal Information:
- Journal Volume: 11; Journal Issue: 3; Journal ID: ISSN 1932-6203
- Publisher:
- Public Library of Science
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; operons; fatty acids; plasmid construction; gene expression; sequence assembly tools; biosynthesis; sequence tagged site anaylsis; anaerobic bacteria
Citation Formats
Javidpour, Pouya, Deutsch, Samuel, Mutalik, Vivek K., Hillson, Nathan J., Petzold, Christopher J., Keasling, Jay D., Beller, Harry R., and Isalan, Mark. Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli. United States: N. p., 2016.
Web. doi:10.1371/journal.pone.0151087.
Javidpour, Pouya, Deutsch, Samuel, Mutalik, Vivek K., Hillson, Nathan J., Petzold, Christopher J., Keasling, Jay D., Beller, Harry R., & Isalan, Mark. Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli. United States. https://doi.org/10.1371/journal.pone.0151087
Javidpour, Pouya, Deutsch, Samuel, Mutalik, Vivek K., Hillson, Nathan J., Petzold, Christopher J., Keasling, Jay D., Beller, Harry R., and Isalan, Mark. Mon .
"Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli". United States. https://doi.org/10.1371/journal.pone.0151087. https://www.osti.gov/servlets/purl/1379122.
@article{osti_1379122,
title = {Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli},
author = {Javidpour, Pouya and Deutsch, Samuel and Mutalik, Vivek K. and Hillson, Nathan J. and Petzold, Christopher J. and Keasling, Jay D. and Beller, Harry R. and Isalan, Mark},
abstractNote = {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.},
doi = {10.1371/journal.pone.0151087},
journal = {PLoS ONE},
number = 3,
volume = 11,
place = {United States},
year = {Mon Mar 14 00:00:00 EDT 2016},
month = {Mon Mar 14 00:00:00 EDT 2016}
}
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Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes
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