大家好，今天为大家带来的文章是2024年5月15日发表在Journal of Agricultural and Food Chemistry上的“Engineering a Carotenoid Cleavage Oxygenase for Coenzyme-Free Synthesis of Vanillin from Ferulic Acid”，通讯作者是陕西师范大学食品学院的孟永宏老师。
Ferulic acid is biosynthesized in one pot without the need for energy and cofactors, adding significant value to the lignin waste stream. However, naturally evolved carotenoid cleaving oxygenase (CCO) has catalytic oxygenation conditions in an extreme alkaline environment, which greatly limits the bioproduction of vanillin in the above pathways. In this paper, the CCO of Thermophilic Thermobacterium (TtCCO) was reasonably designed to have high catalytic activity under neutral pH conditions, and a one-pot synthesis of vanillin was constructed by using TtCCO and Bacillus dwarf ferulic acid decarboxylase. The TtCCO of K192N-V310G-A311T-R404N-D407FN556A mutation (TtCCOM3) was gradually obtained through substrate access channel engineering, catalytic pocket engineering and pocket charge engineering. The results of molecular dynamics simulation showed that TtCCOM3 had high catalytic activity under neutral pH, reduced the site blocking effect in the substrate channel, enhanced the affinity of the substrate in the catalytic pocket, and eliminated the alkaline charge in the pocket. Finally, the one-pot synthesis rate of vanillin in this study was up to 6.89±0.3 mM/h. Thus, the authors' research paves the way for a one-pot biosynthesis process that converts renewable lignin-associated aromatic hydrocarbons into valuable chemicals.
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- 1. DLkcat pre-screening and characterization of FDCs and CCOs
The authors used FDC (AJ278683.1) and CCO (WP013080875.1) as query sequences and selected eight surrogate enzymes from the National Center for Biotechnology Information (NCBIN) database for each of the two catalytic components in the CoQA-independent pathway (Table 1). A phylogenetic tree was constructed for cluster analysis to further elucidate the evolutionary relationships (Fig. 1b). The results showed that FDC was mainly derived from Aspergillus and Bacillus, and the CCO phylogenetic tree contained three classes: γ-Proteobacteria, Thermomicrobia and α-Proteobacteria. Subsequently, the authors used deep learning techniques to predict enzyme turnover number (DLkcat) for pre-screening proteins (Table 1). Among the candidate proteins, Aspergillus luchuensis FDC (AlFDC), Bacillus licheniformis FDC (BlFDC) and BpFDC had the highest kcat values for converting FA to 4-VP. Caulobacter segnis CCO (CsCCO), TtCCO and Serratia sp. CCO (SsCCO) has a high kcat value for converting 4-VP to vanillin. Therefore, the authors expressed these 6 enzymes for subsequent experiments.
Figure 1
Table 1
The authors first identified the enzymatic properties of BpFDC, AlFDC, and BlFDC. The conversion rates for BpFDC, AlFDC, and BlFDC were 89.7, 74.7, and 81.6%, respectively. The optimal catalytic temperatures/pH for BpFDC, AlFDC, and BlFDC are 30 °C/pH = 7, 20 °C/pH = 5, and 30 °C/pH = 5, respectively (Figure 1c, d). The enzyme kinetic parameters of the three FDCs were determined. BpFDC exhibited the highest catalytic efficiency (kcat/km), which was 4.24 and 3.84 times higher than that of AlFDC and BlFDC, respectively (Table 2). Therefore, BpFDC is considered to be the most suitable biocatalyst for the synthesis of vanillin in a CoA-independent pathway. Subsequently, the authors determined the enzymatic properties and kinetic parameters of CsCCO, TtCCO, and SsCCO. Their conversion rates were 23.2%, 48.1%, and 25.3%, respectively. The optimal catalytic pH and temperature for the three CCOs were pH 9 and 30 °C (Figure 1c,d). The catalytic efficiency value (kcat/km) of TtCCO is 19.11/min/mM, which is about 3−4 times that of CsCCO and SsCCO (Table 2). Therefore, TtCCO is also considered as a promising catalyst for the efficient synthesis of vanillin. The optimal catalytic temperature of BpFDC and TtCCO is 30°C, and the optimal catalytic pH is different. The strong alkaline catalytic conditions of TtCCO limit the one-pot green synthesis. In addition, the catalytic efficiency value (kcat/Km) of TtCCO (19.11/min/mM) was significantly lower than that of BpFDC (179.13/min/mM), which limited the production of vanillin.
