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Combining Pro-peptide Engineering and Multisite Saturation Mutagenesis To Improve the Catalytic Potential of Keratinase Chang Su,† Jin-Song Gong,*,† Yu-Xin Sun,† Jiufu Qin,§ Shen Zhai,† Heng Li,† Hui Li,† Zhen-Ming Lu,†,‡ Zheng-Hong Xu,†,‡ and Jin-Song Shi*,†

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Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, P. R. China ‡ National Engineering Laboratory for Cereal Fermentation Technology, School of Biotechnology, Jiangnan University, Wuxi 214122, P. R. China § Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark S Supporting Information *

ABSTRACT: Keratinases are becoming biotechnologically important since they have shown potential in hydrolysis of recalcitrant keratins with highly rigid and strongly cross-linked structures. However, the large-scale application of keratinases has been limited by the inefficient expression level and low enzyme activity. In this work, we employed pro-peptide engineering and saturation mutagenesis to construct excellent keratinase variants with improved activities. It turned out that amino acid substitutions at the pro-peptide cleavage site (P1) could accelerate the release of active mature enzymes, resulting in a 3-fold activity increase. Eighteen sites of the pro-peptide area were targeted for codon mutagenesis, and a multisite saturation mutagenesis library of the six potential sites was generated, achieving a significant improvement of keratinase activity from 179 to 1114 units/mL. Also, the mutants exhibited alterant catalytic properties. Finally, fermentation for keratinase production in a 15 L fermenter was carried out, and the enzyme activity reached up to over 3000 units/mL. Our results demonstrated that propeptide engineering played a crucial role in high expression and engineering of proteases. This study provides a universal route toward improvement of industrial enzymes that were first synthesized as precursors in the form of pre-pro-protein. KEYWORDS: keratinase, expression, pro-peptide engineering, saturation mutagenesis, modification

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the encoding genes is expected to be a powerful tool to improve the expression and activity of keratinases. Like subtilisins, keratinases are first synthesized as precursors in the form of pre-pro-proteins. The pro-peptide, as the intermolecular chaperone, plays an important role in guiding the correct folding of the mature domain. It may directly catalyze the folding reactions or facilitate processes such as structural organization and oligomerization, localization, and sorting. Thus, by mutations at appropriate sites of the propeptide, the folding rate of the protein can be altered to accelerate the maturation of the enzyme,7 increase the amount

eratinases are considered to be special industrially applicable proteolytic enzymes that exhibit the capability of specific hydrolysis of insoluble and recalcitrant keratin wastes such as chicken feathers, animal wool, and hair. Because of their reaction specificity and catalytic performance, keratinases have stood out among normal proteases and gradually showed advantages in applications such as feed and fertilizers,1 detergents,2 leather industries,3 cosmetics,4 and biomedical fields and also exhibited application potential in the preparation of nanomaterials.5 Thus, keratinase mining and production have attracted increasing attention in recent years. However, a potential strain capable of efficiently expressing keratinase with favorable activity is still the main obstacle for industrial applications. With the daily maturation of protein engineering technology, rational or irrational modification of © XXXX American Chemical Society

Received: October 24, 2018

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DOI: 10.1021/acssynbio.8b00442 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology

Figure 1. Deletion and truncation mutagenesis of the pro-peptide region. (a) SDS-PAGE analysis of pro-peptide deletion. M, protein marker; lane 1, pMA5/WB600; lanes 2 and 3: pro-peptide removal mutants; lane 4, KerBp. PK represents precursor keratinase, and MK represents mature keratinase. (b) Analysis and truncation mutagenesis of the pro-peptide secondary structure and results of enzyme activity assays for the truncated mutants.

of enzyme expression,8 or change the structure of the mature enzyme.9 However, the pro-peptide can function as a competitive inhibitor of the mature enzyme by obligatorily bonding with the active site to form a stable and inactive propeptide−enzyme complex.9 Thus, the pro-peptide must be cleaved and degraded after it has finished the guiding process, and then the active mature enzyme is released (Figure S1). On the basis of the folding mechanism mentioned above, pro-peptide engineering was employed for functional modification of the keratinase gene from Bacillus pumilus (kerBp) in this study. First, deletion and truncation of the pro-peptide were performed to verify the crucial role of the pro-peptide area on the folding of the active mature enzyme and investigate the key region. Then the residue at the P1 position was replaced with different amino acids to promote the cleavage efficiency. Finally, in order to further enhance the enzyme activity, 18 sites in the pro-peptide region were screened to identify the most promising single-point mutations, and the dominant sites were subjected to multisite saturation mutagenesis.

