Analysis of Lipid Export in Hydrocarbonoclastic Bacteria of the GenusAlcanivorax: Identification of Lipid Export-Negative Mutants ofAlcanivorax borkumensisSK2 andAlcanivorax jadensisT9

Abstract

Triacylglycerols (TAGs), wax esters (WEs), and polyhydroxyalkanoates (PHAs) are the major hydrophobic compounds synthesized in bacteria and deposited as cytoplasmic inclusion bodies when cells are cultivated under imbalanced growth conditions. The intracellular occurrence of these compounds causes high costs for downstream processing. Alcanivorax species are able to produce extracellular lipids when the cells are cultivated on hexadecane or pyruvate as the sole carbon source. In this study, we developed a screening procedure to isolate lipid export-negative transposon-induced mutants of bacteria of the genus Alcanivorax for identification of genes required for lipid export by employing the dyes Nile red and Solvent Blue 38. Three transposoninduced mutants of A. jadensis and seven of A. borkumensis impaired in lipid secretion were isolated. All isolated mutants were still capable of synthesizing and accumulating these lipids intracellularly and exhibited no growth defect. In the A. jadensis mutants, the transposon insertions were mapped in genes annotated as encoding a putative DNA repair system specific for alkylated DNA (Aj17), a magnesium transporter (Aj7), and a transposase (Aj5). In the A. borkumensis mutants, the insertions were mapped in genes encoding different proteins involved in various transport processes, like genes encoding ( Almost all prokaryotes synthesize lipophilic storage substances as an integral part of their metabolism under limited nitrogen or phosphorus conditions if there is an excess of a suitable carbon source at the same time. The accumulated storage lipids serve as energy and carbon sources during starvation periods, and they are mobilized again under conditions of carbon and energy deficiency. The majority of the members of many genera synthesize hydrophobic polymers, such as poly(3-hydroxybutyrate) (PHB) or other types of polyhydroxyalkanoates (PHAs), whereas the accumulation of triacylglycerols (TAGs; trioxoesters of glycerol and long-chain fatty acids [FAs]) or wax esters (WEs; oxoesters of primary long-chain fatty acids and primary long-chain fatty alcohols) occurs in fewer prokaryotes (66). TAG accumulation has been reported for species of the genera Streptomyces, Mycobacterium, Nocardia, Rhodococcus (4,6,65), and recently also Alcanivorax and other hydrocarbonoclastic marine bacteria (32). Accumulation of WEs has been frequently reported for species of the genus Acinetobacter (66) but also for marine bacteria, such as Marinobacter (50) and Alcanivorax (11,32).In general, the accumulation of at least one type of these compounds occurs intracellularly under imbalanced growth conditions in almost all prokaryotes. The localization of neutral lipids in marine organisms is not restricted to the cell cytoplasm, as extracellular lipid deposition has been shown in studies with Alcaligenes sp. PHY9 and Pseudomonas nautica (24). The production of extracellular wax esters by Alcanivorax jadensis T9 growing on hexadecane was described a few years ago (11). Species of the genus Alcanivorax ...

Neutral lipid production in Alcanivorax borkumensis SK2 and other marine hydrocarbonoclastic bacteria

“…Use of hydrocarbonoclastic bacteria (HCB) to produce lipophilic substances like PHA, TAG, or WE as bulk chemicals from hydrocarbons, was recently reviewed [7]. Alcanivorax borkumensis SK2 seems to be particularly feasible for production of neutral lipids, because this bacterium is also able to export and deposit in the medium the produced neutral lipids like TAG or WE [8]. The latter may be important if the neutral lipids are produced biotechnologically.…”

Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

“…As mentioned above, A. borkumensis SK2 is able to produce and accumulate TAGs as the main storage compound when it is cultivated in the presence of pyruvate as the sole carbon source. Although the export of TAGs has been reported recently (31), in this study we analyzed only the intracellular accumulation of TAGs in order to establish the function of these compounds in A. borkumensis SK2. Recently, it was demonstrated that the amount of accumulated TAGs in this strain diminishes if at least one of the two WS/DGAT (Fig.…”

“…In Acinetobacter species, WEs are degraded into water-soluble molecules and CO 2 during carbon starvation, thereby demonstrating that they serve as an ATP-generating substrate during starvation (16). Accumulation of WEs in species of the genus Acinetobacter (16) but also in marine bacteria such as Marinobacter (25) and Alcanivorax (10,28,31) species has been described previously. Also, marine hydrocarbonoclastic bacteria of the genus Alcanivorax are able to export lipids such as TAGs and WEs (31).…”

“…Accumulation of WEs in species of the genus Acinetobacter (16) but also in marine bacteria such as Marinobacter (25) and Alcanivorax (10,28,31) species has been described previously. Also, marine hydrocarbonoclastic bacteria of the genus Alcanivorax are able to export lipids such as TAGs and WEs (31). Accumulation of at least one type of storage lipid can be found in almost all prokaryotes.…”

Genetic engineering of microorganisms for biodiesel production

“…Indeed, the localization of neutral lipids in organisms is not restricted to the cell cytoplasm, as extracellular lipid deposition has been shown in various strains. There are many strains of bacteria presenting the ability of extracellular TAGs production, like Alcanivorax, 90 Acinetobacter, 91 Alcaligenes and Pseudomonas. 92 It was also reported that similar secretion of fatty acids or TAGs can be achieved by metabolic engineering and mutagenesis, not only in bacterium E. coli, but also in some yeast strains and microalgae.…”