# Patent

## Title
Benzoate-and anthranilate-inducible promoters

## Abstract

Novel benzoate- or anthranilate-inducible promoters, and novel tandem promoters, and variants and improved mutants thereof, useful for commercial prokaryotic fermentation systems, nucleic acid constructs containing the promoters, expression systems using them, methods for expressing proteins by use thereof, and proteins expressed thereby.

## Background
This application is a continuation of co-pending Application No. PCT US2003/020840, published as WO 2004/005211, filed Jul. 3, 2003, which claims priority to U.S. Provisional Application No. 60/393,422, filed Jul. 3, 2002.


REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named “344269 SequenceListing.txt”, created on Jul. 31, 2009, and having a size of 86 kilobytes and is filed as an Amendment to the specification on Jul. 31, 2009. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
BACKGROUND
Gene expression in bacteria, as in any organism, requires that a promoter be present in the regulatory region located 5′ (i.e. upstream) from the coding sequence in order to direct the gene's transcription. Promoters are classified as constitutive promoters and regulated promoters. In commercially useful bacterial expression systems, regulated promoters have proven particularly useful because they permit increase in the organismal biomass while a desired gene(s) is inactive. This allows the host organism to devote maximal energy to cell division and growth. When the regulated promoter is then activated/induced, more cells will be available to express the desired gene(s), thereby increasing the yield of the desired gene product(s).
Regulated promoters include: (1) activatable promoters, i.e. promoters that are inactive until an activator protein binds to the 5′ regulatory region; and (2) repressible promoters, i.e. promoters that are inactive while the 5′ regulatory region is bound by a repressor protein. Some genes or operons are regulated by more than one mechanism. For example, some bacterial genes and operons are subject to both a first, activation or derepression regulatory mechanism, and a second regulatory mechanism, called “catabolite repression.” Catabolite repression, also called “glucose catabolite repression” or “carbon catabolite repression,” is a phenomenon in which gene(s) under the control of a regulated promoter are also maintained in an unexpressed state until the concentration of glucose (the primary carbon source) falls below a threshold level, e.g., until conditions of glucose starvation. In other words, such a gene(s) cannot be expressed until two conditions are met: 1) glucose reduction/starvation and 2) activation or derepression of the regulated promoter. The occurrence of only one or the other condition is not sufficient to achieve expression of such gene(s). Among the genes and operons that have been found subject to catabolite repression are many that encode enzymes and/or pathways needed to utilize non-glucose carbon sources, i.e. alternative carbon sources.
The mechanism by which catabolite repression is effected is still undergoing intense scrutiny. In the case of some catabolite-repressed operons in E. coli, a transcriptional level of control has been assigned, in which catabolite repression is overcome by an “activatable promoter” mechanism. For example, the E. coli lactose operon (lacZYA) is maintained in an untranscribable state until glucose starvation permits a “catabolite activator protein” to bind to the operon's 5′ regulatory region; then, when lactose is present, Lac repressor protein is removed from a separate site(s) (the lac operator(s)) in the 5′ regulatory region, causing derepression, and transcription is initiated. Both conditions, i.e. both glucose starvation and the presence of lactose, are required for formation of lac operon-encoded mRNA in E. coli. 
In some cases, post-transcriptional controls are suspected. For example, there is evidence that, in Pseudomonads and closely related species, catabolite repression involving the crc gene is mediated post-transcriptionally. This is seen from studies of the regulation of bdkR [Ref. 7]. The bdkR protein, a transcriptional activator, is involved in the regulation of expression of branched-chain keto acid dehydrogenase in Pseudomonas putida. The data presented show that, in rich media, there is no bdkR protein detectable in wild type P. putida, despite the presence of bdkR transcripts. However, in a mutant P. putida in which crc is impaired or inactivated, bkdR protein is detected, bdkR transcript levels are slightly lower than those found in the wild type strain, and the transcript of the bdkR-regulated gene, bdkA, is induced about four-fold. Moreover, mutations identified in mutants in which the catabolite repression of bdkR is overcome, have been mapped to the crc gene, or to its cognate gene, vacB. In Shigella flexneri, the vacB protein regulates virulence genes post-transcriptionally; this presents additional, although circumstantial, evidence that crc acts post-transcriptionally [Ref. 13].
In commercial, prokaryotic systems, one of the key technological challenges associated with the production of proteins and chemicals by fermentation is total control of the transgene expression. The promoter selected for use in expressing the transgene of interest should have the following qualities. It should:

    
    
        1. Separate growth from reaction;
        2. Control gene expression for efficient/maximum product yield;
        3. Induce the gene of interest at low/no cost; and
        4. Allow no significant level of transcription in the repressed or non-induced state.
    
    


For these reasons, regulated promoters are relied upon extensively. In particular, the lac promoter (i.e. the lacZ promoter) and its derivatives, especially the tac and trc promoters described in U.S. Pat. No. 4,551,433 to DeBoer, and the related promoters listed in







TABLE 1







Commercial lac Promoters, Derivatives & Relatives













Commercial
Bacterial




Promoter
Inducer
Host Cell(s)
Reference(s)







Ptac16
IPTG
E. coli,
3, 4





Pseudomonads



Ptac17
IPTG
E. coli,
3





Pseudomonads



PlacUV5
IPTG
E. coli,
3





Pseudomonads



Plac
IPTG
E. coli
3, 4



Plac(down)
IPTG
E. coli
3



T7
IPTG
E. coli,
3, 4





Pseudomonads










In a typical commercial, bacterial fermentation system, the host cell contains a construct in which a tac promoter is operably attached to a gene or operon whose expression is desired. The lacI gene, which is a constitutively expressed gene that encodes the Lac repressor protein which binds to the lac operator, is also included in the bacterial host cell (multiple copies of the lacI gene are usually included therein). After growth of a desired quantity or density of biomass, an inducer is added to the culture in order to derepress the tac promoter and permit expression of the desired gene or operon.
In commercial fermentation systems using a lac-type promoter, such as the tac promoter, the gratuitous inducer, IPTG (isopropyl-β-D-1-thiogalactopyranoside, also called “isopropylthiogalactoside”), is almost universally employed. However, IPTG is expensive and must be carefully controlled since it is significantly toxic to biological systems. Standard IPTG preparations are currently available at about US$18 per gram or about US$125 per 10 grams; these IPTG preparations also contain dioxane, which is likewise toxic to biological systems. Dioxane-free IPTG is also available on the market, but costs about twice the price of standard IPTG (i.e. currently about US$36 per gram or about US$250 per 10 grams). In addition to the problems of expense and high toxicity to the fermentation system itself, in situations in which the expression product or fermentation product is to be marketed, environmental and health regulatory issues arise in regard to the presence of IPTG therein, since IPTG also poses toxicity risks to humans, animals, and other biological organisms.
As a result, there is a need for promoters that are both useful for commercial fermentation production systems and activated by non- or low-toxicity inducers.
A further drawback of the use of lac promoters and their derivatives is that these promoters are “leaky” in that, even in a native, repressed state, the promoter permits a relatively high background level of expression. Therefore, multiple copies of the LacI repressor protein gene are usually included within the expression host cell in order to increase the degree of repression of the lac-type promoter. As a result, there is a need for promoters that are both useful for commercial fermentation systems and readily susceptible of being tightly controlled in an inactive state until induced.
In light of these concerns, several other, non-lac-type promoters have been proposed for controlling gene expression in commercial, prokaryotic fermentation systems (see Table 2).