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the correct expression of KerBp. Therefore, pro-peptide engineering was employed to improve the expression level in the subsequent studies. The length of pro-peptide sequences generally varies in different kinds of protease: there are 77 amino acids in the case of subtilisin and 177 residues in neutral metalloprotease.11 The study by Sharma et al.12 revealed that only 187 bp among the whole length of the 520 bp pro-sequence region is required for correct folding of the protein into its active conformation. Thus, the pro-peptide sequence was truncated to explore the critical part and pave the way for site-directed mutations by narrowing the site selection range. Through analysis of the homology model, it was found that the pro-peptide sequence of keratinase consists of five β-sheets, one α-helix, and several random coils. The β-sheets and α-helix were generally considered to play a relatively important role in the secondary structure, and thus, each of the β-sheets and the α-helix were truncated successively to construct different mutants (Figure 1b). However, the results of activity assays showed that different truncations of the pro-peptide led to significant decreases in enzyme activity, which indicated that the full length of the pro-peptide sequence was necessary for the correct folding and functional expression of KerBp. This is in accordance with the conclusion of Sharma et al.12 that the full-length keratinase KerP F1 was more catalytically efficient than the truncated forms. Amino Acid Substitutions at the Cleavage Site of the P1 Position. The cleavage at the P1 position of the propeptide is a crucial rate-limiting step in the formation of mature enzymes.13 It has been reported that the cleavage is more biased toward aromatic hydrophobic amino acids.14 According to Li et al.,15 the L(P1)F mutation improved the expression level of mature Sfp2 with 9 times the specific activity. Takahashi et al.16 substituted Tyr of subtilisin E156Q and G166K at the P1 site with Asp and Glu and achieved

RESULTS AND DISCUSSION

Deletion and Truncation of the Pro-peptide Area. A keratinase coding gene (kerBp) was mined and sequenced in our previous study.10 In silico analysis revealed that the sequence was composed of a signal area along with a propeptide and a core region. In order to ensure the crucial role of the pro-peptide in the activation of the mature keratinase for KerBp, the pro-peptide region was knocked out. The results showed that no keratinase activity was detected, though a protein band (Figure 1a) with a molecular weight of 30 kDa could be observed in SDS-PAGE. This validation test demonstrated that the keratinase could not exhibit activity without the direction of the pro-peptide area, which also confirmed the critical role of the pro-peptide in determining B

DOI: 10.1021/acssynbio.8b00442 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology

Figure 2. Results of substitution at the P1 position. (a) Transparent zones on milk plates produced by mutants after incubation for 12 h in a 37 °C incubator. (b) Activities of mutants after incubation in liquid LB medium for 48 h. (c) SDS-PAGE analysis of KerBp and mutants. M, protein marker; lane 1, pMA5/WB600; lane 2, KerBp; lane 3, Y(P1)F; lane 4, Y(P1)W; lane 5, Y(P1)A; lane 6, Y(P1)D; lane 7, Y(P1)E. PK represents the precursor keratinase, and MK represents the mature keratinase. (d) Analysis of homology models of KerBp and Y(P1)F at the P1 position.

