- Email: [email protected]
Expanding the substrate scope of phenylacetone monooxygenase from Thermobifida fusca towards cyclohexanone by protein engineering
Accepted Manuscript Expanding the substrate scope of phenylacetone monooxygenase from Thermobifida fusca towards cyclohexanone by protein engineering
Guang Yang, Ran Cang, Li-Qun Shen, Feng Xue, He Huang, ZhiGang Zhang PII: DOI: Reference:
S1566-7367(18)30478-3 https://doi.org/10.1016/j.catcom.2018.10.022 CATCOM 5533
To appear in:
Catalysis Communications
Received date: Revised date: Accepted date:
14 September 2018 12 October 2018 20 October 2018
Please cite this article as: Guang Yang, Ran Cang, Li-Qun Shen, Feng Xue, He Huang, Zhi-Gang Zhang , Expanding the substrate scope of phenylacetone monooxygenase from Thermobifida fusca towards cyclohexanone by protein engineering. Catcom (2018), https://doi.org/10.1016/j.catcom.2018.10.022
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ACCEPTED MANUSCRIPT Expanding the Substrate Scope of Phenylacetone Monooxygenase from Thermobifida fusca towards Cyclohexanone by Protein Engineering
Guang Yang a, Ran Cang a, Li-Qun Shen a, Feng Xue b, He Huang a, Zhi-Gang Zhang a* a. School of pharmaceutical sciences, Nanjing Tech University, Nanjing 211800, P. R. China;
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b. School of Marine and Bioengineering, Yancheng Institute of Technology, Yancheng 224051, P. R. China;
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*Email: [email protected]
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Abstract
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Baeyer-Villiger monooxygenases (BVMOs) convert ketones into lactones that have important applications in polymer synthesis. Here we report on expanding the substrate scope of a
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thermostable phenylacetone monooxygenase (PAMO) to cyclohexanone by using site-directed mutagenesis. Several new mutants were found to be active with cyclohexanone for which wild-type PAMO shows no activity. The best mutants could convert 10 mM cyclohexanone
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completely within 12 h, and their catalytic properties were characterized subsequently. In addition
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to cyclohexanone, several other cyclic ketones were also identified as substrate for the new evolved mutants. These results expand the biocatalytic toolbox for further feasible applications.
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Key words: Baeyer-Villiger monooxygenase, Phenylacetone Monooxygenase, Cyclohexanone,
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ε-caprolactone, Biocatalysis, Site-directed Mutagenesis
1. Introduction Baeyer-Villiger Monooxygenases (BVMOs) represent a group of valuable monooxygenases capable of catalyzing Baeyer-Villiger (BV) oxidation of ketones to form lactones or esters that can be used as important building blocks in organic and polymer synthesis [1, 2]. Compared to chemical catalysts, BVMOs generally show high stereoselectivity and activity under mild reaction conditions. So far, only a limited number of BVMOs are available for practical applications [3-6].
ACCEPTED MANUSCRIPT Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIB 9871 as an excellent representative has been extensively studied and found widespread applications [7-9], including the conversion of cyclohexanone to ε-caprolactone (Scheme 1). Caprolactone is an important monomer for the synthesis of biodegradable thermoplastic polylcaprolactone [10], and is a precursor for caprolactam, an important building block which can be further transformed to
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polyamides [11].
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Scheme 1. PAMO BVMO catalyzed biocatalytic oxidation of cyclohexanone to caprolactone
However, the limited stability of CHMO as a long-standing problem has hampered widespread
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applications. Therefore, protein engineering has been employed to improve its stability [12-15]. For example, by applying a rational design strategy, the thermostability and oxidative stability of
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CHMO was improved by Opperman and Reetz [16]. Thereafter, attempts were made to enhance
progress is still limited.
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the thermostability of CHMO by introducing intramolecular disulfide bonds [14, 17]. However,
In order to find stable BVMOs for conversion of cyclohexanone to ε-caprolactone, a different
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strategy was employed in this study, namely, introducing CHMO activity to a relative thermostable BVMO by protein engineering. Thus, we turned to the thermostable Phenylacetone
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Monooxygenase (PAMO) from Thermobifida fusca [18]. It exhibits excellent thermostability and high tolerance to various solvents [19-21]. However, the substrate acceptance of PAMO is rather limited, being active only to small aromatic ketones and no activity to cyclohexanone [22-24]. Several attempts to induce PAMO to accept cyclohexanone by protein engineering methods have been made [12, 25-27]. For instance, by using a homology model of CHMO, a key bulge in the loop near the active site of PAMO was identified. It was shown that eliminating some amino acids in this loop leads to expanded substrate acceptance of the enzyme with essentially no trade off in thermostability [27]. However, cyclohexanone was still not accepted by these PAMO mutants. Based on the thermostable PAMO scaffold, a chimeric PAMO-CHMO enzyme mutant was created
ACCEPTED MANUSCRIPT and its activity against cyclohexanone was tested [28]. Unfortunately, no cyclohexanone activity was detected. Recently, directed evolution was applied to evolve PAMO mutants with activity to cyclohexanone, and indeed, active mutants were obtained following library screening [25, 26]. However, notable screening efforts were required, and resulted mutants in that study led to 100% conversion of 2 mM cyclohexanone within 3 h. While
For a practical industrial process, the
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concentration of accepted substrate by PAMO mutants remained still limited. and For a practical industrial process, further enzyme engineering is needed to obtain a truly active and robust
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biocatalyst.