TABLE 2







Proposed Inducible Commercial non-lac Promoters














Bacterial




Promoter
Inducer
Host Cell(s)
Reference(s)







λPR
High
E. coli,
3, 4




temperature
Pseudomonads



λPL
High
E. coli,
3, 4




temperature
Pseudomonads



Pm
Alkyl- or
Pseudomonads
4, 5




halo-




benzoates



Pu
Alkyl- or
Pseudomonads
5




halo-toluenes



Psal
Salicylates
Pseudomonads
5



Para
Arabinose in
E. coli
5




the absence




of glucose










In regard to the first two promoters listed in Table 2, promoters induced by high temperatures are problematic: since high temperatures can be harmful to the host cell culture; since it is often impractical to generate an even temperature spike throughout the large-scale, commercial fermentation volume; and since it is preferred to operate commercial fermentation equipment at lower temperatures than required for such induction. The other four suggested promoters listed in Table 2 have, to the inventors' knowledge, not been demonstrated to function well in large-scale fermentation conditions; also, the alkyl- and halo-toluene inducers of the Pu promoter are significantly toxic to biological systems.
Thus, there remains a need for promoters that are useful for commercial fermentation production systems, activated by low-cost, low-toxicity chemical inducers, and tightly controlled.
In addition, in order to facilitate control of gene expression for production of proteins (and other expression products) and chemicals (processed by action of the expression products and/or the host cell) in a common fermentation platform using one prokaryotic organism, it is desirable to have a library of expression cassettes. These cassettes would each contain one or more of a variety of promoters that are of differing strengths, and/or induced under different growth conditions or by different chemicals. These expression cassettes would then be linked to various genes of interest to achieve total control of those genes under fermentation friendly conditions. The identification and optimization of a wide variety of growth-phase-dependent or chemically-inducible promoters is thus essential for control of (trans)gene expression during fermentation in such a fermentation platform.
Moreover, the construction of genetic circuits in which activation or induction of a first gene or operon leads to repression or activation of one or more subsequent genes or operons has been suggested as a means for very fine control of gene expression. Both linear (e.g., serial and cascade) and circular (e.g., daisy-chain) genetic circuits have been created. See, e.g., U.S. Patent Pub. No. 20010016354 A1 of Cebolla Ramirez et al. These genetic circuits require a number of different promoters in order to function, and, in commercial fermentation, genetic circuits would need to rely upon promoters that are effective in commercial fermentation conditions. Thus, there is a need in the field of genetic circuits for a greater variety of promoters useful in commercial fermentation.
As noted above, promoters for use in commercial fermentation systems should be tightly regulated so that expression occurs only upon induction, preferably effected late in the fermentation run. The chemicals used to induce the promoters must be low cost, low-toxicity to the host bacterium and other organisms, and must tightly regulate gene expression. In light of the above discussion, there is a need in the art for novel promoters that are tightly regulated and are induced at low cost using low-toxicity inducers.
SUMMARY OF THE INVENTION
The present invention provides novel promoters that are useful for gene expression in commercial fermentation. In a more specific aspect, the invention provides benzoate-inducible promoters, anthranilate-inducible promoters, and tandem promoters that may be employed in bacterial commercial fermentation systems.
The present invention provides:
isolated and/or recombinant benzoate promoter nucleic acids comprising the −35 region of the Pseudomonas fluorescens native benzoate promoter attached upstream of the −10 region thereof, via a 15-20 nucleotide linker; and to the operative promoter nucleic acid segment(s) found in SEQ ID NO:1;
mutant and closely related promoter nucleic acids whose nucleotide sequences are at least 90% homologous to such promoter nucleic acids;
such promoter nucleic acids further comprising a benzoate promoter activator protein (BenR) binding site; and
such promoter nucleic acids further comprising a benzoate promoter activator protein coding sequence, and where such activator protein coding sequences encode a benzoate promoter activator protein having an amino acid sequence at least 90% homologous to any one of the native, mutant, and/or truncated activator protein amino acids sequences presented in SEQ ID NO:2.
The present invention also provides:
isolated and/or recombinant anthranilate promoter nucleic acids comprising the −35 region of the Pseudomonas fluorescens native anthranilate promoter attached upstream of the −10 region thereof, via a 15-20 nucleotide linker; and to the operative promoter nucleic acid segment(s) found in SEQ ID NO:7;
mutant and closely related promoter nucleic acids whose nucleotide sequences are at least 90% homologous to such promoter nucleic acids;
such promoter nucleic acids further comprising an anthranilate promoter activator protein (AntR) binding site; and
such promoter nucleic acids further comprising a anthranilate promoter activator protein coding sequence, and where such activator protein coding sequences encode an anthranilate promoter activator protein having an amino acid sequence at least 90% homologous to any one of the native, mutant, and/or truncated activator protein amino acids sequences presented in SEQ ID NO:9.
The present invention also provides:
tandem promoters comprising a non-catabolite-repressed promoter attached (i.e. covalently attached) to and upstream of a natively catabolite-repressed promoter, either directly or by means of an inter-promoter polynucleotide linker, in which the catabolite repression of the latter promoter is overcome and/or a different improved promoter property is exhibited;
tandem promoters prepared by a process comprising covalently attaching a prokaryotic non-catabolite-repressed promoter to and upstream of a prokaryotic natively catabolite-repressed promoter, either directly or by means of an inter-promoter polynucleotide linker;
such tandem promoters wherein the inter-promoter polynucleotide linker is about 100 or less than 100 nucleotides long;
such tandem promoters in which the component non-catabolite-repressed and natively catabolite-repressed promoters are prokaryotic promoters, or bacterial promoters; and to tandem promoters in which the component promoters are obtained from the same of different species of the Pseudomonads and closely related bacteria, and/or of the genus Pseudomonas, and/or from Pseudomonas fluorescens; and to tandem promoters in which the component promoters are obtained from gene(s) or operon(s) encoding alternative carbon source utilization enzyme(s) or pathway(s); and to tandem promoters in which the non-catabolite-repressed promoter is obtained from an operon encoding an anthranilate degradation pathway and the natively catabolite-repressed promoter is obtained from an operon encoding a benzoate degradation pathway, and/or in which the anthranilate promoter and benzoate promoter are selected from among those summarized in the above paragraphs; and
the operative tandem promoter(s) found in, or constructed from the component promoters shown in, SEQ D NO:13.
The present invention also provides:
altered promoters prepared by a process comprising obtaining at least one polynucleotide having a base sequence at least 90% identical to and heterologous to the base sequence of any one of the claimed promoters or the sequence of any one of at least bases 1275-1307 of SEQ ID NO:1, at least bases 1239-1274 of SEQ ID NO:7, and at least bases 1329-1509 of SEQ ID NO:13; screening the polynucleotide(s) for the ability to direct transcription in a prokaryotic host cell, and optionally for at least one promoter property; and identifying, based on the results, at least one promoter, optionally having at least one improved property; and