Site-Directed Mutagenesis of the Pro-peptide. In order to further explore the potential sites of the pro-peptide region that might have effects on folding of the mature enzyme, site-directed mutagenesis was performed on the basis of analysis and alignment with homologous keratinase coding sequences with high activity and favorable thermostability (Figure S2). Eighteen sites were selected for mutagenesis, including S(42)D, A(49)S, A(51)V, T(52)K, N(54)A, A(60)I, I(62)K, N(64)S, Q(79)K, V(80)A, S(83)D, A(87)L, K(89)E, E(91)K, S(95)D, K(104)V, E(106)H, and Y(108)L. Also, the transparent circles were first observed to preliminarily evaluate the effects of these mutations (Figure 3a). Then a specific activity comparison was performed by enzyme activity assays. Remarkably, the S(42)D, A(49)S, N(54)A, I(62)K, E(91)K, and S(95)D mutants showed increased activity, and especially, a 2.2-fold increase was observed for I(62)K compared with KerBp (from 179 to 402 units/mL) (Figure 3b). The results are in accordance with the study of Uehara et al.,7 who observed that by mutation at appropriate sites of the propeptide, the folding rate of the protein could be altered to accelerate the maturation of the enzyme, thereby increasing the expression level. The results of Rakestraw et al.6 also suggested that engineering of the pre and pro regions may be a more general approach than genomic mutations, which typically enhance productivity for particular proteins. Apart from the enzyme activity, we wondered whether the mutations of pro-peptides could cause changes in the catalytic properties and structure of the mature enzyme. Therefore, the catalytic properties of the mutants were investigated, including the optimal reaction pH, temperature, and substrate specificity. The results showed that the optimum pH for Y(P1)F, A(49)S, N(54)A, and KerBp was pH 10.0, whereas that for T(52)K, A(60)I, I(62)K, K(89)E, and S(95)D was pH 11.0 (Figure 3c). On the other hand, all of the mutants had the same

significant improvement in enzyme activity and yield. In the present study, Tyr at the P1 position of the pro-peptide cleavage site was replaced by two aromatic hydrophobic amino acids (Phe and Trp) and three other residues (Ala, Asp, and Glu) with potential promoting effects for enzyme secretion. The mutants Y(P1)F, Y(P1)W, Y(P1)A, Y(P1)D, and Y(P1)E were successfully constructed and showed improved or decreased enzyme activities to varying degrees, which were first evaluated by the sizes of the clear zones surrounding the transformants on the milk plate (Figure 2a). It could be clearly observed that the transparent circles of Y(P1)F were significantly larger than those of wild-type KerBp and other mutants, while the Y(P1)D mutant could generate only very weak clear zones, indicating a substantial decline in enzyme activity. Then the enzyme activity was quantified with 1% soluble keratin as the substrate. The results were consistent with the observation of transparent zones, among which the Y(P1)F mutant showed an excellent 3-fold activity increase at the same level of cell density (Figure 2b). The SDS-PAGE results showed two protein bands with molecular weights of about 40 and 30 kDa, representing the precursor and mature keratinase, respectively. As shown in Figure 2c, the mature keratinase band of Y(P1)F was darker than that of KerBp, indicating that this mutation at the P1 site accelerated the release efficiency of mature keratinase, and thus, more active mature keratinase was released at the same level of biomass within the same time. On the basis of the above results, it is inferred that the aromatic hydrophobic amino acid Phe is more conducive to the cleavage of the pro-peptide. Through the analysis of homology models, it could be observed that the Y(P1)F mutation reduced the p-hydroxyl on the phenyl ring of the amino acid residue at the P1 position (Figure 2d), which suggested that large P1 residues are inaccessible to the pocket because of steric hindrance. C

DOI: 10.1021/acssynbio.8b00442 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology

Figure 3. Activities and catalytic properties of site-directed mutations of the pro-peptide area. (a) Comparison of transparent circles produced on milk plates of mutants. (b) Comparison of mutant enzyme activities. The inset shows the chromogenic results during the process of the activity assay: the darker the color, the higher the enzyme activity. (c) Optimum pH values for the mutants. (d) Optimum temperatures for the mutants. (e) Thermostabilities of the mutants, as shown by the residual activities of the mutants after incubation at 40 °C for 30, 60, or 90 min. (f) Substrate specifities of the mutants. The activities toward keratinase and casein were tested, and the K:C ratios were compared. (g) Steady-state kinetic analysis of the mutant with the highest activity.

optimal temperature of 40 °C as KerBp (Figure 3d); however, these mutants showed distinct differences in thermostability (Figure 3e). After incubation at 40 °C for 90 min, the Y(P1)F, T(52)K, and S(95)D mutants still retained more than 80% of the activity, which is approximately equivalent to the wild-type KerBp, while other mutants suffered from significant losses. Generally, the ratio of the activities toward keratin and casein

(K:C) could be used to evaluate a keratinase when K:C is higher than 0.5. Here, the K:C ratios of the mutants were compared to characterize the change in the binding capacity. As shown in Figure 3f, A(49)S and I(62)K showed higher K:C ratios. These results indicated that mutation of the pro-peptide area caused conformation alterations between KerBp and the mutants in terms of thermostability and catalytic properties. D

