Effect of product inhibition on the synthesis of PPA
L-pmAAD was cloned and overexpressed in E. coli BL21(DE3) to produce a recombinant strain M0. However, with 70 g/L L-Phe as substrate and 30 g/L whole-cell as biocatalyst, only 42.1 g/L PPA could be produced with a conversion rate of 60.4%. To confirm the product inhibition on L-pmAAD, different concentrations of PPA (0-200 mM) were added to the reaction system before conversion. The maximum reaction rate Vmax values were unchanged, while the Km values increased along with the PPA concentration, demonstrating a competitive inhibition mode of PPA on the cell bioconversion (Supplementary Table 2).
Directed evolution of L-pmAAD by site-saturation mutagenesis
To increase the conformational kinetics of the product binding site and promote the release of the product without perturbing the binding of substrate, the flexible loop regions around the product binding site of L-pmAAD were selected for site-saturation mutagenesis. A homology model of L-pmAAD was constructed based on the crystal structure of P. myxofaciens LAAD (93.7% identity). The product PPA and cofactor FAD were then docked into the active site of L-pmAAD. A total of eight loop regions were identified around the product-binding site (Supplementary Figure 1). Seventeen candidate amino acid residues containing five residues in loop 2 (V411/S412/T414/F415/E417), two residues in loop 3 (T436/V437), five residues in loop 4 (Y97/S98/S102/T105/S106), two residues in loop 5 (D144/E145), one residue in loop 7 (V312) and two residues in loop 8 (L336/L341) were selected for site-saturation mutagenesis.
Then, we substituted the aforementioned candidate residues in the wild-type enzyme (M0) with smaller alanine residues and evaluated the transformed ability of these mutants. As shown in Figure 1, two mutants of M1E145A and M1L341A exhibited 12.1% and 13.3% higher PPA production than the wide type. Therefore, E145A and L341A were combined to construct mutant M2E145A/L341A. The PPA concentration of M2E145A/L341A was 1.41-fold higher than the corresponding value for M0. Furthermore, the activities of M2E145A/L341A increased by 210% compared to that of M0 (Table 1).
Table 1
Effect of 70 g/L L-Phe on deamination reaction of mutants.
Mutant
|
M0
|
M1E145A
|
M1L341A
|
M2E145A/L341A
|
PPA concentration (g/L)
|
42.1
|
47.2
|
47.7
|
59.3
|
Conversion (%)
|
60.4
|
67.8
|
68.5
|
85.2
|
Activity (µmol/min/g)
|
18.5
|
21.2
|
25.1
|
38.9
|
The kinetic parameters and product inhibition constants of the parent M0 and its mutants were also determined (Table 2). The Km value of M1E145A, M1L341A, and M2E145A/L341A was 1.05-, 1.17-, and 1.25-fold higher than the corresponding values for M0, respectively. These mutations seemed to affect the substrate-binding site, resulting in a decrease in the substrate-binding affinity. However, the catalytic efficiency (Kcat/Km) of M2E145A/L341A was 1.62 mM−1·min−1, being 1.35-fold higher than that of M0, which was achieved through compensation by the increased Kcat.
Table 2
Kinetic parameters and the product inhibition constants of L-pmAAD and its mutants.
Enzyme
|
Km (mM)
|
kcat (min−1)
|
kcat/Km (mM−1·min−1)
|
KPI (mM)
|
M0
|
60.4 ± 4.02
|
72.2 ± 4.04
|
1.20
|
119.8 ± 5.91
|
M1E145A
|
63.8 ± 3.14
|
80.4 ± 5.12
|
1.26
|
289.0 ± 11.8
|
M1L341A
|
70.4 ± 2.88
|
94.8 ± 6.89
|
1.35
|
354.6 ± 17.8
|
M2 E145A/L341A
|
75.6 ± 3.98
|
122.4 ± 6.77
|
1.62
|
460.0 ± 13.6
|
Furthermore, the KPI values of M1E145A, M1L341A, and M2E145A/L341A were 1.41-, 1.91-, and 2.84-fold higher than M0, indicating that product inhibition was relieved. In contrast to conventional random mutagenesis (Hegazy et al. 2019; Wang et al. 2019) or rational mutations that focus on engineering the active site (Atreya et al. 2016), herein, we modified the flexible loop regions around the product-binding site to obtain an effective mutant M2E145A/L341A with a 3.84-fold decrease in product inhibition and a 1.35-fold higher catalytic efficiency than the wild-type enzyme.
Computational evaluation of the mutant M2 E145A/L341A
To obtain how the mutation significantly relieves the product inhibition, the relationship between the substituted residues and the surrounding residues was investigated using the AutoDock suite. Based on the structural analysis, the E145 carbonyl group formed a hydrogen bond with the NH2 group of D149 on loop 5 (Figure 2a), and the L341 carbonyl group made a hydrogen bond with the NH2 group of L343 on loop 8 (Figure 2c). The prolonged distance between E145 and D149 increased from 1.8 Å to 3.1 Å when the Glu residue at position 145 was replaced with Ala, and the hydrogen bond between them disappeared (Figure 2b). Similarly, when the Leu residue at position 341 was replaced with Ala, the prolonged distance between L341 and L343 increased from 2.8 Å to 3.4 Å, and then the hydrogen bonds disappeared (Figure 2d).
Furthermore, we determined the PPA binding energy of M0 and its mutants. As shown in Supplementary Table 3, the product binding energy of M2 E145A/L341A was 5.0, 2.5, and 1.7 kJ/mol higher than the corresponding values for M0, ME145A, and M1L341A, respectively, which was in accordance with the product inhibition relief. Consequently, the surrounding amino acids might improve the flexibility owing to the disappearance of hydrogen bonds, thereby enhancing product release. According to previous studies, higher root-mean-square fluctuation (RMSF) values suggested that these two motifs could undergo noticeable movements and were the most likely to influence protein conformation due to their high flexibility (Han et al. 2016; Yang et al. 2017). A remarkable increase in RMSF around region 1 and region 2 was observed from two substitutions of E145 and L341 as a result of modifying the key amino acid residues (Figure 2e), indicating that the two mutation residues to alanine would thus weaken the structural constraint of the region 1 and region 2, leading to a flexible conformation of L-pmAAD and consequently the opening of the product-binding site. A combination of mutation sites might over-regulate the conformational dynamics and result in low enzyme stability (Han et al. 2016). While the increase in beneficial mutation points was accompanied by an increase in PPA production capacity in this study, probably because single-point regulation had a mild effect on conformation and was within the maximum carrying limit of the conformation.
Synthesis of PPA on a 5-L Scale
To provide a better environment for conversion, the bioconversion parameters, including substrate/whole-cell catalyst ratio, bioconversion pH, as well as temperature, were optimized (Figure 3a-c). Under the optimal conditions (the ratio of substrate/whole-cell catalyst was 2.8:1 in pH 8.5 at a conversion temperature of 30°C), the titer, yield, and productivity of PPA could reach 81.2 g/L, 99.0%, and 5.1 g/L/h within 16 h in a 5-L bioreactor (Figure 3d), which was the highest titer reported so far (Coban et al. 2016; Hou et al. 2016b).