In this study, site-directed mutagenesis was applied to evolve PAMO mutants active towards
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cyclohexanone for improved bioconversion. The catalytic properties of the new mutants for conversion of cyclohexanone to ε-caprolactone were characterized. In addition, the substrate scope
2. Materials and Methods
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2.1 General Procedures
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of PAMO mutants was investigated by using several other cyclic ketones as substrates.
cyclopentanone,
cycloheptanone,
4-methylcyclohexanone,
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Cyclohexanone,
2-methylcyclohexanone and L-Arabinose were purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). DpnI restriction enzyme, Site-Directed mutagenesis Kit and Fast
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Site-Directed Mutagenesis kit were obtained from TIANGEN biotech (Beijing, China). Primers was synthesized and purified by PAGE from GENEWIZ, Inc. (Nanjing, China). AxyPrepTM
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Plasmid Miniprep Kit was obtained from Axygen Biosciences (America). NADPH was purchased from Meryer (Shanghai) Chemical Technology Co., Ltd. (Shanghai, China). Wild-type and mutants PAMO were purified with CapturemTM His-Tagged Purification Maxiprep Kit from Takara (China). TB medium and LB medium were prepared following standard protocols.
2.2 Preparation of PAMO Mutants by Site-Directed Mutagenesis The PAMO mutants were prepared by using Fast Site-Directed Mutagenesis kit from Tiangen Biotech (Beijing, China) following the provided manual. The primers used are summarized (Supporting Information, Table S1). The mutation of PAMO mutants was verified by DNA
ACCEPTED MANUSCRIPT sequencing service in BioSune biotechnology company (Shanghai, China).
2.3 Whole Resting cells-catalyzed Biotransformation of Substrates Wild-type or mutant PAMO glycerol stock was inoculated into fresh LB media containing 100 µg/mL carbenicillin for cultivation under 37 ℃, 200 rpm. After overnight growing, 1 mL of culture was transferred into 100 mL of TB media supplemented with 100 µg/mL carbenicillin for
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further cultivation at 37 ℃, 200 rpm. When the OD600 reached 0.6-0.8, 0.1 g/100 mL of Arabinose
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was added as inducer for enzyme expression. After 20 h, resting cells were prepared as described before, briefly, cell pellets were harvested by centrifugation and washed two times with 50 mM,
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pH 8.0, phosphate sodium buffer.
Four hundred milligrams of prepared resting cell pellets were resuspended in 2 mL of 50 mM,
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pH8.0, phosphate sodium buffer containing specific amount of cyclohexanone as substrate in a 50 mL Falcon tube. The reaction mixture was shaken at 1000 rpm and 42 ℃ for given interval times.
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The product was extracted by adding ethyl acetate into the reaction mixture. The conversion of cyclohexanone was determined by GC equipped with HP-5MS column (30 m×0.32 mm×0.25 µm).
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Other cyclic ketones with similar structure were used as substrates in Baeyer-Villiger oxidation
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reactions under the same reaction conditions.
2.4 Thermostability of PAMO mutants
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The ThermoFAD method was employed to examine the thermostability of PAMO mutants[29]. Experiments were performed with a StepOnePlus™ Real-Time PCR System. Unfolding curves
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were generated using a temperature gradient from 20 to 90 ℃, performing a fluorescence measurement after every 0.5 ℃ increase after a 10s delay for signal stabilization. The Tm values were determined by the peaks of the derivatives of the fluorescence intensity. In the experiments, 20 µL samples of wild-type PAMO and mutants in 50 mM Tris-HCl buffer (pH 8.0) were used.
2.5 Steady-state kinetic analysis The activity of evolved PAMO mutants was measured by spectrophotometrically monitoring the decrease of NADPH in the reaction mixture at 25 ℃ and 340 nm. The steady-state kinetic parameters were measured using purified enzyme. The 200 µL reaction system contained 0.5-0.6
ACCEPTED MANUSCRIPT µM enzyme, 100 µM NADPH, 1 % ( v/v) acetonitrile and 50 mM Tris-HCl (pH 8.0). The data was measured after every 15s in 3 minutes. The experiments were performed with a SpectraMax M3.
3. Results and Discussion 3.1 Initial screening result for PAMO activity to cyclohexanone
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To engineer PAMO mutants with high activity for the BV oxidation of cyclohexanone, several mutants previously evolved for asymmetric sulfoxidation of thioethers were tested (Supporting
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Information, Table S2) [30]. Considering NADPH regeneration for roughly assessing activity,
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whole E. coli resting cells were applied as before [25, 31]. In order to improve the conversion and thus activity, some reaction parameters were modified. As PAMO exhibits high thermostability
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and activity at 50 ℃, cyclohexanone oxidation was performed at 42 ℃ with an agitation speed of 1000 rpm and 0.2 g/mL of wet resting cell as catalyst (Supporting Information, Table S3) [23].
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The preliminary results suggested that mutants evolved for sulfoxidation also show considerable activity to cyclohexanone under the given reaction conditions. In addition, several single mutants
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(P440F, P440W) showed high activity to cyclohexanone as well. However, it seems like that
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adding 20 mM of glucose did not improve the conversion (Supporting Information, Table S4). In a recent paper Parra et al. mentioned that the P440F mutant was found to be inactive on cyclohexanone [25]. The possible reason for this disagreement is that higher reaction temperature
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and catalyst dosage were used in this study. Meanwhile,this This result further verified that
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position P440 is indeed a “hot spot” (Table 1Fig. 1) [26, 32, 33].
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Fig. 1. Previously evolved PAMO mutants tested for activity in cyclohexanone oxidation.
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Table 1. Previously evolved PAMO mutants tested for activity in cyclohexanone oxidation. PAMO mutants
Conv. (%) a
WT PAMO
0
I67C/P440F/A442F/L443D
14.5
I67C/P440Y
24.3
4
I67Y/P440Y
56.3
5
P440F
48.3
6
P440W
33.5
7
P440H
6.2
8
P440Y
4.4
9
P440I
2.4
Entry
2
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a. Reaction condition: 0.2 g/mL wet cell weight, 2 mM cyclohexanone, 1 % (v/v) acetonitrile, 50 mM phosphate sodium buffer (pH 8.0), shaking at 1000 rpm under 42 ℃ for 4 h. The product was determined by GC.
