Elsevier

Journal of Biotechnology

Volume 331, 10 April 2021, Pages 74-82
Journal of Biotechnology

Efficient secretory expression of Bacillus stearothermophilus α/β-cyclodextrin glycosyltransferase in Bacillus subtilis

https://doi.org/10.1016/j.jbiotec.2021.03.011Get rights and content

Highlights

  • B. stearothermophilus α/β-CGTase was efficiently expressed in B. subtilis.

  • The optimal signal peptide was obtained from 173 signal peptides.

  • Inclusion bodies were significantly reduced by replacement of N-terminal amino acids.

  • The expression level reached 249.35 U⋅ mL−1, which was 2.3 times that of the control.

  • The fermentation process could be more simply regulated by knocking out ppsE and sfp.

Abstract

Bacillus stearothermophilus α/β-cyclodextrin glycosyltransferase (α/β-CGTase) is an excellent transglycosylase with broad potential for food application, but its expression level is low in Bacillus subtilis. In this study, the optimal signal peptide for α/β-CGTase expression was screened from 173 signal peptides in B. subtilis WS11. The α/β-CGTase activity in a 3-L fermentor reached 151.93 U⋅ mL−1, but substantial amounts of inclusion bodies were produced. The N-terminal 12 amino acids of α/β-CGTase were then replaced with the N-terminal 15 amino acids of a β-CGTase from the same family that has a high percentage of disorder-promoting amino acids. As a result, the inclusion bodies were significantly reduced, and the enzyme activity increased to 249.35 U mL−1, 2.3 times that of the strain constructed previously. Finally, the ppsE and sfp genes of B. subtilis WS11, which are related to lipopeptide biosurfactant synthesis, were knocked out to produce B. subtilis WS13. When B. subtilis WS13 was used to produce α/β-CGTase in a 3-L fermentor, 70 % less defoaming agent was required than with B. subtilis WS11. Furthermore, enzyme production and growth of WS13 were equivalent to those of WS11. This study is of great significance for future research to efficiently scale-up production of α/β-CGTase.

Introduction

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19), which belongs to the α-amylase family (glycosyl hydrolase family 13), is a multifunctional enzyme capable of catalyzing three transglycosylation reactions (disproportionation, cyclization, and coupling) as well as hydrolysis (van der Veen et al., 2000). The cyclization reaction involves an intramolecular transglycosylation in which the non-reducing glycosides of the maltooligosaccharide donors are used as receptors to produce cyclodextrins. The coupling reaction (in which the donor is cyclodextrin) and disproportionation reaction (in which the donors are maltodextrin, oligosaccharides, etc.) use a variety of small molecular sugars and their derivatives or analogs as receptors, and produce a variety of glycosylation products that have improved physical and chemical properties and higher added value. Therefore, CGTase is widely used in the food, medicine, and cosmetics industries. The preparation of CGTase itself has also drawn the attention of researchers (Han et al., 2014; Qi and Zimmermann, 2005).

Effective methods that improve CGTase expression include natural screening and heterologous recombinant expression. The yields of CGTases produced by heterologous recombinant expression were generally significantly higher than those of wild-type strains (Elbaz et al., 2015; Han et al., 2014; Man et al., 2016; Tao et al., 2020; Zhang et al., 2017). So far, most heterologous recombinant expression procedures used Escherichia coli as the host, and some high yields have been obtained, such as those of Bacillus sp. G1 β-CGTase (69.15 U⋅ mL−1), Paenibacillus macerans α-CGTase (275.3 U⋅ mL−1), and B. stearothermophilus NO2 α/β-CGTase (1904 U⋅ mL−1, disproportion activity) (Cheng et al., 2011; Deng et al., 2018; Han et al., 2014; Low et al., 2011; Tao et al., 2020). However, when CGTases are used in the food industry for the production of bread, α-cyclodextrin, modified starch, glycosylated stevioside, trehalose, etc. (Han et al., 2014; Ji et al., 2020; Mukai et al., 1997), safety issues are particularly prominent. E. coli is generally considered to present a security problem due to the production of endotoxin. Bacillus subtilis is a Gram-positive bacterium that produces neither toxins nor thermogenic or sensitizing proteins, and it has been approved as a safe strain for use in food industry by the US Food and Drug Administration and the Ministry of Agriculture of China. As B. subtilis is simple to culture, grows rapidly, and has a strong protein-secreting capacity, it has been widely used in the production of industrial enzyme preparations (Guan et al., 2015; Wu et al., 1991; Zhou et al., 2019). However, only a few papers have reported the expression of CGTase in B. subtilis. Gimenez et al. expressed the CGTase of Bacillus firmus strain 37 in B. subtilis WB800 with the activity of 1.33 μmol β-CD/min/mL (Gimenez et al., 2019). Li et al. expressed Bacillus circulans β-CGTase in B. subtilis WB600 and the β-CGTase enzyme activity reached 36.9 U⋅ mL−1 after optimization of the fermentation conditions (Li et al., 2016). In our previous work, we obtained the B. circulans β-CGTase activity of 30.5 U⋅ mL−1 and P. macerans α-CGTase activity of 9.5 U⋅ mL−1 expressed in B. subtilis CCTCC M 2016536 during shake-flask cultivation, and a β-CGTase activity of 571.2 U⋅ mL−1 (2.5 mg⋅ mL−1) in a 3-L fermentor (Zhang et al., 2017).

