Functions and export mechanism of extracellular triacylglycerols and wax esters in Alcanivorax sp..
Final Report Abstract
The project addressed and investigated the bacterial export of triacylglycerols (TAG) and wax esters (WE). Besides the known and widespread intracellular accumulation of these hydrophobic molecules as lipid bodies, strains of the genus Alcanivorax produce significant amounts of extracellular TAGs and WEs. The project aims were as follows: (i) The physiology of lipid export in Alcanivorx sp. should be investigated. Furthermore, the biological role of the excreted lipids should be determined. (ii) In parallel, the proteins and genes involved in biosynthesis and export of neutral lipids in Alcanivorax sp. should be identified and characterized at the molecular level. This aspect should be in particular investigated if the intra- and extracellular lipids were synthesized basically by the same or altered pathways. (iii) Investigations on the controlled expression of the respective proteins in strains, which are still able to accumulate intracellular lipids but are unable to export lipids, should provide insights into the molecular mechanisms of bacterial lipid export. (iv) Later, the biochemical and biophysical properties of the purified lipid export protein(s) should be investigated by in vitro and in vivo experiments. We were able to deepen our understanding on the bacterial biosynthesis and export of neutral lipids. In addition, we considered to deal with Alcanivorax sp. with regard to biotechnological processes. "Lipid biotechnology" covers the microbial production and biotechnological transformation of lipids and lipid-soluble compounds. Storage lipids like TAGs and different fatty acid types are the main targets for biotechnological product development, while phospholipids, sphingolipids, glycolipids, sterols, carotenoids as well as other lipid-soluble compounds are utilized for the production of bioactive molecules in cosmetics and pharmaceuticals or for nutrition. Since TAGs and fatty acids are precursors of biodiesel, their production has recently attracted considerable attention. Screening for bacteria able to produce high amounts of TAGs is the first and essential step in the search for oleaginous microorganisms. Bacteria, which accumulate more of 20 % of their weight as lipid, are referred to as oleaginous. TAG accumulation by A. borkumensis SK2 of up to 23 % of its cell dry weight has already been reported. However, additional studies are necessary to further increase the content of TAGs in A. borkumensis SK2, before this strain can be considered as candidate for biotechnological production of neutral lipids. Since the hydrocarbon metabolism of bacteria is complex, the feasibility of bioremediation as technology of oil polluted sites is still controversially discussed, and new applications of hydrocarbon-degrading bacteria should be studied. Improved systems for biodegradation of petroleum components are being commercialized with positive and environmental advantages. The ability of OHCB, especially of A. borkumensis, to metabolize almost exclusively hydrocarbons can be also used to produce storage compounds like TAGs, and/or WEs as bulk chemicals, while the extracellular deposition of these compounds could represent a major advantage concerning the downstream processes, if they are produced biotechnologically.
Publications
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(2006) Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2. J. Bacteriol. 188:8452-8459
Sabirova J. S., M. Ferrer, H. Lünsdorf, V. Wray, R. Kalscheuer, A. Steinbüchel, K. N. Timmis, and P. N. Golyshin
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(2007) Analysis of storage lipid accumulation in Alcanivorax borkumensis: Evidence for alternative triacylglycerol biosynthesis routes in bacteria. J. Bacteriol. 189:918-928
Kalscheuer R., T. Stöveken, U. Malkus, R. Reichelt, P. N. Golyshin, J. S. Sabirova, M. Ferrer, K. N. Timmis, and A. Steinbüchel
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(2007) Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: Properties, function and occurrence of wax ester synthases/acyl-CoA:diacylglycerol acyl transferases. Biochemie 89:230-242
Wältermann, M., T. Stöveken, and A. Steinbüchel
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(2008) Bacterial acyltransferases as an alternative for lipase-catalyzed acylation for the production of oleochemicals and fuels. Angew. Chem. (Int. Ed.) 47:3688-3694
Stöveken, T., and A. Steinbüchel
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(2008) Bakterielle Acyltransferasen als Alternative für lipasekatalysierte Acylierungen zur Produktion von Oleochemikalien und Brennstoffen. Angew. Chem. (deutsche Ausgabe) 120:3746-3752
Stöveken, T., and A. Steinbüchel
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(2010) Analysis of lipid export in hydrocarbonoclastic bacteria of the genus Alcanivorax: identification of lipid export-negative mutants of Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9. J. Bacteriol. 192:643-656
Manilla-Pérez E., C. Reers, M. Baumgart, S. Hetzler, R. Reichelt, U. Malkus, R. Kalscheuer, M. Wältermann, and A. Steinbüchel
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(2010) Cell surface analysis of the lipid-discharging, obligate hydrocarbonoclastic species of the genus Alcanivorax. Eur J Lipid Sci Technol. 112:681-691
Lange A. B., K. B. Tenberge, H. Robenek, and A. Steinbüchel
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(2010) Isolation and characterization of a mutant of the marine bacterium Alcanivorax borkumensis SK2 defective in lipid biosynthesis. Appl. Environ. Microbiol. 76:2884-2894
Manilla-Pérez E., A. B. Lange, S. Hetzler, M. Wältermann, R. Kalscheuer, and A. Steinbüchel
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(2010) Occunence, production, and export of lipophilic compounds by hydrocarbonoclastic marine bacteria and their potential use to produce bulk chemicals from hydrocarbons. Appl. Microbiol. Biotechnol. 86:1693-1706
Manilla-Pérez E., A. B. Lange, S. Hetzler., and A. Steinbüchel