DOI: 10.1021/acssynbio.8b00442 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology

Figure 4. Results of multisite saturation mutagenesis. (a) Initial screening of mutant enzyme activity. Blue represents the enzyme activity of the wild-type (WT) strain, and green represents the activities of the mutants chosen for fermentation and further enzyme assay. (b) Fermentative enzyme production results. Blue represents the enzyme activity of the WT strain, and green represents the activities of the top mutants with the most obvious improvements. (c) SDS-PAGE analysis of multisite saturation mutants. Keratinases of 36 mutants were randomly selected from the fermentation supernatant for SDS-PAGE analysis. However, only a single band of about 30 kDa was detected in the unpurified fermentation broth, which is the mature keratinase. It is assumed that keratinase degraded the other proteins in the fermentation broth because of its strong protease activity.

The previous studies by Takagi et al.9 suggested that point mutations within the pro-peptide can affect the structure and activity of the mature enzyme. Shinde et al.17 also proposed the “protein memory” theory, in which an identical polypeptide can fold into an altered conformation with different secondary structure, stability, and substrate specificities through a mutated pro-peptide sequence. Multisite Saturation Mutagenesis. After the sites that might have potential effects on the viability and catalytic performance of the mature keratinase were successfully identified, saturation mutagenesis of the specific sites (42, 49, 54, 62, 91, and 95) was performed to explore the optimal amino acid at each site. However, NNN, NNK, or NNS degenerate codons were utilized for randomizing saturation mutagenesis in most literature reports, which resulted in overlapping mutation and the formation of premature stop codons. This phenomenon has led to a great screening burden. Therefore, the “22c-trick” approach was attempted in this study, which uses a mixture of three primers per randomized positionnamely, NDT, VHG, and TGG (where D = A/G/T, V = A/C/G, H = A/C/T), in a 12:9:1 molar ratioresulting in a zero probability for premature stop codons and an almost uniform amino acid distribution: 2/22 for Leu and Val and 1/ 22 for each of the remaining 18 amino acids.18 Herein a strategy of multisite saturation mutagenesis was carried out by combining simultaneous and iterative saturation mutagenesis to achieve a random recombination of mutated amino acid residues to generate a focused mutant library. The strategy made a superior mutation effect with relatively short working time and less screening pressure. Libraries were constructed on the basis of grouping of mutation sites and two rounds of fusion PCR (Figure S3).

After the successful construction of the mutant libraries, a highly efficient screening method was particularly important. The first round of screening was conducted on the basis of the speed and size of transparent circle production. A total of 160 (65 and 95) mutant transformants were first picked out and inoculated into liquid LB medium for a second round of screening via activity assays. According to the two rounds of screening (Figure 4a), a total of 46 (28 and 18) mutants with enhanced enzyme activity were further selected for fermentation and enzyme assays. As a result, 15 strains exhibited higher activities of over 800 units/mL compared with the wild-type keratinase, and the highest reached 1114 units/mL, representing a 6-fold improvement (Figure 4b). Meanwhile, the cell density of the mutant showed an equivalent level with the wild type. The SDS-PAGE protein electrophoresis analysis of the fermentation supernatant of mutants showed a single band at about 30 kDa (Figure 4c). It was hypothesized that the keratinase could degrade other heteroproteins, and thus, the target protein band was relatively single. These mutants with improved activity were sequenced, which revealed that all of the sequenced clones harbored at least one mutant codon, and the mutation efficiency reached 100% (Figure S4). In order to directly observe the amino acid changes of the mutation sites that conferred higher expression levels, models of KerBp and the top mutant (M7) were acquired via homology modeling. They were constructed on the basis of the crystal structure of the un-autoprocessed form of IS1-inserted pro-subtilisin E (PDB ID 3whi.1.A), which shares 70% sequence identity. The acquired models were visualized and further analyzed using PyMOL software (Figure S5). Among the six mutation sites, five amino acids were changed in mutant M7 (S(42)D, N(54)F, I(62)E, E(91)N and E

DOI: 10.1021/acssynbio.8b00442 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology

Figure 5. Growth and enzyme production of mutant M7 in a 15 L biofermenter. (a) Growth and enzyme production curve. (b) SDS-PAGE analysis of the supernatant of the fermentation broth after culturing for 44 h.

combination of pro-peptide engineering and multisite saturation mutagenesis strategies. When the fermentation process was expanded in a 15 L fermentor, the highest activity reached 3040 units/mL under the same fermentation conditions. The results proved that the pro-peptide area has a great influence on the expression and conformation of the active mature keratinase. As a means of molecular modification, pro-peptide engineering can improve the change of the catalytic center to achieve a synergistic effect. Overall, we constructed a genetically engineered strain for high expression of keratinase with commercial value and provided a universal route toward improvement of the self-processed enzyme.