Many strategies, including protease knockout, chaperone co-expression, promoter and signal peptide optimization, and others, have been investigated to improve the level of recombinant protein expression (Guan et al., 2016; Gupta and Rao, 2014; Song et al., 2016; Zhou et al., 2019). In these strategies, signal peptide is an important regulatory element in the secretion of proteins, and a good signal peptide can greatly enhance the secretion of the target protein (Brockmeier et al., 2006; Kang et al., 2020; Yao et al., 2019). Brockmeier et al. constructed a signal peptide screening system in B. subtilis using cutinase and cytoplasmic esterase as reporter proteins. Using this system, it became possible to explore the effect of the Sec pathway signal peptide on the transport of heterologous proteins. The results showed that all signal peptides had the potential to transport heterologous proteins, but each heterologous protein had a specific optimal signal peptide (Brockmeier et al., 2006). Ling et al. selected 14 signal peptides to fuse with CGTase for expression and secretion in E. coli. They found that the use of the GlcNAc-binding protein A (GAP) signal peptide increased extracellular and periplasmic CGTase activity by 7.35 and 2.02 times, respectively (Ling et al., 2017). Sonnendecker et al. compared the effects of different signal peptides on CGTase expression in Bacillus sp. G825−6, and found that DacD signal peptide-mediated expression was 3.9- and 3.1-times higher than expression mediated by the PelB signal peptide and the native signal peptide, respectively (Sonnendecker et al., 2017). These studies showed that expression levels can be increased by screening for signal peptides that match heterologous proteins. However, a particular preferred signal peptide is not suitable for the expression of all heterologous proteins. Therefore, it is especially important to identify a signal peptide suitable for the expression of the target protein from a large number of samples using a high-throughput platform (Freudl, 2018; Peng et al., 2019).

The Sec pathway is the most important protein secretion pathway in B. subtilis. It causes the secretion of cellular proteins that are in an unfolded state, and the unfolded and translocation-competent state is of great importance (Harwood and Cranenburgh, 2008; Terpe, 2006). Li et al. developed predictors of disordered protein by analyzing more than 6000 amino acid sequence attributes (Li et al., 2000). Campen et al. ranked the amino acids from order-promoting to disorder-promoting (Campen et al., 2008). Wang further explored the differences in N-terminal amino acid composition between exported and cytoplasmic proteins. He found that the N-termini of exported proteins are dominated by disorder-promoting amino acids, which may provide binding sites for key proteins that aid protein secretion. By contrast, the N-termini of cytoplasmic proteins are mainly composed of order-promoting amino acids, which tend to form higher-order structures that hinder extracellular secretion (Wang, 2013).

Bacillus stearothermophilus α/β-CGTase is among the most widely used enzymes in industrial production due to its excellent transglycosylation efficiency and high specificity (Tao et al., 2018). Our team constructed a recombinant α/β-CGTase-producing strain derived from B. subtilis WS11. After promoter screening and optimization, its activity in a 3-L fermentor reached 110.4 U/mL (Li et al., 2018; Zhang et al., 2018). In this study, α/β-CGTase expression was further improved by screening for the optimal signal peptide and modifying its N-terminal amino acid sequence. Finally, in order to reduce the production of foam during fermentation and facilitate regulation of the fermentation process, the genes in the B. subtilis WS11 genome involved in lipopeptide production were knocked out.

Section snippets

Strains and plasmids

E. coli JM109 variants harboring expression plasmids pHY-α/βcgt and pHY-βcgt, as well as the expression host B. subtilis WS11, were constructed in our laboratory and stored in 15 % glycerol at -80 °C (Table 1) (Li et al., 2018; Zhang et al., 2017, 2018). The cloning host E. coli JM109, the cloning plasmid pMD19-T-simple, and the B. subtilis Secretory Protein Expression System kit were purchased from Takara (Dalian, China) Bioengineering Co., Ltd.

Signal peptide screening and construction of recombinant bacteria

Using plasmid PHY-α/βcgt as a template, the α/βcgt

Effect of the signal peptide on heterologous expression of α/β-CGTase in B. Subtilis

Different signal peptides have different effects on the expression of heterologous proteins, so it is especially important to screen a large number of samples to select a signal peptide suitable for α/β-CGTase (Brockmeier et al., 2006). In this study, the expression plasmid pBE-S-spα/βcgt, which contained the B. subtilis Secretory Protein Expression System signal peptide library, was constructed and then inserted into the host strain B. subtilis RIK1285. The recombinant strains were cultured in

Conclusions

Cyclodextrin glycosyltransferase is capable of preparing a variety of high value-added products via transglycosylation reactions and is widely used in many fields. Since B. subtilis is simple to culture, grows rapidly, and has strong protein-secreting capacity and a good safety profile, it has been widely used in the production of industrial enzyme preparations. The aim of this study was to extend our previous host strain construction, promoter screening, and optimization efforts (Li et al.,

Author statement

Lingqia Su: Conceptualization, Data Curation, Funding acquisition, Methodology, Project administration, Writing - Original Draft, Writing - Review & Editing. Yunfei Li: Validation, Formal analysis, Data Curation, Investigation, Writing - Original Draft. Jing Wu: Conceptualization, Resources, Funding acquisition, Project administration, Resources, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The study was financed by the National Natural Science Foundation of China (31730067, 31771916), the Natural Science Foundation of Jiangsu Province (BK20180082), the Science and Technology Project of Jiangsu Province - Modern Agriculture (BE2018305), and the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-03).

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