Improved Efficiency of the Desulfurization of Oil Sulfur Compounds in

May 27, 2019 - (16,21) Not only to obtain as much as possible the sulfur-free HBP ... (21) In addition, the dszC gene was placed into a separate casse...
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Research Article Cite This: ACS Synth. Biol. 2019, 8, 1441−1451

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Improved Efficiency of the Desulfurization of Oil Sulfur Compounds in Escherichia coli Using a Combination of Desensitization Engineering and DszC Overexpression

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Lu Li, Yibo Liao, Yifan Luo, Guangming Zhang, Xihao Liao, Wei Zhang, Suiping Zheng, Shuangyan Han, Ying Lin,* and Shuli Liang* Guangdong Key Laboratory of Fermentation and Enzyme Engineering, Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China S Supporting Information *

ABSTRACT: The 4S pathway of biodesulfurization, which can specifically desulfurize aromatic S-heterocyclic compounds without destroying their combustion value, is a low-cost and environmentally friendly technology that is complementary to hydrodesulfurization. The four Dsz enzymes convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). Of these four enzymes, DszC, the first enzyme in the 4S pathway, is the most severely affected by the feedback inhibition caused by HBP. This study is the first attempt to directly modify DszC to decrease its inhibition by HBP, with the results showing that the modified protein is insensitive to HBP. On the basis of the principle that the final HBP product could show a blue color with Gibbs reagent, a high-throughput screening method for its rapid detection was established. The screening method and the combinatorial mutagenesis generated the mutant AKWC (A101K/W327C) of DszC. After the IC50 was calculated, the feedback inhibition of the AKWC mutant was observed to have been substantially reduced. Interestingly, the substrate inhibition of DszC had also been reduced as a result of directed evolution. Finally, the recombinant BL21(DE3)/ BADC*+C* (C* represents AKWC) strain exhibited a specific conversion rate of 214.84 μmolHBP/gDCW/h, which was 13.8-fold greater than that of the wild-type strain. Desensitization engineering and the overexpression of the desensitized DszC protein resulted in the elimination of the feedback inhibition bottleneck in the 4S pathway, which is practical and effective progress toward the production of sulfur-free fuel oil. The results of this study demonstrate the utility of desensitization of feedback inhibition regulation in metabolic pathways by protein engineering. KEYWORDS: biodesulfurization, 4S pathway, DszC, feedback inhibition, directed evolution, desensitization

A

sulfone monooxygenase), and DszB (2-hydroxybiphenyl-2sulfinate (HBPS) desulfinase) (Figure 1A). DszD (NADH:FMN oxidoreductase) provides the FMNH2 molecules that are required for DszC and DszA enzymatic activity.13−15 For natural bacteria or recombinant strains that harbor the dszABC, considerable efforts have been made to increase the BDS rate. Resting cells of Escherichia coli DH10B (pDSR2, pDSR3),16 Acidovorax delafieldii R-8,17 Pseudomonas putida CECT5279,18 Sphingomonas subarctica 17b,19 and Ralstonia eutropha PTCC16520 have been shown to efficiently perform the desulfurization process, with the P. putida KT2440 (pIZdszB1A1C1-D1) strain21 exhibiting the highest specific conversion rate (21 μmolHBP /g DCW/h). However, this desulfurization rate still cannot meet the industrial requirements for the development of a commercial BDS process.21

t present, global society is moving toward the use of ultralow-sulfur or sulfur-free fuel oil because of the environmental pollution and health hazards resulting from the combustion of sulfur-containing fuels.1−3 Moreover, the aromatic S-heterocyclic compounds present in petroleum, such as dibenzothiophene (DBT) and its alkylated derivatives, are difficult to completely desulfurize using the conventional hydroresulfurization (HDS) method.4−6 The 4S pathway of biodesulfurization (BDS), which was first identified and studied extensively in the Gram-positive Rhodococcus erythropolis strain IGTS8,7 can specifically cleave the C−S bond of DBT while leaving its carbon skeleton and combustion value intact.8−10 This property makes BDS an attractive, low-cost, and environmentally friendly technology for use as a complementary strategy to HDS.11,12 The 4S pathway transforms DBT into 2-hydroxybiphenyl (HBP) and sulfite via a series of four reactions that are catalyzed by DszC (DBT monooxygenase), DszA (DBT © 2019 American Chemical Society

Received: March 23, 2019 Published: May 27, 2019 1441

DOI: 10.1021/acssynbio.9b00126 ACS Synth. Biol. 2019, 8, 1441−1451

Research Article

ACS Synthetic Biology

Figure 1. Basis and workflow of the high-throughput screening method. (A) The workflow of the high-throughput screening method was used to rapidly screen for potential positive DszC mutants. The arrows indicate the direction of gene transcription. The restriction enzyme recognition sites are abbreviated as follows: E, EcoRI; X, XbaI; N, NheI; H, HindIII; S, SpeI; and P, PstI. The Ptac and Plac sites are indicated by dark gray and light gray arrows, respectively. T represents the transcription terminator. (B) Different concentrations of DBT were converted to HBP by resting E. coli BL21(DE3)/pSB4A5-BADC cells within 48 h. The error bars represent the standard deviation of triplicate biological determinations. (C) Gibbs reaction results of a portion of the samples at 24 h (B). The negative control contained only resting cells harboring the dszABCD genes without the addition of DBT as a substrate. The positive control was performed to test whether the Gibbs reagent was functional and contained only 0.1 mM HBP.

by DBT and HBPS, which also limits the desulfurization rate.23 Although some strategies have been implemented to increase the BDS rate, such as elimination of the overlap between dszA and dszB, rearrangement of dsz genes,24 directed evolution of Dsz enzymes,25−28 and studies about the “extended 4S pathway”,29−32 no direct efforts have been made to decrease the feedback inhibition of DszC by HBP. Therefore, to

The existence of many bottlenecks in the desulfurization process is responsible for the limitation of the BDS rate,22 and the feedback inhibition caused by HBP is one of the major bottlenecks of the 4S pathway.22,23 DszC is the key initiating enzyme of the desulfurization process, and compared with the other three Dsz enzymes, the inhibition of DszC by HBP is the most severe.23 Moreover, the activity of DszC is also inhibited 1442

