Enhanced production of iturin A in Bacillus amyloliquefaciens by genetic engineering and medium optimization

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Enhanced production of iturin A in Bacillus amyloliquefaciens by genetic engineering and medium optimization Yuxiang Xua, Dongbo Caia, Hong Zhangd, Lin Gaoa,c, Yong Yanga, Jiaming Gaoe, Yanyan Lib, Chunlei Yangb, Zhixia Jid, Jun Yub,*, Shouwen Chena,* a State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan, 430062, China b Tobacco Research Institute of Hubei Province, Wuhan 430062, China c Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, China d State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China e Tobacco Company of Hubei Province, Wuhan 430062, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacillus amyloliquefaciens Iturin A Promoter replacement AbrB Medium optimization

Brown spot disease, caused by Alternaria alternate, is one of the most destructive leaf spot diseases that made a major impact on tobacco growing. Biocontrol has attracted increasing attentions as its features of environmental friendship, promoting plant growth, etc. Iturin A, with broad spectrum antifungal activity, is a kind of biosurfactant that mainly produced by Bacillus. In this study, the promoter of iturin A synthetase cluster of Bacillus amyloliquefaciens HZ-12 was respectively replaced by promoters P43, PbacA, PsrfA and Pylb, our results implied that transcriptional level of gene ituD and iturin A titer showed consistent change trends, and PbacA was proven as the most efficient promoter, iturin A titer of which reached 950.08 ± 19.43 mg/L. Furthermore, regulator gene abrB was deleted to release the repression effect of AbrB on PbacA, ituD transcriptional level and iturin A titer were increased by 133.25 % and 20.88 %, respectively. Then, the maximum iturin A titer reached 2013.43 ± 32.86 mg/L by optimizing fermentation medium, increased by 392.15 % compared to the original (408.97 ± 21.35 mg/L). Finally, our results demonstrated that enhancing iturin A synthetic capability benefited the suppression of A. alternate. Collectively, this study provided a promising strain with an efficient fermentation medium for large-scale industrial production of iturin A.

1. Introduction Soil-borne diseases, consisting of sclerotinia, fusarium wilt, bacterial wilt, black shank disease etc., are an important class of plant diseases that caused serious impacts on modern agriculture, and further constrained the development of efficient and green agriculture [1]. Brown spot disease is one of the most destructive leaf spot diseases that occur worldwide, and it mainly occurs in the remarkable range of plants, including in tobacco, apple, pear, tomato etc. [2–4]. Chemical control has the features of high efficiency and low cost in control of plant diseases, however, there is no chemical pesticide that has substantial inhibitory effect on the outbreak of brown spot diseases [5], and “3R effects” (Resistance, Resurgence and Residue) caused by longterm pesticide application is also not conducive to the modification of soil environment. Biocontrol is regarded as the promising practice for preventing plant diseases, as it owns the advantages of promoting plant

⁎

growth, environmental friendship, etc [1]. Recently, several kinds of biocontrol microorganisms targeted to brown spot disease have been identified, including in Actinomycetes [6], Trichoderma [7], Bacillus species [8], etc. Among them, several Bacillus species have been made into commercial biocontrol agent, as Bacillus could produce a broad spectrum of bioactive peptides, such as iturin, surfactin and fengycin, and all these bioactive lipopeptides own the potentially inhibitory effects on phytopathogens [9,10]. Iturin A is one of the most effective ingredients in iturin family, and it has the broad-spectrum antifungal activity, and might be acted as the efficient biocontrol agent for suppressing outbreak of A. alternate, the pathogen of brown spot disease. Iturin A is a kind of biosurfactant that mainly produced by Bacillus, and it is synthesized by non-ribosomal polypeptide synthetase (NRPS). Iturin A contains β-amino fatty acids (14–17 carbon atoms) ringed with 7 amino acids, L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser, and this special amphiphilic structure confers it the broad-spectrum

Corresponding authors at: 368 Youyi Avenue, Wuchang District, Wuhan 430062, Hubei, China. E-mail addresses: [email protected] (J. Yu), [email protected] (S. Chen).

https://doi.org/10.1016/j.procbio.2019.11.017 Received 9 August 2019; Received in revised form 7 November 2019; Accepted 18 November 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Yuxiang Xu, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.11.017

