Positively regulated bacterial expression systems

Abstract

Regulated promoters are useful tools for many aspects related to recombinant gene expression in bacteria, including for high-level expression of heterologous proteins and for expression at physiological levels in metabolic engineering applications. In general, it is common to express the genes of interest from an inducible promoter controlled either by a positive regulator or by a repressor protein. In this review, we discuss established and potentially useful positively regulated bacterial promoter systems, with a particular emphasis on those that are controlled by the AraC-XylS family of transcriptional activators. The systems function in a wide range of microorganisms, including enterobacteria, soil bacteria, lactic bacteria and streptomycetes. The available systems that have been applied to express heterologous genes are regulated either by sugars (L-arabinose, L-rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone-related compounds, ε-caprolactam, propionate, thiostrepton, alkanes or peptides. It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species. Based on bioinformatics analyses, the AraC-XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/P(BAD), RhaR-RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.

Figure 1

Binding of AraC to P_BAD in the absence (A) and presence (B) of the inducer molecule l‐arabinose (closed circle) (modified with permission from Schleif, 2003). O₂, I₁ and I₂ are DNA binding sites for AraC. The RNAP is depicted in light colour. For details see text section The AraC/P_BADregulator/promoter system.

Figure 2

AraC‐XylS family regulators' characteristic HTH DNA binding motif is shown by using the member MarA as a model (α‐helixes 3 and 6 in red colour). Conserved amino acid residues are depicted in the bottom row. The alignment was derived from the full‐length primary sequences of the given TFs by using the PROMALS3D web server (Pei et al., 2008). Parameters were left at default values. The figure was prepared by using PyMOL (DeLano, 2003). Note that MarA binds DNA as a monomer.

Figure 3

Mechanism of induction of the RhaR‐RhaS/rhaBAD system (modified with permission from Altenbuchner and Mattes, 2005). l‐rhamnose is actively transported into the cells and it binds to RhaS which then becomes activated and stimulates transcription of rhaT (encoding l‐rhamnose transporter protein) and the rhaBAD operon (encoding l‐rhamnose catabolic enzymes). RhaR is also activated by l‐rhamnose and stimulates transcription of the rhaSR operon (encoding RhaR and RhaS). The catabolite repression is indicated. For further details see text section The RhaR‐RhaS/rhaBAD system shares many expression properties with AraC/P_BAD.

Figure 4

The XylS/Pm expression system. Inducer molecules enter cells passively and bind to XylS which then becomes activated. The activated XylS stimulates transcription from Pm. For details see text section Broad‐host‐range expression systems based on XylS/Pm.

Figure 5

Fermentation course of recombinant E. coli cells expressing totally 2.1 g L⁻¹ of single‐cell antibody fragment scFv‐phOx (P) by using the XylS/Pm expression system. Lines: 1, growth curve; 2, soluble P in periplasm; 3, soluble P in growth medium; 4, insoluble P in cytoplasm; 5, total soluble + insoluble P (data imported from Sletta et al., 2004; 2007).