Bacterial strains, plasmids and materials
Compounds DHAP, DHA, l-GAL, AP from potato, isopropyl-β-d-thiogalactopyranoside (IPTG), polyphosphate, ATP, l-fructose, l-tagatose, glycerol, glucose, and antibiotics were purchased from Sigma-Aldrich. All restriction enzymes and DNA ligase were purchased from Novagen (Darmstadt, Germany). Ni–NTA affinity chromatography column was purchased from QIAGEN. The yeast extract and tryptone were purchased from OXOID LID, and brain heart infusion (BHI) was purchased from Becton, Dickinson and Company. All bacterial strains and plasmids are listed in Table 4.
Vectors and strains construction
The genes fucA from E. coli and dhaK from Citrobacter freundii were amplified from genome and cloned into pET-21a(+) to obtain pET21-FucA and pET21-DhaK, respectively. The plasmid pET21-PPK containing the gene of ppk from Rhodobacter sphaeroides was kindly provided by Professor Chun You in our institute. For the construction of plasmid pXRTYH and pXFucTYH, gene hdpA was amplified from C. glutamicum 13032 genome. The amplified fragments were ligated into plasmid previous constructed plasmids pXRTY [40] and pXFucTY [46] at the SmaI and SacI sites to obtain pXRTYH and pXFucTYH, respectively. The constructed plasmids were then electroporated into the recombinant strain SY6, in which the gene tpi has been eliminated, to generate strains SY6(pXRTYH) and SY6(pXFucTYH). Plasmid pEFDK containing genes glpF, dhaD and dhaK [46] and pXRTY were co-transformed into SY6 strain to obtain SY6(pXRTY/pEFDK).
Recombinant proteins expression and purification
Escherichia coli BL21(DE3) strains harboring expression plasmids were cultured at 37 °C in 1 L LB medium containing 100 mg/L ampicillin to an optical density OD600 of 0.6. 0.5 mM IPTG was added into the culture to induce protein expression and the temperature was adjusted to 16 °C to avoid inclusion body formation. After incubation for an additional 20 h, cells were harvested, washed twice and suspended in 50 mM triethanolamine (TEA) (pH 7.5) buffer. The suspension cells were then lysed by sonication and centrifuged at 14,000×g and 4 °C for 10 min. Clear supernatant was collected and loaded onto an Ni2+-NTA-agarose column pre-equilibrated with binding buffer (50 mM TEA buffer, 300 mM NaCl, 20 mM imidazole, pH 7.5). The retained proteins were recovered with elution buffer (50 mM TEA buffer, 300 mM NaCl, 300 mM imidazole, pH 7.5). The eluted fraction containing purified protein was dialyzed to eliminate buffer, salt and imidazole. The purified enzymes were freeze dried using a vacuum pump and stored at − 20 °C.
Enzyme activity assay
The activity of PPK was assayed in a reaction mixture (200 μL) containing PPK (0.05 mg), 25 mM TEA buffer (pH 7.0), 25 mM DHA, 10 mM ADP, 10 mM polyphosphate and 5 mM MgCl2·6H2O. After the reaction at 30 °C for 30 min, the reaction was stopped by the addition of 10% H2SO4 (0.5 μL). Product was determined via high-performance liquid chromatography (HPLC). One unit of enzyme activity was defined as the enzyme amount catalyzing the consumption of 1 μmol DHA per min.
The activity of FLS was assayed in a reaction mixture (200 μL) containing FLS (2 mg), 25 mM TEA buffer (pH 7.0), 25 mM FALD, 1 mM MgSO4 and 0.1 mM TPP. After the reaction at 30 °C for 45 min, the reaction was stopped by the addition of 10% H2SO4 (0.5 μL). One unit of enzyme activity was defined as the enzyme amount catalyzing the formation of 1 μmol of total DHA and GA per min.
Steady-state kinetic parameters of RhaD and FucA to DHA and l-GAL
Reaction: aldol addition of DHAP to l-GAL. To a solution containing freshly neutralized DHAP (60 mM) and RhaD (0.05 mg powder) in 50 mM TEA buffer pH 7.5 at 25 °C, different amounts of l-GAL (0.2, 0.5, 2, 5, 10, 20, 40, 60 mM) were added. The final volume was 400 μL. Samples (40 μL) were withdrawn at different times (0, 2, 5, 10, 20, 30 min) and the reaction was stopped by the addition of 10% H2SO4 (0.5 μL). Samples were then analyzed by HPLC to measure the l-GAL consumption. One mmol of l-GAL consumed was equivalent to 1 mmol of l-fructose-1-phosphate formed.