improved promoters prepared by a process of: utilizing a promoter polynucleotide, having a base sequence of any one of the claimed promoters or the sequence of any one of at least bases 1275-1307 of SEQ ID NO:1, at least bases 1239-1274 of SEQ ID NO:7, and at least bases 1329-1509 of SEQ ID NO:13, as a hybridization probe for sequence-altered polynucleotide(s) at least 90% homologous thereto, or of performing mutagenesis and/or recombination upon said promoter polynucleotide to generate said sequence-altered polynucleotide(s), or of utilizing an information string representing the base sequence of the promoter polynucleotide to perform a search for a heterologous string at least 90% homologous thereto and providing a sequence-altered polynucleotide having the base sequence represented by said heterologous string; or of modifying such an information string into such a heterologous string and utilizing said modified string to identify an information string identical thereto and then providing a sequence-altered polynucleotide having the base sequence represented by said information string; followed by screening the sequence-altered polynucleotide(s) for the ability to direct transcription in a prokaryotic host cell, and for at least one promoter property; and identifying, based on the results, at least one promoter having at least one improved property.
The present invention also provides:
isolated nucleic acid molecules comprising a nucleic acid sequence whose complement hybridizes under stringent hybridization and wash conditions to a nucleobase polymer molecule having a base sequence of any one of the claimed promoters or of any one of at least bases 1275-13b7 of SEQ ID NO:1, at least bases 1239-1274 of SEQ ID NO:7, and at least bases 1329-1509 of SEQ ID NO:13, wherein said isolated nucleic acid molecule can function as a promoter in a prokaryotic cell; and
isolated nucleobase polymer molecules having the base sequence of a prokaryotic promoter polynucleotide molecule having a base sequence at least 90% identical to the base sequence of any one of the claimed promoters or of any one of at least bases 1275-1307 of SEQ ID NO:1, at least bases 1239-1274 of SEQ ID NO:7, and at least bases 1329-1509 of SEQ ID NO:13
The present invention also provides:
recombinant nucleic acid molecules that can function as expression construct(s) in a prokaryotic cell, comprising a promoter containing a base sequence at least 90% identical to the base sequence of any one of the claimed promoters or of any one of at least bases 1275-1307 of SEQ ID NO:1, at least bases 1239-1274 of SEQ ID NO:7, and at least bases 1329-1509 of SEQ ID NO:13; such recombinant expression constructs comprising an mRNA-encoding sequence; such recombinant expression constructs wherein the expression construct is a vector; such recombinant expression constructs wherein the vector is a plasmid; genetically engineered prokaryotic host cells containing any such a recombinant expression construct, and preferably also at least one, and more preferably more than one, copy of a gene encoding the relevant activator protein for the promoter of said recombinant expression construct (and where said gene is expressed in the host cell); expression systems comprising such a genetically engineered prokaryotic host cell that preferably contains at least one, and more preferably more than one, copy of a gene encoding the relevant activator protein for the promoter of said recombinant expression construct (and where said gene is expressed in the host cell); and such expression systems wherein the promoter is a benzoate-inducible promoter and the activator protein has an amino acid sequence of either any one of residues 1-335 or 21-335 of SEQ ID NO:2, optionally containing Asn152; and such expression systems wherein the promoter is an anthranilate-inducible promoter and the activator protein has an amino acid sequence of either residues 1-330 of SEQ ID NO:9or residues 1-330 of SEQ ID NO:9 containing Ala268.
The present invention also provides:
a process for preparing a transcription product comprising growing such a genetically engineered prokaryotic host cell, and inducing the recombinant expression construct therein, thereby expressing the transcription product encoded thereby; and a process for preparing a polypeptide comprising expressing an mRNA transcription product, by use of such a process for preparing a transcription product, and further permitting the host cell to translate the mRNA into the polypeptide encoded thereby.
The present invention also provides transcriptional activator proteins operative in prokaryotic cells. These include a transcriptional activator protein having an amino acid sequence at least 90% homologous to that of any one of residues 1-335 of SEQ ID NO:2, residues 1-335 of SEQ ID NO:2containing Asn152, residues 21-335 of SEQ ID NO:2, and residues 21-335 of SEQ ID NO:2 containing Asn152; and a transcriptional activator protein having an amino acid sequence at least 90% homologous to that of any one of residues 1-330 of SEQ ID NO:9, or of residues 1-330 of SEQ ID NO:9 containing Ala268. The present invention also provides polynucleotide molecules containing a base sequence encoding such transcriptional activator proteins.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 presents a plasmid map of pDOW1019, a vector comprising Pben509, a benzoate-inducible promoter according to the present invention.
FIG. 2 presents a plasmid map of pDOW1028, a vector comprising Pben278, a benzoate-inducible promoter according to the present invention.
FIG. 3 presents a plasmid map of pDOW1035, a vector comprising an anthranilate-inducible promoter according to the present invention, including the coding sequence of the activator protein therefor.
FIG. 4 presents a plasmid map of pDOW1057, a vector comprising an anthranilate-benzoate tandem promoter according to the present invention.
FIG. 5 presents a comparison of the nucleotide sequences of the Pben509 (nucleotides c994-c1502 of SEQ ID NO:1) and Pben278 (nucleotides g1228-c1502 of SEQ ID NO:1) benzoate -inducible promoters. The TTA translational stop site of the upstream ORF, and the putative−35 region (TTGACG, nucleotides 1275 through 1280 of SEQ: ID NO:1), −10 region (TACGGT, nucleotides 1296 through 1301 of SEQ ID NO:1), and “C” transcription start site are underlined. The “C” double-underlined in the Pben278 sequence indicates a mutation in Pben278, which differs from Pben509 and the genomic sequence; this mutation was introduced during PCR amplification.
FIG. 6 presents a bar chart showing β-galactosidase induction under the control of Pben509 (pDOW1019) or Pben278 (pDOW1028), in the presence of 0 mm (□) or 10 nM (▪) sodium benzoate. pDOW1017 is a vector with no promoter. Results shown are average measurements from triplicate wells assayed at 24 hours post-induction (host cells were grown in a defined salts medium for 24 hours prior to induction).
FIG. 7 presents a comparison of the nucleotide sequences of four Pant promoters: Pant+AntR (a1-cl395 of SEQ ID NO:13), Pant713 (nucleotides c592-c1304 of SEQ ID NO:7), Pant705 (nucleotides c592-g1288 of SEQ ID NO:7), and Pant311 (nucleotides c994-c1304 of SEQ ID NO:7). The double-underlined nucleotide triplets (TCA and CAT)respectively indicate the stop and start codons of the AntR ORF, which is encoded by the strand complementary to that shown. The lone double-underlined “A” indicates a mutation from the genomic sequence, confirmed on both strands, which lies within the AntR coding sequence; this change results in an expressed change from alanine to serine in the AntR protein. The putative −35 region (ATAGCC, nucleotides 1238 through 1243 of SEQ ID NO:7), −10 region (CTTAAT, nucleotides 1263 through 1267 of SEQ ID NO:7), and transcription start site (“A”), and the complement of the putative ribosome binding site (GGAGG, nucleotides 1817 through 1821 of SEQ ID NO:7) are all underlined.
FIG. 8 presents a graph comparing the activity of a Pant713 construct (pDOW1029) versus an antR/Pant construct (pDOW1035), when induced with either 5 mM anthranilate or 2 mM 6-chloroanthranilate. In all constructs, the promoter was fused to a β-galactosidase-encoding sequence. The activity of the β-galactosidase reporter was followed over an 8 hour time course post-induction. The results show the activity of pDOW1035 following induction occurs much faster and is much higher than that of pDOW1029.