DOI: 10.1021/acssynbio.9b00126 ACS Synth. Biol. 2019, 8, 1441−1451

ACS Synthetic Biology

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improve the metabolic efficiency of the 4S pathway, decreasing the feedback inhibition on DszC and increasing its expression may be reasonable strategies. Protein engineering is a practical method for reducing the effects of feedback inhibition on an enzyme. The X-ray crystal structures of DszC and its homologous proteins in Rhodococcus sp. XP, Rhodococcus erythropolis D-1, and Paenibacillus sp. A112 have been solved, and the binding sites of DBT/FMN have been identified.33−36 Abinfuentes et al. stated that there was a noncompetitive inhibition of DszC by HBP,23 showing that HBP has one or several binding sites on DszC that are different from the DBT substrate binding site. However, the binding site of HBP to DszC is still unknown, hindering efforts to modify DszC using rational design. In contrast, a combination of a well-designed library and an efficient screening method could also be an effective strategy for enzyme engineering. Generally, distinguishable phenotypes are crucial for developing highthroughput screening methods. The Gibbs reagent can react with HBP to show a blue color, and the absorbance of the blue compound is proportional to the HBP concentration within a certain range.28,37,38 Although Gibbs colorimetry is less accurate than high-performance liquid chromatography (HPLC), it can quickly filter out negative and undesired mutants. Therefore, using a combination of a rapid, highthroughput screening method and the docking results of HBP with DszC in silico, it is possible to identify mutants with reduced feedback inhibition. For strain improvement, it is often necessary to overexpress key enzymes in the pathway for a desired product.39 In addition to desensitization engineering of DszC, elevating the expression of the most sensitive enzyme is another effective approach that can be used to overcome metabolic bottlenecks. In a previous study, the rate of DBT removal was enhanced in a recombinant E. coli strain harboring the dszABC genes by overexpressing a flavin-reductase from Vibrio harveyi.16 In another study, a rate-limiting step that resulted from low DszB activity was overcome by overexpressing the dszB gene.40 In a more recent study, the addition of DszB derived from crude cell extracts to the P. putida KT2440 (pIZdszB1A1C1-D1) cultures improved the conversion of DBT into HBP.21 These results led us to examine whether the overexpression of DszC, resulting from an increase in gene dosage, could also serve as a valid method for overcoming the metabolic bottleneck in the 4S pathway. In the present study, we aimed to improve the desulfurization rate of the 4S pathway in a recombinant E. coli strain by simultaneously relieving feedback inhibition and enhancing the expression of DszC. On the basis of the results of high-throughput screening and ligand docking analysis, a final DszC mutant was generated by iterative saturation mutagenesis. The IC50 value of the mutant protein increased from the level of micromoles per liter to millimoles per liter, indicating its insensitivity to HBP. In addition, the desensitized DszC mutant was subsequently overexpressed to improve the specific conversion rate to the highest level ever reported in the literature (214.84 μmolHBP/gDCW/h). During this study, it was a priority to make the upstream metabolic flux as large as possible and to avoid feedback inhibition. The results presented in this research exhibit significant improvements of the BDS rate that were obtained from a combination of desensitization engineering and overexpression of the key enzyme DszC.

Research Article

RESULTS AND DISCUSSION

Development of a Rapid and High-Throughput Screening Method Based on the Detection of Blue Coloration. To quickly and efficiently obtain potential positive mutants, a suitable high-throughput screening method was required. Gibbs reagent is able to react with HBP generated by the 4S pathway to produce a blue color that can be visualized and detected at 610 nm.28 Therefore, a rapid, high-throughput screening method was developed involving a catalytic module mediated by the resting cells of recombinant strains harboring the dszABCD genes as well as a colorrendering module utilizing the reaction of Gibbs reagent and HBP. Prior to the development of a screening system, a functional E. coli BL21 (DE3) recombinant strain containing the plasmid of pSB4A5-BADC was constructed (Figure 1A). To ensure sufficient desulfurization capacity, several modifications and rearrangements of the native dsz genes from the R. erythropolis IGTS8 strain were made. All of the dsz genes were optimized according to E. coli codon usage and were used for subsequent plasmid construction. The genetic overlap between dszA and dszB41 was removed, and the expression of DszB, which had been shown to be the rate-limiting step of the 4S pathway,23 was enhanced by the Ptac promoter. Previous reports have shown that the removal rate of DBT is increased by enhancing the expression level of FMN:NADH oxidoreductase, but the efficiency of HBP formation is decreased.16,21 Not only to obtain as much as possible the sulfur-free HBP retaining the combustion value but also to produce more HBP for the desulfurization recombinant strain to facilitate screening of DszC mutants desensitized to HBP, the expression of DszD was weakened by using the weaker Plac promoter and by including double terminators according to the reported plasmid construction protocol.21 In addition, the dszC gene was placed into a separate cassette so that it could be easily replaced with mutant versions. The plasmid pSB4A5-BADC allowed the dsz genes to be regulated by the IPTG-dependent lacI/Ptac and lacI/Plac system rather than the native sulfurdependent signaling, which prevented the suppression of transcription by sulfate.42 The resting cells of E. coli BL21(DE3)/pSB4A5-BADC could functionally convert DBT to HBP (Figure 1B), which further reacted with Gibbs reagent to produce a blue coloration (Figure 1C). Moreover, when the pH of the Gibbs reaction is properly controlled, the blue color remained sufficiently stable to allow for measurements to be made within 2 h (Figure S2 and Table S3). The degree of HBP production was reflected in the depth of the blue color, indicating that the amount of HBP was proportional to the degree of color development within a certain range and could be feasibly used for the high-throughput screening assay.28,37,38 The DBT concentration was the key factor in the development and implementation of the screening method. To minimize the preference of the screening results toward “reduced substrate inhibition”, a DBT concentration of 0.25 mM was selected instead of 0.5 mM. On the one hand, when using a DBT concentration of 0.25 mM, the resting BL21(DE3)/BADC cells could completely degrade DBT within 24 h, whereas there was still residual DBT when a concentration of 0.5 mM was used (Figure S3). On the other hand, the HBP concentration generated using 0.25 mM DBT was sufficiently high to inhibit DszC,23 and the blue coloration 1443

DOI: 10.1021/acssynbio.9b00126 ACS Synth. Biol. 2019, 8, 1441−1451

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ACS Synthetic Biology

Figure 2. Screening of DszC mutants and analysis of potential binding sites for HBP on DszC. (A) The 13 potential positive mutants obtained from the first round of preliminary screening were screened in a second round in 96-well plates. The numbers in the figure represent the average of the absorbance values of two parallel assays. (B) Culturing of the A101V mutant and WT strains by shake flask fermentation and preparation of resting cells that were subjected to whole cell catalysis of DBT to produce HBP. The amount of HBP was determined by HPLC. Asterisks indicate significant differences between the WT and A101V mutant strains, ** = 0.001 < p < 0.01 by t-test. The error bars represent the standard deviation of triplicate biological determinations. (C) The pocket containing A101 and W327 was predicted using Discovery Studio 3.0. HBP is shown in green, the π bond is shown in orange, and A101 is shown in red.

Figure 3. Verification and evaluation of DszC mutants in vivo and in vitro. (A) and (B) Description of the enzymatic activity of DszC mutants subject to HBP (A) and DBT (B) inhibition. The enzymatic activities shown in (A) were measured in the presence of 1.5 μM DBT. All experimental conditions used to measure the enzymatic activities of the mutants were identical to those of the WT protein. Asterisks represent significant differences between WT and A101V, *** = 0.0001 < p < 0.001 by two-way ANOVA. The error bars represent the standard deviation of triplicate biological determinations. (C) The yield of HBP obtained from the BL21(DE3)/BAD+AKWC and BL21(DE3)/BADC strains at different DBT concentrations. The error bars represent the standard deviation of triplicate biological determinations. (D) The yield of HBP produced by the mutant strains in the presence of 0.5 mM DBT. Asterisks represent significant differences between the WT strain and the A101V or A101K strains, * = p < 0.05, ** = 0.001 < p < 0.01 by t-test. The error bars represent the standard deviation of triplicate biological determinations. All of the working recombinant strains mentioned in (C) and (D) were resting cells.

observed at this HBP concentration was also within a suitable range. The amount of HBP produced by the DBT-degrading BL21(DE3)/BADC resting cells within 48 h using a DBT concentration of 0.1−0.5 mM was measured. As shown in Figure 1B, excessive levels of DBT (0.5 mM) resulted in lower

