Improvement of iturin A production inBacillus subtilisZK0 by overexpression of thecomAandsigAgenes

Zhang, Z.; Ding, Zhong-Tao; Zhong, Juan; Zhou, Jinyan; Shu, Dan; Luo, Dejun; Yang, Jie; Hong, Tao

doi:10.1111/lam.12739

Cited by 36 publications

(15 citation statements)

References 17 publications

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“…ComA and DegU, as signal regulators in the two-component system, control many processes of cells, including biofilm and competence formation, bacterial motility and antibacterial substances synthesis (Hamoen et al 2003;Harshey 2003;Kobayashi 2007;Koumoutsi et al 2007;Verhamme et al 2007;Mariappan et al 2012;Wang et al 2015;Zhang et al 2017). As an important protein controlling spore formation, Spo0A played a key regulation role in the synthesis of iturin A and bacilysin (Yazgan Karatas ß et al 2003; Rahman et al 2006;Boguslawski et al 2015).…”

Section: Discussionmentioning

confidence: 99%

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CodY , ComA , DegU and Spo0A controlling lipopeptides biosynthesis in Bacillus amyloliquefaciens fmbJ

Sun

Liu

Lin

et al. 2021

J of Applied Microbiology

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Aim: In the study, we investigated the regulatory effects of these genes (codY, comA, degU and spo0A) on the biosynthesis of three lipopeptides (bacillomycin D, fengycin and surfactin) in Bacillus amyloliquefaciens. Methods and Results: The codY, comA, degU and spo0A genes in B. amyloliquefaciens fmbJ were knocked out. The results showed that the productions of bacillomycin D were significantly reduced compared with that of fmbJ. Their deletion induced great changes in the levels of transcripts specifying metabolic pathways, quorum sensing system and substance transport system in fmbJ. Moreover, overexpression of these genes improved the productions of bacillomycin D. In particular, the overexpression of spo0A enhanced bacillomycin D yield up to 648Á9 AE 60Á9 mg l À1 from 277Á3 AE 30Á5 mg l À1 . In addition, the yields of surfactin in fmbJDcodY and fmbJDdegU were significantly improved, and the regulatory factor CodY had no significant effect on the synthesis of fengycin. Conclusions: These genes positively regulated the expression of bacillomycin D and fengycin synthase genes in strain fmbJ. However, codY and degU negatively regulated surfactin biosynthesis. Moreover, it was found that CodY had a concentration dependence on bacillomycin D synthesis. Spo0A might play a direct regulatory role in the synthesis and secretion of bacillomycin D. Significance and Impact of the Study: This study indicated that genetic engineering of regulatory genes was an effective strategy to improve the yields of antimicrobial lipopeptides and provided promising strains for industrial production of lipopeptides.

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Section: Discussionmentioning

confidence: 99%

“…The construction of expression vectors was based on pHT43 (Zhang et al 2017). The primers used for construction of expression vectors are presented in Table S2.…”

Section: Construction Of Strainsmentioning

confidence: 99%

CodY , ComA , DegU and Spo0A controlling lipopeptides biosynthesis in Bacillus amyloliquefaciens fmbJ

Sun

Liu

Lin

et al. 2021

J of Applied Microbiology

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“…Second, positive or negative regulators of cyclic lipopeptides, such as the ComP/ComA two-component system, DegU, Sfp or YczE, PerR, and Rap proteins and Phr peptides, might be involved in a mechanism that increases iturin production in the surfactin-less mutant strain. ComP/ComA, Sfp, PerR, and Phr are positive regulators of srfA transcription [ 11 , 45 – 48 ], and overexpression of the comA and sigA genes was shown to improve iturin production [ 49 ]. In addition, DegU and YczE positively regulate the synthesis of bacillomycin D in Bacillus amyloliquefaciens strain [ 50 ].…”

Section: Discussionmentioning

confidence: 99%

Organization and characterization of genetic regions in Bacillus subtilis subsp. krictiensis ATCC55079 associated with the biosynthesis of iturin and surfactin compounds

