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

Xu, Yuxiang; Cai, Dongbo; Hong, Zhang; Gao, Lin; Yang, Yong; Gao, Jun; Li, Yanyan; Yang, Chunlei; Ji, Zhixia; Yu, Jae Hyeon; Chen, Shouwen

doi:10.1016/j.procbio.2019.11.017

Cited by 48 publications

(28 citation statements)

References 42 publications

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“…There was a 6.7-fold increase in production in comparison with wild iturin A, which yielded 8.86 g/L under optimized conditions (crude oil, 4.0%; yeast extract, 0.7%; NaNO 3 , 3.0%), as reported in our previous studies [ 23 ]. In a recent study [ 26 ], the recombinant strain HZ-PbacA showed high iturin A production owing to the replacement of the promoter PbacA in place of the promoter of the iturin A synthetase cluster. 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.…”

Section: Resultsmentioning

confidence: 99%

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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Section: Resultsmentioning

confidence: 99%

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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“…Therefore, iturin A possess bright application prospects in the biological control of plant fungal pathogens ( Despite this advantage, the high production cost coupled with low original yield limit the further industrial application of iturin A, thus, developing inexpensive materials is necessary to cheaper product iturin A (Dang et al 2019;Shi et al 2018). In current studies, various agro-industrial wastes or by-products such as rapeseed meal (RSM) (Chen et al 2021), rapeseed cake (RSC) (Chen et al 2019a), soybean curd residue (Mizumoto et al 2006), soybean meal (Xu et al 2020), sun ower oil cake (SOC) (Kumar et al 2017) have been targeted as substrates for iturin A production. These results have greatly stimulated the interest in exploring the degradation of FW to product iturin A.…”

Section: Introductionmentioning

confidence: 99%

Artificial Consortia of Bacillus Amyloliquefaciens HM618 and Bacillus Subtilis for Utilizing Food Waste to Synthetize Iturin A

Miao

Wang

Qiao

et al. 2022

Preprint

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Food waste is a kind of biodegradable organic resource. It can be degraded into the small energy molecules for providing precursors to synthetize iturin A, which is a kind of lipopeptide with broad-spectrum antifungal activity for widely applying in biocontrol of the plant disease. However, the lower yield and higher cost during producing iturin A limit its application. In this study, the recombinants of Bacillus subtilis producing lipase and amylase were constructed to utilize food waste for producing iturin A under the consortium with Bacillus amyloliquefaciens HM618 produced iturin A and the engineering B. subtilis. The results showed that recombinant strain WB-A13 had the highest amylase activity of 23406.4 U/mL, and the lipase activity of WB-L01 was 57.5 U/mL. When HM618 was co-cultured with the engineering strain WB800N-AmyE or WB800N-Lip, the relatively highest yield of iturin A reached to 7.66 mg/L in co-cultured fermentation of consortium with HM618 and WB-A14, which was increased 32.9% compared to that of HM618 under pure culture. Under three-strains consortium containing HM618, WB-L02 and WB-A14, the yield of iturin A reached 8.13 mg/L, which was 38.8% higher than the control when the initial size OD600 of above three strains was 0.2, 0.15 and 0.15 respectively. Taken together, artificial consortium of B. amyloliquefaciens and the recombinant B. subtilis can better bioconvert food waste into iturin A, which provides a new strategy for the high-value utilization of food waste.

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“…To face these obstacles, a first efficient approach is to lower the cost of the substrate by targeting cheap raw materials and optimizing culture media Symbols: K, foamability; a, coefficient of the linear model of biomass concentration in the foam (ND); b, coefficient of the linear model of biomass concentration in the foam (g•L −1 ); α, coefficient of the linear model of outgoing waste flow rate (ND); β, coefficient of the linear model of overflowing flow rate (ND); µ, specific growth rate (h −1 ); D, dilution rate (h −1 ). composition (Das and Mukherjee, 2007;Mizumoto and Shoda, 2007;Wang et al, 2008;Marti et al, 2015;Dang et al, 2019;Xu et al, 2020). A complementary approach is to increase lipopeptide productivity of the natural biocatalysts with the screening of natural strains or with applying metabolic and genetic engineering strategies (Leclere et al, 2005;Béchet et al, 2013;Coutte et al, 2015;Dang et al, 2019;Wu et al, 2019;Xu et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…composition (Das and Mukherjee, 2007;Mizumoto and Shoda, 2007;Wang et al, 2008;Marti et al, 2015;Dang et al, 2019;Xu et al, 2020). A complementary approach is to increase lipopeptide productivity of the natural biocatalysts with the screening of natural strains or with applying metabolic and genetic engineering strategies (Leclere et al, 2005;Béchet et al, 2013;Coutte et al, 2015;Dang et al, 2019;Wu et al, 2019;Xu et al, 2020). As the biological activity of the lipopeptide mixture strongly depends on the lipopeptide family or isoform produced, the selectivity of the production was also addressed within several studies of environmental parameters as oxygen transfer (Guez et al, 2008;Fahim et al, 2012), temperature (Fickers et al, 2008), and amino acid feeding (Fickers et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

New Continuous Process for the Production of Lipopeptide Biosurfactants in Foam Overflowing Bioreactor

Guez

Vassaux

Larroche

et al. 2021

Front. Bioeng. Biotechnol.

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In this work, an original culture process in bioreactor named overflowing continuous culture (O-CC) was developed to produce and recover continuously mycosubtilin, a lipopeptide antifungal biosurfactant of major interest. The lipopeptide production was first investigated in shake conical flasks in different culture media [ammonium citrate sucrose (ACS), Difco sporulation medium (DSM), and Landy], followed by a pH condition optimization using 3-(N-morpholino)propanesulfonic acid (MOPS) and 2-(N-morpholino)ethanesulfonic acid (MES) buffered media. A simple theoretical modeling of the biomass evolution combined with an experimental setup was then proposed for O-CC processed in stirred tank reactor at laboratory scale. Seven O-CC experiments were done in modified Landy medium at the optimized pH 6.5 by applying dilution rates comprised between 0.05 and 0.1 h–1. The O-CC allowed the continuous recovery of the mycosubtilin contained in the foam overflowing out of the reactor, achieving a remarkable in situ product removal superior to 99%. The biomass concentration in the overflowing foam was found to be twofold lower than the biomass concentration in the reactor, relating advantageously this process to a continuous one with biomass feedback. To evaluate its performances regarding the type of lipopeptide produced, the O-CC process was tested with strain BBG116, a mycosubtilin constitutive overproducing strain that also produces surfactin, and strain BBG125, its derivative strain obtained by deleting surfactin synthetase operon. At a dilution rate of 0.1 h–1, specific productivity of 1.18 mg of mycosubtilin⋅g–1(DW)⋅h–1 was reached. Compared with other previously described bioprocesses using almost similar culture conditions and strains, the O-CC one allowed an increase of the mycosubtilin production rate by 2.06-fold.

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

Cited by 48 publications

References 42 publications

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

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

Artificial Consortia of Bacillus Amyloliquefaciens HM618 and Bacillus Subtilis for Utilizing Food Waste to Synthetize Iturin A

New Continuous Process for the Production of Lipopeptide Biosurfactants in Foam Overflowing Bioreactor

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