HBP production, which may be due to the combination of the substrate inhibition of DBT on DszC and the feedback inhibition of HBP on the entire 4S pathway.23 Moreover, the enzymatic activity of purified DszC in the presence of increasing concentrations of DBT and HBP was measured, and inhibition of DszC by DBT and HBP was observed 1444

DOI: 10.1021/acssynbio.9b00126 ACS Synth. Biol. 2019, 8, 1441−1451

Research Article

ACS Synthetic Biology (Figure 3A,B). The above results indicated the feasibility of the color-based high-throughput screening assay to rapidly screen positive mutants of DszC, including the mutants with reduced feedback inhibition. Directed Evolution of DszC Based on Random Mutation. A DszC mutant library was generated via errorprone PCR and was transformed into E. coli BL21 (DE3) cells. Approximately 10 000 colonies were preliminary screened in a 96-well plate format, and 13 mutant strains showing a higher absorbance (darker blue color) than the wild-type strain were obtained. The 13 isolates were rescreened in two parallel wells in 96-well plates (Figure 2A), the results of which demonstrated the whole-cell catalysis of DBT by these strains, with the presented values representing the average absorbances of the two parallel wells. The mutant strains showing a higher absorbance than the wild-type strain were again selected and cultured in shake flasks, and the phenotype was finally confirmed by HPLC measurement. The A101V strain was observed to have an HBP yield increase of 27.60% compared with the wild-type (WT) strain (Figure 2B). For the purified A101V protein, when HBP was used as an inhibitor, its enzymatic activity based on the observation of DBTO2 formation is higher than that of wild-type DszC in the presence of 0−25 μM HBP, and it was approximately 40−45% higher than the WT protein in the presence of 5−15 μM HBP (Figure 3A). The IC50 of HBP for the A101V mutant was increased 23.80% compared with the WT protein (3.95 ± 0.24 to 4.89 ± 0.19 μM) (Table 2). The reduced feedback inhibition on the A101V mutant may be primarily due to steric hindrance, as valine is larger than alanine. After mutating A101 to valine in silico using Discovery Studio 3.0 (DS), the protein was docked with HBP, and the free binding energy was calculated. The binding free energy of HBP with A101V was higher than that of the WT protein, increasing from −66.68 to −41.58 kcal/mol. This ancillary result indicated that the binding of A101V with HBP was more unstable than that of the WT protein, as the degree of HBP binding to A101V was lower than that observed for the WT protein. This result was consistent with the results of in vitro experiments. Interestingly, the inhibition of A101V by DBT was simultaneously mitigated (Figure 3B, Table 2), which may be due to a structural change caused by the mutation. On the basis of the above data, it could be inferred that the modification on the 101st residue of DszC effectively alleviated the feedback and substrate inhibition of this enzyme. Docking of the ligand HBP to DszC was also performed in silico using DS to predict potential binding sites. Among the 21 predicted binding pockets (Supporting Information Table S2), the residue A101 was located in only one. The goal of using ligand docking in silico was to guide which of the residues should be modified next in this potential binding pocket. The results showed that a π bond is only formed between W327 and HBP (Figure 2C); thus, W327 is likely to be a more crucial residue, and the subsequent directed evolution of DszC focused on A101 and W327. Further Saturation Mutagenesis for the Directed Evolution of DszC. Saturation mutagenesis of the 101st amino acid of DszC was performed, and the enzymatic activity based on observing DBTO2 was measured in the presence of 1.5 μM DBT (substrate) and 5 μM HBP (inhibitor) (Table 1). After the first round of saturation mutagenesis, the enzymatic activity of the A101K mutant was observed to be the highest, and it was 57.51% higher than that of the WT protein. It was speculated that because lysine has a larger R group than

Table 1. Relative Enzymatic Activities of DszC Mutants under the Inhibition of HBP mutant

relative enzymatic activity (%)a

WT A101C A101D A101E A101F A101G A101H A101I A101K A101L A101M A101N A101P A101Q A101R A101S A101T A101V A101W A101Y

100.00 102.46 ± 9.81 80.93 ± 2.74 103.39 ± 11.33 106.49 ± 8.56 95.97 ± 12.58 99.03 ± 7.76 96.81 ± 10.88 157.51 ± 10.92 100.93 ± 11.08 107.53 ± 12.79 112.85 ± 4.08 109.58 ± 11.84 103.71 ± 10.21 98.33 ± 10.54 103.16 ± 11.41 91.32 ± 5.35 145.53 ± 2.89 98.11 ± 5.29 94.93 ± 12.26

mutants

relative enzymatic activity (%)a

A101K+W327A A101K+W327C A101K+W327D A101K+W327E A101K+W327F A101K+W327G A101K+W327H A101K+W327I A101K+W327 K A101K+W327L A101K+W327M A101K+W327N A101K+W327P A101K+W327Q A101K+W327R A101K+W327S A101K+W327T A101K+W327 V A101K+W327Y W327C W327Y

191.96 ± 11.94 331.16 ± 20.92 76.49 ± 4.82 116.69 ± 1.21 87.17 ± 5.14 108.05 ± 2.09 118.27 ± 6.57 89.37 ± 2.33 100.88 ± 14.40 81.11 ± 11.67 82.76 ± 2.37 115.56 ± 17.19 74.10 ± 10.87 81.20 ± 10.18 91.74 ± 2.15 79.17 ± 7.94 100.11 ± 7.02 117.85 ± 6.32 260.49 ± 17.33 251.45 ± 7.70 179.29 ± 20.94

a The enzymatic activities of all mutants were measured in the presence of 1.5 μM DBT and 5 μM HBP. All data presented are the average values of three biological replicates.

alanine, there was a resulting increase in steric hindrance that prevents HBP from entering the binding pocket. The in silico ligand docking results showed that the two loops of W327 and the loop of HBP form stable π bonds in the pocket (Figure 2C), which may be crucial for the binding of HBP to DszC. Therefore, iterative saturation mutagenesis of W327 based on the A101K mutant was performed. Under the same assay conditions (1.5 μM DBT and 5 μM HBP), approximately half of the obtained mutants exhibited higher enzymatic activity than the WT protein (Table 1). In particular, the A101K/W327C (AKWC) and A101K/W327Y (AKWY) mutants exhibited 3.3- and 2.6-fold higher enzymatic activity, respectively, than the WT and had the highest enzymatic activities of all the mutants. Interestingly, most of the amino acids in the binding pocket were observed to be hydrophobic. In this hydrophobic pocket, the most critical hydrophobic residue, W327, was mutated to cysteine and tyrosine, which are the most polar and hydrophilic amino acids of all the uncharged polar amino acids. This mutation is likely to lead to difficulties in the ability of the water-insoluble HBP to bind DszC at this site, and more importantly, the two mutants lose the π bond that interacts with HBP. The contribution of a single substitution at W327 to changes in feedback inhibition of DszC by HBP was further investigated. The enzymatic activities of the W327C and W327Y mutants were 2.5- and 1.8-fold higher than that of the WT protein, suggesting that substitution at position 327 greatly contributed to desensitization of feedback inhibition. When DszC was mutated to AKWC in silico, the result of docking HBP with it was shown as “no ligands docked”, and the same result appeared on the W327C mutant (see Reports of Ligand Docking in Supporting Information). The docking results correspond to the above experimental results. The enzymatic 1445