By careful analysis we found that single mutant P440Y gave very low conversion in cyclohexanone oxidation, however, double mutant I67C/P440Y and I67Y/P440Y showed
ACCEPTED MANUSCRIPT significantly increased conversion. This result implies that position I67 may also play an important role in cyclohexanone acceptance of PAMO. In addition, previous studies suggested that the position I67 could be part of the active site of PAMO [32]. Based on the information mentioned above, and using P440F as starting point, position I67 was targeted for further focused
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mutagenesis (Fig. 2).
Fig. 2. Potential radomization amino acid sites in PAMO.The flavin and NADP cofactors are shown in yellow and
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orange. Based on the crystal structure of PAMO (PDB: 2YXL).
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3.2 Randomization of position I67 using P440F as template Using mutant P440F as a template, mutants at position P67 I67 were produced by site-directed mutagenesis, thereby replacing isoleucine proline by the 19 other canonical amino acids. Rather
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than applying traditional NNK-based saturation mutagenesis, we chose this strategy because it requires no screening. Interestingly, most of resulting mutants exhibited improved activity against
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cyclohexanone (Supporting Information, Table S5 4), implying that position I67 is indeed a “hot spot”. Of the obtained mutants, the best one proved to be I67Y/P440F which could convert substrate with 97.8% conversion after 4 h. The other three selected double mutants also gave high activities and good conversions from 87% to 95% (Table 1 2).
Table 1 2. Obtained new mutants for cyclohexanone oxidation Entry a
PAMO mutant
Conv. (%)
1
WT PAMO
0
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50.2
3
I67Y/P440F
97.8
4
I67A/P440F
95.1
5
I67C/P440F
92.4
6
I67G/P440F
87.5
7
I67Y
0
8
I67A
0
9
I67C
0
10
I67G
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2
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0
a. Reaction condition and measurement conditions: as in Fig. 1Table 1.
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With the aim of investigating the interaction of mutation at position I67 and P440 in the obtained double mutants, single mutants were also prepared by site-directed mutagenesis and tested as
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catalysts in cyclohexanone oxidation. Surprisingly, four single-mutation variants at position I67 did not show any activity on cyclohexanone even with prolonged reaction time. In contrast, when combining single mutations at position 67 with P440F to form the respective double mutants,
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higher activity was detected. This result implies that there is possible additive or cooperative effect
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existing between I67 and P440, the phenomenon being reported before [34]. The same applies when deconvoluting the double mutant I67Y/P440F. All results are summarized in Table 1 2.
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3.3 High substrate concentration Catalytic performance of PAMO mutants The catalytic property of evolved mutants in cyclohexanone oxidation was tested with increased
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substrate concentration. As shown in Table 2 3, all the mutants convert cyclohexanone to carprolactone within 8 h at the substrate concentrate of 5 mM. In order to enhance the process efficiency, the substrate concentration was increased up to 10 mM. Remarkably, more than 95% of conversion was achieved by all four double mutants upon prolonging the reaction time to 12 h. The results show that new evolved PAMO mutants are able to convert cyclohexanone at considerable high concentration, indicating their potential applications in organic and polymers synthesis.
Table 2 3. Catalytic activity of PAMO mutants with increased substrate concentration.
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Entry a
PAMO mutant
1
WT PAMO
2
P440F
Conv. (%) b
Concentration(mM) 5
0
5
63.6
10
44.4
5
>99
10
>99
I67Y/P440F
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Substrate
5 4
>99
I67A/P440F
>99
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10
I67C/P440F 10 5 I67G/P440F
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6
>99
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5 5
10
>99 >99 95.1
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a. Reaction condition: 0.2 g/mL wet cell weight, 1 % (v/v) acetonitrile, 50 mM phosphate sodium buffer (pH 8.0), shaking at 1000 rpm under 42 ℃, 8 h for 5 mM and12 h for 10 Mm cyclohexanone. The product was determined
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by GC.
3.4 Steady-state kinetic parameters of evolved PAMO mutants The steady-state kinetic parameters for starting mutant and new evolved mutants were determined
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in the production of lactone from cyclohexanone by using purified mutant enzymes (Supporting Information, Fig. S1). As shown in Table 3 4 , a double mutant I67Y/P440F has a similar kcat value
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as variant P440F, while the other three double mutants have 2-3 times higher kcat values than that of P440F. The increased kcat values indicate higher specific enzymatic activities than starting mutant P440F. The Km values of the four double mutants are 2-4 times higher than that of P440F. However, the kcat /Km value of I67Y/P440F I67/P440F is 3 times lower than that of P440F P400F; the other three double mutants have similar kcat /Km values as the mutant P440F, implying that the catalytic efficiency of the evolved double mutants is of the same order of magnitude as that of starting mutant P440F. In addition, the kcat /Km value of our best mutant in this study is comparable to that of PAMO mutants reported by Parra et al., but still much lower than that of CHMO [14, 25].
ACCEPTED MANUSCRIPT Since WT PAMO does not accept cyclohexanone, the actual improvements starting from the natural form are considerable enormous.
Table 3 4 . Steady-state kinetic parameters of evolved PAMO mutants PAMO mutants
Km (mM)
kcat (s-1)
kcat /Km (s-1M-1)
1
P440F
0.1037
0.0653
633
2
I67Y/P440F
0.2107
0.0456
3
I67A/P440F
0.3472
0.1364
4
I67C/P440F
0.4412
0.198
5
I67G/P440F
0.2595
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Entry
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393
510
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0.1324
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Reaction condition: The 200 µL reaction mixture contained 0.5-0.6 µM enzyme, 100 µM NADPH, 1% (v/v)
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acetonitrile and 50 mM Tris-HCl (pH 8.0).