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Table S1 (seeing in the Supplementary Materials). B. amyloliquefaciens HZ-12 was acted as the original strain for constructing recombinants [17], and Escherichia coli DH5α was acted as the host for plasmid construction. B. amyloliquefaciens and E. coli were respectively cultivated at 28 °C and 37 °C, and Lysogeny Broth (LB) medium was applied as basic medium for strain cultivation, and corresponding antibiotic (20 mg/L kanamycin or 20 mg/L tetracycline) was added when necessary. The medium for iturin A production contained (g/L): 30 corn starch, 70 soybean meal, 1.0 K2HPO4·3H2O, 1.0 MgSO4·7H2O, 1.0 FeSO4·7H2O, 0.01 MnSO4·H2O (natural pH), and pH was not controlled during fermentation process. The seed was cultivated in a 250 mL flask containing 30 mL LB medium for 10 h, and transferred (1 mL) into a 250 mL flask containing 25 mL iturin A production medium, and further cultivated at 28 °C, 230 rpm for 72 h, and the stirring radius of shaker was 26 mm. All the fermentation experiments were performed in triplicate.

antimicrobial activity, thus, iturin A can be used as the potential biocontrol agent against harmful plant pathogens [9]. The synthetase cluster of iturin A contains four open reading frame (ORF), ituD, ituA, ituB and ituC, among these genes, ituD owns the function of malonylCoA transacylase, which relates to fatty acid synthesis, and ituA relates to the synthesis of β-amino fatty acid, ituB and ituC encode peptide synthetases, and all of these four ORFs are driven by promoter Pitu [11]. Enhancing the synthetic capability of bioactive peptide plays an important role in the suppression of plant pathogen [12], and several researches have been reported for enhancement production of iturin A. Mizutomo et al. optimized the fermentation condition of Bacillus subtilis RB14-CS for iturin A production by response surface analysis, and iturin A titer was increased to 5.591 mg/g (Wet weight) [13]. Jin et al. developed a novel two-stage glucose-feeding strategy, and the maximum iturin A titer produced by B. subtilis reached 1.12 g/L, increased by 1.67-fold [14]. In addition, several genetic engineering tactics have been developed for iturin A production. Tsuge et al. replaced the promoter of iturin A synthetase cluster Pitu with PrepU from pUB110, which led to a 3-fold increase of iturin A production, reached 330 mg/L [11]. Through promoter replacement, fermentation condition optimization and DegQ overexpression, the iturin A titer produced by B. amyloliquefaciens reached 113.1 mg/L [15]. Overexpression of transcriptional factors SigA and ComA was also proven as an efficient strategy, and iturin A titer produced by B. subtilis ZK0 was improved to 215 mg/L, increased by 42-fold [16]. B. amyloliquefaciens HZ-12 (CCTCC M2015234) is a wild-type strain with high-level production of α-glycosidase inhibitor 1Deoxynojirimycin [17]. Through genome sequencing, we confirmed that B. amyloliquefaciens HZ-12 possesses the whole iturin A synthetase cluster, and it might have the capability of iturin A synthesis. The aim of this study wants to increase iturin A titer by genetic engineering and fermentation medium optimization, and further evaluate the iturin A enhancement on suppression effectiveness of A. alternate, the pathogen of brown spot disease during tobacco growing.

2.2. Construction of promoter replacement strains The vector T2(2)-Ori was applied for constructing the promoter replacement strains of B. amyloliquefaciens, according to our previously reported research [18], and the construction procedure of recombinant HZ-PbacA was served as an example. In brief, the up-stream and downstream homogenous arms of promoter Pitu from B. amyloliquefaciens HZ-12, promoter PbacA from Bacillus licheniformis DW2 were amplified by corresponding primers (Table S1), and fused by Splicing Overlap Extension (SOE)-PCR. The fused fragment was digested that inserted into T2(2)-Ori at restriction sites XbaI/SacI, diagnostic PCR and DNA sequence confirmed that the promoter replacement plasmid (T2-PbacA) was constructed successfully. Then, T2-PbacA was transformed into B. amyloliquefaciens HZ-12 by electroporation, and the positive transformant was then cultivated in LB medium with 20 mg/L kanamycin at 45 °C to promote single-cross transformation, and then transferred into kanamycin-free medium at 37 °C for several generations. The kanamycin-sensitive colonies were picked, and further verified by diagnostic PCR and DNA sequencing, and the positive recombinant was named as HZ-PbacA. Similarly, the iturin A synthetase operon driven by promoters P43, PsrfA and Pylb from B. subtilis 168 were constructed successfully, named as B. amyloliquefaciens HZ-P43, HZ-PsrfA and HZ-Pylb, respectively.