Reaction: aldol addition of DHAP to DHA. To a solution containing freshly neutralized DHAP (60 mM) and RhaD (0.05 mg powder) in 50 mM TEA buffer pH 7.5 at 25 °C, different amounts of DHA (2, 5, 10, 20, 40, 60, 80 mM) were added. The final volume was 400 μL. Samples (40 μL) were withdrawn at different times (0, 2, 5, 10, 20, 30 min) and the reaction was stopped by the addition of 10% H2SO4 (0.5 μL). Samples were then analyzed by HPLC to measure the DHA consumption. One mmol of DHA consumed was equivalent to 1 mmol of adduct formed.
Reaction: aldol addition of DHAP to l-GAL. To a solution containing freshly neutralized DHAP (60 mM) and FucA (0.1 mg powder) in 50 mM TEA buffer pH 7.5 at 25 °C, different amounts of l-GAL (0.5, 2, 5, 10, 20, 40, 60 mM) were added. The final volume was 400 μL. Samples (40 μL) were withdrawn at different times (0, 2, 5, 10, 20, 30 min) and the reaction was stopped by the addition of 10% H2SO4 (0.5 μL). Samples were then analyzed by HPLC to measure the l-GAL consumption. One millimole of l-GAL consumed was equivalent to 1 mmol of l-tagatose-1-phosphate formed.
Reaction: aldol addition of DHAP to DHA. To a solution containing freshly neutralized DHAP (60 mM) and FucA (0.1 mg powder) in 50 mM TEA buffer pH 7.5 at 25 °C, different amounts of DHA (2, 5, 10, 20, 40, 60, 80, 100, 200 mM) were added. The final volume was 400 μL. Samples (40 μL) were withdrawn at different times (10, 20, 40, 60, 90, 120, 180 min) and the reaction was stopped by the addition of 10% H2SO4 (0.5 μL). Samples were then analyzed by HPLC to measure the DHA consumption. One mmol of DHA consumed was equivalent to 1 mmol of adduct formed.
Aldol reactions with DHAP and DHA as substrates
The reaction mixture (1 mL) contained freshly neutralized 50 mM DHAP solution, 50 mM DHA, 50 mM TEA buffer (pH 7.5) and RhaD (1 mg) or FucA (2 mg). The reaction mixture was transferred to a 1.5-mL Eppendorf tube and shaken at 25 °C and 120 rpm for 24 h. Then, the pH of the mixture was adjusted to 4.5–5.5 using 10% H2SO4, and 2 U AP was supplemented. The dephosphorylation reaction was performed at 30 °C for another 24 h.
Molecular modeling
Models of the dimer structures of FucA complex and RhaD complex were generated as follows: the monomer structure of FucA is derived from the previous study (PDB code 4FUA). However, there is no available polymer structure of FucA by searching the PDB database. TM-align program [49] was used to search for FucA homologies; in the top 10 hits ranked by TM-score, 2OPI crystalized in polymers was used as template. In the case of RhaD, the X-ray structure (PDB code 1GT7) was directly used as the basis for dimer creation of RhaD. The coordinates of the donor DHAP and the acceptor DHA/L-GAL in constructed dimers were superimposed with those from the PDB codes 1OJR and 4FUA.
Based on the catalytic mechanism of class II Aldolase, a specific residue in the adjacent monomer (Tyr113′ in FucA, and Glu171′ in RhaD) plays a key role on the protonation of the carbonyl oxygen of ketone acceptors. Therefore, dimer models of enzyme–substrate complexes (dubbed as “FucA–DHAP–DHA”, “FucA–DHAP–GAL”, “RhaD–DHAP–DHA” and “RhaD–DHAP–GAL”) were built to reflect the catalytic mechanism.
During the simulations, a constant force of 10 kcal/mol between the nucleophile C-atom and the electrophile C-atom was constructed via the consideration of Van der Waals’ force. To estimate the stability of 100 ns trajectories of the four systems, root-mean-square deviation (RMSD) for all Cα atoms was analyzed and no significant structure difference was observed. Furthermore, root-mean-square fluctuation (RMSF) which could reflect the stability of individual residue of protein was also evaluated; most residues are stable except for the terminations between two monomers.