S(95)D), leading to a higher expression level of mature keratinase. This may refer to the transitions from α-helices to random coils. The transitions made the overall structure of the pro-peptide area less rigid and more flexible, which was more favorable for guiding the folding of mature enzymes. Analysis of the far-UV circular dichroism spectra also showed similar but different trendlines for the wild-type enzyme and the mutant, proving that the mutation of the pro-peptide changed the secondary structure of the mature keratinase. Fermentation of Recombinant Keratinase in a 15 L Fermenter. In order to characterize the fermentation process of recombinant keratinase and lay the foundation for its industrial production, scale-up fermentation from a shaking flask to a 15 L fermenter was carried out for mutant M7. The cell density and activities were detected at regular intervals, showing the growth and enzyme production progress as a function of time (Figure 5). It was observed that the strain thrived without efficient accumulation of enzyme during the first 8 h. At the 24th hour, the cell density reached the maximum, and in this phase keratinase was continuously produced and accumulated. However, the cell density began to remain nearly stable after 24 h, while the amount of enzyme continued to rise, reaching the maximum at the 32nd hour with an activity of 3040 units/mL. Recently, various efforts to improve the keratinase expression and production have been reported in the literature. Hu et al.19 expressed the keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli with two vectors, pET30a and pET32a, which achieved specific keratinase activities of 9.8 units/mg (total activity of 26.8 units/mL) and 7.2 units/mg (19.4 units/mL), respectively. Nahar et al.20 cloned the keratinase gene from B. licheniformis strain MZK05 and subsequently expressed it in E. coli BL21, resulting in an increased keratinolytic activity of 196 units/mL. Dong et al.21 overexpressed a novel keratinase from Bacillus polyfermenticus B4 in recombinant Bacillus subtilis via the T7 promoter, which reached a high activity of 473 ± 20 units/mL. It might be concluded that the mutant M7 has great potential in industrial applications, and in subsequent works detailed optimization studies toward keratinase production should be carried out in succession. In summary, although the unique importance of the degradation ability and catalytic application potential of keratinase has attracted increasing attention from scholars and entrepreneurs, the enzyme activity and expression level are still unsatisfactory. Herein we achieved a significant improvement of keratinase activity from 179 to 1114 units/mL via the

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MATERIALS AND METHODS Materials. The keratinase gene kerBp was obtained in our previous study. E. coli JM109 and B. subtilis WB600, preserved in our laboratory, were used as the cloning host and expression host, respectively. The pMD19-T simple vector was obtained from TaKaRa (China). The expression vector, pMA5, was preserved in our laboratory. Taq and PrimerSTAR HS DNA polymerase, T4 DNA ligase, and restriction enzymes were purchased from TaKaRa. The substrate for the enzyme assay, 5% soluble keratin, was purchased from J&K (China). Plasmid extraction and gel elution kits were purchased from Generay Biotechnology (China). Host cells were cultivated at 37 °C for 12 h in LB medium and TB medium for fermentation. Expression of KerBp in the Bacillus subtilis System. KerBp was amplified from the genome of B. pumilus with genespecific primers designed according to the encoding sequence. The restriction sites BamH I and Mlu I were also introduced by the primers. The PCR products were gel-purified and ligated with the pMD19-T simple vector, after which the ligation product was transformed into the cloning host E. coli JM109 and incubated at 37 °C on LB agar containing 100 μg/mL ampicillin for 12 h. The positive colonies were inoculated into 10 mL of LB with addition of ampicillin and cultivated for 10− 12 h. Plasmids were extracted from positive transformants and verified by DNA sequencing (Sangon Biotech). The cloning plasmids and the expression vector, pMA5, were digested by the restriction endonucleases BamH I and Mlu I. They were subsequently ligated using T4 DNA ligase at 16 °C for 8 h to construct the expression plasmid kerBp-pMA5, which was then transformed into the cloning host E. coli JM109 with a heat-shock procedure. Plasmids of the transformants were extracted and transformed into B. subtilis WB600 for keratinase expression. F