2. Materials and methods 2.1. Strains, plasmids and cultivation conditions The strains and plasmids used in this research were listed in Table 1. The primers used for strain construction and RT-qPCR were listed in Table 1 The strains and plasmids used in this study. Strains

Relevant genotype/description

Source

B. subtilis 168 B. licheniformis DW2 B. amyloliquefaciens HZ-12 HZ12-PbacA HZ12-P43 HZ12-PsrfA HZ12-Pylb HZ12△abrB HZ12-PbacA△abrB HZ-12-PbacA/pHY-AbrB HZ-12-PbacA/pHY-300 E. coli DH5a Plasmids T2(2)-Ori T2-PbacA T2-P43 T2-Pylb T2-Psrf T2-△abrB pHY300PLK pHY-AbrB

Wild type CCTCC M2011344

(Wang et al., 2018) CCTCC

Iturin A production strain, Wild-Type (CCTCC M2015234) A derivative of HZ-12, with itu operon driven by bacitracin synthetase promoter PbacA A derivative of HZ-12 with itu operon driven by promoter P43 A derivative of HZ-12, with itu operon driven by surfactin synthetase promoter Psrf A derivative of HZ-12, with itu operon driven by promoter Pylb A derivative of HZ-12, deletion of abrB A derivative of HZ-PbacA, deletion of abrB A derivative of HZ-PbacA, harboring plasmid pHY-AbrB A derivative of HZ-PbacA, harboring plasmid pHY300PLK F– Φ80d/lacZΔM15, Δ(lacZYA-argF) U169, recA1, endA1, hsdR17 (rK–, mK+), phoA, supE44, λ–, thi-1, gyrA96, relA1

CCTCC This work This work This work This work This work This work This work This work (Wang et al., 2018)

E. coli-Bacillus shuttle vector, OripUC/Orits, Kanr PbacA with the upstream and downstream homogenous arms of Pitu inserted into T2(2)-ori P43 with the upstream and downstream homogenous arms of Pitu inserted into T2(2)-ori Pylb with the upstream and downstream homogenous arms of Pitu inserted into T2(2)-ori PsrfA with the upstream and downstream homogenous arms of Pitu inserted into T2(2)-ori T2(2)-Ori derivative containing homologous arms of gene abrB, to delete abrB E. coli–Bacillus shuttle vector; Ampr in E. coli, Tcr in both E. coli and B. subtilis Plasmid pHY300PLK harboring P43 promoter, gene abrB and amyL terminator

(Wang et al., 2018) This work This work This work This work This work (Wang et al., 2018) This work

2

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Fig. 1. The construction procedure and verification of recombinant strain HZ-PbacA. A: Construction procedure of the recombinant strain HZ-PbacA, and the recombinant strains were constructed by homologous double crossover; B: Verification of HZ-PbacA. M: DL5000 Marker (5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); Lane 1: PCR product of the genome DNA of HZ-PbacA by primers Ver-KYF/PbacA-R (1140 bp), Lane 2: PCR product of the genome DNA of HZ-12 by primers Ver-KYF/PbacA-R (Negative control), Lane 3: PCR product of the genome DNA of HZ-PbacA by primers PbacA-F/VerKYR (1103 bp), Lane 4: PCR product of the genome DNA of HZ-12 by primers PbacA-F/ Ver-KYR (Negative control).

surface of PDA solid medium, and the same amount of A. alternate was picked and inoculated on PDA solid medium, which harboring fermentation supernatant of B. amyloliquefaciens, and cultivated for 5 days. Finally, the growth statuses were measured to evaluate the antifungal effects of iturin A on A. alternate.

2.3. Construction of gene abrB deletion strain of B. amyloliquefaciens The construction procedure of abrB deletion strain was similar to that of promoter replacement strain, with minor modification [19]. Briefly, the up-stream and down-stream homogenous arms of gene abrB were amplified and fused, and then inserted into T2(2)-Ori to obtain the vector T2-abrB. Then, T2-abrB was electro-transferred into B. amyloliquefaciens, the abrB deletion strain was attained by homologous double crossover, and further confirmed by diagnostic PCR and DNA sequence.

2.6. Statistical analyses All samples were analyzed in triplicate, and all data were conducted to analyze the variance at P < 0.05 and P < 0.01, and a t test was applied to compare the mean values using the software package Statistica 6.0.