FIG. 9 presents bar charts showing induction of a Pant-Pben tandem promoter construct, and induction of component promoter constructs. In all constructs, the promoter was fused to a β-galactosidase-encoding sequence. Induction of a Pant-Pben tandem promoter construct (pDOW1057) with either benzoate or anthranilate is presented in comparison to that of constructs containing one of the component promoters: a Pben promoter construct (pDOW1028), containing Pben278, and a Pant promoter construct (pDOW1035), containing the antR/Pant. FIG. 9A presents results at 5 hours post-induction and FIG. 9B presents results at 24 hours post-induction.
FIG. 10 presents a bar chart comparison of Pben509 activity to that of improved mutants 2d3 and 21b5, created by error prone PCR of Pben509. Expression constructs were formed by fusing promoter-containing fragments to a phoA reporter gene. Cultures containing the constructs were induced with 10 mM benzoate and alkaline phosphatase activity was measured at 24 hours post-induction.
FIG. 11 presents a comparison of the nucleotide sequences of Pben509 (nucleotides c994-c1502 of SEQ ID NO:1) and two improved mutants thereof created by means of error prone PCR: mutant 2d3 (nucleotides c994-c1502 of SEQ ID NO:1 with a1106→t1106) and mutant 21b5 (nucleotides c994-c1502 of SEQ ID NO:1 with c1223→t1223 and g1302→a1302). The TTA translational stop site of the upstream ORF, and the putative −35 region (TTGACG, nucleotides 1275 through 1280 of SEQ ID NO:1), −10 region (TACGGT, nucleotides 1296 through 1301 of SEQ ID NO:1), and “C” transcription start site are underlined. Mutations are shown double-underlined.
FIG. 12 presents a graph of anthranilate-induced expression in 20 L fermentation conditions. An antR/Pant construct pDOW1035 (▪) and a tandem promoter construct pDOW1057 (▴) were induced with 5 mM sodium anthranilate plus a 1 mM/hour anthranilate feed. The activity level for each 20 L fermentation run is shown.
FIG. 13 presents graphs demonstrating improved anthranilate-induced expression from a tandem promoter construct (pDOW1057) in an antA knockout host created by means of a single crossover knockout of the antA gene in the host cell (Pseudomonas fluorescens). Results are shown for 20 L fermentations induced at a target level of about 5 mM sodium anthranilate and followed over a 48 hour time course post-induction. No anthranilate feed was required. FIG. 13A presents the activity of β-galactosidase expressed from the reporter gene construct; FIG. 13B shows the maintenance of anthranilate concentration, demonstrating that the knockout host cell does not metabolize anthranilate.
FIG. 14 presents a bar chart showing induction of Pben −10 mutants. The alkaline phosphatase activity of P. fluorescens containing the indicated Pben::phoA fusions is shown following 24-hour induction with 0 mM (□) or 5 mM (▪) sodium benzoate. Representative experiment of triplicate samples is shown.
FIG. 15 presents a bar chart showing induction of Pant −10 mutants. The alkaline phosphatase activity of P. fluorescens antA::kanR containing the indicated Pant::phoA fusions is shown following 24-hour induction with 0 mM (D) or 5 mM (▪) anthranilate. Representative experiment of triplicate samples is shown.
FIG. 16 presents bar charts indicating the effect of Pben88-10 mutations on the activity of the antR-Pant311-Pben tandem promoter. The β-galactosidase activity of P. fluorescens containing the indicated tandem promoter::lacZ fusions is shown following 24-hour induction with 0 mM (□) or 5 mM (▪) sodium benzoate (FIG. 16A) or anthranilate (FIG. 16B). Representative experiment of triplicate samples is shown
FIG. 17 presents a graph of benzoate consumption during 20 L fermentation when benR is present in multicopy with pDOW1090. Data shown are HPLC analyses measuring the concentration of benzoate throughout the induction of two 20 L fermentors in duplicate fermentations, labeled run “030211I” (●) and run “030211K” (▪). The cultures were induced with 5 mM sodium benzoate.
FIG. 18 presents bar chart comparisons of tandem promoter construct activity. The β-galactosidase activity of P. fluorescens containing the indicated tandem promoter::lacZ fusions (except for pDOW1035, which is ant activator-Pant311::lacZ; and pDOW1126, which is ben activator Pben88−10consensus::lacZ) is shown following induction with 0 or 5 mM sodium benzoate (FIGS. 18A and 18B for 2, 4, and 24 hour time-points) or anthranilate (FIGS. 18C and 18D, for 0, 2, 6, and 24 hour time-points). Representative experiment of triplicate samples is shown. Time points are indicated as “post-induction” by the letter “I”.
FIG. 19 presents a bar chart demonstrating an analysis of Pben activity in the benAB knock-out strain. β-galactosidase activity of a P. fluorescens MB101 benAB knock-out strain carrying pDOW1019 (Pben278::lacZ) is shown as a result of induction with either 0 mM (□) or 5 mM (▪) sodium benzoate for up to 24 hours. Cells were grown in LB medium.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention provides commercially useful benzoate-inducible promoters, anthranilate-inducible promoters, and tandem promoters that may be employed in bacterial commercial fermentation systems. Preferred bacterial host cells for use in such systems include Pseudomonads and closely related bacteria The chemical inducers of these promoters include benzoic and anthranilc acids, their effective chemical analogs, and biologically acceptable salts thereof.
Benzoic and anthranilic acids and biologically acceptable salts, preferably sodium or potassium salts, thereof, are inexpensive chemicals with low toxicity that can be utilized as (alternative) carbon sources by bacterial host cells, including Pseudomonads and closely related bacteria For example, these chemical inducers are available on the market at less than about US$0.15 per gram (versus about US$18 or US$36 per gram for IPTG).
The present inventors have isolated, sequenced, and characterized the native promoters responsible for expression of the P. fluorescens benzoate (benABCD) degradative genes, which may be induced with benzoate in the absence of glucose, and of the P. fluorescens (antABC) degradative genes, which may be induced with anthranilate. (The expression products of these operons are catabolic pathway enzymes responsible for degradation of benzoate and anthranilate, respectively, in Pseudomonas fluorescens biotype A.) These promoters have been found capable of inducing expression of exogenous genes about 250-fold, for the benzoate promoter, and about 25- to about 35-fold, for the anthranilate promoter, when induced with 5 mM sodium benzoate and 5 mM sodium anthranilate, respectively. The present inventors have found these promoters to be sufficiently inducible for use in commercial fermentation systems to produce proteins and chemicals in bacterial host cells, including Pseudomonads and closely related bacteria
In addition, the present inventors have created tandem promoter constructs in which a non-catabolite-repressed promoter is linked upstream of a natively catabolite-repressed promoter, thereby surprisingly overcoming the catabolite repression of the latter promoter and/or thereby exhibiting a different improved property (e.g., increased strength of induction or increased tightness of regulation). At least one example of a tandem promoter construct has been described for expression of foreign genes [6]. However, this example is a tandem arrangement of two copies of the same promoter, Plac, and the reference presents no evidence to suggest that the tandem Plac-Plac promoter has advantages over a single Plac promoter. Likewise, dual promoter constructs are known, e.g., for use in shuttle vectors, in which two promoters operative in different species or genera are both operably attached to the same gene so that the gene can be expressed in either of the two different species or genera.
In contrast to these tandem and dual promoter constructs, the present creation of tandem promoter constructs, in which two non-identical promoters are placed in tandem arrangement, has surprisingly been found to retain advantageous features of both promoters.
