Optimal secretion of thermostable Beta-glucosidase in Bacillus subtilis by signal peptide optimization

Khadye, Vishwanath S.; Sawant, Sneha; Shaikh, Kurshedaktar M.; Srivastava, R K; Chandrayan, Sanjeev K.; Odaneth, Annamma A.

doi:10.1016/j.pep.2021.105843

Cited by 16 publications

(3 citation statements)

References 30 publications

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“…The secretion and expression of α-LA in B. subtilis were successfully realized by screening the SP library, and Arnett et al also successfully expressed active moringa oleifera coagulant protein in the same way and screened strains linked to YngK SP to maximize their yield [33] . When the same signal peptide library was used to screen for signal peptides that promote β-glucosidase secretion, only 15% of the strains had significantly higher β-glucosidase secretions than the basal strains [34] . This indicates that the secretion-promoting effect of signal peptides varies with proteins.…”

Section: Selection Of Sps To Facilitate the Efficient Secretion Of α-Lamentioning

confidence: 99%

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Zhu,

Sun,

et al. 2024

Food Science and Human Wellness

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Alpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA was identifi ed in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant. The original strain most likely only produced α-LA intracellular, but not extracellular. To improve the expression and secretion of α-LA in RIK1285, a library of 173 homologous SPs from the B. subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285. SP YjcN was determined to be the best signal peptide. Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purifi ed by nickel column to calculate the highest yield signal peptide. In addition, different promoters (P aprE , P 43 , and P glv ) were compared and applied. The results indicated that the strain RIK1285-pBE-P glv -YjcN-LALBA had the highest α-LA yield, reaching 122.04 μg/mL. This study demonstrates successful expression and secretion of human α-LA in B. subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

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Section: Selection Of Sps To Facilitate the Efficient Secretion Of α-Lamentioning

confidence: 99%

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Zhu,

Sun,

et al. 2024

Food Science and Human Wellness

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“…Another shuttle vector lately fashionable is pBE-S, successfully used for the secretion of thermostable α-glucosidase by B. subtilis RIK1285 [23].…”

Section: Autonomously Replicating Vectorsmentioning

confidence: 99%

Cloning Systems in Bacillus: Bioengineering of Metabolic Pathways for Valuable Recombinant Products

Arsov,

Armenova,

Gergov

et al. 2024

Fermentation

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Representatives of the genus Bacillus have been established as one of the most important industrial microorganisms in the last few decades. Genetically modified B. subtilis and, to a lesser extent, B. licheniformis, B. amyloliquefaciens, and B. megaterium have been used for the heterologous expression of numerous proteins (enzymes, vaccine components, growth factors), platform chemicals, and other organic compounds of industrial importance. Vectors designed to work in Bacillus spp. have dramatically increased in number and complexity. Today, they provide opportunities for genetic manipulation on every level, from point mutations to systems biology, that were impossible even ten years ago. The present review aims to describe concisely the latest developments in the shuttle, integrative, and CRISPR-Cas9 vectors in Bacillus spp. as well as their application for large-scale bioengineering with the prospect of producing valuable compounds on an industrial scale. Genetic manipulations of promoters and vectors, together with their impact on secretory and metabolic pathways, are discussed in detail.

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“…The optimization of the signal peptide of a specific enzyme was generally carried out using high-throughput screening of the signal peptide library or semi-rational design based on the signal peptide composition [ 23 , 24 ]. Using an optimized CitH signal peptide identified in a signal peptide library, the activity of a thermostable glucosidase was increased from 1.00 to 5.20 U/mL in the culture supernatant, by expressing the enzyme as a secretory form in B. subtilis [ 25 ]. An increase in the titer of an alkaline polygalacturonate lyase produced in B. subtilis was achieved using a semi-rational strategy with six signal peptides [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

Enhanced Extracellular Production and Characterization of Sucrose Isomerase in Bacillus subtilis with Optimized Signal Peptides

Guo

Jiang

et al. 2022

Foods

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Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, which is an important functional sugar widely used in the food industry. However, the lack of safe and efficient expression systems for recombinant SIase has impeded its production and application. In this study, enhanced expression of a SIase from Klebsiella sp. LX3 (referred to as KsLX3-SIase) was achieved in Bacillus subtilis WB800N, by optimizing the signal peptides. First, 13 candidate signal peptides were selected using a semi-rational approach, and their effects on KsLX3-SIase secretion were compared. The signal peptide WapA was most efficient in directing the secretion of KsLX3-SIase into the culture medium, producing a specific activity of 23.0 U/mL, as demonstrated by shake flask culture. Using a fed-batch strategy, the activity of KsLX3-SIase in the culture medium was increased to 125.0 U/mL in a 5-L fermentor. Finally, the expressed KsLX3-SIase was purified and was found to have maximum activity at 45 °C and pH 5.5. Its Km for sucrose was 267.6 ± 18.6 mmol/L, and its kcat/Km was 10.1 ± 0.2 s−1mM−1. These findings demonstrated an efficient expression of SIase in B. subtilis, and this is thought to be the highest level of SIase produced in a food-grade bacteria to date.

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Optimal secretion of thermostable Beta-glucosidase in Bacillus subtilis by signal peptide optimization

Cited by 16 publications

References 30 publications

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Cloning Systems in Bacillus: Bioengineering of Metabolic Pathways for Valuable Recombinant Products

Enhanced Extracellular Production and Characterization of Sucrose Isomerase in Bacillus subtilis with Optimized Signal Peptides

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