Using inexpensive substrate to achieve high-level lipase A secretion by Bacillus subtilis through signal peptide and promoter screening

Wu, Fang–Jing; Ma, Jiayuan; Cha, Yaping; Lu, De-Lin; Li, Zhiwei; Zhuo, Min; Luo, Xiao‐Chun; Li, Shuang; Zhu, Ming‐Jun

doi:10.1016/j.procbio.2020.08.010

Cited by 16 publications

(9 citation statements)

References 69 publications

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“…For plasmids expressed by fusion of mCherry with different HlyA truncates, the pRSF-mCherry-HlyA218 plasmid was used as a template to amplify hlyA158 with primers hlyA218-F/hlyA158-R, hlyA112 with primers hlyA112-F/hlyA158-R, hlyA60 with primers hlyA60-F/hlyA218-R, H1 with primers H1–F/H1-R, H2 with primers H2–F/H2-R, and H12 with primers H1–F/H2-R, and the obtained fragment was digested by Sal I and Xho I. Digested fragments of different HlyA truncates were ligated to the same digested pRSF-mCherry-hlyA218 vector. To obtain plasmids pRSF-AXE-HlyA60 and pRSF-LipA-HlyA60, the axe gene encoding acetyl xylan esterase (AXE, GenBank: AJ249957) was amplified with primers AXE-F/AXE-R and the lipA gene fragment (sequence is shown in Table S2) encoding lipase was amplified from pMAL1 with LipA-F/LipA-R and recombined with the pRSF-mCherry-hlyA60 vector digested using Nco I and Sac I. The recombinant strains were named AXEA60 and LipAA60 after transformation of pRSF-AXE-HlyA60 and pRSF-LipA-HlyA60 into E.…”

Section: Methodsmentioning

confidence: 99%

“…Digested fragments of different HlyA truncates were ligated to the same digested pRSF-mCherry-hlyA218 vector. To obtain plasmids pRSF-AXE-HlyA60 and pRSF-LipA-HlyA60, the axe gene encoding acetyl xylan esterase (AXE, GenBank: AJ249957) was amplified with primers AXE-F/AXE-R and the lipA gene fragment (sequence is shown in Table S2) encoding lipase was amplified from pMAL1 26 with LipA-F/LipA-R and recombined with the pRSF-mCherry-hlyA60 vector digested using NcoI and SacI. The recombinant strains were named AXEA60 and LipAA60 after transformation of pRSF-AXE-HlyA60 and pRSF-LipA-HlyA60 into E. coli BL21 (DE3), respectively.…”

Section: Plasmid and Strain Constructionmentioning

confidence: 99%

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Discovery and Identification of a Novel Tag of HlyA60 for Protein Active Aggregate Formation in Escherichia coli

Ma,

Liu,

Cai

et al. 2023

J. Agric. Food Chem.

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The strategy of active aggregation tag fusion expression with target proteins can solve the problems of restricted expression, inefficient purification, and laborious immobilization faced in the production of recombinant proteins in Escherichia coli. We localized a novel active aggregation peptide HlyA60 from the hemolysin A secretion system, which can effectively induce aggregate formation with satisfactory protein activities in E. coli after fusion expression with the protein of interest. Based on structural prediction and surface properties, the process of active aggregation of HlyA60 through electrostatic interactions and hydrophobic interactions was analyzed. To investigate the potential application of HlyA60 as an efficient aggregation tag, it was fused with acetyl xylan esterase and lipase A, separately. The resulting fusion proteins demonstrated active aggregation rates of 97.6 and 66.7%, respectively, leading to 1.9-fold and 1.7-fold increases in bacterial density at the end of fermentation. The AXE-HlyA60 fusion protein, which exhibited superior performance, was subjected to purification and immobilization. It was able to achieve column-free purification with an impressive 98.8% recovery and in situ immobilization; the immobilization enabled 30 cycles of reactions to take place with 85% residual activity maintained. Our findings provide a novel tool for efficiently producing recombinant proteins in E. coli.

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Section: Methodsmentioning

confidence: 99%

Section: Plasmid and Strain Constructionmentioning

confidence: 99%

Discovery and Identification of a Novel Tag of HlyA60 for Protein Active Aggregate Formation in Escherichia coli

Ma,

Liu,

Cai

et al. 2023

J. Agric. Food Chem.

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“…The Gram-positive bacterium B. subtilis is Generally Recognized As Safe (GRAS) microorganism due to its lack of pathogenicity and absence of endotoxins as well as its safe use as food and feed probiotics. It is one of the most important industrial hosts for the production of numerous proteins, especially homologous enzymes, such as α-amylase (Chen et al 2015 ), protease (Dong et al 2017 ; Wenzel et al 2011 ), xylanase (Helianti et al 2016 ; Rashid and Sohail 2021 ), lipase (Lu et al 2010 ; Wu et al 2020 ), β-glucanase (Niu et al 2018 ), and so on (Schallmey et al 2004 ; Terpe 2006 ). Also, a few heterogeneous enzymes have been over-expressed by B. subtilis by using different promoters (Harwood et al 2002 ).…”

Section: Introductionmentioning

confidence: 99%

A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis

Bai

et al. 2022

Bioresour. Bioprocess.