For example, the tandem arrangement of the anthranilate promoter, Pant, and the benzoate promoter, Pben, has resulted in formation of a tandem promoter that, when induced with anthranilate under fermentation conditions, exhibits both freedom from catabolite repression (a desirable feature of Pant, not shared by Pben) and improved strength of induction (a desirable feature of Pben, not shared by Pant). Thus, the tandem promoters of the present invention permit retention of desirable properties of the individual promoter elements, so that the resulting tandem promoter can exhibit improved properties: e.g., increased strength of induction, or increased tightness of regulation (i.e. transcription only when contacted with the relevant inducer of the promoter's activator or repressor protein); and/or lack of catabolite repression.
GLOSSARY
A### (Absorption)
As used herein in regard to analytical detection, terms such as “A450” mean “absorption at a wavelength of 450 nm.”
A and An (Indefinite Articles)
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a host cell” literally defines both those embodiments employing only a single host cell, those employing a plurality of host cells of a single type, and those employing a plurality of host cells of a plurality of types.
* (Asterisk)
As used herein in regard to calculations, the “*” symbol (asterisk) indicates the mathematical multiplication function.
BCIP
5-Bromo4-chloro-3-indolyl phosphate, e.g., a divalent salt thereof, such as a disodium salt. This is used in conjunction with a, e.g., tetrazolium salt, such as: a halide salt of Nitroblue Tetrazolium (NEBT), e.g., bis-[2-(4-yl-2-methoxyphenyl)3-(4-nitrophenyl)-5-phenyl-tetrazolium chloride]; or of Iodo-Nitro-Tetrazolium (INT), also called Iodoblue Tetrazolium, e.g., 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride.
Comprising
As used herein, the term “comprising” means that the subject contains the elements enumerated following the term “comprising” as well as any other elements not so enumerated. In this, the term “comprising” is to be construed as a broad and open-ended term; thus, a claim to a subject “comprising” enumerated elements is to be construed inclusively, i.e. construed as not limited to the enumerated elements. Therefore, the term “comprising” can be considered synonymous with terms such as, e.g., “having,” “containing,” or “including.”
The invention, as described herein, is spoken of using the terms “comprising” and “characterized in that.” However, words and phrases having narrower meanings than these are also useful as substitutes for these open-ended terms in describing, defining, or claiming the invention more narrowly.
Corresponding
As used herein in reference to a sequence record's “corresponding to” a polynucleotide source, the term “corresponding” means that a given base sequence contained, as an information string, within the sequence record, is present in the form of a physical nucleobase sequence-containing molecule within the polynucleotide source.
ddH2O
As used herein, ddH2O refers to distilled, deionized water purified through a Milli-Q gradient system with Q-GARD purification pack (Miripore, Bedford, Mass.).
dNTPs
Except where otherwise indicated, as used herein in regard to reagents for polynucleotide synthesis reactions, the term “dNTPs” means an equimolar solution of each of the four deoxyribonucleotide triphosphates (dGTP, dCTP, dATP, dTTP). Thus, e.g., reference to 10 mM dNTPs indicates a solution containing 10 mM each of dGTP, dCTP, dATP, and dTTP.
Exogenous and Foreign
The term “exogenous” means “from a source external to” a given cell or molecule. In the present application, as is common use in the art, this term is used interchangeably with the term “foreign,” as synonyms. Both of these terms are used herein to indicate that a given object is foreign a given the cell or molecule (e.g., a promoter polynucleotide), i.e. not found in nature in the cell and/or not found in nature with or connected to the molecule.
Heterologous
As used herein, the term “heterologous mean “non-identical” in sequence (not 100% identical in base sequence).
In and On
As used herein in regard to growing organisms by use of a growth medium, the organisms may be said to be grown “in” or “on” the medium. In the expression systems of the present invention, the medium is preferably a gel or liquid medium, more preferably an aqueous gel or liquid medium, and still more preferably an aqueous liquid medium. Thus, in this context, the terms “in” and “on” are used synonymously with one another to indicate growth of the host cells in contact with the medium.
Information String
As used herein, the phrase “information string” means a series of data elements (e.g., bits, bytes, or alphanumeric characters), which series represents the information of a given series of nucleobases.
IPTG
Isopropyl-β-D-1-thiogalactopyranoside.
ONPG
O-Nitrophenyl-β-D-galactopyranoside, also known as 2-Nitrophenyl-β-D-galactopyranoside.
ORF
Open reading frame.
PNPP
para-Nitrophenyl phosphate, e.g., a divalent salt thereof, such as a disodium salt. Also referred to as 4-nitrophenyl phosphate.
Polynucleotide Length
As used herein, the term “nucleotides” is used to describe the length of polynucleotides. However, in this context, the terminology is meant to refer both to length in nucleotides per se in regard to single stranded polynucleotides, and to length in base pairs in regard to double stranded polynucleotides.
Polynucleotide Source
As used herein, the phrase “polynucleotide source” means any source of a physical embodiment of a nucleic acid containing a given nucleobase sequence, such as a nucleic acid sample, clone, or native cell containing such a polynucleotide molecule.
Promoter Activator Protein Terminology
The native activator of a given promoter is designated with an “R” as, e.g., BenR, AntR for the native activators of the Pben and Pant promoters, respectively.
Promoter Terminology
“Pant”: Promoter for the anthranilate operon of P. fluorescens. 
“Pben”: Promoter for the benzoate operon of P. fluorescens. 
“Plac”: Promoter for the lactose operon of E. coli. 
“Ptandem” and “tandem promoter”: a tandem arrangement of promoters in which a non-catabolite-repressed promoter is attached to and upstream of, by means of a sequence of 0 to about 100 nucleotides, a catabolite-repressed promoter. This is exemplified herein by Pant-Pben tandem promoter constructs.
“Promoter”: a polynucleotide comprising at least 25 nucleotides, more commonly about 30 nucleotides, containing a prokaryotic “−35 region through −10 region.” Preferably, this “−35 region through −10 region” is a “−35 region through −10 region” obtained from a single gene, or a combination of a −35 region and a −10 region obtained from cognate genes, the gene(s) being obtained from at least one prokaryote, more preferably at least one organism of the “Pseudomonads and closely related species.” Preferably, the “−35 region through −10 region” is a σ70 “−35 region through −10 region,” and the “−35 region” and the “−10 region” in the combination are, respectively, a σ70 −35 region and a σ70 −10 region.
The −35 region is linked upstream of the −10 region by an intra-promoter polynucleotide of preferably about 15 to about 20 nucleotides. More preferably, a promoter according to the present invention comprises about 35 nucleotides, which contains, in addition to the “−35 region through −10 region,” a segment of about 5 to about 10 immediate downstream nucleotides, more preferably 6 to 7 immediate downstream nucleotides, terminating in a transcription start site nucleotide. In a preferred embodiment, this segment is obtained from the same gene as provides at least one of the −35 or −10 region. In a particularly preferred embodiment, the promoter will also contain an immediate upstream region of about 20 to about 250, more preferably about 40 to about 150, nucleotides comprising a promoter activator protein binding site, preferably an AraC/XylS-class binding site. In a preferred embodiment, the binding site region is obtained from the same gene as provides at least one of the −35 or −10 region.
As used herein, the term “−35 region” or “minus 35 region”, indicates a 5-6 nucleotide sequence beginning approximately 35 nucleotides upstream (i.e. in a 5′ direction from) a transcription start site, the transcription start site being numbered as “+1.”
Likewise, the term “−10 region” or “minus 10” region” indicates a 5-6 nucleotide sequence beginning approximately 10 nucleotides upstream (i.e. in a 5′ direction from) a transcription start site, the transcription start site being numbered as “+1.”
RNA Terms
As used herein, the following RNA terms have the definitions recited below.