2.4. Construction of AbrB overexpression strain The regulator gene abrB overexpression strain was constructed according to our previous research [20]. P43 promoter from B. subtilis 168, gene abrB from B. amyloliquefaciens HZ-12 and amyL terminator from B. licheniformis WX-02 were amplified and fused by SOE-PCR. The fused fragment was inserted into pHY300 at restriction sites EcoRI/ XbaI, diagnostic PCR and DNA sequence confirmed that AbrB expression was constructed successfully, named as pHY-AbrB. The vector pHY-AbrB was transferred into B. amyloliquefaciens HZ-PbacA by electroporation, named as HZ-PbacA/pHY-AbrB.

3. Results 3.1. Establishment of recombinant strains by promoter replacement and their effects on iturin A production Based on the genome sequence of B. amyloliquefaciens HZ-12, the whole iturin A synthetase gene cluster is existed in HZ-12 [17], moreover, our fermentation results implied that HZ-12 possesses the capability of iturin A synthesis, according to the peak maps of culture supernatant of HZ-12 and standard iturin A in Fig. S1 (seeing in the Supplementary Material). Furthermore, to improve the synthetic capability of iturin A, the promoter of iturin A synthetase operon Pitu was replaced by bacitracin synthetase cluster promoter PbacA from B. licheniformis DW2, promoters P43, Pylb and PsrfA from B. subtilis 168, respectively. Previously, P43 promoter is widely regarded as a strong promoter [23], PsrfA showed the better performance on lichenysin production [18], Pylb was confirmed as a strong promoter, especially at stable period [24], and the promoter of bacitracin synthetase gene cluster PbacA was confirmed as a strong promoter in our previous research [25]. Thus, these above promoters were chose to construct the recombinant strain by promoter replacement. The procedure for constructing recombinant strain with promoter PbacA was showed in Fig. 1, diagnostic PCR and DNA sequence confirmed that the recombinant strain was constructed successfully, named as HZ-PbacA. Following the same procedure, the iturin A synthetase gene cluster driven by promoters P43, Pylb and PsrfA were constructed, resulting in the recombinant strains HZ-P43, HZ-Pylb and HZ-PsrfA, respectively.

2.5. Analytical methods To determine the concentration of iturin A, the volume of 0.3 mL fermentation supernatant was mixed with 1.2 mL methanol, shaken for 1 h and centrifuged at 10,000 g for 10 min, and the supernatant was attained for iturin A determination. The iturin A concentration was measured on an Agilent HPLC 1260 (Agilent Technologies, USA), which equipped with Agilent Lichrospher C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase was 10 mmol/L ammonium acetate/acetonitrile = 65:35 (V/V), and flow rate was 1.0 mL/min. The injection volume was 10 μL and detection wavelength was 210 nm, and concentration of iturin A was calculated by the standard curve made by standard iturin A (Sigma, CAS 52229-90-0, purity 95 %) [21]. Cell biomass was measured by dilution coating. The transcriptional levels of gene ituD were determined according to the previous research, and 16S rRNA was served as the reference gene [22]. To measure the antifungal effect of iturin A on A. alternate, 0.1 mL fermentation supernatant of B. amyloliquefaciens was coated on the 3

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Fig. 2. Effects of replacing promoter PituD by different promoters (PbacA, P43, Pylb and PsrfA) on iturin A titer and transcriptional level of ituD. A: Iturin A titers of B. amyloliquefaciens HZ-12, HZ-PbacA, HZ-P43, HZ-Pylb and HZPsrfA (72 h), B: Transcriptional levels of ituD of strains HZ-12, HZ-PbacA, HZ-P43, HZ-Pylb and HZ-PsrfA (42 h) (∗, P < 0.05; and ∗∗, P < 0.01 indicate the significance levels between recombinant strains and control strain).

3.3. Optimization of medium compositions for enhanced production of iturin A

Then, these strains were cultivated in iturin A production medium, as well as the control strain HZ-12. Based on the results of Fig. 2, HZ-12 produced 408.97 ± 21.35 mg/L iturin A, and the recombinant strain HZ-PbacA produced the highest iturin A titer (950.08 ± 19.43 mg/L), which was 132.15 % higher than that of HZ-12. The iturin A yield of HZ-PbacA reached 13.20 mg/L/h, significant higher than that of HZ-12 (5.68 mg/L/h). HZ-PsrfA produced the lowest titer of iturin A (51.15 ± 3.43 mg/L), decreased by 87.49 %. In addition, the iturin A concentrations produced by HZ-P43 and HZ-Pylb were 735.12 ± 21.04 mg/L and 673.28 ± 14.42 mg/L, increased by 79.75 % and 64.63 % compared to HZ-12, respectively. Meanwhile, the expression level of gene ituD was measured at mid-log phase (42 h), our results showed that transcriptional levels of ituD were correlated with iturin A titers among these strains, and the promoter strengths for iturin A synthesis were as follows: PbacA > P43 > Pylb > Pitu > PsrfA. Collectively, these above results demonstrated that enhancement production of iturin A in HZ-PbacA was attributed to the increase of iturin A synthetase expression level, and promoter PbacA was the most effective promoter for iturin A synthesis.