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To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25–40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation. Graphical Abstract

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“…It has some advantages, such as greater homogeneity of the culture medium and greater ease of controlling variable parameters such as temperature and pH. In addition, recovery and purification of extracellular lipases by SMF are simple, and agro-industrial residue substrates can also be used as carbon and nitrogen sources (Bharathi & Rajalakshmi, 2019;Geoffry & Achur, 2018;Wu et al, 2020).…”

Section: Streptomyces Violascensmentioning

confidence: 99%

“…Recent reports exemplify the use of agro-industrial residues as substrates for the production of microbial lipases. Wu et al (2020) showed the viability of industrial production of lipase A by Bacillus subtilis using glycerol and chicken feather hydrolysate as an inexpensive source of carbon and nitrogen. Knob et al (2020) isolated lipolytic yeasts from slaughterhouse refrigerator effluent and oil mill effluent.…”

Section: Streptomyces Violascensmentioning

confidence: 99%

Aplicação de microrganismos lipolíticos em alimentos e na biodegradação de poliuretanos

Salgado

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Os microrganismos são considerados fontes inestimáveis de compostos extracelulares, como enzimas, tornando-os o centro da investigação científica, tanto nos aspectos de biodeterioração em que estão envolvidos, quanto para aplicação biotecnológica. As lipases são enzimas responsáveis principalmente, pela hidrólise dos triacilgliceróis e amplamente exploradas comercialmente. Além da hidrólise, conseguem catalisar a reação reversa, a esterificação e, também reações como transesterificação, interesterificação, acidólise e aminólise. Dese modo, as lipases são reconhecidas como catalisadores promissores no melhoramento da qualidade sensorial de diversos produtos alimentícios além de, biodegradarem diversos substratos. A lipase de Serratia liquefaciens L135 foi identificada como uma poliuretanase, ou seja, uma enzima com a capacidade de biodegradar poliuretanos (PU). Este trabalho foi desenvolvido com o objetivo de explorar o potencial desta enzima na indústria de alimentos e na biodegradação de poliuretanos por S. liquefaciens isoladamente ou em consórcio microbiano. Para isso buscou-se isolar potenciais biodegradadores de PU do intestino de larvas de Galleria mellonella e avaliar o efeito da associação com S. liquefaciens na biodegradação de PU in vitro. O primeiro capítulo apresenta uma revisão de literatura que abrange as aplicações de lipases em indústrias de laticínios; óleos e gorduras; panificação e confeitaria; carnes; sabores e aromas; e outras indústrias alimentícias. Além disso, aborda as técnicas de fermentação de baixo custo e engenharia de proteínas, como promissoras para produção de lipases microbianas. No segundo capítulo são apresentados resultados de estudos in silico e in vitro do potencial biodegradador de PU por S. liquefaciens L135 e sua poliuretanase. Para o estudo in silico, foram construídos monômeros e tetrâmeros de PU para realizar o docking molecular com a poliuretanase modelada e validada de S. liquefaciens. Foi possível verificar que os PU ligaram ao resíduo de aminoácido S207, que faz parte da tríade catalítica e é responsável pela ação hidrolítica da poliuretanase. Os monômeros apresentaram melhores scores de ligações, quando comparados com os tetrâmeros, devido às interações estéricas repulsivas. Os PU, Impranil® e poli[4,4'-metilenobis(fenilisocianato)-alt-1,4- butanodiol/di(propilenoglicol)/policaprolactona] (PCLMDI), foram usados para as análises in vitro. A biodegradação do Impranil® por S. liquefaciens e sua poliuretanase foi confirmada em ágar pela formação de halos transparentes. Além disso, foram preparados discos de Impranil® e filmes de PCLMDI e a biodegradação durante o cultivo de S. liquefaciens foi avaliada por microscopia eletrônica de varredura (MEV). Foi possível averiguar a formação de rachaduras e poros na superfície dos PU, configurando o processo de biodegradação. No terceiro capítulo, foi feito o isolamento da bactéria Staphylococcus warneri do intestino da larva de G. mellonella, com potencial de biodegradação de PU. O isolado S. warneri, denominado de UFV_01.21, foi avaliado em cultura pura e em consórcio com S. liquefaciens L135 para biodegradação de PU e, em ambas condições, o Impranil® foi utilizado como única fonte de carbono. Com seis dias de incubação, suspensões de Impranil® em caldo Luria-Bertani (LB) inoculadas com S. liquefaciens, S. warneri e com consórcio microbiano, apresentaram 88, 96 e 76% de biodegradação, respectivamente. Tanto nos discos de Impranil®, quanto em filmes de PCLMDI, S. warneri em cultura pura e em consórcio com S. liquefaciens foi capaz de aderir e formar biofilmes. Por MEV, foi possível confirmar a biodegradação devido a formação de rachaduras, sulcos, poros e rugosidades nas superfícies dos PU avaliados. Esses resultados reforçam o potencial dos microrganismos e suas enzimas para biodegradação de PU. Palavras-chave: Biocatalisadores. Biodegradação. Consórcio Microbiano. Docking Molecular.

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Using inexpensive substrate to achieve high-level lipase A secretion by Bacillus subtilis through signal peptide and promoter screening

Cited by 16 publications

References 69 publications

Discovery and Identification of a Novel Tag of HlyA60 for Protein Active Aggregate Formation in Escherichia coli

Discovery and Identification of a Novel Tag of HlyA60 for Protein Active Aggregate Formation in Escherichia coli

A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis

Aplicação de microrganismos lipolíticos em alimentos e na biodegradação de poliuretanos

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