Molecular dynamics (MD) simulations
The initial structures used for MD simulation were obtained from modeling analysis. Each apo-protein was protonated at pH 7.5 using H++ webserver. The Amber ff14SB force field was employed for the protein in all the MD simulations [50]. Na+ ions were added to neutralize the system, and the TIP3P water model was used to solvate each system, ensuring a solvent layer of at least 10 Å from any point on the protein surface. Charges and parameters for ligands were generated with the Antechamber module using the AM1-BCC charge model along with the amber GAFF force field. The force field of zinc ion and its neighboring atoms (cutoff was set as 2.8 Å) were parameterized using ‘MCPB.py’ modeling, using a hybrid bonded/restrained nonbonded model. As a result, the three histidine residues (H92, H94 and H155 in FucA, and H141, H143 and H212 in RhaD) were attached to zinc ion by coordinate bonds, whereas the two oxygen atoms of donor were attached to zinc ion by applying harmonic restraint (100 kcal/mol). After proper parameterizations and setup, the resulting system’s geometries were minimized (5000 steps for steepest conjugate and 5000 steps for conjugate gradient) to remove poor contacts and relax the system. The systems were then annealed from 0 to 300 K (≈ 27 °C) to mimic experimental temperature under the constant amount of substance (N), volume (V) and temperature (T) (NVT ensemble) for 50 ps. Subsequently, the systems were maintained for 25 ps of density equilibration under constant amount of substance (N), pressure (P) and temperature (T) (NPT ensemble) at constant temperature of 300 K and pressure of 1.0 atm using Langevin-thermostat (ntt = 3) with collision frequency of 2 ps−1 and pressure relaxation time of 1 ps. The heating and density equilibrations were carried out with a weak restraint of 20 kcal mol−1 Å−2 performed on all the residues. The systems were further equilibrated for 250 ps to get well settled pressure and temperature for conformational and chemical analyses. After proper minimizations and equilibrations, a productive MD run of 100 ns was performed for each system. During all MD simulations, the covalent bonds containing hydrogen were constrained using SHAKE algorithm [51], with a MD time step of 2 fs. The trajectory file was written every 1000 steps. All the above MD simulations were performed with GPU version of Amber 16 package. The generated trajectories (interval = 200, a total of 500 frames for each case) were used for the relative binding energy evaluation.
In vitro cascade reaction
To synthesize dendroketose in vitro, the reaction mixture (2 mL) containing 50 mM TEA (pH 7.5), 40 mM FALD, 0.1 mM TPP, FLS (1.5 U, 16 mg) was initially carried out at 25 °C and 120 rpm for 16 h. Then, DhaK (0.5 U, 0.025 mg), PPK (0.5 U, 0.03 mg), RhaD (0.5 U, 2 mg), YqaB (0.9 U, 1.5 mg), polyphosphate (20 mM), ATP (0.5 mM), and MgSO4 (10 mM) were added into the reaction system, and it performed for another 24 h. Samples (100 μL) were captured very two hours, treated with 10% H2SO4, centrifuged (22,000 rpm, 20 min) and analyzed by HPLC.
Shake flask scale cultivation
For precultivation of recombinant strain, a single clone was grown in 5 mL of BHI medium. After incubation for approximately 15 h, cells were inoculated into a 500-mL shake flask containing 100 mL BHI medium and cultivated at 25 °C in a rotatory shaker at 220 rpm. When the cell OD600 reached 0.8, 1 mM IPTG was added to induce enzyme expression. Subsequently, the cells were harvested by centrifugation (8000×g, 10 min, 4 °C) and were suspended in CGXII medium [52]. Then, 50 mL cells were transferred into a 250-mL shake flask with an initial OD600 of approximately 30. When appropriate, 10 mg/L chloramphenicol and 25 mg/L kanamycin were added. The fermentation process was carried out at 30 °C and 200 rmp.
If glycerol and DHA were used as substrates, the concentration of glycerol and DHA was assigned to 220 mM and 110 mM, respectively. To produce dendroketose from glucose, 220 mM glucose was supplemented into the medium. For the fed-batch fermentation, 220 mM glucose was supplemented again into the medium after fermentation for 6 h. Samples were collected every 2 h and centrifuged at 14,000×g for 20 min. The resulting supernatants were analyzed by HPLC. The desired product was separated by a chromatographic column filled with Ca2+ ion exchange resin, identified by a refractive index detector and then collected by a fraction collector. The purified products were analyzed by NMR.
Analytical methods
Cell density was determined by measuring the optical density at 600 nm (OD600) with a UV–Vis spectrophotometer (TU-1901, Persee, Beijing, China). Cell dry weight (CDW, g/L) of E. coli was calculated from OD600 values using the experimentally determined correlation factor of 0.25 g cells (dry weight [DW])/liter for an OD600 of 1. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard. HPLC system (Agilent 1100 series, Hewlett-Packard) equipped with a refractive index detector and fitted with chromatographic column (Bio-Rad Aminex HPX-87H column or Waters Sugar-Pak I column) was used to qualitative and quantitative analysis of substrates and products.