3.5 Thermostability of PAMO mutants
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Thermostability of WT PAMO and the variants was examined by measuring the Tm values with
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the ThermoFAD method following the protocol as described previously [29]. The results shows that the Tm value for WT PAMO is 58 ℃; Tm values for P440F and the four best mutants are 55.5 ℃
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(P440F), 53 ℃ (I67C/P440F), 51 ℃ (I67A/P440F), 51 ℃ (I67Y/P440F) and 51 ℃ (I67G/P440F)
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respectively, indicating that the introduced mutations decreased thermostability slightly (Fig. S2).
Figure 2. Thermostability of WT PAMO and evolved mutants as measured by the ThermoFAD protocol.
3.6 Substrate scope of evolved PAMO mutants The substrate scopes of the evolved mutants were investigated by using several other cyclic ketones for which WT PAMO shows no activity. Interestingly, it was found that all tested ketones are accepted by the mutants with moderate to high conversion, except for cycloheptanone which has less than 10% conversion (Table 4 5). Cyclopentanone and 2-methylcyclohexanone were transformed to the corresponding lactones by four double mutants with conversion of >95%,
ACCEPTED MANUSCRIPT which is better than that catalyzed by single mutant P440F. However, 4-methylcyclohexanone was converted to lactone with maximal conversion of 40% in which less than 5% of 4-methylcyclohexanol formed (data not shown). The results indicate that the substrate scope of our new mutants was expanded successfully.
Conv. (%) a Substrate WT
P440F
I67Y/P440F
I67A/P440F
0
57.5
97.8
96.7
0
88.4
91.0
0
3.9
O
2
45.1
96.7
96.5
92.7
11.4
40.3
36.8
2.4
9.0
3.2
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3
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97.3
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I67G/P440F
98.1
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1
I67C/P440F
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Entry
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Table 4 5. Additional cyclic ketones used as substrates in PAMO-catalyzed BV oxidation
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O
0
0.44
9.0
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a. Reaction condition: 0.2 g/mL wet cell weight, 2 mM cyclohexanone, 1 % (v/v) acetonitrile, 50 mM phosphate
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sodium buffer (pH 8.0), shaking at 1000rpm under 42 ℃ for 4 h. The product was determined by GC.
4. Conclusions
In summary, protein engineering to expand the thermostable PAMO for acceptance of cyclohexanone with high activity to form ε-caprolactone from cyclohexanone was achieved by applying a protein engineering strategy based on iterative site-specific mutagenesis. An alternative approach based on iterative saturation mutagenesis (ISM) at randomization sits sites lining the binding pocket (CASTing) is likely to provide even better variants, but this requires the screening of medium-sized or small libraries [35]. In our approach, several mutants with excellent activity to
ACCEPTED MANUSCRIPT cyclohexanone were obtained and the catalytic properties were characterized. The best mutant I67Y/P440F converts cyclohexanone with a conversion of 97.8% within 4 h; 10 mM with >99% conversion under prolonged reaction time to 12 h. However, it was found that the introduced mutations lead to a slight decrease of enzyme thermostability. In addition, evolved PAMO mutants also show high activity to structurally different cyclic ketones. Our results provide new and
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practical biocatalysts for organic synthesis and biotechnologies.
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Conflicts of interest
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The authors declare no conflict of interest.
Acknowledgements
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This work was financially supported by the National Science Foundation of China (21776134, 21646014, and 21606192), the program of Jiangsu Synergetic Innovation Center for Advanced
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Bio-Manufacture (XTE1851).
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Supplementary data
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Appendix A. Supplementary data
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oxidation of racemic ketones in aqueous–organic media catalyzed by phenylacetone monooxygenase, Tetrahedron: Asymmetry 19 (2008) 197-203. [21] G. de Gonzalo, C. Rodríguez, A. Rioz-Martínez, V. Gotor, Improvement of the biocatalytic properties of one phenylacetone monooxygenase mutant in hydrophilic organic solvents, Enzyme Microb. Technol. 50 (2012) 43-49. [22] G.d. Gonzalo, D.E.T. Pazmiño, G. Ottolina, M.W. Fraaije, G. Carrea, Oxidations catalyzed by phenylacetone monooxygenase from Thermobifida fusca, Tetrahedron: Asymmetry 16 (2005) 3077-3083. [23] C. Rodríguez, G. de Gonzalo, M.W. Fraaije, V. Gotor, Enzymatic kinetic resolution of racemic ketones catalyzed by Baeyer–Villiger monooxygenases, Tetrahedron: Asymmetry 18 (2007) 1338-1344. [24] A.T.P. Carvalho, D.F.A.R. Dourado, T. Skvortsov, M. de Abreu, L.J. Ferguson, D.J. Quinn, T.S. Moody, M. Huang, Spatial requirement for PAMO for transformation of non-native linear substrates, Phys. Chem. Chem. Phys. 20 (2018) 2558-2570.
ACCEPTED MANUSCRIPT [25] L.P. Parra, J.P. Acevedo, M.T. Reetz, Directed evolution of phenylacetone monooxygenase as an active catalyst for the baeyer–villiger conversion of cyclohexanone to caprolactone, Biotechnol. Bioeng. 112 (2015) 1354-1364. [26] H.M. Dudek, M.J. Fink, A.V. Shivange, A. Dennig, M.D. Mihovilovic, U. Schwaneberg, M.W. Fraaije, Extending the substrate scope of a Baeyer–Villiger monooxygenase by multiple-site mutagenesis, Appl. Microbiol. Biotechnol. 98 (2014) 4009-4020. [27] D.E.T. Pazmiño, R. Snajdrova, D.V. Rial, M.D. Mihovilovic, M.W. Fraaije, Altering the Substrate Specificity and Enantioselectivity of Phenylacetone Monooxygenase by Structure-Inspired Enzyme Redesign, Adv. Synth. Catal. 349 (2007) 1361-1368.