DOI: 10.1021/acssynbio.9b00126 ACS Synth. Biol. 2019, 8, 1441−1451

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ACS Synthetic Biology Table 2. Primary Properties of the Purified DszC Mutants in This Work mutants

stock concn (mg/mL)

WT A101V W327C AKWC

3.27 3.66 4.12 4.04

kcat (min−1)a 2.84 3.47 4.17 4.66

± ± ± ±

0.12 0.19 0.25 0.42

Km (μM)a

kcat/Km (μM−1 min−1)

± ± ± ±

4.60 5.15 5.63 5.75

0.62 0.68 0.74 0.81

0.010 0.011 0.008 0.012

KI (μM)a

IC50 (HBP)

± ± ± ±

3.95 ± 0.24 μM 4.89 ± 0.19 μM 80.38 ± 2.52 mM 83.00 ± 2.10 mM

0.66 0.71 0.77 0.84

0.010 0.011 0.021 0.019

a

The parameters were calculated by MATLAB using the Edward model of substrate inhibition.44,45

the presence of 0.5 mM DBT. In addition, strains containing single-point mutations (A101V, A101K, W327C, and W327Y) were also constructed. Resting cell assays were performed for all of the mutant strains in the presence of a DBT substrate with a concentration of 0.5 mM. All of the recombinant strains expressing mutant DszC proteins generated more HBP than that of the strain expressing the WT protein (Figure 3D). The production of HBP by the strains expressing the W327C and W327Y mutants was between that observed for the strains expressing the A101V/A101K and AKWC/AKWY mutants, which was approximately 2-fold of that observed for the strain expressing the WT protein. These results showed that the mutation of the 327th residue had a greater influence on the desensitization of DszC than the 101st residue, which is consistent with the results of the in vitro experiments (Table 1 and Table 2). On the basis of the above results, the loss of DBT and the formation of HBP during the degradation of 0.025 mM DBT by the strains expressing the WT, A101K, and AKWC DszC proteins were further evaluated (Figure S4). The advantage of the inhibition-resistant AKWC mutant was clearly observed compared with the less-effective single-point mutants and the wild-type DszC. These results indicated that the desulfurization efficiency of recombinant strains harboring DszC mutants that are insensitive to feedback inhibition was significantly improved. The data presented in Table 2 shows that the major contribution of the DszC mutants to increasing the BDS rate came from their desensitization to HBP. Thus, the desensitization engineering of DszC was an effective method to improve the desulfurization efficiency. Further Enhancement of the Desulfurization Efficiency Using a Combination of Desensitization Engineering and the Overexpression of DszC. The desulfurization efficiency of recombinant strains overexpressing each of the dsz genes was measured (Figure 4A). DszB is the ratelimiting enzyme within the 4S pathway,40,46,47 and the desulfurization efficiency of the recombinant strain with the overexpressed dszB gene was 2.3-fold greater than that of the WT strain. However, the desulfurization efficiency was observed to be the highest when the dszC gene was overexpressed, as this strain exhibited a 5.9-fold greater desulfurization efficiency than that observed for the WT strain. The first enzyme in a metabolic pathway is typically the most severely subjected to feedback inhibition,23,48−50 and DszC has been shown to be subject to the most severe feedback inhibition in the presence of HBP in the 4S pathway and is also inhibited by the substrate DBT.23 On the other hand, the amount of DszC expression in the 4S system in this study may have been less than that observed in other studies or that occurring in desulfurization bacteria in nature. Consequently, the overexpression of DszC was a feasible method that could be used to improve desulfurization efficiency. To further improve the desulfurization efficiency, the mutant AKWC was overexpressed in E. coli. When a higher DBT concentration of 0.5 mM was used, the BL21(DE3)/

activity of the A101K mutant only increased by approximately 58% compared with that of the WT enzyme. These results indicated that the 327th residue of DszC is more crucial than the 101st residue for feedback inhibition by HBP. The degree of desensitization of the DszC mutants was further evaluated at different concentrations of HBP and DBT. The AKWC and W327C mutants retained greater than 82 and 57% of their activity in the presence of 5 to 10 μM HBP, whereas the WT and A101V mutant were acutely inhibited by HBP (Figure 3A). When the HBP concentration exceeded 20 μM, a considerable decrease in enzymatic activity occurred for the three mutants as well as the WT protein; however, the enzymatic activity of AKWC mutant remained approximately 2-fold higher than that of the other three mutants. Furthermore, the IC50 values for the W327C and AKWC mutants increased from 3.95 ± 0.24 μM to 80.38 ± 2.52 mM and 83.00 ± 1.12 mM, respectively (Table 2). The results also indicated that the modification of the 327th residue was crucial for the desensitization of DszC to HBP, while the mutation of the 101st residue played a supporting role. Overall, the feedback inhibition of the AKWC mutant by HBP was significantly desensitized. It should be noted that the feedback-insensitive mutant AKWC simultaneously exhibited decreased substrate inhibition. When the DBT substrate concentration was increased, the highest enzymatic activity of the AKWC mutant was approximately 19 U/mg at a substrate concentration of 2−2.5 μM DBT, an increase of approximately 36% compared with the highest enzymatic activity observed for the WT protein (Figure 3B). The KI values of the DszC mutants were slightly higher than that of the WT protein, indicating that their substrate inhibition was alleviated (Table 2). The DszC substrate binding pocket has been previously described in detail,33−36,43 and the HBP binding site should not be the same as the substrate binding site because the inhibition of DszC by HBP is noncompetitive.23 Although the true HBP binding site could not be verified in this study, the acquisition of the AKWC mutant made a significant contribution to the feedback inhibition desensitization of DszC. Thus, it was inferred that the mutation of DszC may also result in conformational changes in the DBT-DszC-FMN complex as well as changes in substrate inhibition. In future research, protein engineering will be used to further alleviate the substrate inhibition of DszC to improve the desulfurization efficiency. The desulfurization efficiency of the desensitized DszC mutants in the 4S pathway was evaluated in vivo to verify their effects. As shown in Figure 3C, the HBP yield of the strain containing the WT protein decreased when the DBT concentration was higher than 0.25 mM, which may be due to the presence of bottlenecks22 in the processes involved in DBT and HBP inhibition, transmembrane transport, and other potential factors. However, the recombinant strain carrying the AKWC mutant achieved the maximum rate of HBP production, 4.4-fold greater than that of the WT strain in 1446