In order to further improve iturin A titer of HZ-PbacA△abrB, the medium compositions including carbon source, nitrogen source and inorganic salt were optimized. To screen the optimum carbon and nitrogen sources for iturin A production, different carbon and nitrogen sources with the equal C and N contents were applied, respectively (Fig. 4A and Fig. 4C). Based on our results of Fig. 4, corn starch was proven as the efficient carbon source for iturin A production, and 40 g/ L corn starch resulted in the highest iturin A titer. Although glucose is recognized as the most commonly used carbon source, it might not work well with soybean meal, and low iturin A titer was attained when glucose was served as carbon source, although the cell biomass reached the highest (Fig. 4A-B). Soybean meal was the best nitrogen source for iturin A synthesis, although the cell biomass was the lowest, compared to other nitrogen sources (Fig. 4C), and the highest iturin A titer was achieved at 90 g/L soybean meal (Fig. 4D). In addition, the optimum concentrations of inorganic salt K2HPO4·3H2O, MgSO4·7H2O, FeSO4·7H2O, MnSO4·H2O were investigated, and our results showed that the concentrations of 1.0 g/L, 0.15 g/L, 0.5 g/L and 0.015 g/L resulted in the highest iturin A titers (Fig. 4E-H), respectively. In addition, the initial pHs of fermentation medium and stirring speeds of shaker were further optimized. Based on our results, the initial pHs (natural, 6.80, 7.20 and 7.50) showed no significant difference on iturin A production, and 230 rpm was the most reasonable choice of stirring speed for iturin A production (Fig. S2). Based on these above single factor optimization experiments, corn starch, soybean meal, K2HPO4·3H2O and FeSO4·7H2O were proven to serve as the critical roles on iturin A production. Then, these four compositions were chose for orthogonal array experiment, and Minitab 16 was applied for statistical analysis of fermentation data. Based on the results of Table 2, K2HPO4·3H2O was found to be the most significant factor, followed by corn starch, FeSO4·7H2O and soybean meal, and the optimum medium for iturinA production was (g/L) 50 corn starch, 70 soybean meal, 1.0 K2HPO4·3H2O, 0.2 FeSO4·7H2O, 0.5 MgSO4·7H2O, 0.015 MnSO4·H2O. Then, the fermentation process of HZ-PbacA△abrB was measured in the optimized medium. Based on our results of Fig. 5, after a short lag phase, HZ-PbacA△abrB rapidly entered into exponential phase, and the maximum cell biomass reached (164.23 ± 6.53)*108 CFU/mL at 24 h. Then, the biomass was significantly dropped after 24 h, due to cell autolysis. Meanwhile, iturin A was synthesized from 24 h, and the synthetic rate was decreased after 60 h, and this might due to the cell viability and nutrients insufficient at the late stage of fermentation. The iturin A titer and yield reached the highest level (2013.43 ± 32.86 mg/L and 27.96 ± 0.74 mg/L/h) at 72 h, and decreased when the fermentation extended beyond 72 h (Table S3). Since

3.2. Deletion of regulator gene abrB improved iturin A production Based on the previous research of our group, AbrB was proven as the negative regulator of bacitracin synthetase operon, which directly banded to promoter PbacA in B. licheniformis DW2 [26]. In this study, to further improve the expression level of iturin A synthetase, gene abrB was deleted in HZ-PbacA, attaining the mutant strain HZ-PbacA△abrB. As shown in Fig. 3, although the maximum biomass of HZ-PbacA△abrB ((145.42 ± 6.04)*108 CFU/mL) showed the 8.68 % decrease compared to HZ-PbacA ((159.24 ± 7.48)*108 CFU/mL) (Fig. 3A), the iturin A titer produced by HZ-PbacA△abrB reached 1148.43 ± 27.05 mg/L, increased by 20.88 %, and the iturin A yield reached 15.95 ± 0.63 mg/L/h. Moreover, deletion of abrB increased the expression level of ituD by 133.25 % (Fig. 3B), which might be the reason for iturin A enhancement in HZ-PbacA△abrB. In addition, since gene ituD transcriptional level was enhanced significantly in abrB deletion strain, the iturin A titer was only increased by 19.80 %, indicating the limiting factor for high-level production of iturin A at this time was not the expression level of iturin A syntherase, but the supplies of precursors, cell growth, etc. In addition, overexpression of AbrB was not conducive to iturin A synthesis, and the iturin A titer was decreased by 19.80 % compared to HZ-PbacA/pHY300 (Fig. 3C). Therefore, it was suggested that deletion of abrB released the repression effect of AbrB on promoter PbacA, which further increased the expression of iturin A synthetase and iturin A titer, and the strain HZ-PbacA△abrB with the highest iturin A titer (1148.43 ± 27.05 mg/L) was served as the candidate for fermentation medium optimization. 4