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[28] H.L. van Beek, G.d. Gonzalo, M.W. Fraaije, Blending Baeyer–Villiger monooxygenases: using a robust BVMO as a scaffold for creating chimeric enzymes with novel catalytic properties, Chem. Commun. 48
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(2012) 3288-3290.
[29] F. Forneris, R. Orru, D. Bonivento, L.R. Chiarelli, A. Mattevi, ThermoFAD, a Thermofluor®-adapted flavin
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ad hoc detection system for protein folding and ligand binding, FEBS J. 276 (2009) 2833-2840. [30] Z.-G. Zhang, R. Lonsdale, J. Sanchis, M.T. Reetz, Extreme Synergistic Mutational Effects in the Directed Evolution of a Baeyer–Villiger Monooxygenase as Catalyst for Asymmetric Sulfoxidation, J. Am. Chem. Soc.
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136 (2014) 17262-17272.
[31] J. Wachtmeister, D. Rother, Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale, Curr. Opin. Biotechnol. 42 (2016) 169-177.
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[32] H.M. Dudek, G. de Gonzalo, D.E. Torres Pazmiño, P. Stępniak, L.S. Wyrwicz, L. Rychlewski, M.W. Fraaije, Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis, Appl. Environ. Microbiol. 77 (2011) 5730-5738. [33] E. Malito, A. Alfieri, M.W. Fraaije, A. Mattevi, Crystal structure of a Baeyer–Villiger monooxygenase, Proc.
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Natl. Acad. Sci. U. S. A. 101 (2004) 13157-13162. [34] M.T. Reetz, The Importance of Additive and Non-Additive Mutational Effects in Protein Engineering,
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Angew. Chem. Int. Ed. 52 (2013) 2658-2666. [35] M.T. Reetz, J.D. Carballeira, Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional
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enzymes, Nat. Protoc. 2 (2007) 891-903.
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Graphical Abstract:
ACCEPTED MANUSCRIPT Highlights: Hihg active PAMO mutants towards cyclohexanone was obtaind by protein engineering.
10 mM cyclohexanone was converted by mutants with 99% yield after 12h.
Mutants showed broadened substrate scope towards other cyclic ketones as well.
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Guang Yang, Ran Cang, Li-Qun Shen, Feng Xue, He Huang, ZhiGang Zhang PII: DOI: Reference:
S1566-7367(18)30478-3 https://doi.org/10.1016/j.catcom.2018.10.022 CATCOM 5533
To appear in:
Catalysis Communications
Received date: Revised date: Accepted date:
14 September 2018 12 October 2018 20 October 2018
Please cite this article as: Guang Yang, Ran Cang, Li-Qun Shen, Feng Xue, He Huang, Zhi-Gang Zhang , Expanding the substrate scope of phenylacetone monooxygenase from Thermobifida fusca towards cyclohexanone by protein engineering. Catcom (2018), https://doi.org/10.1016/j.catcom.2018.10.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Expanding the Substrate Scope of Phenylacetone Monooxygenase from Thermobifida fusca towards Cyclohexanone by Protein Engineering
Guang Yang a, Ran Cang a, Li-Qun Shen a, Feng Xue b, He Huang a, Zhi-Gang Zhang a* a. School of pharmaceutical sciences, Nanjing Tech University, Nanjing 211800, P. R. China;
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b. School of Marine and Bioengineering, Yancheng Institute of Technology, Yancheng 224051, P. R. China;
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*Email: [email protected]
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Abstract
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Baeyer-Villiger monooxygenases (BVMOs) convert ketones into lactones that have important applications in polymer synthesis. Here we report on expanding the substrate scope of a
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thermostable phenylacetone monooxygenase (PAMO) to cyclohexanone by using site-directed mutagenesis. Several new mutants were found to be active with cyclohexanone for which wild-type PAMO shows no activity. The best mutants could convert 10 mM cyclohexanone
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completely within 12 h, and their catalytic properties were characterized subsequently. In addition
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to cyclohexanone, several other cyclic ketones were also identified as substrate for the new evolved mutants. These results expand the biocatalytic toolbox for further feasible applications.
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Key words: Baeyer-Villiger monooxygenase, Phenylacetone Monooxygenase, Cyclohexanone,
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ε-caprolactone, Biocatalysis, Site-directed Mutagenesis
1. Introduction Baeyer-Villiger Monooxygenases (BVMOs) represent a group of valuable monooxygenases capable of catalyzing Baeyer-Villiger (BV) oxidation of ketones to form lactones or esters that can be used as important building blocks in organic and polymer synthesis [1, 2]. Compared to chemical catalysts, BVMOs generally show high stereoselectivity and activity under mild reaction conditions. So far, only a limited number of BVMOs are available for practical applications [3-6].
ACCEPTED MANUSCRIPT Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIB 9871 as an excellent representative has been extensively studied and found widespread applications [7-9], including the conversion of cyclohexanone to ε-caprolactone (Scheme 1). Caprolactone is an important monomer for the synthesis of biodegradable thermoplastic polylcaprolactone [10], and is a precursor for caprolactam, an important building block which can be further transformed to
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polyamides [11].
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Scheme 1. PAMO BVMO catalyzed biocatalytic oxidation of cyclohexanone to caprolactone
However, the limited stability of CHMO as a long-standing problem has hampered widespread
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applications. Therefore, protein engineering has been employed to improve its stability [12-15]. For example, by applying a rational design strategy, the thermostability and oxidative stability of
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CHMO was improved by Opperman and Reetz [16]. Thereafter, attempts were made to enhance
progress is still limited.
