Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli

Triglycerides (TAGs), the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT). With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3- fold in the monounsaturated fatty acids (MUFA) occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids

Chemoenzymatic Synthesis of alpha-Hydroxy-beta-methyl-gamma-hydroxy Esters: Role of the Keto-Enol Equilibrium To Control the Stereoselective Hydrogenation in a Key Step

alpha-Hydroxy-beta-methyl-gamma-hydroxy esters not only are found in many natural products and potent drugs but also are useful intermediates in organic synthesis due to their highly functionalized skeleton that can be further manipulated and applied in the synthesis of many compound with remarkable biological activities. This work was based on a chemoenzymatic approach to obtain these molecules with three contiguous stereogenic centers in a highly enantio- and diastereoselective way. Two distinct linear routes were proposed in which the key steps in both routes consisted of initial stereocontrolled ketoester bioreduction followed by unsaturated carbonyl bioreduction or reduction with Pd-C. Other key reactions in the synthesis include a Wasserman protocol for chain homologation and a Mannnich-type olefination with maintenance of enantiomeric excess for all intermediates during the sequence. Whereas route A gave exclusively the skeleton with 3R,4R,5S configuration (99% ee and 11.5% global yield after 7 steps), route B gave the skeleton with 3R,4R,5S and 3R,4S,5R configurations (dr 1:12, 98% ee and 20% global yield after 5 steps).Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Computer Modeling Explains the Structural Reasons for the Difference in Reactivity of Amine Transaminases Regarding Prochiral Methylketones

Amine transaminases (ATAs) are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze the transfer of an amino group from an amino donor to an aldehyde and/or ketone. In the past decade, the enzymatic reductive amination of prochiral ketones catalyzed by ATAs has attracted the attention of researchers, and more traditional chemical routes were replaced by enzymatic ones in industrial manufacturing. In the present work, the influence of the presence of an α,β-unsaturated system in a methylketone model substrate was investigated, using a set of five wild-type ATAs, the (R)-selective from Aspergillus terreus (Atr-TA) and Mycobacterium vanbaalenii (Mva-TA), the (S)-selective from Chromobacterium violaceum (Cvi-TA), Ruegeria pomeroyi (Rpo-TA), V. fluvialis (Vfl-TA) and an engineered variant of V. fluvialis (ATA-256 from Codexis). The high conversion rate (80 to 99%) and optical purity (78 to 99% ee) of both (R)- and (S)-ATAs for the substrate 1-phenyl-3-butanone, using isopropylamine (IPA) as an amino donor, were observed. However, the double bond in the α,β-position of 4-phenylbut-3-en-2-one dramatically reduced wild-type ATA reactivity, leading to conversions of 99% ee. Computational docking simulations showed the differences in orientation and intermolecular interactions in the active sites, providing insights to rationalize the observed experimental results

Ultracentrifugation experiments reveal tDGAT remains associated to the neutral lipids accumulated by the recombinant bacteria.

<p><b>A and B, cell lysate of <i>E</i>. <i>coli</i> C41 (DE3) cells expressing tDGAT were sonicated and separated into phases by centrifugation. Samples from 1 to 3 correspond to consecutive centrifugations of the same sample at 2,000 g, 10,000 g and 55,000 g respectively</b>. (A) Electrophoretic 8% SDS-PAGE gel analysis of the supernatants (S1-S3) and pellets (P1-P3) of the different centrifugation fractions. tDGAT forming inclusion bodies falls at 2,000g. Lane M: Low Range SDS-PAGE Molecular Weight Standards (BioRad). (B) Thin layer chromatography (TLC) of lipid fractions extracted from 50 ml cultures expressing tDGAT. Lane +: Full pellet centrifuged at 55,000g. Lanes 1–4: Centrifugation pellets from P1 to P3. C and D, lipid bodies purification by sucrose gradient. (C) TLC of lipid fractions extracted from a cell lysate of <i>E</i>. <i>coli</i> C41 (DE3) cells expressing tDGAT: Lane M, triolein; lane 1, pellet obtained after centrifugation at 3,000 g and lane 2, the supernantant of the sucrose gradient centrifugation at 180,000 g. (D) Electrophoretic 12% SDS-PAGE gel analysis of the total <i>E</i>. <i>coli</i> C41 (DE3) cell lysate (1), proteins in the pellet obtained at 3,000 g (2) and proteins in the supernantant of the sucrose gradient centrifugation (3). Lane M shows the PageRuler Plus Prestained Protein Ladder (ThermoFisher). Position of TAGs are marked with black arrows in the TLC images. White arrowheads correspond to the protein tDGAT.</p

FA profile in <i>E</i>. <i>coli</i> cells expressing tDGAT.