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Fig. 3. Effects of abrB deletion and overexpression on iturin A titer and ituD transcriptional level. A: Iturin A titers of B. amyloliquefaciens HZ-12, HZ-PbacA and HZPbacA△abrB (72 h), B: Gene ituD transcriptional levels of HZ-12, HZ-PbacA and HZPbacA△abrB (42 h) (∗, P < 0.05; and ∗∗, P < 0.01 indicate the significance levels between recombinant strains and control strain).

disease. Previously, Mizumoto et al. confirmed that additional iturin A efficiently suppressed Rhizoctonia solani [12], and iturin A also showed a significant control on the yeast population during orange juice storage [28]. In this research, our results confirmed that the efficient antifungal activity of iturin A against A. alternate, the pathogen of brown spot disease during tobacco growing, moreover, enhancing the synthetic capability of iturin A was conducive to the improvement of antifungal effectiveness. However, our results implied that the pathogen A. alternate can still grow under the optimal combination condition (HZ-PbacA△abrB with the optimum fermentation condition), indicated that the iturin A titer should be improved or several other kinds of lipopeptides should be matched with iturin A to improve the suppression effectiveness. Recent years, several articles focused on the strategies on surfactant enhancement [29], and the common strategies for enhancing surfactant production were (i) strengthening synthetase operon [15,18,30,31], (ii) enhancing precursor supply [32–34], (iii) reduction of by-product formation [35], (iv) enhancing the surfactant efflux [36], (v) manipulation of transcriptional factor [16,35], (vi) optimization of fermentation medium [13,18]. Among these above tactics, promoter replacement was served as the common strategy to enhance biosurfactant production. Previously, Qiu et al. replaced the promoter of lichenysin synthetase operon Plch by PsrfA, which led to 5.5-fold increase of lichenysin titer [18], also, PsrfA was engineered as an autoinducible promoter to improve aminopeptidase and nattokinase production in B. subtilis [37]. However, this promoter showed poor performance in this research, suggested that promoter function is closely related to the target gene. Jiao et al. developed a novel strong promoter Pg3 for surfactin production, and the surfactin titer was increased to 9.74 g/L [30]. The surfactin synthetase operon promoter PsrfA was replaced by the constitutive promoter Pveg, and the surfactin titer was increased from 0.07 g/L to 0.26 g/L [38]. In addition, the iturin A synthetase operon promoter Pitu was replaced by PrepU, which resulted in a 3-fold increase of iturin A titer [11]. In this study, our results found that the promoter PbacA from B. licheniformis DW2 was the most effective promoter for iturin A synthesis, and iturin A titer produced by HZ-PbacA was increased to 950.08 ± 19.43 mg/L by 132.13 %. Meanwhile, in the previous research of our group, promoter PbacA also showed the

theconcentrations of corn starch and soybean meal were difficult to measure during the fermentation process, we have not determined these data here. 3.4. Effect of iturin A enhancement on the suppression of A. alternate To evaluate the antifungal effect of iturin A on A. alternate, the pathogen of brown spot disease, culture supernatant of HZ-12 and HZPbacA△abrB cultivated by the original and optimized mediums were coated on the surface of PDA soild medium, and the sterilization medium was served as the control. Then, the pathogen A. alternate were picked and inoculated on the PDA solid medium and cultivated for 5 days. Based on our results of Fig. 6, A. alternate grew well in control group (A), and addition of supernatant harboring iturin A suppressed A. alternate growing, and the strongest antifungal effect was obtained by HZ-PbacA△abrB with the optimized medium (E). Thus, our results suggested that enhancing iturin A synthesis was conducive to the suppression of A. alternate for the occurrence of brown spot disease. 4. Discussion Brown spot disease is the main disease during leaf maturity, which makes the significant losses to the normal growth of tobacco [5]. Bacillus is served as the important biocontrol agent [8], and iturin A has the broad spectrum antifungal activity [9], which might have the significant effect on the suppression of plant fungal disease. In this study, iturin A titer was significantly enhanced by replacing the promoter of iturin A synthetase cluster with PbacA and deletion of regulator gene abrB, as well as fermentation medium optimization, moreover, plate confrontation analysis implied that enhancing iturin A synthetic capability is an effective strategy to suppress the growth of A. alternate. Brown spot disease is one of the most destructive leaf spot diseases that occurred worldwide, which brought serious economic loss during agriculture production, however, there is no chemical pesticide that has the substantial inhibitory effect on the outbreak of brown spot diseases [5]. Biocontrol of soil-borne diseases has attracted considerable attention [27]. Iturin A produced by Bacillus has showed the better biological activity on fungal, and generally applied in the suppression of fungal 5