    
    
        aRNA: anti-sense RNA
        cRNA: cytoplasmic RNA
        gRNA: guide RNA (for editing mitochondrial pre-mRNAs)
        hnRNA: heteronuclear RNA
        mRNA: messenger RNA
        miRNA: microRNA (for regulating mRNA expression)
        mtRNA: mitochondrial RNA
        nRNA: nuclear RNA
        ncRNA: non-coding RNA
        pRNA: packaging RNA (for virus and phage particle assembly)
        rRNA: ribosomal RNA
        satRNA: satellite RNA
        scRNA: small cytoplasmic RNA
        siRNA: small interfering RNA
        snRNA: small nuclear RNA
        snoRNA: small nucleolar RNA
        srpRNA: signal recognition particle RNA
        stRNA: small temporal RNA (a subgroup of miRNA)
        tRNA: transfer RNA
        tmRNA: transfer-messenger-like RNA (for marking, for subsequent degradation, nascent polypeptides in stalled ribosomes)
        vRNA: viral (and/or phage) RNA

Searching

    
    


As used herein in regard to seeking for information, the term “search” means performing (by manual, visual, or automated means) one or more comparisons between a known information string and other information strings in order to identify an identical or non-identical information string.
Sequence Record
As used herein, the phrase “sequence record” means a stored embodiment of one or more information strings, such as a computer readable record or a paper record.
−10 Variant Promoter Designations

    “−10 con”: indicates a variant promoter in which the native −10 region has been substituted with the consensus −10 region sequence ‘tataat.’
    “−10 ben”: indicates a variant promoter in which the native −10 region has been substituted with the −10 region from P. fluorescens Pben ‘tacggt.’
    “−10 benAc”: indicates a variant promoter in which the native −10 region has been substituted with the −10 region from Acinetobacter Pben ‘taaggt.’
    “−10 wt”: indicates truncated native promoter retaining the wild-type −10 sequence.

 ̃ (Tilde)