<p>(A) Bar graphs showing the MUFA percentage (dark orange) among the whole FA pool in diverse <i>E</i>. <i>coli</i> strains [1: WT <i>E</i>. <i>coli</i> C41 (DE3); 2: <i>E</i>. <i>coli C41</i> (pET29c::<i>tDGAT</i>); 3: WT <i>E</i>. <i>coli</i> BW27783; 4: <i>E</i>. <i>coli</i> BW27783 (pBAD33::<i>tDGAT</i>)] (B) Circular graphs showing a more detailed comparison among the whole lipid extractions from <i>E</i>. <i>coli</i> C41 (DE3) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176520#pone.0176520.ref001" target="_blank">1</a>] and <i>E</i>. <i>coli</i> C41 (pET29c::<i>tDGAT</i>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176520#pone.0176520.ref002" target="_blank">2</a>] with the TAG-TLC spot from <i>E</i>. <i>coli</i> C41 (pET29c::<i>tDGAT</i>) (C). MUFA are presented in dark orange, saturated FA in blue, confirmed cyclopropyl FA in grey and unidentified FA (inferred from literature) in yellow.</p

Detection of TAG production in the <i>E</i>. <i>coli</i> C41 (DE3) strain carrying the enzyme tDGAT and correlation with its expression.

<p>(A) TLC showing the analysis of the lipid fractions extracted from cultures of <i>E</i>. <i>coli</i> C41 (DE3) with the plasmid constructions pET29c::<i>Ma1</i> (lane 3), pET29c::<i>Ma2</i> (lane 4), pET29c::<i>Ab</i> (lane 5), pET29c::<i>tDGAT</i> (lane 6) and the naked plasmid pET29c (lane 7). Oleic acid (lane 1) and trioleoylglycerol (lane 2) were loaded as control standards. Positions of TAGs and WEs are marked by black arrows. (B) TLC plate showing TAG production in the engineered strain <i>E</i>. <i>coli</i> C41 (pET29c::<i>tDGAT</i>) along time after IPTG induction. Lipid extractions from 6.5 mg of dry biomass collected 0, 30, 60, 90 and 120 minutes after induction with 100 mM IPTG at OD_600nm = 0.5 were loaded in lanes 1–5, respectively. A similar lipid extraction from wild type <i>E</i>. <i>coli</i> was loaded in lane 6. Different amounts of purified commercial TAGs (4.5; 18; 36 and 72 μg) were loaded in lanes 7–10 in order to estimate the TAG production by densitometric assay. The black arrows point to the migration distance of the TAGs and WEs. (C) SDS-PAGE electrophoretic gel of whole cell lysates of <i>E</i>. <i>coli</i> (pET29c::<i>tDGAT</i>) collected after induction periods of 0 and 90 minutes (lanes 1 and 2). Molecular masses (in kilodaltons) are indicated at the left. The band indicated by the white arrowhead corresponds to the protein tDGAT.</p

¹H NMR spectra (CDCl₃, 300.19 MHz) from lipid mixtures show an increase in the proportion of double bonds.

<p>Comparison of the 0.0–5.5 ppm regions of the ¹H NMR spectra from the whole lipid extract of engineered <i>E</i>. <i>coli</i> C41 (DE3) cells expressing the tDGAT (B, low green spectrum) and the whole lipid extract of the same bacteria without any heterologous protein (A, upper red spectrum). Peaks are named from right to left and attributions are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176520#pone.0176520.s009" target="_blank">S1 Table</a>. Black arrows point to the peaks indicating an increment in the proportion of double bonds in the recombinant strain. For more clarity, the spectrum is zoomed and only the region where all signals appear is shown. The only signal outside this region is the CDCl₃ solvent residual peak (7.26 ppm, data not shown).</p

Representative proteins of the WS/DGAT family.

<p>Representative proteins of the WS/DGAT family.</p

tDGAT belongs to WS/DGAT family.

<p>Structural prediction analysis of the tDGAT protein revealed a monomer with a two-domain structure: N-terminal domain is colored in blue, C-terminal domain in red and the connecting loops in green. Beta sheets and alpha helices are shown in darker colors and labeled with the same notation used in the alignment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176520#pone.0176520.s001" target="_blank">S1 Fig</a>. The active site HHxxxDG is also marked with a black star and colored in yellow. Other important conserved motifs are labeled (mI and mII).</p

Correspondence between fluorescence and TAG production in Nile Red -stained cells.

<p>(A) Fluorescence microscopy images corresponding to the Nile Red -stained strains <i>E</i>. <i>coli</i> C41 (DE3) (left) and <i>E</i>. <i>coli</i> C41 (pE29c::<i>tDGAT</i>) (right) acquired with different wavelengths: red filter, 543 nm; green filter, 488 nm. Merged images of both fluorescence scans acquired and zoomed areas with fewer bacteria are displayed. The white arrow points to a lipid inclusion in the engineered tDGAT-expressing <i>E</i>. <i>coli</i> C41 (DE3) strain. (B) TLC plate showing the neutral lipid profile of the lipid extracts from the cultures analysed in the fluorescence experiments. Black arrow points to the TAGs. (C) Bar charts showing red (620 nm) average fluorescence spectroscopy measurements from both stained cultures. Error bars correspond to three experiments. AFU: Arbitrary Fluorescence Units. (D) Images of cytometry analysis of <i>E</i>. <i>coli</i> C41 (DE3) (left) and <i>E</i>. <i>coli</i> C41 (pE29c::<i>tDGAT</i>) (right) stained with Nile Red 20 h after induction of tDGAT expression with IPTG. An increase higher than 30% of the events were detected as fluorescent in the recombinant strain accumulating TAGs.</p