Table 2
- 2. Substrate channel engineering of TtCCO
Remodeling the substrate channel of TtCCO can make it easier for 4-VP to enter the catalytic bag. Therefore, carotenoid oxygenase 1 (PDB ID: 7T8P) was used as a template to construct the TtCCO structure by AlphaFold2 and further docked with 4-VP. Using caver script33 (Figure 2), the authors predicted that 4-VP enters the active center of Fe2+ binding in the catalytic pocket through a narrow channel. The substrate entry channel of WT-TtCCO consists of 20 residues (N145, V147, F148, H190, P191, K192, I193, M200, R404, I405, D407, V410, E497, H554, G555, N556, W557, A189, V310 and S403). Noting that the Debye-Waller factor (b-factor) provides the basis for the mobility and flexibility of the internal atoms, residues, side chains, and ring regions of proteins, the authors used the docking results of TtCCO with 4-VP within 100 ns for molecular dynamics (MD) simulations. Residues with RMSF fluctuations of less than 0.05 nm were selected to enhance the flexibility of the substrate channels. Subsequently, residues with coarse side chains (N145, F148, S403, N556, and W557) mutate to residues with shorter side chains, such as alanine, valine, and glycine. The N145A, S403A, and N556A-TtCCO variants exhibit higher conversion rates under whole-cell catalysis. Further enzyme kinetic analysis showed that the mutant N556ATtCCO had the highest catalytic efficiency of 57.19/min/mM, which was 2.99 times higher than that of WT-TtCCO (Table 3). The catalytic efficiencies of the mutants N145A and S403A are 33.14 and 25.69/min/mM, respectively (Table 3). Subsequently, the authors combined the above-mentioned single-point mutation sites for iterative mutagenesis and analyzed the enzyme kinetics of their purified enzymes separately (Table 3). The relative enzyme activity of the second iteration of the TtCCO variant and the higher iteration of the TtCCO variant was not higher than that of the N556A-TtCCO variant. The catalytic efficiencies of the double mutants N145A-S403A, N145A-N556A, and S403A-N556A were 35.89, 33.16, and 29.93/min/mM, respectively (Table 3). At the same time, the catalytic efficiency of the triple mutant N145A-S403A-N556A is 35.24/min/mM (Table 3). Therefore, N556A-TtCCO (TtCCOM1) was chosen as the best template for subsequent enzyme engineering.
Figure 2
Table 3
3.Engineering design of TtCCO catalytic bag
The catalytic center of TtCCO is formed by the coordination of Fe2+ with H190, H244, H309 and H554. TtCCO utilizes Fe2+4 × His catalytic center and oxygen molecules to facilitate the cleavage of olefin double bonds to form aldehydes. Oxygen molecules attach to non-heme ferrous centers to form superoxide domains. The Fe2+-bound oxygen atom is close to the alkenyl radical and forms two C−O bonds with the substrate. Subsequently, the electron rearrangement in the Fe2+-bound oxygenated intermediate facilitates the cleavage of the C-C bond to produce vanillin and small molecule formaldehyde. The residues in the catalytic pocket act as a conformational regulation, introduce oxygen molecules, and provide electrons to facilitate the catalytic reaction. MD simulations (100 ns) of the TtCCOM1 and 4-VP complexes showed that D407, V310, and A311 helped to interact with the vinyl group of the 4-VP close to the Fe2+ center (Figure 3). D407, V310 and A311 were used to construct a single-point mutation library by site saturation mutagenesis. D407F-TtCCOM1, V310G-TtCCOM1, and A311T-TtCCOM1 had higher conversion rates of 81.4, 82.2, and 80.1%, respectively (Table 3). The enzyme kinetic parameters of single-point mutants were further determined. The catalytic efficiencies of D407F-TtCCOM1, V310G-TtCCOM1, and A311T-TtCCOM1 were 70.94, 72.08, and 64.45/min/mM, respectively (Table 3). Subsequently, iterative mutagenesis is performed to generate triple and quadruple mutants. Finally, D407F/A311T/V310GTtCCOM1 (TtCCOM2) was obtained, and the catalytic efficiency was improved. The catalytic efficiency of TtCCOM2 is 90.36/min/mM, which is 4.73 times higher than that of WT-TtCCO (19.11/min/mM).
Figure 3
- 4. Efficient catalysis of the pocket charge of the engineered TtCCO under neutral conditions
Changing the charge within the catalytic pocket of the enzyme can adjust the catalytic pH of the enzyme, therefore, the authors tried to achieve green catalysis by changing the internal charge environment of the catalytic groove. To clearly describe the surface charge gradient of the enzyme bag, the authors plotted a potential map depicting the surface charge of TtCCOM2. The key residues Asp192 and Arg404 in 6Å were identified as potential keys for TtCCOM2 catalytic pH (Figure 4a). The conversion rates of the single-point mutants K192N and R404N of TtCCOM2 reached 86.6 and 87.3%, respectively (Table 3). Their optimal pH has been expanded to the neutral range, and the consumption rate of 4-VP has remained largely unchanged. The authors used an iterative approach to find neutral and effective mutants and identified the mutant K192N/R404N-TtCCOM2 (TtCCOM3). The catalytic efficiency of TtCCOM3 is 6.71 times higher than that of the wild type. The catalyst retains considerable activity in the pH range of 7 ~ 9 (Figure 4b).