The  ̃ symbol (the tilde) is used herein to indicate “about”.
× (Times)
The × symbol (the times symbol), as used herein in regard to the concentration of a solution, means, e.g., that a 5× preparation is five times as concentrated as a 1× preparation, for the same solution composition (ie. for the same relative amounts of all components therein).
Tris
The term “Tris” as used herein means Tris (hydroxymethyl) aminomethane (available from Fisher Scientific, Pittsburgh, Pa.).
X-gal
5-bromo4-chloro-3-indolyl-β-D-galactopyranoside
General Materials & Methods
Unless otherwise noted, standard techniques, vectors, control sequence elements, and other expression system elements known in the field of molecular biology are used for nucleic acid manipulation, transformation, and expression. Such standard techniques, vectors, and elements can be found, for example, in: Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); and Bukhari et al. (eds.), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY).
The promoters of the present invention include the benzoate promoter from Pseudomonas fluorescens, the anthranilate promoter from Pseudomonas fluorescens, and their derivatives. The promoters of the present invention also include tandem promoters having a non-catabolite-repressed promoter linked upstream of a natively catabolite-repressed promoter, in which the catabolite repression of the latter promoter is overcome and/or a different improved promoter property is exhibited.
The promoters of the present invention are typically in the form of DNA when in use in an expression system. However, the nucleobase sequence of the promoters may be present in the form of DNA, RNA, or any nucleic acid analog known in the art, e.g., peptide nucleic acid (PNA).
Benzoate Promoters
In a preferred embodiment, a benzoate promoter of the present invention is the Pseudomonas fluorescens native benzoate promoter or an improved mutant thereof. The present inventors have found this promoter to be inducible with benzoic acid, benzoic acid analogs (e.g., m-toluic acid), and biologically acceptable salts thereof (e.g., sodium benzoate).
In a preferred embodiment, a benzoate promoter of the present invention comprises the −35 region of the Pseudomonas fluorescens native benzoate promoter attached upstream of the −10 region of this native promoter, via a 15-20 nucleotide linker. In a preferred embodiment, the linker is 15 nucleotides long.
In a preferred embodiment, a benzoate promoter of the present invention comprises nucleotides 1275-1280 of SEQ ID NO:1 attached upstream of nucleotides 1296-1301 of SEQ ID NO:1, via a 15-20 nucleotide linker. In a preferred embodiment, the linker is 15 nucleotides long. In a particularly preferred embodiment, the linker is nucleotides 1281-1295 of SEQ ID NO:1, a benzoate promoter of this preferred embodiment thereby comprising nucleotides 1275-1301 of SEQ ID NO:1. In a preferred embodiment, a benzoate promoter of the present invention comprises nucleotides 1275-1301 of SEQ ID NO:1 attached immediately upstream of a spacer segment of about 6 nucleotides, preferably of 6 nucleotides, in length, and terminating with a nucleotide that functions as a transcription start site. In a preferred embodiment, the spacer segment is nucleotides 1302-1307 of SEQ ID NO:1, a benzoate promoter of this preferred embodiment thereby comprising nucleotides 1275-1307 of SEQ ID NO:1.
In a preferred embodiment, a benzoate promoter of the present invention comprises both a “−35 to −10 region” and a benzoate promoter activator (or repressor) protein binding site, preferably an activator protein binding site. In a preferred embodiment, a benzoate promoter of the present invention comprises nucleotides 1275-1301 of SEQ ID NO:1 attached immediately downstream of a spacer region of about 50 nucleotides in length. In a preferred embodiment, a benzoate promoter of the present invention comprises nucleotides 1275-1301 of SEQ ID NO:1 attached immediately downstream of a spacer region of about 45 nucleotides in length. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:1, beginning about 50 nucleotides upstream of nucleotide 1275 and ending with nucleotide 1274. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:1, beginning about 45 nucleotides upstream of nucleotide 1275 and ending with nucleotide 1274. In a preferred embodiment, the spacer region has the sequence of nucleotides 1228-1274 of SEQ ID NO:1, a benzoate pronmoter of this preferred embodiment thereby comprising the sequence of nucleotides 1228-1301.
In a preferred embodiment, a benzoate promoter of the present invention comprises nucleotides 1275-1301 of SEQ ID NO:1 attached immediately upstream of said spacer segment and attached immediately downstream of said spacer region. In a preferred embodiment, a benzoate promoter of the present invention comprises nucleotides 1228-1307 of SEQ ID NO:1.
In a preferred embodiment, in expression systems in which a benzoate promoter according to the present invention is used, the host cell will also contain and express at least one nucleic acid encoding a benzoate promoter activator protein. Even more preferred is the use therein of multiple expressed copies of such a Pben activator protein-encoding nucleic acid. In a preferred embodiment, the Pben activator protein will have an amino acid sequence of SEQ ID NO:2 or the residue 152 (Asn) variant thereof, or an amino acid sequence of residues 21-335 of SEQ ID NO:2 or the residue 152 (Asn) variant thereof. In a preferred embodiment, the nucleic acid encoding the Pben activator protein will contain the sequence of bases 285-1229 of SEQ ID NO:1 or the base 679 mutant variant thereof, or the sequence of bases 225-1229 of SEQ ID NO:1 or the base 679 mutant variant thereof; or the complement thereof of any of these; or the RNA equivalent of any of these.
Anthranilate Promoters
In a preferred embodiment, an anthranilate promoter of the present invention is the Pseudomonas fluorescens native anthranilate promoter or an improved mutant thereof. The present inventors have found this promoter to be inducible with anthranilic acid, anthranilic acid analogs (e.g., haloanthranilic acids), and biologically acceptable salts thereof (e.g., sodium anthranilate); and with o-toluate (o-toluate has been found to induce this promoter as well as does anthranilate).
In a preferred embodiment, an anthranilate promoter of the present invention comprises the −35 region of the Pseudomonas fluorescens native anthranilate promoter attached upstream of the −10 region of this native promoter, via a 15-20 nucleotide linker, more preferably a 16-19 nucleotide linker. In a preferred embodiment, the linker is 19 nucleotides long.
In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1244 of SEQ ID NO:7 attached upstream of nucleotides 1264-1268 of SEQ ID NO:7, via a 15-20 nucleotide linker. In a preferred embodiment, the linker is 19 nucleotides long. In a particularly preferred embodiment, the linker is nucleotides 1245-1263 of SEQ ID NO:7, an anthranilate promoter of this preferred embodiment thereby comprising nucleotides 1239-1268 of SEQ ID NO:7. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately upstream of a spacer segment of about 6 nucleotides, preferably of 6 nucleotides, in length, and terminating with a nucleotide that functions as a transcription start site. In a preferred embodiment, the spacer segment is nucleotides 1269-1274 of SEQ ID NO:7, an anthranilate promoter of this preferred embodiment thereby comprising nucleotides 1239-1274 of SEQ ID NO:7.
In a preferred embodiment, an anthranilate promoter of the present invention comprises both a “−35 to −10 region” and an anthranilate promoter activator (or repressor) protein binding site, preferably an activator protein binding site. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 250 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 200 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 150 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 120 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 110 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 100 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 85 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 80 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 75 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 70 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 65 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 60 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 55 nucleotides in length. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1268 of SEQ ID NO:7 attached immediately downstream of a spacer region of about 50 nucleotides in length.
In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:7, beginning about 100 nucleotides upstream of nucleotide 1239 and ending with nucleotide 1238. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:7, beginning about 85 nucleotides upstream of nucleotide 1239 and ending with nucleotide 1238. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:7, beginning about 80 nucleotides upstream of nucleotide 1239 and ending with nucleotide 1238. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:7, beginning about 75 nucleotides upstream of nucleotide 1239 and ending with nucleotide 1238. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:7, beginning about 70 nucleotides upstream of nucleotide 1239 and ending with nucleotide 1238. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:7, beginning about 65 nucleotides upstream of nucleotide 1239 and ending with nucleotide 1238. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:7, beginning about 60 nucleotides upstream of nucleotide 1239 and ending with nucleotide 1238. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:7, beginning about 55 nucleotides upstream of nucleotide 1239 and ending with nucleotide 1238. In a preferred embodiment, the spacer region has the sequence of the region shown in SEQ ID NO:7, beginning about 50 nucleotides upstream of nucleotide 1239 and ending with nucleotide 1238. In a preferred embodiment, the spacer region has the sequence of nucleotides 1139-1238 of SEQ ID NO:7, an anthranilate promoter of this preferred embodiment comprising nucleotides 1139-1238 of SEQ ID NO:7.