DOI: 10.1021/acssynbio.8b00442 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology Culture Conditions. The recombinant strains were first cultured in 10 mL of LB medium (100 μg/mL kanamycin) at 37 °C for 12 h. Subsequently, the seed liquor was inoculated into 50 mL of TB medium (100 μg/mL kanamycin) for further cultivation at 37 °C for 48 h. B. subtilis WB600 carrying pMA5, used as the control group, was also cultured under the same conditions. The fermentation broths were centrifuged, and the supernatants were collected for activity assay and SDS-PAGE analysis. Keratinase Activity Assay. The enzymatic activity was determined at pH 9.0 and 40 °C using 1% soluble keratin as the substrate according to the method of Yamamura et al.22 with modifications. The reaction mixture contained 1 mL of enzyme and 1 mL of 1% soluble keratin diluted in Tris-HCl buffer (0.05 M, pH 9.0). The reaction proceeded by incubation of the mixture at 40 °C for 15 min and was terminated with 2 mL of 5% TCA. The mixture was centrifuged at 12000g for 5 min to remove residual substrate after standing for 10 min. A 1 mL aliquot of the supernatant was mixed with 1 mL of Folin phenol reagent and 5 mL of 0.4 M Na2CO3 at 40 °C for 20 min. The keratinase activity was measured at 660 nm against a control group, the reaction of which was immediately terminated after addition of the enzyme. One unit of keratinase activity is defined as an increase of 0.01 in the optical density (OD) value at 660 nm in 15 min. All of the assays were performed in triplicate. SDS-PAGE Analysis. The supernatants of fermentation broths were collected by centrifugation at 8000g for 20 min at 4 °C. Protein concentration was determined by the Coomassie brilliant blue G-250 dye-binding method with bovine serum albumin as the standard. The fermentation supernatants were mixed with 5× loading buffer, and the mixtures were boiled for 5 min and then subjected to SDS-PAGE. After electrophoresis, gels were stained with Coomassie blue R-250. Verification of the Key Role of the Pro-peptide. The pro-peptide region was deleted to verify its effect on the mature keratinase. The signal and mature keratinases were amplified via the primers signal (F), signal (D) and ker (F), ker (D), respectively (Table S1), with the kerBp-pMA5 plasmid as the template. The PCR products were gel-purified and taken as the template for a round of fusion PCR to obtain the signal− mature keratinase (SMF) gene with signal (F) and ker (D) as primers (Figure S6). The SMF gene fragment was ligated into the pMA5 vector by the restriction enzymes BamH I and Mlu I as described above to construct the expression plasmid SMFpMA5, which was then transformed into the B. subtilis WB600 host cells for the expression of keratinase. Truncation of the Pro-peptide. The kerBp-pMA5 plasmid was used as the template to amplify the signal peptide sequence and the truncated mutation sequence by designing primers with different homologous arms (Table S2), and then the signal peptide was added to the N-terminus of the mature enzyme with different lengths of pro-peptide through a round of fusion PCR. Different mutant gene fragments were also ligated into the pMA5 vector by restriction enzymes and T4 DNA ligase. The ligation products were then transformed into the Bacillus subtilis WB600 host cells. The recombinant strains were cultured, and the supernatant of the fermentation broth was obtained for detection of the enzyme activity. Amino Acid Substitutions at the Cleavage Site of the P1 Position. Site-directed mutagenesis was performed using the one-step reverse PCR method according to the Quick Change site-directed mutagenesis kit provided by Stratagene