et al. 2017

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Bacillus subtilis subsp. krictiensis ATCC55079 produces the cyclic lipopeptide antibiotics iturin A–F as well as several surfactins. Here, we analyzed and characterized the biosynthetic genes associated with iturin and surfactin production in this strain. We aligned the sequences of each iturin and surfactin synthetase ORF obtained from a genomic library screen and next generation sequencing. The resulting 37,249-bp and 37,645-bp sequences associated with iturin and surfactin production, respectively, contained several ORFs that are predicted to encode proteins involved in iturin and surfactin biosynthesis. These ORFs showed higher sequence homologies with the respective iturin and surfactin synthetase genes of B. methylotrophicus CAU B946 than with those of B. subtilis RB14 and B. subtilis ATCC6633. Moreover, comparative analysis of the secondary metabolites produced by the wild-type and surfactin-less mutant (with a spectinomycin resistance cassette inserted into the srfAB gene within the putative surfactin gene region) strains demonstrated that the mutant strain showed significantly higher antifungal activity against Fusarium oxysporum than the wild-type strain. In addition, the wild-type strain-specific surfactin high performance liquid chromatography (HPLC) peaks were not observed in the surfactin-less mutant strain. In contrast, the iturin A peak detected by HPLC and liquid chromatography-mass spectrometry (LC/MS) in the surfactin-less mutant strain was 30% greater than that in the wild-type strain. These results suggested that the gene cluster we identified is involved in surfactin biosynthesis, and the biosynthetic pathways for iturin and surfactin in Bacillus strains producing both iturin and surfactin may utilize a common pathway.

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“…Additionally, an attempt was made to delete the regulator gene abrB to enhance the expression of iturin A synthetase by relieving the repression caused by AbrB on PbacA. As per previous studies, iturin A production from B. subtilis ZK0 and wild-type B. subtilis ZEB01 was found to be 0.005 g/L, which increased to 0.215 g/L by the simultaneous overexpression of the comA and sigA regulatory genes in B. subtilis ZK0 [ 27 ]. Dang et al (2019) [ 28 ] reported 0.037 g/L of iturin A when a strong promoter was inserted into the upstream of itu operon for the transcription enhancement of iturin A genes.…”

Section: Resultsmentioning

confidence: 83%

Gene Expression and Characterization of Iturin A Lipopeptide Biosurfactant from Bacillus aryabhattai for Enhanced Oil Recovery

Yaraguppi

Bagewadi

Mahanta

et al. 2022

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Biosurfactants are eco-friendly surface-active molecules recommended for enhanced oil recovery techniques. In the present study, a potential lipopeptide (biosurfactant) encoding the iturin A gene was synthesized from Bacillus aryabhattai. To improvise the yield of the lipopeptide for specific applications, current research tends toward engineering and expressing recombinant peptides. An iturin A gene sequence was codon-optimized, amplified with gene-specific primers, and ligated into the pET-32A expression vector to achieve high-level protein expression. The plasmid construct was transformed into an E. coli BL21 DE3 host to evaluate the expression. The highly expressed recombinant iturin A lipopeptide was purified on a nickel nitrilotriacetic acid (Ni-NTA) agarose column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the purity and molecular mass of iturin A was 41 kDa. The yield of recombinant iturin A was found to be 60 g/L with a 6.7-fold increase in comparison with our previously published study on the wild strain. The approach of cloning a functional fragment of partial iturin A resulted in the increased production of the lipopeptide. When motor oil was used, recombinant protein iturin A revealed a biosurfactant property with a 74 ± 1.9% emulsification index (E24). Purified recombinant protein iturin A was characterized by mass spectrometry. MALDI-TOF spectra of trypsin digestion (protein/trypsin of 50:1 and 25:1) showed desired digested mass peaks for the protein, further confirming the identity of iturin A. The iturin A structure was elucidated based on distinctive spectral bands in Raman spectra, which revealed the presence of a peptide backbone and lipid. Recombinant iturin A was employed for enhanced oil recovery through a sand-packed column that yielded 61.18 ± 0.85% additional oil. Hence, the novel approach of the high-level expression of iturin A (lipopeptide) as a promising biosurfactant employed for oil recovery from Bacillus aryabhattai is not much reported. Thus, recombinant iturin A demonstrated its promising ability for efficient oil recovery, finding specific applications in petroleum industries.

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Improvement of iturin A production inBacillus subtilisZK0 by overexpression of thecomAandsigAgenes

Cited by 36 publications

References 17 publications

CodY , ComA , DegU and Spo0A controlling lipopeptides biosynthesis in Bacillus amyloliquefaciens fmbJ

CodY , ComA , DegU and Spo0A controlling lipopeptides biosynthesis in Bacillus amyloliquefaciens fmbJ

Organization and characterization of genetic regions in Bacillus subtilis subsp. krictiensis ATCC55079 associated with the biosynthesis of iturin and surfactin compounds

Gene Expression and Characterization of Iturin A Lipopeptide Biosurfactant from Bacillus aryabhattai for Enhanced Oil Recovery

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