Occurrence, production, and export of lipophilic compounds by hydrocarbonoclastic marine bacteria and their potential use to produce bulk chemicals from hydrocarbons

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bWax esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for wax ester production is the bifunctional wax ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four different bacteria: Marinobacter aquaeolei VT8, Acinetobacter baylyi, Rhodococcus jostii RHA1, and Psychrobacter cryohalolentis K5. The results from kinetic studies of isolated enzymes reveal a differential activity based on the order of substrate addition and reveal subtle differences between the substrate selectivity of the different enzymes. These in vitro results are compared to the wax ester and triacylglyceride product profiles obtained from each organism grown under neutral lipid accumulating conditions, providing potential insights into the role that the WS/DGAT enzyme plays in determining the final wax ester products that are produced under conditions of nutrient stress in each of these bacteria. Further, the analysis revealed that one enzyme in particular from M. aquaeolei VT8 showed the greatest potential for future study based on rapid purification and significantly higher activity than was found for the other isolated WS/DGAT enzymes. The results provide a framework to test prospective differences between these enzymes for potential biotechnological applications such as high-value petrochemicals and biofuel production.

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Differences in Substrate Specificities of Five Bacterial Wax Ester Synthases

Barney

Wahlen

Garner

et al. 2012

168

“…Besides a store of carbon and energy, these lipophilic compounds serve as a sink for reducing equivalents in microorganisms (6,7). These lipophilic materials are ideal for energy storage because of their minimal space requirements, higher caloric values compared to proteins or carbohydrates, and lack of cellular toxicity (8). In contrast, nonesterified fatty acids (i.e., free fatty acids [FFAs]) are toxic due to their amphiphilic nature (9).…”

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confidence: 99%

An Oleaginous Bacterium That Intrinsically Accumulates Long-Chain Free Fatty Acids in its Cytoplasm

Katayama

Kanno

Morita

et al. 2014

Medium-and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the family Erysipelotrichaceae in the phylum Firmicutes but is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited ␤-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.

“…1) (5). Together with the fatty acyl-CoA reductase and fatty aldehyde reductase enzymes (6)(7)(8) which provide the requisite fatty alcohol, the WS/DGAT enzyme is proposed to produce wax esters in bacteria from the fatty acyl-CoA pool (4). The WS/ DGAT from Acinetobacter has been extensively studied, while WS/DGAT enzymes from additional species have been characterized to a lesser extent (5,(9)(10)(11)(12).…”

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confidence: 99%

“…While common in many higher organisms such as plants and animals, wax esters are produced in only a small selection of bacteria (4). In bacteria, the enzyme responsible for catalyzing the esterification of a fatty acyl-coenzyme A (CoA) and a fatty alcohol is referred to as the wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) (EC 2.3.1.75; Fig.…”

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confidence: 99%

Identification of a Residue Affecting Fatty Alcohol Selectivity in Wax Ester Synthase

Barney

Mann

Ohlert

2013

The terminal enzyme in the bacterial wax ester biosynthetic pathway is the bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT), which utilizes a fatty alcohol and a fatty acyl-coenzyme A (CoA) to synthesize the corresponding wax ester. In this report, we identify a specific residue in WS/DGAT enzymes obtained from Marinobacter aquaeolei VT8 and Acinetobacter baylyi that alters fatty alcohol selectivity and kinetic parameters when modified to alternative residues.