(San Diego, CA, USA). It was carried out using the thermostable DNA polymerase PrimeSTAR HS with high fidelity. The kerBp-pMA5 plasmid from E. coli JM109 was taken as a DNA template. One-step reverse PCR (the system is shown in Table S3, and the program is shown in Table S4) was carried out by using partially complementarity primers (Table S5) containing the mutation sites to amplify the mutated linear plasmids. The template plasmid DNA that can be methylated was then digested with Dpn I restriction enzyme. Subsequently, the enzymatically digested product was directly transformed into E. coli JM109, which has a repair function to repair and circularize linear mutant plasmids. The plasmids extracted from the mutant transformants were then transformed into B. subtilis WB600 for expression of keratinase. Site-Directed Mutagenesis of Pro-peptide Area. The amino acid residues were selected for mutation according to the comparative analysis of homologous sequences and introduced by one-step reverse PCR as described above. Mutation plasmids were obtained with the recombinant plasmid of kerBp-pMA5 from E. coli JM109 as the template and a pair of partially complementary oligonucleotide sequences containing the mutation site as primers (Table S6). Afterward, the methylated template DNA was digested with Dpn I enzyme. The amplified mutant plasmids were transferred to E. coli JM109 for cyclization repair and then to B. subtilis WB600. Multisite Saturation Mutagenesis. According to the results of site-directed mutagenesis, the mutation sites were grouped into two, and the primers were designed as shown in Table S7. The procedures are shown in Figure S3. The methods of transparent zones and colorimetric determination of enzyme activity were employed for screening of the libraries. The positive clones were picked out according to the generated transparent zones and subsequently cultured in LB broth supplemented with 50 μg/mL kanamycin. The secondary screening was performed with 1% soluble keratin as the substrate for determining the keratinase activity. Then the improved mutants were cultured in 50 mL of TB fermentative medium for expression of keratinase. Investigation of the Keratinase Expression Level in the 15 L Fermenter. Cultivations of mutant M7 expressing keratinase in the 15 L fermentor were studied in order to investigate its industrialization potential. The target strains were first activated on solid LB plates. A single colony was picked into 10 mL of liquid LB medium and cultured at 220 rpm for 10 h at 37 °C. The seed liquor was then transferred into 50 mL of fermentation medium (TB medium) by 3% inoculum for further cultivation at 37 °C for 10−12 h. Then the culture broth was inoculated with a 5% inoculum into the 15 L fermenter, which contained 9 L of TB medium. The culture temperature and pH were set at 37 °C and pH 6.8−7.2, respectively, with a stirring speed of 400 rpm and aeration of 1.5 vvm. The cell density and enzyme activity were determined every 4 h for drawing the growth process curve.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00442. Illustration of the effects of pro-peptide on mature keratinase (Figure S1); selection of mutation sites by G

DOI: 10.1021/acssynbio.8b00442 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

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comparison of homologous sequences (Figure S2); procedures of saturation and combinatorial mutations (Figure S3); sequencing results and transcriptional level analysis (Figure S4); structure comparison between wild-type and mutant keratinases (Figure S5); deletion of pro-peptide sequence by a round of fusion PCR (Figure S6); sequences of the primers used for deletion of the pro-peptide (Table S1); sequences of the primers used for truncation of the pro-peptide (Table S2); onestep reverse PCR system (Table S3); one-step reverse PCR program (Table S4); sequence of the primers used for site-directed mutagenesis at the P1 position (Table S5); sequences of the primers used for site-directed mutagenesis of pro-peptide (Table S6); sequences of the primers used for multisite saturation mutagenesis (Table S7) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel./Fax: +86-510-85328177. E-mail: [email protected] (J.-S. Shi). *E-mail: [email protected] (J.-S. Gong). ORCID

Zheng-Hong Xu: 0000-0003-1340-6838 Jin-Song Shi: 0000-0001-8514-3112 Author Contributions

C.S. and J.-S.G. conceived the study and designed the experiments. C.S. performed all of the experiments and analyzed the data. Y.-X.S. and S.Z. participated in the genetic engineering and activity assay. J.Q., He.L., Hu.L., and Z.-M.L assisted with data analysis, interpretation, and paper editing. C.S. and J.-S.G. wrote the manuscript. The study was supported and directed by Z.-H.X. and J.-S.S. All of the authors revised and approved the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21676121), the Jiangsu Key Research & Development Plan (BE2018622), the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-18), and the Top-Notch Academic Programs Project of Jiangsu Higher Education Institutions (PPZY2015B146).

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REFERENCES

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DOI: 10.1021/acssynbio.8b00442 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology (22) Yamamura, S., Morita, Y., Hasan, Q., Rao, S. R., Murakami, Y., Yokoyama, K., and Tamiya, E. (2002) Characterization of a new keratin-degrading bacterium isolated from deer fur. J. Biosci Bioeng 93 (6), 595−600.

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DOI: 10.1021/acssynbio.8b00442 ACS Synth. Biol. XXXX, XXX, XXX−XXX