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Fig. 4. Optimization of the fermentation medium for iturin A production, and the cultivation time is 72 h. A: Carbon resources (30 g/L corn starch, 30 g/L soluble starch, 33.33 g/L Glucose, 31.67 g/L maltose, 34.07 g/L glycerol and 31.67 g/L sucrose), B: Concentrations of corn starch (20 g/L, 30 g/L, 40 g/L, 50 g/L and 60 g/L), C: Nitrogen resources (70 g/L soybean meal (Total nitrogen 7.2 %), 70 g/L rapeseed meal (Total nitrogen 7.2 %), 70 g/L cottonseed meal (Total nitrogen 7.2 %), 63 g/L beef extract (Total nitrogen 8 %), 39.69 g/L tryptone (Total nitrogen 12.7 %), 39.69 g/L peptone (Total nitrogen 12.7 %) and 44.80 g/L yeast extract (Total nitrogen 11.25 %)), D: Concentrations of soybean meal (30 g/L, 50 g/L, 70 g/L, 90 g/L and 110 g/L), E: K2HPO4·3H2O concentrations (0.5 g/L, 1 g/L, 1.5 g/L and 2 g/L), F: MgSO4·7H2O concentrations (0.05 g/L, 0.1 g/L, 0.15 g/L and 0.2 g/L), G: FeSO4·7H2O concentrations (0.25 g/L, 0.5 g/L, 0.75 g/L and 1 g/L), H: MnSO4·H2O concentrations (0.01 g/ L, 0.02 g/L, 0.03 g/L and 0.04 g/L) (∗, P < 0.05; and ∗∗, P < 0.01 indicate the significance levels between the optimum condition and control).

Table 2 Orthogonal test analysis. 　

Corn Strach

K2HPO4·3H2O

FeSO4•7H2O

　

g L−1

Soybean meal g L−1

　

g L−1

g L−1

　

1 2 3 4 5 6 7 8 9 K1 K2 K3 R Optimal level

30 (1) 30 (1) 30 (1) 40 (2) 40 (2) 40 (2) 50 (3) 50 (3) 50 (3) 4829.76 4902.06 5301.95 472.19 A3

70 (1) 80 (2) 90 (3) 70 (1) 80 (2) 90 (3) 70 (1) 80 (2) 90 (3) 5255.86 4940.25 4837.65 418.21 B1

0.5 (1) 1.0 (2) 1.5 (3) 1.0 (2) 1.5 (3) 0.5 (1) 1.5 (3) 0.5 (1) 1.0 (2) 4811.54 5366.25 4855.98 554.71 C2

0.10 (1) 0.15 (2) 0.20 (3) 0.20 (3) 0.10 (1) 0.15 (2) 0.15 (2) 0.20 (3) 0.10 (1) 4748.3 5111.9 5173.57 425.27 D3

1537.23 1738.13 1554.4 1887.99 1470.94 1543.13 1830.64 1731.18 1740.13 Range analysis

Fig. 5. The fermentation process curve (iturin A titer and biomass) of HZPbacA△abrB in the optimum medium during iturin A production (50 g/L corn starch, 70 g/L soybean meal, 1.0 g/L K2HPO4·3H2O, 0.2 g/L FeSO4·7H2O, 0.5 g/ L MgSO4·7H2O, 0.015 g/L MnSO4·H2O).