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the thermostability of CHMO by introducing intramolecular disulfide bonds [14, 17]. However,
In order to find stable BVMOs for conversion of cyclohexanone to ε-caprolactone, a different
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strategy was employed in this study, namely, introducing CHMO activity to a relative thermostable BVMO by protein engineering. Thus, we turned to the thermostable Phenylacetone
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Monooxygenase (PAMO) from Thermobifida fusca [18]. It exhibits excellent thermostability and high tolerance to various solvents [19-21]. However, the substrate acceptance of PAMO is rather limited, being active only to small aromatic ketones and no activity to cyclohexanone [22-24]. Several attempts to induce PAMO to accept cyclohexanone by protein engineering methods have been made [12, 25-27]. For instance, by using a homology model of CHMO, a key bulge in the loop near the active site of PAMO was identified. It was shown that eliminating some amino acids in this loop leads to expanded substrate acceptance of the enzyme with essentially no trade off in thermostability [27]. However, cyclohexanone was still not accepted by these PAMO mutants. Based on the thermostable PAMO scaffold, a chimeric PAMO-CHMO enzyme mutant was created
ACCEPTED MANUSCRIPT and its activity against cyclohexanone was tested [28]. Unfortunately, no cyclohexanone activity was detected. Recently, directed evolution was applied to evolve PAMO mutants with activity to cyclohexanone, and indeed, active mutants were obtained following library screening [25, 26]. However, notable screening efforts were required, and resulted mutants in that study led to 100% conversion of 2 mM cyclohexanone within 3 h. While
For a practical industrial process, the
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concentration of accepted substrate by PAMO mutants remained still limited. and For a practical industrial process, further enzyme engineering is needed to obtain a truly active and robust
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biocatalyst.
In this study, site-directed mutagenesis was applied to evolve PAMO mutants active towards
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cyclohexanone for improved bioconversion. The catalytic properties of the new mutants for conversion of cyclohexanone to ε-caprolactone were characterized. In addition, the substrate scope
2. Materials and Methods
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2.1 General Procedures
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of PAMO mutants was investigated by using several other cyclic ketones as substrates.
cyclopentanone,
cycloheptanone,
4-methylcyclohexanone,
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Cyclohexanone,
2-methylcyclohexanone and L-Arabinose were purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). DpnI restriction enzyme, Site-Directed mutagenesis Kit and Fast
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Site-Directed Mutagenesis kit were obtained from TIANGEN biotech (Beijing, China). Primers was synthesized and purified by PAGE from GENEWIZ, Inc. (Nanjing, China). AxyPrepTM
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Plasmid Miniprep Kit was obtained from Axygen Biosciences (America). NADPH was purchased from Meryer (Shanghai) Chemical Technology Co., Ltd. (Shanghai, China). Wild-type and mutants PAMO were purified with CapturemTM His-Tagged Purification Maxiprep Kit from Takara (China). TB medium and LB medium were prepared following standard protocols.
2.2 Preparation of PAMO Mutants by Site-Directed Mutagenesis The PAMO mutants were prepared by using Fast Site-Directed Mutagenesis kit from Tiangen Biotech (Beijing, China) following the provided manual. The primers used are summarized (Supporting Information, Table S1). The mutation of PAMO mutants was verified by DNA
ACCEPTED MANUSCRIPT sequencing service in BioSune biotechnology company (Shanghai, China).
2.3 Whole Resting cells-catalyzed Biotransformation of Substrates Wild-type or mutant PAMO glycerol stock was inoculated into fresh LB media containing 100 µg/mL carbenicillin for cultivation under 37 ℃, 200 rpm. After overnight growing, 1 mL of culture was transferred into 100 mL of TB media supplemented with 100 µg/mL carbenicillin for
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further cultivation at 37 ℃, 200 rpm. When the OD600 reached 0.6-0.8, 0.1 g/100 mL of Arabinose
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was added as inducer for enzyme expression. After 20 h, resting cells were prepared as described before, briefly, cell pellets were harvested by centrifugation and washed two times with 50 mM,
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pH 8.0, phosphate sodium buffer.
Four hundred milligrams of prepared resting cell pellets were resuspended in 2 mL of 50 mM,
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pH8.0, phosphate sodium buffer containing specific amount of cyclohexanone as substrate in a 50 mL Falcon tube. The reaction mixture was shaken at 1000 rpm and 42 ℃ for given interval times.
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The product was extracted by adding ethyl acetate into the reaction mixture. The conversion of cyclohexanone was determined by GC equipped with HP-5MS column (30 m×0.32 mm×0.25 µm).
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Other cyclic ketones with similar structure were used as substrates in Baeyer-Villiger oxidation
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reactions under the same reaction conditions.
2.4 Thermostability of PAMO mutants
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The ThermoFAD method was employed to examine the thermostability of PAMO mutants[29]. Experiments were performed with a StepOnePlus™ Real-Time PCR System. Unfolding curves
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were generated using a temperature gradient from 20 to 90 ℃, performing a fluorescence measurement after every 0.5 ℃ increase after a 10s delay for signal stabilization. The Tm values were determined by the peaks of the derivatives of the fluorescence intensity. In the experiments, 20 µL samples of wild-type PAMO and mutants in 50 mM Tris-HCl buffer (pH 8.0) were used.
2.5 Steady-state kinetic analysis The activity of evolved PAMO mutants was measured by spectrophotometrically monitoring the decrease of NADPH in the reaction mixture at 25 ℃ and 340 nm. The steady-state kinetic parameters were measured using purified enzyme. The 200 µL reaction system contained 0.5-0.6
ACCEPTED MANUSCRIPT µM enzyme, 100 µM NADPH, 1 % ( v/v) acetonitrile and 50 mM Tris-HCl (pH 8.0). The data was measured after every 15s in 3 minutes. The experiments were performed with a SpectraMax M3.