DOI: 10.1021/acssynbio.9b00126 ACS Synth. Biol. 2019, 8, 1441−1451

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ACS Synthetic Biology

BL21(DE3)/BADC+C strain and 23.6-fold higher than that of the BL21(DE3)/BADC strain (Figure 4B). The reaction solution of BL21(DE3)/BADC*+C* was confirmed by Liquid Chromatograph Mass Spectrometer (LC-MS) to validate the formation of the product HBP (Figure S5). Additionally, the specific conversion rate of the WT strain was 15.56 μmolHBP/ gDCW/h, while the BADC*+C* strain achieved a rate of 214.84 μmolHBP/gDCW/h (Table 3), which should be the highest value of the engineered resting cells in the aqueous phase reported to date.21 These results showed that the desulfurization efficiency was dramatically increased by combining the desensitization engineering and the overexpression of DszC. It should be noted that the rate of conversion of DBT to HBP was low in the presence of a high concentration of DBT (greater than 0.25 mM), which may be mainly due to substrate inhibition of DszC. During desulfurization engineering of resting cells in the aqueous phase, the conversion rate was only 50 and 7.5% in the presence of 0.4 and 1.2 mM DBT, respectively.16,51 When the DBT concentration was as low as 0.025 mM, the conversion rate could reach 100% after 0.5−1 h.18,21 The conversion rate of the BL21(DE3)/BADC*+C* strain in the presence of low concentrations of DBT (0.025 to 0.1 mM) was determined (Figure 4C), with DBT almost completely converted to HBP within 30 min at a concentration of 0.025 mM. The conversion rate reached 100% within 120 min even when the DBT concentration was increased to 0.075 mM. Although the conversion rate was reduced when the DBT concentration was 0.1 mM, reaching approximately 70% within 120 min, the engineered strains exhibited excellent desulfurization efficiency and application potential that was achieved through a combination of desensitization engineering and the overexpression of DszC. In contrast, the BL21(DE3)/BADC +C strain could not completely convert DBT within 2 h in the same reaction conditions. When a higher DBT concentration was used, there was a higher residual amount of DBT and a lower conversion rate (Figure S6). This finding also showed the obvious advantages of the strains containing the AKWC mutant, which was achieved via desensitization engineering.

Figure 4. Desulfurization efficiency and the conversion rate of various recombinant strains. (A) The yield of HBP produced by recombinant strains containing additional doses of each dsz gene in the presence of 0.25 mM DBT; the strains included BL21(DE3)/pSB4A5-BADC +pSB1C3-dszA, BL21(DE3)/pSB4A5-BADC+pSB1C3-dszB, and BL21(DE3)/pSB4A5-BADC+pSB1C3-dszC. (B) The yield of HBP produced by the BL21(DE3)/pSB4A5-BADC+pSB1C3-dszC and BL21(DE3)/pSB4A5-BADC*+pET28a-C* strains (C* represents AKWC) in the presence of different DBT concentrations. The pET28a plasmid containing the strong T7 promoter was used to express additional AKWC, as this plasmid can increase the expression of heterologous protein to a greater degree than pSB4A5 and pSB1C3. (C) The rate at which the BL21(DE3)/BADC*+C* strains converted DBT to HBP in the aqueous phase at different DBT concentrations and different reaction times. All of the working recombinant strains described here were resting cells. All of the error bars shown in Figure 4 represent the standard deviation of triplicate biological determinations.

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CONCLUSIONS In this study, the mutant AKWC of DszC was shown to be desensitized to HBP via protein engineering, which combined the high-throughput screening method and combinatorial mutagenesis. Interestingly, the substrate inhibition of DszC also decreased during the directed evolution. In addition, the specific conversion rate of the recombinant strain BL21(DE3)/ BADC*+C* was further increased and reached a much higher HBP yield (214.84 μmolHBP/gDCW/h) through enhancing the expression of the insensitive DszC mutant AKWC. This result demonstrated that the desensitization engineering and the overexpression of the critical enzyme DszC improved the desulfurization efficiency of the 4S pathway. The use of desensitizing engineering when the binding site of an inhibitor to the enzyme is unknown has not been well studied. Given the advantages of E. coli as a host for expressing heterologous genes, although it is intolerant to organic phase,

BADC*+C* (C* represents AKWC) strain produced an HBP yield of 33.0 μM, which was 2.2-fold higher than that of the Table 3. Specific Conversion Rate of the Superior Strains strain

WT

AKWC

BADC+C

BADC*+C*

EBDS (μmolHBP/gDCW/h)

15.56 ± 1.31

32.81 ± 2.76

98.05 ± 3.46

214.84 ± 8.10

a

a

The specific conversion rate (E = cfinalHBP/CX/tBDS) was only determined for resting cells. 1447

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ACS Synthetic Biology

dszC using the NheI and HindIII sites. The resulting dszC mutant library was transformed into E. coli BL21 (DE3). High-Throughput Screening Method and Resting Cell Assays. E. coli BL21 (DE3) colonies carrying the mutant library were added individually into a 96-well plate, cultured at 37 °C and 200 rpm, and incubated overnight to obtain the parent well plates. All individual colonies were replicated via transfer from the parent well plates to second-generation deep well plates and were cultured again overnight. Each well was induced for 4 h with 0.2 mM IPTG under the same culture conditions. DBT (0.25 mM) was then added and the plate was incubated at 30 °C for 24 h in a 1050 rpm plate shaker to perform the whole-cell catalysis. The same amount of fermentation broth was taken from each well of the secondgeneration deep well plate and added to a 96-well enzyme plate to measure the OD600 to determine the cell concentration. The second-generation deep well plates were then centrifuged, and 200 μL of the supernatant was transferred to another 96-well enzyme plate for the Gibbs reaction. An appropriate amount of 10% Na2CO3 was added to each well to adjust the pH to 9−10, and 1 mg/mL Gibbs ethanol solution was added to each well. The 96-well plate was scanned at 610 nm, and strains that produced more HBP per unit of bacterial count (A610/A600) than the WT strain were selected for the second round of wellplate screening and shake flask rescreening. The resting cell assays primarily consisted of shake flask rescreening tests and in vivo verification tests. After the first culture was produced from a single colony on a plate, IPTG was added to the second culture when the OD600 reached 0.6− 0.8 and was induced at 16 °C and 180 rpm for 16 h. The cells were subsequently collected, washed and resuspended in PBS to an OD600 of 2.0, 5 mL of which was then mixed with DBT at a concentration required by the test. After whole cell catalysis was performed at 30 °C and 250 rpm, the samples were processed and measured by HPLC. Discovery Studio 3.0. The 3D space conformation of the small molecule ligands was constructed using Sketching. The DszC protein structure (ID:4JEK) was downloaded from the PDB database and prepared by cleaning the protein. The proteins were defined as receptor molecules to identify potential binding sites in the receptor. After the parameters were determined, the molecular docking calculations were performed using the Libdock method. Any amino acid in DszC could be mutated to the remaining 19 amino acids in silico, after which the binding free energy after docking with small molecules could be calculated. DszC Enzyme Assays. For the DszC enzyme assays, 400 μM NADH, 50 μM FMN, 10 μM DszD, 10 μM DszC, and variable concentrations of DBT and HBP were added to binding buffer (pH 4.0) to a final volume of 1 mL. The data for the optimization of the reaction temperature and pH for the DszC enzyme assay are shown in Figure S1. The reaction was performed for 5 min at 16 °C on a shaker at 1000 rpm. An equal volume of ethyl acetate was then added, and the mixture was shaken thoroughly prior to extraction of the ethyl acetate layer and subsequent HPLC analysis. The specific enzyme activity of DszC was defined as nanomoles of DBTO2 produced per minute per milligram of DszC at 16 °C and pH 4. Iterative Saturation Mutagenesis for DszC. Using pET28a-dszC as a template, site-directed mutagenesis primers and the KOD neo DNA polymerase, saturation mutagenesis of the 101st residue in DszC was performed. The PCR product

the results of this study may provide strategies for the practical application of crude oil desulfurization using solvent-tolerant hosts in the future. The results of this study have improved the biodesulfurization 4S pathway and have taken a major step toward the production of sulfur-free fuel oil. Furthermore, a successful case has been made for the desensitization of key enzymes in metabolic pathways that are subject to feedback inhibition.