　

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Fig. 6. Effect of iturin A enhancement on the suppression of A. alternate (0.1 mL fermentation supernatant was coated on the surface of PDA solid medium, and the same amount of A. alternate was picked and inoculated on PDA solid medium, and then cultivated for 5 days. Acting as the control, 0.1 mL sterilized water was applied). A: Control, B: HZ-12 with original medium, C: HZ-12 with optimum medium, D: HZ-PbacA△abrB with original medium, E: HZ-PbacA△abrB with optimum medium.

5. Conclusions

best performance on the synthesis of short branched-chain fatty acids [25]. Since B. licheniformis DW2 is an industrial strain for bacitracin production [39], our results suggested that promoter PbacA might act as an universal strong promoter for further research. Moreover, iturin A is a kind of secondary metabolite, which mainly synthesized at the middle and late stages during fermentation, therefore, the transcriptional levels of gene ituD were measured at 42 h, although the expression profiles of these promoters (PbacA, P43, PsrfA and Pylb) were not consistent in protein production. Transcriptional factor AbrB was known as a global transition state transcription factor in Bacillus, and it was proven to be a negative regulator for antibiotic synthesis, competent formation, protease expression etc [40]. Meantime, our previous research implied that AbrB directly regulated bacitracin synthesis via binding to the promoter of bacitracin synthetase operon PbacA, and deletion of abrB released the inhibition effect of PbacA and improved the synthetic capability of bacitracin in B. licheniformis [26]. Based on our results of Fig. 3, deletion of gene abrB in B. amyloliquefaciens also benefited PbacA transcription, which led to a 20.88 % increase of iturin A production. Since regulator AbrB from B. amyloliquefaciens HZ-12 showed 96 % identities with that of B. licheniformis DW2 at amino acid level, therefore, the regulator AbrB of B. amyloliquefaciens HZ-12 might also repress PbacA expression in B. amyloliquefaciens, and this was the reason for the increases of ituD transcriptional level and iturin A titer in abrB deletion strain HZ-PbacA△abrB. Additionally, previous research implied that deletion of abrB had no effect on the production of bacillomycin and fengycin in B. amyloliquefaciens SQR9 [41], additionally, Qian et al. demonstrated that AbrB had no significant effect on bacillomycin D production [42]. In this study, the regulator gene abrB was deleted in the original strain HZ-12, our results showed that the iturin A titer was decreased by 9.67 % in abrB deficient strain, maybe due to the decrease of cell biomass, and the expression level of ituD showed no difference between abrB deletion strain with control (Fig. S3). Since bacillomycin and iturin A both belong to iturin family [10], and AbrB could also affect iturin A synthesis through other physiological metabolism, therefore, we suggested that iturin A synthesis is not regulated by AbrB in HZ-12. Moreover, the iturin A titer produced by HZ-PbacA△abrB was compared with other iturin A producers in Table S2, different from the reported high titer of surfactin, the iturin A titers usually at a low level, and this might due to different amino acid compositions between surfactin and iturin A. In addition, the iturin A titer attained in this research reached a relatively high level, only behind B. subtilis RB14.

Iturin A could be applied in the suppression of brown spot disease, as it owns the broad spectrum antifungal activity. In this study, our results implied that promoter of iturin A synthetase operon served as the critical role on iturin A synthesis, and PbacA was the most efficient promoter for iturin A production. Deletion of regulator gene abrB of B. amyloliquefaciens released the repression effect of AbrB on PbacA, which further led to a 20.88 % increase of iturin A titer. Meanwhile, fermentation medium optimization was also an efficient tactics to enhance iturin A production, and the maximum iturin A titer reached 2013.43 ± 32.86 mg/L, increased by 392.15 % compared to the original. Moreover, enhancing iturin A synthetic capability was beneficial for the suppression of A. alternate, the pathogen of brown spot disease. Taken together, this study provided a promising strain with an efficient fermentation technology for large-scale industrial production of iturin A. Athour’s contribution D Cai, J Yu and S Chen designed the study. Y Xu and H Zhang carried out the molecular biology studies and construction of engineering strains. Y Xu, D Cai, L Gao, Y Yang, Y Li and J Yu carried out the fermentation studies. Y Xu, D Cai, C Yang, Z Ji, J Yu and S Chen analyzed the data and wrote the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the National Key Research and Development Program of China (2018YFA090039), the Technical Innovation Special Fund of Hubei Province (No. 2018ACA149), the Science and Technology Project of Hubei Tobacco Company (027Y2019-018) and the Key Technology Project of China National Tobacco Corporation (110201502014, 110201502018). Appendix A. Supplementary data Supplementary material related to this article can be found, in the 7

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online version, at doi:https://doi.org/10.1016/j.procbio.2019.11.017. References

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