There is a need in the field of microbial metabolic engineering for tunable promoters and novel regulatory switches for the inducible expression of heterologous proteins. The “old standard” lac promoter/Lac repressor system is still widely used. However, the commonly used inducer, isopropylthio-beta-D-galactoside (“IPTG”), is expensive and thus not practical for large-scale bioprocesses. Additionally, higher concentrations of IPTG increase the metabolic burden on the cell, in turn reducing the maximal expression of the target gene (Donovan et al., J. Ind. Microbiol. 16:145-154 (1996)). The few available alternatives also have limitations.
One possible solution to this problem is to appropriate existing genetic systems of transcriptional regulators to enhance heterologous gene expression. A number a of transcriptional regulators are known. For example, the LysR family of transcriptional regulators is one of the largest groups of transcriptional regulators in prokaryotes (Schell, Annu. Rev. Microbiol. 47:597-626 (1993)). Currently, there are over 80 known members of this regulator family. Proteins having greater than 20% amino acid identity with another LysR family member or having the consensus sequence of the N-terminal region of the LysR family are considered to be members of this regulator family. LysR family members are also commonly found in the size range of 276 to 324 amino acids, bind to similar DNA sequences in the absence of inducers, have promoters that are located close to or overlapping those of the regulated target gene, and most can repress their own transcriptional levels 3- to 10-fold. Activation of the regulated target gene occurs in the presence of inducer and usually results in a 6- to 200-fold increase in regulated target gene transcription. Regulated target genes are diverse and have numerous functions.
Recently, the gene encoded by open reading frame (“ORF”) b3243 in Escherichia coli (“E. coli”) has been demonstrated to function via quorum sensing (Sperandio et al., Infect. Immun. 70:3085-3093 (2002)). Quorum sensing is the ability of bacteria cells to communicate with one another through perception of the accumulation of signaling molecules based on bacteria cell density. The gene encoded by ORF b3243 is up-regulated via quorum sensing resulting in a 23-fold increase in transcription of the gene. The protein produced by the b3243 ORF was not purified. The gene was found to have a role in the regulation of the LEE genes involved in a type III secretion system, a pathogenecity system that serves to translocate, upon contact with eukaryotic host cells, proteins from the bacteria cytoplasm into the host cell cytoplasm. As the b3243 ORF gene is a putative regulator of the LysR family, the b3243 ORF gene itself was able to induce a four-fold induction of LEE1 transcription. No inducer of the b3243 ORF gene was identified. One in the art will appreciate that this inefficient induction prevents the b3243 ORF gene from being a viable promoter/regulator system without the identification of its inducer.
The use of promoter/reporter gene constructs is well known in the art (Serebriiskii and Golemis, Anal. Biochem. 285:1-15 (2000); Spergel et al., Prog. Neurobiol. 63:673-686 (2001); Yarranton, Curr. Opin. Biotechnol. 3:506-511 (1992)). Particularly, reporter systems utilizing β-galactosidase, green florescent protein, luciferase, and chloramphenicol acetyl transferase (CAT) are all commonly used in the art. Additionally, reporter systems such as luxCDABE are useful because this operon contains all of the genes required for bioluminescent reporting ((Van Dyk et al., Proc. Nat. Acad. Sci. USA 98:2555-2560 (2001)).
The luxCDABE operon has been utilized to create a collection of random gene fusions, comprising 27% of the known or predicted transcriptional units of E. coli (Van Dyk et al., J. Bacteriol. 183:5496-5505 (2001)). Treatment of E. coli cells containing these gene fusions with nalidixic acid, a quinolone, results in selective up-regulation of ten genes. Some of these up-regulated genes are LexA-regulated SOS genes, while others are not generally induced by DNA damage.
Aromatic compounds such as aromatic carboxylic acids are usually toxic to microorganisms. Numerous bacterial strains resistant to aromatic compounds, however, are known in the prior art (Diaz et al., Microbiol. Mol. Biol. Rev. 65:523-569 (2001)). Additionally, a LysR family member from Acinetobacter, BenM, is responsive to synergistic induction by benzoic acid, an aromatic carboxylic acid, and muconic acid (Bundy et al., Proc. Natl. Acad. Sci. USA 99:7693-7698 (2002)). Benzoic acid alone, however, produces minimal, if any, induction of BenM activity. Even the synergistic response with muconic acid produces only a four-fold increase in BenM activity.
U.S. Pat. No. 5,292,643 issued to Shibano et al. on Mar. 8, 1994 describes genes related to fusaric acid resistance in variety of microorganisms. Specifically, genes capable of decomposing or detoxifying fusaric acid are disclosed. One of the genes postulated to be involved in fusaric acid resistance, fusB, shares some homology with the putativTe efflux transporter (PET) yhcP gene (Paulsen et al., FEMS Microbiol. Lett. 156:1-8 (1997)). Applicants incorporate by reference the co-owned and concurrently filed application entitled “PET Family of Efflux Proteins”, U.S. Patent Application No. 60/440,760, which describes new proteins efflux proteins whose expression may alter the expression of carboxylic acids.
The problem to be solved therefore is to discover facile and inexpensive methods of inducible expression of heterologous genes. Applicants have solved the stated problem through the discovery that the promoter elements of the yhcRQP operon are responsive to the expression of the yhcS regulator (a member of the LysR family of transcriptional regulators) whose expression may be induced by an inexpensive cadre of aromatic carboxylic acid inducers.