Figure 4
5. Molecular simulation of 4-VP oxidation catalyzed by TtCCO mutants
The authors performed structural analysis and computational analysis of the mechanism of TtCCOM3 activity enhancement through molecular docking and MD simulation. First, compared with WTTtCCO, the N556A-mutated TtCCOM1 obtained by substrate access channel engineering, was modified from an s-shaped to an l-shape, mainly caused by N556A (Fig. 5a, b).
Figure 5
The authors further analyzed the average substrate throughput for TtCCOM1 (0.55) and WT-TtCCO (0.39). The substitution of the 556-bit residue accelerates the substrate transfer efficiency. The authors also used MD simulations to analyze the b-factor values for each residue at 100 ns in WT-TtCCO and TtCCOM1. The root mean square fluctuation (b-factor) values in the region around N556A are relatively high, showing greater flexibility than WTTtCCO. The increased flexibility promotes the movement of 4-VP radicals in the substrate pathway, thereby increasing the rate at which 4-VP binds to the active center of TtCOCO.
The modification of the TtCCO substrate pocket involves spatial structure alteration and charge modification, which also brings new intermolecular forces to 4-VP. Therefore, the authors used molecular docking and MD simulations to analyze TtCCOM3 (K192N−V310G-A331T-R404N-D407F-TtCCOM1) and substrate complexes. First, the angle of C8-C9-Fe2+ and the distance from C9 to Fe2+ in the protein-substrate complex were measured within 100 ns (Figure 5c). The angle between C8 and C9 and Fe2+ is 60°, and the distance from wild type C9 to Fe2 is 5±0.5 Å. In the TtCCOM3 mutant, the vinyl oxide group of 4-VP is vertically exposed to the Fe2+ catalytic center, and the distance between the vinyl group of 4-VP and the Fe2+ catalytic center is 3.8±0.2 Å. The authors also measured the energy decomposition of residues before and after mutations. The new interaction energies of N192, T311, and N404 with 4-VP were 15.8, 34.9, and 8.9 kJ/mol, respectively (Fig. 5d). F407 forms a strong repulsive force of 24.3 kJ/mol with 4-VP, compressing the conformation of 4-VP in the catalytic bag closer to the Fe2+ catalytic center (from 5±0.5 to 3.8±0.2 Å) (Fig. 5c,d). A distinct conformational change in binding to 4-VP was found in the mutant (Figure 5e,f). The mean distances at loci 556 and 310 for TtCCOM3 and 4-VP were 4.9 and 6.6 Å, respectively, and for wild types were 3.6 and 2.2 Å, respectively. The increased average distance can reduce spatial obstacles. The increased polarity of TtCCOM3 in D407F/V310G/A311T promotes electron transfer between 4-VP and Fe2+ catalytic centers. H190 forms a π-cationic bond with the center of the benzene ring of 4-VP and interacts with the styrene group of 4-VP π-alkyl group. H309 and H554 produce π-alkyl hydrophobic interactions with the styrene group of 4-VP (Figure 5g, h). The enhanced hydrophobic interaction with the styrene group promoted the transfer of electrons to olefins, promoted the recognition and anchoring of TtCCOM3 4-VP, and improved the catalytic activity of TtCCO.
N145 and D245 of TtCCOM3 form new hydrogen bonds with 4-OH and are conjugated with a central double bond (Figure 6), and the polar residue D245 attacks the olefin opposite the hydroxyl group of 4-VP. The iron(III) superoxide radical initiates a nucleophilic attack, inserting an oxygen atom into a carbon atom. The activation of the 4-OH group by D245 and N145 results in the migration of the electron cloud towards the central double bond and the Fe(III) superoxide region. Hydrogen bonding and other intermolecular forces enhance the stability of intermediate radicals and promote the formation of C−O bonds and the cleavage of O−O bonds.
Figure 6
6. One-pot biosynthesis of vanillin driven by a coenzyme-dependent pathway under neutral conditions
The authors combined BpFDC with TtCCOM3 to obtain a neutral and efficient catalyst. Subsequently, pETDuet-BpFDC-TtCCO and pETDuet-BpFDC-TtCCOM3 were constructed and expressed in Escherichia coli BL21 (DE3) to produce vanillin in one pot. Fermentation at 30 °C and pH 7 for 12 h to measure vanillin yield (Figure 7a, b). The results showed that the molar conversion of the pETDuet-BpFDC-TtCCOM3 group was 98%. The maximum yield of the novel E. coli catalytic system was 6.89±0.3 mM/h, which was about 6.15 times that of the pETDuet-BpFDC-TtCCO group. As a result, enzyme engineering enables more efficient vanillin synthesis.
Figure 7