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METHODS Media and Culture Conditions. LB medium (1% NaCl, 1% peptone and 0.5% yeast extract) was used to prepare E. coli competent cells and for shake flask fermentation. LB medium containing 50 μg/mL kanamycin, 100 μg/mL ampicillin, or 25 μg/mL chloramphenicol was used to screen for positive transformants containing the corresponding resistance marker. For shake flask fermentation, a single colony was picked from an agar plate containing the corresponding antibiotic and inoculated into 5−10 mL of LB medium containing the same antibiotic, which was then incubated at 37 °C in a shaker at 200 rpm for 13−16 h to obtain the seed culture for the first culture. The seed culture (1−2 mL) was inoculated into 100 mL of LB medium containing the same antibiotic, and the secondary culture was grown under the same culture conditions. Construction of Plasmids and Strains. Supporting Information Table S1 lists the plasmids (synthesized by Generay in Shanghai, China) that were used in this study. The plasmid pSB4A5 was used to express the dszA, dszB, dszC, and dszD genes (Figure 1A). PCR amplification was performed using the KOD or KOD neo DNA polymerase (Takara, Dalian, China) according to the manufacturer’s protocol. All of the restriction enzymes and the T4 ligase used in this study were purchased from Thermo Fisher Scientific (Shanghai, China). All kits used for DNA manipulation were purchased from Magen (Guangzhou, China). To produce the strains, the chemical thermal shock method was used to transform the adapter-ligated fragments or plasmids into competent E. coli cells. The transformants were selected for using the antibiotic corresponding to the resistance marker in the plasmid. All of the strains used in this study are listed in Supporting Information Table S1. Protein Expression and Purification. The plasmids were transformed into E. coli BL21 (DE3). Single colonies were picked from agar plates containing the correct antibiotics, which were used to produce the first (5−10 mL) and secondary (100 mL) cultures in liquid LB containing the corresponding antibiotic. When the OD600 reached 0.6−0.8, 0.2 mM IPTG was added to induce the culture, which was then incubated at 16 °C and 180 rpm for 16 h. The cells were collected, washed, and then suspended in 10 mL of binding buffer and disrupted by sonication. After centrifugation at high speed and low temperature, the supernatant containing the crude protein was loaded onto a Ni-NTA His-Bind Resin, and the protein was eluted with elution buffer containing 0.25 M imidazole. In the final step, the desired protein was collected. Error-Prone PCR and Construction of the E. coli Mutant Library. Mutations were introduced into the dszC gene using error-prone PCR52 with rTaq polymerase (Takara, Dalian, China) and 0.64 mM MnSO4. In the pSB4A5-BADC plasmid, the wild-type dszC gene was replaced with mutated 1448

DOI: 10.1021/acssynbio.9b00126 ACS Synth. Biol. 2019, 8, 1441−1451

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ACS Synthetic Biology Notes

was digested with DpnI and transformed into competent E. coli BL21 (DE3) cells. The strain containing the A101 K mutation, which was verified by sequencing, was used as template. The same experimental procedures were used for saturation mutagenesis of the 327th residue in DszC. Finally, the sequences of the site-directed mutations in DszC were determined. Analytical Methods. Absorbances at 600 and 610 nm (A600 and A610, respectively) were measured using an ultraviolet−visible spectrophotometer (Thermo Scientific; type: 354). The DBT, DBTO2 and HBP concentrations were determined via HPLC with a C18 column (Agilent, Zorbax SBC18, 250 × 4.6 mm, 5 μm particles), a mobile phase of acetonitrile−water (7:3), a flow rate of 0.7 mL/min, and a detection wavelength of 280 nm. Calibration was performed using highly purified standards for each compound. LC-MS Analysis. Chromatographic separation was achieved on C18 column (Agilent, Eclipse plus, 100 × 2.1 mm, 3.5 μm particles). The mobile phase consisted of acetonitrile−water (9:1) at a flow rate of 0.6 mL/min and run time of 10 min. The LC system was coupled with a triple quadrupole mass spectrometer (AB SciEx QTRAP4500) equipped with an electrospray ionization source (ESI). Other parameters of the mass spectrometer were: MS range: 100− 500; Mode of analysis: Negative; Curtain gas: 40 psi; Ion spray voltage: 4500 V; Source temperature: 450 °C; Ion source gas (gas1): 50 psi; Ion source gas (gas 2): 50 psi; Declustering potential: 100 V.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Grant No. 31470159), the National Science Foundation for Young Scientists of China (Grant No. 31400062), the Science and Technology Planning Project of Guangzhou City (Grant No. 201607010307), and the Fundamental Research Funds for the Central Universities (Grant No. 2017MS103).

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ABBREVIATIONS HDS, hydrodesulfurization; BDS, biodesulfurization; DBT, dibenzothiophene; HBP, 2-hydroxybiphenyl; DBTO, dibenzothiophene sulfoxide; DBTO2, dibenzothiophene sulfone; HBPS, 2-(2-hydroxybiphenyl) sulfinic acid salt; AKWC, the A101K/W327C mutant of DszC; AKWY, the A101K/W327Y mutant of DszC; C*, AKWC; LC-MS, liquid chromatograph mass spectrometer; HPLC, high-performance liquid chromatography

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(1) Su, T., Su, J., Liu, S., Zhang, C., He, J., Huang, Y., Xu, S., and Gu, L. (2018) Structural and Biochemical Characterization of BdsA from Bacillus subtilis WU-S2B, a Key Enzyme in the “4S” Desulfurization Pathway. Front. Microbiol. 9, 231. (2) Khosravinia, S., Mahdavi, M. A., Gheshlaghi, R., Dehghani, H., and Rasekh, B. (2018) Construction and Characterization of a New Recombinant Vector to Remove Sulfate Repression of dsz Promoter Transcription in Biodesulfurization of Dibenzothiophene. Front. Microbiol. 9, 1578. (3) Bhanjadeo, M. M., Rath, K., Gupta, D., Pradhan, N., Biswal, S., Mishra, B., and Subudhi, U. (2018) Differential desulfurization of dibenzothiophene by newly identified MTCC strains: Influence of Operon Array. PLoS One 13, e0192536. (4) Rahpeyma, S. S., Mohammadi, M., and Raheb, J. (2017) Biodesulfurization of Dibenzothiophene by Two Bacterial Strains in Cooperation with Fe3O4, ZnO and CuO Nanoparticles. J. Microb. Biochem. Technol. 9, 587−591. (5) Todescato, D., Maass, D., Mayer, D. A., Vladimir Oliveira, J., de Oliveira, D., Ulson de Souza, S. M. A. G., and Ulson de Souza, A. A. (2017) Optimal Production of a Rhodococcus erythropolis ATCC 4277 Biocatalyst for Biodesulfurization and Biodenitrogenation Applications. Appl. Biochem. Biotechnol. 183, 1375. (6) Khosravinia, S., Mahdavi, M. A., Gheshlaghi, R., and Dehghani, H. (2018) Author Correction: Characterization of Truncated dsz Operon Responsible for Dibenzothiophene Biodesulfurization in Rhodococcus sp. FUM94. Appl. Biochem. Biotechnol. 184, 897−897. (7) Denome, S. A., Olson, E. S., and Young, K. D. (1993) Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8. Appl. Environ. Microbiol. 59, 2837−2843. (8) Ferreira, P., Sousa, S. F., Fernandes, P. A., and Ramos, M. J. (2017) Improving the Catalytic Power of the DszD Enzyme for the Biodesulfurization of Crude Oil and Derivatives. Chem. - Eur. J. 23, 17231−17241. (9) Mishra, S., Panda, S., Pradhan, N., Satapathy, D., Biswal, S. K., and Mishra, B. K. (2017) Insights into DBT biodegradation by a native Rhodococcus strain and its sulphur removal efficacy for two Indian coals and calcined pet coke. Int. Biodeterior. Biodegrad. 120, 124−134. (10) Akhtar, N., Akhtar, K., and Ghauri, M. A. (2018) Biodesulfurization of Thiophenic Compounds by a 2-Hydroxybiphenyl-Resistant Gordonia sp. HS126−4N Carrying dszABC Genes. Curr. Microbiol. 75, 597−603.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.9b00126.

