The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5His 6 WS/DGAT comprising an N-terminal His 6 tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5His 6 atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein ؊1 min ؊1 was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5His 6 WS/DGAT with 1-hexadecanethiol and palmitoylCoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1⍀Km, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1⍀Km in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.Polyhydroxyalkanoic acids (PHA) in bacteria represent the most abundant group of neutral storage lipids, which serve as intracellular carbon and energy storage compounds (23). In addition, triacylglycerols (TAGs) and wax esters also occur as bacterial storage lipids, though at a much lower frequency than in eukaryotic microorganisms (22). Significant TAG accumulation was found, particularly in species belonging to the class Actinomycetes (1), as was reported for the genera Mycobacterium (3), Nocardia, and Streptomyces (2). TAGs are synthesized by the acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (DGAT) (13), which catalyzes the esterification of diacylglycerol with long-chain acyl-CoA. Wax esters are oxoesters of primary long-chain fatty alcohols and long-chain fatty acids. Occurrence of wax esters has been frequently reported for various Acinetobacter species (6). Under growthlimiting conditions, Acinetobacter calcoaceticus accumulates wax esters intracellularly as insoluble inclusions. The chemical structure of the wax esters synthesized by A. ca...

Recently, we could identify a bifunctional enzyme from this strain exhibiting at the same time WS as well as DGAT activity [10]. Experiments with a knockout mutant demonstrated the key role of the WS/DGAT for storage lipid accumulation in A. calcoaceticus ADP1 since virtually the complete wax ester and most of the TAG biosynthesis is mediated by this bifunctional enzyme. Biochemi- Acinetobacter calcoaceticus ADP1 possesses a bifunctional wax ester synthase/acylCoA:diacylglycerol acyltransferase (WS/DGAT) catalyzing the biosynthesis of wax esters and triacylglycerols. The unspecificity of WS/DGAT was used for in vitro and in vivo biosynthesis of wax diesters consisting of 1,16-hexadecanediol esterified with longchain fatty acids. An in vitro assay employing the membrane fraction of recombinant Escherichia coli XL1-Blue expressing wax/dgat coding for WS/DGAT and using 1,16-hexadecanediol and 1-14 C-palmitoyl-CoA as substrates resulted in the production of 2 radiolabeled substances as revealed by autoradiography suggesting the acylation of one or both hydroxy groups of 1,16-hexadecanediol by WS/DGAT. For in vivo biosynthesis of wax diesters, the knock-out mutant A. calcoaceticus ADP1acr1Ω Km was generated by disruption of acr1 coding for acyl-CoA reductase which caused the inability to synthesize fatty alcohols and, thus in consequence, wax esters. Co-cultivation of A. calcoaceticus ADP1acr1Ω Km on gluconate and 1,16-hexadecanediol in nitrogen-limited mineral salts medium resulted in the accumulation of a mixture of wax diesters of 1,16-hexadecanediol esterified with palmitic and oleic acid as revealed by electron impact ionization mass spectrometry. 1-Monopalmitoylglycerol could also be utilized as an alternative acyl acceptor by the unspecific WS/DGAT in vitro resulting in the synthesis of 1,2-and 1,3-dipalmitoylglycerol, whereas 1-oleoylglycerol-3-phosphate (lysophosphatidic acid) was not accepted.

Heterologous protein production is widely used in industrial biotechnology. However, using non-native production hosts can lead to enzymes with altered post-translational modifications, such as glycosylation. We have investigated how production in a non-native host affects the physicochemical properties and enzymatic activity of a feruloyl esterase from Myceliophthora thermophila , Mt Fae1a. The enzyme was produced in two microorganisms that introduce glycosylation ( M. thermophila and Pichia pastoris ) and in Escherichia coli (non-glycosylated). Mass spectrometric analysis confirmed the presence of glycosylation and revealed differences in the lengths of glycan chains between the enzymes produced in M. thermophila and P. pastoris . The melting temperature and the optimal temperature for activity of the non-glycosylated enzyme were considerably lower than those of the glycosylated enzymes. The three Mt Fae1a versions also exhibited differences in specific activity and specificity. The catalytic efficiency of the glycosylated enzymes were more than 10 times higher than that of the non-glycosylated one. In biotechnology, immobilization is often used to allow reusing enzyme and was investigated on mesoporous silica particles. We found the binding kinetics and immobilization yield differed between the enzyme versions. The largest differences were observed when comparing enzymes with and without glycosylation, but significant variations were also observed between the two differently glycosylated enzymes. We conclude that the biotechnological value of an enzyme can be optimized for a specific application by carefully selecting the production host. Electronic supplementary material The online version of this article (10.1186/s13568-019-0852-z) contains supplementary material, which is available to authorized users.

Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and L-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier L-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.

SummaryThe global demand for crude oil is expected to continue to rise in future while simultaneously oil production is currently reaching its peak. Subsequently, rising oil prices and their negative impacts on economy, together with an increased environmental awareness of our society, directed the focus also on the biotechnological production of fuels. Although a wide variety of such fuels has been suggested, only the production of ethanol and biodiesel has reached a certain economic feasibility and volume, yet. This review focuses on the current state and perspectives of biotechnological production of biodiesel‐like compounds. At present by far most of the produced biodiesel is obtained by chemical transesterification reactions, which cannot meet the demands of a totally ‘green’ fuel production. Therefore, also several biotechnological biodiesel production processes are currently being developed. Biotechnological production can be achieved by purified enzymes in the soluble state, which requires cost‐intensive protein preparation. Alternatively, enzymes could be immobilized on an appropriate matrix, enabling a reuse of the enzyme, although the formation of by‐products may provide difficulties to maintain the enzyme activity. Processes in presence of organic solvents like t‐butanol have been developed, which enhance by‐product solubility and therefore prevent loss of enzyme activity. As another approach the application of whole‐cell catalysis for the production of fatty acid ethyl esters, which is also referred to as ‘microdiesel’, by recombinant microorganisms has recently been suggested.