3. Results and Discussion 3.1 Initial screening result for PAMO activity to cyclohexanone
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To engineer PAMO mutants with high activity for the BV oxidation of cyclohexanone, several mutants previously evolved for asymmetric sulfoxidation of thioethers were tested (Supporting
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Information, Table S2) [30]. Considering NADPH regeneration for roughly assessing activity,
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whole E. coli resting cells were applied as before [25, 31]. In order to improve the conversion and thus activity, some reaction parameters were modified. As PAMO exhibits high thermostability
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and activity at 50 ℃, cyclohexanone oxidation was performed at 42 ℃ with an agitation speed of 1000 rpm and 0.2 g/mL of wet resting cell as catalyst (Supporting Information, Table S3) [23].
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The preliminary results suggested that mutants evolved for sulfoxidation also show considerable activity to cyclohexanone under the given reaction conditions. In addition, several single mutants
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(P440F, P440W) showed high activity to cyclohexanone as well. However, it seems like that
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adding 20 mM of glucose did not improve the conversion (Supporting Information, Table S4). In a recent paper Parra et al. mentioned that the P440F mutant was found to be inactive on cyclohexanone [25]. The possible reason for this disagreement is that higher reaction temperature
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and catalyst dosage were used in this study. Meanwhile,this This result further verified that
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position P440 is indeed a “hot spot” (Table 1Fig. 1) [26, 32, 33].
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Fig. 1. Previously evolved PAMO mutants tested for activity in cyclohexanone oxidation.
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Table 1. Previously evolved PAMO mutants tested for activity in cyclohexanone oxidation. PAMO mutants
Conv. (%) a
WT PAMO
0
I67C/P440F/A442F/L443D
14.5
I67C/P440Y
24.3
4
I67Y/P440Y
56.3
5
P440F
48.3
6
P440W
33.5
7
P440H
6.2
8
P440Y
4.4
9
P440I
2.4
Entry
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a. Reaction condition: 0.2 g/mL wet cell weight, 2 mM cyclohexanone, 1 % (v/v) acetonitrile, 50 mM phosphate sodium buffer (pH 8.0), shaking at 1000 rpm under 42 ℃ for 4 h. The product was determined by GC.
By careful analysis we found that single mutant P440Y gave very low conversion in cyclohexanone oxidation, however, double mutant I67C/P440Y and I67Y/P440Y showed
ACCEPTED MANUSCRIPT significantly increased conversion. This result implies that position I67 may also play an important role in cyclohexanone acceptance of PAMO. In addition, previous studies suggested that the position I67 could be part of the active site of PAMO [32]. Based on the information mentioned above, and using P440F as starting point, position I67 was targeted for further focused
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mutagenesis (Fig. 2).
Fig. 2. Potential radomization amino acid sites in PAMO.The flavin and NADP cofactors are shown in yellow and
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orange. Based on the crystal structure of PAMO (PDB: 2YXL).
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3.2 Randomization of position I67 using P440F as template Using mutant P440F as a template, mutants at position P67 I67 were produced by site-directed mutagenesis, thereby replacing isoleucine proline by the 19 other canonical amino acids. Rather
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than applying traditional NNK-based saturation mutagenesis, we chose this strategy because it requires no screening. Interestingly, most of resulting mutants exhibited improved activity against
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cyclohexanone (Supporting Information, Table S5 4), implying that position I67 is indeed a “hot spot”. Of the obtained mutants, the best one proved to be I67Y/P440F which could convert substrate with 97.8% conversion after 4 h. The other three selected double mutants also gave high activities and good conversions from 87% to 95% (Table 1 2).
Table 1 2. Obtained new mutants for cyclohexanone oxidation Entry a
PAMO mutant
Conv. (%)
1
WT PAMO
0
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50.2
3
I67Y/P440F
97.8
4
I67A/P440F
95.1
5
I67C/P440F
92.4
6
I67G/P440F
87.5
7
I67Y
0
8
I67A
0
9
I67C
0
10
I67G
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a. Reaction condition and measurement conditions: as in Fig. 1Table 1.
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With the aim of investigating the interaction of mutation at position I67 and P440 in the obtained double mutants, single mutants were also prepared by site-directed mutagenesis and tested as
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catalysts in cyclohexanone oxidation. Surprisingly, four single-mutation variants at position I67 did not show any activity on cyclohexanone even with prolonged reaction time. In contrast, when combining single mutations at position 67 with P440F to form the respective double mutants,
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higher activity was detected. This result implies that there is possible additive or cooperative effect
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existing between I67 and P440, the phenomenon being reported before [34]. The same applies when deconvoluting the double mutant I67Y/P440F. All results are summarized in Table 1 2.
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3.3 High substrate concentration Catalytic performance of PAMO mutants The catalytic property of evolved mutants in cyclohexanone oxidation was tested with increased
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substrate concentration. As shown in Table 2 3, all the mutants convert cyclohexanone to carprolactone within 8 h at the substrate concentrate of 5 mM. In order to enhance the process efficiency, the substrate concentration was increased up to 10 mM. Remarkably, more than 95% of conversion was achieved by all four double mutants upon prolonging the reaction time to 12 h. The results show that new evolved PAMO mutants are able to convert cyclohexanone at considerable high concentration, indicating their potential applications in organic and polymers synthesis.
Table 2 3. Catalytic activity of PAMO mutants with increased substrate concentration.