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REFERENCES

Bacterial strains and plasmids used in this study, possible pockets on DszC that may accommodate HBP, absorbance values of Gibbs reactions under different conditions, optimum conditions for the DszC enzyme assay, photographs of the Gibbs reaction under various conditions within 2 h, HPLC chromatogram of the resting cells reaction solution, disappearance of DBT and the production of HBP, LC-MS results using the reaction solution of the resting cells, the rate of conversion of DBT to HBP, and the ligand docking reports of HBP with AKWC and W327C (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail for S.L.: [email protected]. Tel./Fax: +86-02039380698. *E-mail for Y.L.: [email protected]. ORCID

Lu Li: 0000-0001-8957-5246 Shuli Liang: 0000-0002-9224-0049 Author Contributions

L.L., Y.L., and S.L.L. designed the research; L.L., Y.B.L., Y.F.L., G.M.Z., X.H.L., and W.Z. performed the experiments; X.H.L., S.P.Z., S.Y.H., Y.L., and S.L.L. provided guidance and experience; L.L. analyzed the data; L.L. and S.L.L. wrote the manuscript. All authors read and approved the final manuscript. 1449

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ACS Synthetic Biology (11) Yu, Y., Fursule, I. A., Mills, L. C., Englert, D. L., Berron, B. J., and Payne, C. M. (2017) CHARMM force field parameters for 2′hydroxybiphenyl-2-sulfinate, 2-hydroxybiphenyl, and related analogs. J. Mol. Graphics Modell. 72, 32−42. (12) Geronimo, I., Nigam, S., and Payne, C. (2017) Desulfination by 2’-hydroxybiphenyl-2-sulfinate desulfinase proceeds via electrophilic aromatic substitution by the Cysteine-27 proton. Chemical Science 8, 5078. (13) Kilbane, J. J., and Stark, B. (2016) Biodesulfurization: a model system for microbial physiology research. World J. Microbiol. Biotechnol. 32, 1−9. (14) Agarwal, M., Dikshit, P. K., Bhasarkar, J. B., Borah, A. J., and Moholkar, V. S. (2016) Physical insight into ultrasound-assisted biodesulfurization using free and immobilized cells of Rhodococcus rhodochrous MTCC 3552. Chem. Eng. J. 295, 254−267. (15) Martinez, I., Santos, V. E., Gomez, E., and Garcia-Ochoa, F. (2016) Biodesulfurization of dibenzothiophene by resting cells of Pseudomonas putida CECT5279: Influence of the oxygen transfer rate in the scale-up from shaken flask to stirred tank reactor. J. Chem. Technol. Biotechnol. 91, 184−189. (16) Reichmuth, D. S., Hittle, J. L., Blanch, H. W., and Keasling, J. D. (2000) Biodesulfurization of dibenzothiophene in Escherichia coli is enhanced by expression of a Vibrio harveyi oxidoreductase gene. Biotechnol. Bioeng. 67, 72−79. (17) Luo, M. F., Xing, J. M., Gou, Z. X., Li, S., Liu, H. Z., and Chen, J. Y. (2003) Desulfurization of dibenzothiophene by lyophilized cells of Pseudomonas delaf ieldii R-8 in the presence of dodecane. Biochem. Eng. J. 13, 1−6. (18) Calzada, J., Heras, S., Alcon, A., Santos, V. E., and Garciaochoa, F. (2009) Biodesulfurization of Dibenzothiophene (DBT) Using Pseudomonas putida CECT 5279: A Biocatalyst Formulation Comparison. Energy Fuels 23, 5491−5495. (19) Gunam, I. B., Yamamura, K., Sujaya, I. N., Antara, N. S., Aryanta, W. R., Tanaka, M., Tomita, F., Sone, T., and Asano, K. (2013) Biodesulfurization of dibenzothiophene and its derivatives using resting and immobilized cells of Sphingomonas subarctica T7b. J. Microbiol. Biotechnol. 23, 473. (20) Dejaloud, A., Vahabzadeh, F., and Habibi, A. (2017) Ralstonia eutropha as a biocatalyst for desulfurization of dibenzothiophene. Bioprocess Biosyst. Eng. 40, 969−980. (21) Martínez, I., Mohamed, E. S., Rozas, D., García, J. L., and Díaz, E. (2016) Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds. Metab. Eng. 35, 46. (22) Martínez, I., El-Said Mohamed, M., Santos, V. E., García, J. L., Garcíaochoa, F., and Díaz, E. (2017) Metabolic and process engineering for biodesulfurization in Gram-negative bacteria. J. Biotechnol. 262, 47−55. (23) Abinfuentes, A., Mohamed, E. S., Wang, D. I. C., and Prather, K. L. J. (2013) Exploring the Mechanism of Biocatalyst Inhibition in Microbial Desulfurization. Appl. Environ. Microbiol. 79, 7807−7817. (24) Li, G. Q., Li, S. S., Zhang, M. L., Wang, J., Zhu, L., Liang, F. L., Liu, R. L., and Ma, T. (2008) Genetic rearrangement strategy for optimizing the dibenzothiophene biodesulfurization pathway in Rhodococcus erythropolis. Appl. Environ. Microbiol. 74, 971−976. (25) Coco, W. M., Levinson, W. E., Crist, M. J., Hektor, H. J., Darzins, A., Pienkos, P. T., Squires, C. H., and Monticello, D. J. (2001) DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat. Biotechnol. 19, 354. (26) Ohshiro, T., Ohkita, R., Takikawa, T., Manabe, M., Lee, W. C., Tanokura, M., and Izumi, Y. (2007) Improvement of 2-Hydroxybiphenyl-2-sulfinate Desulfinase, an Enzyme Involved in the Dibenzothiophene Desulfurization Pathway, from Rhodococcus erythropolis KA2−5-1 by Site-Directed Mutagenesis. Biosci., Biotechnol., Biochem. 71, 2815−2821. (27) Kamali, N., Tavallaie, M., Bambai, B., Karkhane, A. A., and Miri, M. (2010) Site-directed mutagenesis enhances the activity of NADHFMN oxidoreductase (DszD) activity of Rhodococcus erythropolis. Biotechnol. Lett. 32, 921−927.

