An auto-inducible phosphate-controlled expression system of Bacillus licheniformis

Abstract

Background: A promoter that drives high-level, long-term expression of the target gene under substrate limited growth conditions in the absence of an artificial inducer would facilitate the efficient production of heterologous proteins at low cost. A novel phosphate-regulated expression system was constructed using the promoter of the phytase encoding gene phyL from Bacillus licheniformis for the overexpression of proteins in this industrially relevant host.

Results: It is shown that the phyL promoter enables a strong overexpression of the heterologous genes amyE and xynA in B. licheniformis when cells were subjected to phosphate limitation. Whether B. licheniformis can use phytate as an alternative phosphate source and how this substrate influences the PphyL controlled gene expression under growth conditions with limited inorganic phosphate concentrations were also investigated. It is shown that B. licheniformis cells are able to use sodium phytate as alternative phosphate source. The addition of small amounts of sodium phytate (≤ 5 mM) to the growth medium resulted in a strong induction and overexpression of both model genes in B. licheniformis cells under phosphate limited growth conditions.

Conclusions: The PphyL controlled expression of the investigated heterologous genes in B. licheniformis is strongly auto-induced under phosphate limited conditions. The proposed PphyL expression system enables an overexpression of target genes in B. licheniformis under growth conditions, which can be easily performed in a fed-batch fermentation process.

Keywords: Bacillus licheniformis; Heterologous gene expression; Phosphate starvation; Phytate.

Fig. 1

Comparison of the promoter regions of the phyL gene from B. licheniformis and the phyC gene from B. amyloliquefaciens FZB42. Consensus sequences (− 35 and − 10) are underlined. The boxes indicate the putative ribosome binding sites. Grey shading indicates the putative PhoP binding boxes. The structure of the phyC promoter of B. amyloliquefaciens FZB42 was investigated in detail by Makarewicz et al. [26]

Fig. 2

The phyL promoter-dependend expression of the recombinant amylase and xylanase encoding genes of the B. licheniformis strains TH3 (a) and TH4 (b) under phosphate limited growth conditions (n = 3 independent cultivations). Lines indicate cell growth, while bars indicate enzyme activity. Growth: triangles TH3, squares TH4. Enzyme activity: grey bars amylase, black bars xylanase. c and d are SDS-PAGE separations of extracellular proteins in B. licheniformis strains TH3 and TH4, respectively. Each sample contained 20 μL of culture medium after removal of the cells by centrifugation. Protein bands are indicated by arrows

Fig. 3

The expression of the recombinant amylase and xylanase driven by the phyL promoter (n = 3 independent cultivations) of the B. licheniformis strains TH3 (a) and TH4 (b) without and with the addition of sodium phytate. Sodium phytate was added at an OD_{500 nm} of 1.0 (after 8 h cultivation). Lines indicate cell growth, while bars indicate enzyme activity. Growth: Open symbols TH3, filled symbols TH4; triangles without the addition of sodium phytate, circles with 0.5 mM sodium phytate, squares with 5 mM sodium phytate. Enzyme activity: white bars without the addition of sodium phytate, grey bars with 0.5 mM sodium phytate, black bars with 5 mM sodium phytate. SDS-PAGE separation of extracellular proteins in 20 μL of culture supernatant of strain TH4 without addition of sodium phytate (c) and with 0.5 mM sodium phytate (d). Sodium phytate was added at an OD _{500 nm} of 1.0 (after 8 h cultivation). Protein bands are indicated by arrows

Fig. 4

Northern-blot analysis of the amyE gene expression of the B. licheniformis strain TH3 without (a) and with (b) 0.5 mM sodium phytate (added at an OD_{500 nm} of 1.0). RNA samples were isolated from exponentially growing cells at an OD of 0.4 (1), at an OD of 1.0 (2) and 1, 2, 3 and 4 h after an OD of 1.0 (3, 4, 5 and 6). 10 μg of total RNA was used for each sample