Efficient constitutive expression of thermostable 4-α-glucanotransferase in Bacillus subtilis using dual promoters

Kang, Hee-Kwon; Jang, Jun-Hyuck; Shim, Jae‐Hoon; Park, Jong-Tae; Kim, Young-Wan; Park, Kwan-Hwa

doi:10.1007/s11274-010-0351-5

Cited by 27 publications

(18 citation statements)

References 12 publications

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“…A previous report demonstrated the expression level of AP in B. subtilis WB600 under the control of P HpaII . However, the expression and secretion levels were incomparable to those of other proteins [ 22 , 30 , 31 ]. Plasmid pBSG06 was introduced into B. subtilis 168, generating recombinant strain BSG107.…”

Section: Resultsmentioning

confidence: 99%

Construction and development of an auto-regulatory gene expression system in Bacillus subtilis

et al. 2015

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BackgroundBacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA).ResultsThe promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the −10 and −35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level.ConclusionThe auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression system that has an expression pattern that is split between cell-growth and over-expression, leading to an increase in cell density and elevating the overall expression levels of heterologously expressed proteins. The broad applicability of this system and inducer-free expression property in B. subtilis facilitate the industrial scale-up and medical applications for the over-production of a variety of desired proteins.

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Section: Resultsmentioning

confidence: 99%

Construction and development of an auto-regulatory gene expression system in Bacillus subtilis

et al. 2015

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“…Numerous efforts have been carried out for enhanced hosts from the single factor to the network level (Anne, Economou, & Bernaerts, 2017; Song et al, 2015; van Dijl & Hecker, 2013; Westers et al, 2004), such as knockout of protease genes (Westers, Dijkstra, Westers, Van Dijl, & Quax, 2006; Wu, Lee, Tran, & Wong, 1991), fine‐tuning of gene expression at the levels of both transcription and translation (H.‐K. Kang et al, 2010; Phan, Nguyen, & Schumann, 2012), minimization of the dispensable genomic regions (Morimoto et al, 2008; Tanaka et al, 2013), and so on. In perspective of the industrial production, the optimized secretory pathway and engineered B. subtilis hosts would be combined to lead to next‐generation super‐secreting Bacillus cell factories.…”

Section: Discussionmentioning

confidence: 99%

Efficient secretory production of large‐size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution

Liu

Wang

Zhu

et al. 2020

Biotech & Bioengineering

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Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large‐size heterologous enzymes with a special focus on 83‐kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5‐IA was improved by directed evolution with two novel double‐layer Petri‐dish‐based high‐throughput screening (HTS) methods. The high‐sensitivity HTS relied on the detection of high‐activity Cel5 on the carboxymethylcellulose/Congo‐red assay. The second modest‐sensitivity HTS focused on the detection of low‐activity IA on the amylodextrin‐I2 assay. After six rounds of HTS, a secretory Cel5‐IA level was increased to 234 mg/L, 155 times the wild‐type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large‐size heterologous proteins in B. subtilis.

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“…Widner [33] found that gene expression was distinctly increased under the control of two or three tandem promoters in contrast to one alone in B. subtilis . Kang [34] also reported that two expression systems constructed by sequential alignment of a constitutive promoter for either α-amylase from B. subtilis NA64 or maltogenic amylase from B. licheniformis downstream of the Hpa II promoter elevated the TSaGT productivity by 11- and 12-fold, respectively, in comparison with the single Hpa II promoter system. Li [35] tried to construct multiple core-tac-promoters (MCPtacs) in tandem and found that integration of the phaCAB genes with the 5 copies of core-tac-promoters resulted in an engineered E. coli that can accumulate 23.7% polyhydroxybutyrate of the cell dry weight in batch cultivation.…”

Section: Discussionmentioning

confidence: 99%

Generation of an Artificial Double Promoter for Protein Expression in Bacillus subtilis through a Promoter Trap System

Yang

Zhang

et al. 2013

PLoS ONE

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Bacillus subtilis is an attractive host for production of recombinant proteins. Promoters and expression plasmid backbones have direct impacts on the efficiency of gene expression. To screen and isolate strong promoters, a promoter trap vector pShuttleF was developed in this study. Using the vector, approximately 1000 colonies containing likely promoters from Bacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09 exhibited the highest β-Gal activities in both Escherichia coli and B. subtilis. The activity of pShuttle-09 in B. subtilis was eight times of that of the P43 promoter, a commonly used strong promoter for B. subtilis. A sequence analysis showed that pShuttle-09 contained PluxS and truncated luxS in-frame fused with the reporter gene as well as another fragment upstream of PluxS containing a putative promoter. This putative promoter was a hybrid promoter and its β-Gal activity was higher than PluxS. Reconstructing the hybrid promoter from pShuttle-09 to PlapS further improved the β-Gal production by 60%. The usefulness of our promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. Thus, our data provide additional evidence to support the concept of using a promoter trap system to create new promoters.

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Efficient constitutive expression of thermostable 4-α-glucanotransferase in Bacillus subtilis using dual promoters

Cited by 27 publications

References 12 publications

Construction and development of an auto-regulatory gene expression system in Bacillus subtilis

Construction and development of an auto-regulatory gene expression system in Bacillus subtilis

Efficient secretory production of large‐size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution

Generation of an Artificial Double Promoter for Protein Expression in Bacillus subtilis through a Promoter Trap System

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