(28) Pan, J., Wu, F., Wang, J., Yu, L., Khayyat, N. H., Stark, B. C., and Kilbane, J. J. (2013) Enhancement of desulfurization activity by enzymes of the Rhodococcus dsz operon through coexpression of a high sulfur peptide and directed evolution. Fuel 112, 385−390. (29) Xu, P., Yu, B., Li, F. L., Cai, X. F., and Ma, C. Q. (2006) Microbial degradation of sulfur, nitrogen and oxygen heterocycles. Trends Microbiol. 14, 398−405. (30) Bordoloi, N. K., Rai, S. K., Chaudhuri, M. K., and Mukherjee, A. K. (2014) Deep-desulfurization of dibenzothiophene and its derivatives present in diesel oil by a newly isolated bacterium Achromobacter sp. to reduce the environmental pollution from fossil fuel combustion. Fuel Process. Technol. 119, 236−244. (31) Akhtar, N., Ghauri, M. A., Anwar, M. A., and Akhtar, K. (2009) Analysis of the dibenzothiophene metabolic pathway in a newly isolated Rhodococcus spp. FEMS Microbiol. Lett. 301, 95−102. (32) Akhtar, N., Ghauri, M. A., Anwar, M. A., and Heaphy, S. (2015) Phylogenetic characterization and novelty of organic sulphur metabolizing genes of Rhodococcus spp. (Eu-32). Biotechnol. Lett. 37, 837−847. (33) Liu, S., Zhang, C., Su, T., Wei, T., Zhu, D., Wang, K., Huang, Y., Dong, Y., Yin, K., Xu, S., Xu, P., and Gu, L. (2014) Crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å. Proteins: Struct., Funct., Genet. 82, 1708−1720. (34) Guan, L. J., Lee, W. C., Wang, S., Ohshiro, T., Izumi, Y., Ohtsuka, J., and Tanokura, M. (2015) Crystal structures of apo- and FMN-bound DszC from Rhodococcus erythropolis D-1. FEBS J. 282, 3126−3135. (35) Hino, T., Hamamoto, H., Suzuki, H., Yagi, H., Ohshiro, T., and Nagano, S. (2017) Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11−2, reveal potential for expanding its substrate selectivity. J. Biol. Chem. 292, jbc.M117.788513. (36) Zhang, L., Duan, X., Zhou, D., Dong, Z., Ji, K., Meng, W., Li, G., Li, X., Yang, H., Ma, T., and Rao, Z. (2014) Structural insights into the stabilization of active, tetrameric DszC by its C-terminus. Proteins: Struct., Funct., Genet. 82, 2733−2743. (37) Raheb, J., and Hajipour, M. J. (2011) The Effect of Elimination of the dszC Gene in Energy Consuming of Biodesulfurization in Broken 4S Pathway. Energy Sources, Part A 33, 1814−1821. (38) Konishi, J., Onaka, T., Ishii, Y., and Suzuki, M. (2000) Demonstration of the carbon-sulfur bond targeted desulfurization of benzothiophene by thermophilic Paenibacillus sp. strain A11−2 capable of desulfurizing dibenzothiophene. FEMS Microbiol. Lett. 187, 151−154. (39) Zhang, Y., Meng, Q., Ma, H., Liu, Y., Cao, G., Zhang, X., Zheng, P., Sun, J., Zhang, D., Jiang, W., and Ma, Y. (2015) Determination of key enzymes for threonine synthesis through in vitro metabolic pathway analysis. Microb. Cell Fact. 14, 86. (40) Reichmuth, D. S., Blanch, H. W., and Keasling, J. D. (2004) Dibenzothiophene biodesulfurization pathway improvement using diagnostic GFP fusions. Biotechnol. Bioeng. 88, 94−99. (41) Li, G.-q., Ma, T., Li, S.-s., Li, H., Liang, F. L., and Liu, R. L. (2007) Improvement of Dibenzothiophene Desulfurization Activity by Removing the Gene Overlap in the dsz Operon. Biosci., Biotechnol., Biochem. 71, 849−854. (42) Li, M. Z., Squires, C. H., Monticello, D. J., and Childs, J. D. (1996) Genetic analysis of the dsz promoter and associated regulatory regions of Rhodococcus erythropolis IGTS8. J. Bacteriol. 178, 6409− 6418. (43) Barbosa, C. C., Neves, R. P. P., Sousa, S. F., Ramos, M. J., and Fernandes, P. A. (2018) Mechanistic Studies of a Flavin Monooxygenase: Sulfur Oxidation of Dibenzothiophenes by DszC. ACS Catal. 8, 9298−9311. (44) Nsoe, M. N., Kofa, G. P., Ndi, K. S., Mohammadou, B., Heran, M., and Kayem, G. J. (2018) Biodegradation of Ammonium Ions and Formate During Ammonium Formate Metabolism by Yarrowia lipolytica and Pichia guilliermondii in a Batch Reactor. Water, Air, Soil Pollut. 229, 162. 1450

DOI: 10.1021/acssynbio.9b00126 ACS Synth. Biol. 2019, 8, 1441−1451

Research Article

ACS Synthetic Biology (45) Dutta, K., Dasu, V. V., Mahanty, B., and Prabhu, A. A. (2015) Substrate Inhibition Growth Kinetics for Cutinase Producing Pseudomonas cepacia Using Tomato-peel Extracted Cutin. Chem. Biochem. Eng. Q. 29, 437−445. (46) Gray, K. A., Pogrebinsky, O. S., Mrachko, G. T., Xi, L., Monticello, D. J., and Squires, C. H. (1996) Molecular mechanisms of biocatalytic desulfurization of fossil fuels. Nat. Biotechnol. 14, 1705− 1709. (47) Santos, V. E., Alcón, A., Martı ́n, A. B., Gómez, E., and GarciaOchoa, F. (2007) Desulfurization of dibenzothiophene using the 4S enzymatic route: Influence of operational conditions on initial reaction rates. Biocatal. Biotransform. 25, 286−294. (48) Maita, N., Okada, K., Hirotsu, S., Hatakeyama, K., and Hakoshima, T. (2001) Preparation and crystallization of the stimulatory and inhibitory complexes of GTP cyclohydrolase I and its feedback regulatory protein GFRP. Acta Crystallogr., Sect. D: Biol. Crystallogr. 57, 1153−1156. (49) Chitour, Y., Grognard, F., and Bastin, G. (2014) Stability analysis of a metabolic model with sequential feedback inhibition. In International Symposium on Positive Systems (Benvenuti, L., De Santis, A., and Farina, L., Eds.), Vol. 294, Springer, Berlin, Heidelberg. (50) Malykh, E. A., Butov, I. A., Ravcheeva, A. B., Krylov, A. A., Mashko, S. V., and Stoynova, N. V. (2018) Specific features of l -histidine production by Escherichia coli concerned with feedback control of AICAR formation and inorganic phosphate/metal transport. Microb. Cell Fact. 17, 42. (51) Meesala, L., Balomajumder, C., Chatterjee, S., and Roy, P. (2008) Biodesulfurization of dibenzothiophene using recombinant Pseudomonas strain. J. Chem. Technol. Biotechnol. 83, 294−298. (52) Cirino, P. C., Mayer, K. M., and Umeno, D. (2003) Generating Mutant Libraries Using Error-Prone PCR. Methods Mol. Biol. 231, 3.

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DOI: 10.1021/acssynbio.9b00126 ACS Synth. Biol. 2019, 8, 1441−1451