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Entry a
PAMO mutant
1
WT PAMO
2
P440F
Conv. (%) b
Concentration(mM) 5
0
5
63.6
10
44.4
5
>99
10
>99
I67Y/P440F
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5 4
>99
I67A/P440F
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10
>99 >99 95.1
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a. Reaction condition: 0.2 g/mL wet cell weight, 1 % (v/v) acetonitrile, 50 mM phosphate sodium buffer (pH 8.0), shaking at 1000 rpm under 42 ℃, 8 h for 5 mM and12 h for 10 Mm cyclohexanone. The product was determined
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3.4 Steady-state kinetic parameters of evolved PAMO mutants The steady-state kinetic parameters for starting mutant and new evolved mutants were determined
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in the production of lactone from cyclohexanone by using purified mutant enzymes (Supporting Information, Fig. S1). As shown in Table 3 4 , a double mutant I67Y/P440F has a similar kcat value
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as variant P440F, while the other three double mutants have 2-3 times higher kcat values than that of P440F. The increased kcat values indicate higher specific enzymatic activities than starting mutant P440F. The Km values of the four double mutants are 2-4 times higher than that of P440F. However, the kcat /Km value of I67Y/P440F I67/P440F is 3 times lower than that of P440F P400F; the other three double mutants have similar kcat /Km values as the mutant P440F, implying that the catalytic efficiency of the evolved double mutants is of the same order of magnitude as that of starting mutant P440F. In addition, the kcat /Km value of our best mutant in this study is comparable to that of PAMO mutants reported by Parra et al., but still much lower than that of CHMO [14, 25].
ACCEPTED MANUSCRIPT Since WT PAMO does not accept cyclohexanone, the actual improvements starting from the natural form are considerable enormous.
Table 3 4 . Steady-state kinetic parameters of evolved PAMO mutants PAMO mutants
Km (mM)
kcat (s-1)
kcat /Km (s-1M-1)
1
P440F
0.1037
0.0653
633
2
I67Y/P440F
0.2107
0.0456
3
I67A/P440F
0.3472
0.1364
4
I67C/P440F
0.4412
0.198
5
I67G/P440F
0.2595
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0.1324
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Reaction condition: The 200 µL reaction mixture contained 0.5-0.6 µM enzyme, 100 µM NADPH, 1% (v/v)
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acetonitrile and 50 mM Tris-HCl (pH 8.0).
3.5 Thermostability of PAMO mutants
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Thermostability of WT PAMO and the variants was examined by measuring the Tm values with
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the ThermoFAD method following the protocol as described previously [29]. The results shows that the Tm value for WT PAMO is 58 ℃; Tm values for P440F and the four best mutants are 55.5 ℃
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(P440F), 53 ℃ (I67C/P440F), 51 ℃ (I67A/P440F), 51 ℃ (I67Y/P440F) and 51 ℃ (I67G/P440F)
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respectively, indicating that the introduced mutations decreased thermostability slightly (Fig. S2).
Figure 2. Thermostability of WT PAMO and evolved mutants as measured by the ThermoFAD protocol.
3.6 Substrate scope of evolved PAMO mutants The substrate scopes of the evolved mutants were investigated by using several other cyclic ketones for which WT PAMO shows no activity. Interestingly, it was found that all tested ketones are accepted by the mutants with moderate to high conversion, except for cycloheptanone which has less than 10% conversion (Table 4 5). Cyclopentanone and 2-methylcyclohexanone were transformed to the corresponding lactones by four double mutants with conversion of >95%,
ACCEPTED MANUSCRIPT which is better than that catalyzed by single mutant P440F. However, 4-methylcyclohexanone was converted to lactone with maximal conversion of 40% in which less than 5% of 4-methylcyclohexanol formed (data not shown). The results indicate that the substrate scope of our new mutants was expanded successfully.
Conv. (%) a Substrate WT
P440F
I67Y/P440F
I67A/P440F
0
57.5
97.8
96.7
0
88.4
91.0
0
3.9
O
2
45.1
96.7
96.5
92.7
11.4
40.3
36.8
2.4
9.0
3.2
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I67G/P440F
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I67C/P440F
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Table 4 5. Additional cyclic ketones used as substrates in PAMO-catalyzed BV oxidation
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0.44
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a. Reaction condition: 0.2 g/mL wet cell weight, 2 mM cyclohexanone, 1 % (v/v) acetonitrile, 50 mM phosphate
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sodium buffer (pH 8.0), shaking at 1000rpm under 42 ℃ for 4 h. The product was determined by GC.
4. Conclusions
In summary, protein engineering to expand the thermostable PAMO for acceptance of cyclohexanone with high activity to form ε-caprolactone from cyclohexanone was achieved by applying a protein engineering strategy based on iterative site-specific mutagenesis. An alternative approach based on iterative saturation mutagenesis (ISM) at randomization sits sites lining the binding pocket (CASTing) is likely to provide even better variants, but this requires the screening of medium-sized or small libraries [35]. In our approach, several mutants with excellent activity to
ACCEPTED MANUSCRIPT cyclohexanone were obtained and the catalytic properties were characterized. The best mutant I67Y/P440F converts cyclohexanone with a conversion of 97.8% within 4 h; 10 mM with >99% conversion under prolonged reaction time to 12 h. However, it was found that the introduced mutations lead to a slight decrease of enzyme thermostability. In addition, evolved PAMO mutants also show high activity to structurally different cyclic ketones. Our results provide new and
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practical biocatalysts for organic synthesis and biotechnologies.
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Conflicts of interest
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The authors declare no conflict of interest.
Acknowledgements
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This work was financially supported by the National Science Foundation of China (21776134, 21646014, and 21606192), the program of Jiangsu Synergetic Innovation Center for Advanced
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Bio-Manufacture (XTE1851).
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Supplementary data
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Appendix A. Supplementary data
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ACCEPTED MANUSCRIPT
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Graphical Abstract:
ACCEPTED MANUSCRIPT Highlights: Hihg active PAMO mutants towards cyclohexanone was obtaind by protein engineering.
10 mM cyclohexanone was converted by mutants with 99% yield after 12h.
Mutants showed broadened substrate scope towards other cyclic ketones as well.
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