Elsevier

Journal of Biotechnology

Volume 157, Issue 1, January 2012, Pages 167-172
Journal of Biotechnology

Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements

https://doi.org/10.1016/j.jbiotec.2011.10.006Get rights and content

Abstract

Transcription efficiency of inducible promoters remains a bottleneck in recombinant protein production in Bacillus subtilis cells. Here, we present experimental data how to generate strong IPTG-inducible promoters by optimization of nucleotides at the conserved regions of the groESL promoter including the UP element, the −35, −15, −10 and the +1 region. Combination of these changes into one promoter enhanced the amount of recombinant proteins accumulating intracellularly up to about 30% of the total cellular protein.

Highlights

► A strong intracellular expression system for Bacillus subtilis is described. ► Five different elements of the promoter of the groESL operon have been systematically optimized. ► The amount of recombinant proteins accumulated up to 30% of the total cellular proteins.

Introduction

Bacteria have not evolved to produce a single polypeptide chain in huge amounts. On the contrary, their genetic systems have adapted in such a way to synthesize only the number of protein molecules they really need. One example is the Lac repressor of Escherichia coli cells which is present only in about ten copies per wild-type cell (Gilbert and Müller-Hill, 1966). Nevertheless, based on results obtained from basic research studies dealing with transcription and translation, expression signals have been optimized to obtain high level of gene expression resulting in up to 50% of recombinant protein as part of the total cellular protein content (Miroux and Walker, 1996). In bacteria, almost all the expression systems contain inducible promoters. Therefore, tightly regulated, expression systems with a low basal activity are important tools in molecular biology. So far, most inducible systems have been constructed for E. coli to produce recombinant proteins in high quantities and at low production costs (Mergulhao et al., 2005, Valdez-Cruz et al., 2010, Brautaset et al., 2009, Hannig and Makrides, 1998, Baneyx, 1999). The amount of recombinant proteins produced is dependent on four major factors: efficiency of transcription, mRNA stability, efficiency of translation and stability of the protein.

Bacillus subtilis is a versatile microorganism for the production of recombinant proteins. Its well-known genetics in addition to simple, fast and cost-effective high-density cultivation are important reasons for the use of B. subtilis for heterologous gene expression (Pohl and Harwood, 2010, Schallmey et al., 2004, Schumann, 2007, Westers et al., 2004, Zweers et al., 2008). This microorganism has several advantages over E. coli. First, it is non-pathogenic and does not produce any endotoxin leading to the GRAS (generally regarded as safe) status by the FDA, which means that B. subtilis can be used during food production. Second, due to only one membrane, it easily secretes extracellular proteins. And third, there is no pronounced codon bias. All these advantages are in favour for this Gram-positive bacterium instead of E. coli. Its wide use for the overproduction of recombinant proteins is hampered by the fact that less has been done to engineer B. subtilis cells for high level production of recombinant protein production. The very first inducible promoter has been designated as Pspac and induction occurs by addition of IPTG (Yansura and Henner, 1984). The second promoter system is based on xylose as inducer (Kim et al., 1996). Other systems are based on phosphate (Lee et al., 1991), citrate (Yamamoto et al., 2000), tetracycline (Geissendorfer and Hillen, 1990), subtilin (Bongers et al., 2005), glycine (Phan and Schumann, 2007) as inducer and one system is based on the T7 RNA polymerase (Chen et al., 2010).

Using the E. coliB. subtilis shuttle vector pMLBs72, which has been shown to replicate stably in both species (Titok et al., 2003), we have first developed several expression vectors based on established induction systems (Nguyen et al., 2005). Then, we introduced a new expression system based on the strong promoter of the groESL operon which was fused to the lac operator resulting in the Pgrac promoter. We could demonstrate that this new expression system can result in up to 16% recombinant protein of the total cellular protein (Phan et al., 2006). Here, we attempted to improve the wild-type groESL promoter first by systematically altering its different DNA elements namely the UP element, the −35 and −10 elements and the transcriptional start site, and second to combine the improved elements into one single promoter. To this end, we could enhance the amount of recombinant protein up to about 30%.

Section snippets

Bacterial strains, plasmids and growth conditions

E. coli strains DH10B or XL1 Blue (Stratagene) were used as recipient in all cloning experiments. B. subtilis 1012 (Saito et al., 1979) was used as recipient for all plasmids. Plasmid pHT01 contains the Pgrac promoter and the lacI gene (Nguyen et al., 2007), while the promoter-probe vector pHT06 contains the lacI gene and the lac operator (Phan et al., 2010). Cells were routinely grown in Luria broth (LB) at 37 °C under aeration. Antibiotics were added where appropriate (ampicillin at 100 μg/ml

The strong synthetic promoter Pgrac

Recently, we reported on a new synthetic promoter called Pgrac also termed P01 (Phan et al., 2006). To obtain Pgrac, we fused the lac operator immediately downstream of the groESL promoter to make it controllable by IPTG (Fig. 1A). When the genes bgaB, htpG and pbpE were fused downstream of Pgrac, the recombinant proteins accumulated to 10, 12 and 16% of the total cellular protein, respectively. This promoter also turned out to be 30 (data not shown) and 60 times stronger than PxylA and Pspac,

Conclusions

It has been suggested that the groESL operon of B. subtilis is preceded by an UP element enhancing the activity of the promoter to provide a sufficient amount of GroESL chaperones (Schmidt et al., 1992). Here, we have proven that the AT-rich DNA sequence upstream of the groESL operon indeed functions as an UP element to enhance expression of a transcriptional fusion. We could further show that increasing the AT-richness of the UP element resulted in an about 200% increase in its activity. We

Acknowledgements

P. T. P. Trang was supported by a PhD scholarship from the Bavarian Science Foundation, H. D. Nguyen by the DLR (VNB02/B03) and the MOST (Life Science-643204).

References (39)

  • S. Pohl et al.

    Heterologous protein secretion by Bacillus species: from the cradle to the grave

    Adv. Appl. Microbiol.

    (2010)
  • W. Schumann

    Production of recombinant proteins in Bacillus subtilis

    Adv. Appl. Microbiol.

    (2007)
  • M.A. Titok et al.

    Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector

    Plasmid

    (2003)
  • L. Westers et al.

    Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism

    Biochim. Biophys. Acta

    (2004)
  • S.E. Aiyar et al.

    Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase alpha subunit

    Proc. Natl. Acad. Sci. U.S.A.

    (1998)
  • R.S. Bongers et al.

    Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin

    Appl. Environ. Microbiol.

    (2005)
  • T. Brautaset et al.

    Positively regulated bacterial expression systems

    Microbiol. Biotechnol.

    (2009)
  • T. Caramori et al.

    The UP element of the promoter for the flagellin gene, hag, stimulates transcription from both SigD- and SigA-dependent promoters in Bacillus subtilis

    Mol. Gen. Genet.

    (1998)
  • P.T. Chen et al.

    Construction of chromosomally located T7 expression system for production of heterologous secreted proteins in Bacillus subtilis

    J. Agric. Food Chem.

    (2010)
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