In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1239-1274 of SEQ ID NO:7 attached immediately upstream of said spacer segment and attached immediately downstream of said spacer region. In a preferred embodiment, an anthranilate promoter of the present invention comprises nucleotides 1139-1274 of SEQ ID NO:7.
In a preferred embodiment, in expression systems in which an anthranilate promoter according to the present invention is used, the host cell will also contain and express at least one nucleic acid encoding an anthranilate promoter activator protein. Even more preferred is the use of multiple expressed copies of such a Pant activator protein-encoding nucleic acid. In a preferred embodiment, the Pant activator protein will have an amino acid sequence of SEQ ID NO:9 or the residue 268 (Ala) variant thereof. In a preferred embodiment, the nucleic acid encoding the Pant activator protein will contain the sequence of bases 1-990 of SEQ ID NO:8 or the base 802 variation thereof; or the complement thereof of any of these; or the RNA equivalent of any of these.
Mutant and Closely Related Activator Proteins and Polynucleotides Encoding Them The same methods as described below for use in obtaining mutant promoters may similarly be used to obtain mutant activator proteins and the coding sequences and genes thereof In this case, at least a portion of the gene encoding a given activator protein, e.g., all or part of the coding sequence thereof, may be used as, or be used to form a probe for use in hybridization probing; or may provide a base sequence to be used in the form of an information string, identical or at least 90% identical thereto, to search a database for structurally related sequences for testing. Likewise all or part of the amino acid sequence of the activator protein may be used as an information string to perform such searching. The resulting sequences identified by hybridization or bioinformatic searching are then tested for promoter activation activity and/or for improved properties.
Thus, also included within the present invention are transcriptional activator proteins having an amino acid sequence at least 90% identical to and heterologous to that of: a Pben activator protein having an amino acid sequence of any one of residues 1-335 of SEQ ID NO:2, residues 1-335 of SEQ ID NO:2 containing Asn152, residues 21-335 of SEQ ID NO:2, and residues 21-335 of SEQ ID NO:7 containing Asn152; and a Pant activator protein having an amino acid sequence of any one of residues 1-330 of SEQ ID NO:9 and residues 1-330 of SEQ ID NO:9 containing Ala268. The present invention also includes polynucleotides encoding said mutant and closely related transcriptional activator proteins.
Tandem Promoters
In a preferred embodiment, a tandem promoter of the present invention comprises a (natively) non-catabolite-repressed promoter attached upstream of a natively catabolite-repressed promoter, in which the catabolite repression of the latter promoter is overcome and/or a different improved promoter property is exhibited.
In a preferred embodiment, both the non-catabolite-repressed promoter and the natively catabolite-repressed promoter are selected from the prokaryotes. In a preferred embodiment, both the non-catabolite-repressed promoter and the natively catabolite-repressed promoter are selected from the bacteria. In a preferred embodiment, both the non-catabolite-repressed promoter and the natively catabolite-repressed promoter are selected from the Proteobacteria; preferably Gram negative Proteobacteria. In a preferred embodiment, both the non-catabolite-repressed promoter and the natively catabolite-repressed promoter are selected from the “Pseudomonads and closely related bacteria” or from a Subgroup thereof, as defined below.
In a preferred embodiment, both promoters are selected from the same species. In a preferred embodiment, both promoters are obtained from the same species in a genus selected from among the “Pseudomonads and closely related bacteria” or among a Subgroup thereof, as defined below. In a preferred embodiment, both promoters are selected from organisms of the genus Pseudomonas. In a preferred embodiment, both promoters are selected from the same species in the genus Pseudomonas. In a preferred embodiment, both promoters are selected from Pseudomonas fluorescens. In a preferred embodiment, both promoters are selected from Pseudomonas fluorescens biotype A.
The individual promoters selected for use in a tandem promoter according to the present invention may be activatable promoters, repressible promoters, or a combination thereof. In a preferred embodiment at least one of, and preferably both of, the individual promoters will be activatable promoters. Where a repressible promoter is present as a promoter element in such a tandem promoter, preferably the cell in which the tandem promoter is utilized will also contain at least one, and preferably more than, one copy of an expressible coding sequence for a repressor protein that mediates the regulation of the promoter. Where an activatable promoter is present as a promoter element in such a tandem promoter, preferably the cell in which the tandem promoter is utilized will also contain at least one, and preferably more than, one copy of an expressible coding sequence for an activator protein that mediates the regulation of the promoter.
In a preferred embodiment, both promoters are obtained as native promoters of genes or operons encoding enzyme(s) and/or pathway(s) capable of enabling a cell to utilize (e.g., to import, export, transport, or metabolize) alternative carbon source(s). In a preferred embodiment, the non-catabolite-repressed promoter is a native promoter of a gene or operon encoding enzyme(s) and/or pathway(s) capable of biocatalytically degrading anthranilate, i.e. an “anthranilate promoter.” In a preferred embodiment, the natively catabolite-repressed promoter is a native promoter of a gene or operon encoding enzyme(s) and/or pathway(s) capable of biocatalytically degrading benzoate, i.e. a “benzoate promoter.” In a preferred embodiment, the anthranilate promoter is an anthranilate promoter as described above. In a preferred embodiment, the benzoate promoter is a benzoate promoter as described above.
In a preferred embodiment, a tandem promoter of the present invention is a construct formed by linking the Pseudomonas fluorescens native anthranilate promoter to, and upstream of, the Pseudomonas fluorescens native benzoate promoter. The present inventors have found such a promoter arrangement to be inducible with anthranilic acid, anthranilic acid analogs (e.g., haloanthranilic acids), and biologically acceptable salts thereof (e.g., sodium anthranilate); with benzoic acid and biologically acceptable salts thereof; and with o-toluate (o-toluate has been found to induce this promoter as well as does anthranilate).
In a preferred embodiment, the non-catabolite-repressed promoter is attached immediately upstream of the natively catabolite-repressed promoter. This attachment may be made directly between the promoters (or directly between native nucleic acid segments containing the promoters) or by means of an, e.g., polynucleotide linker connecting the promoters (or segments) to one another. In a preferred embodiment, the non-catabolite-repressed promoter is attached upstream of the natively catabolite-repressed promoter, via an inter-promoter linker. Preferably, the inter-promoter linker will be a polynucleotide, provided that that polynucleotide linker contains no sequence that functions as a transcription termination signal. In a preferred embodiment the inter-promoter linker is a polynucleotide of about 100 nucleotides in length. In a preferred embodiment, the inter-promoter linker is less than 100 nucleotides in length. In a preferred embodiment the inter-promoter linker is a polynucleotide of length equal to or less than 90 nucleotides. In a preferred embodiment the inter-promoter linker is a polynucleotide of length equal to or less than 80 nucleotides. In a preferred embodiment the inter-promoter linker is a polynucleotide of length equal to or less than 70 nucleotides. In a preferred embodiment the inter-promoter linker is a polynucleotide of length equal to or less than 60 nucleotides. In a preferred embodiment the inter-promoter linker is a polynucleotide of length equal to or less than 50 nucleotides. In a preferred embodiment the inter-promoter linker is a polynucleotide of length equal to or less than 40 nucleotides. In a preferred embodiment the inter-promoter linker is a polynucleotide of length equal to or less than 30 nucleotides. In a preferred embodiment the inter-promoter linker is a polynucleotide of length equal to or less than 20 nucleotides. In a preferred embodiment, the inter-promoter linker is equal to or less than 10 nucleotides. In a preferred embodiment the inter-promoter linker is a polynucleotide at least about 5 nucleotides, or at least about 10 nucleotides, or at least about 20 nucleotides, or at least about 30 nucleotides, or at least about 40 nucleotides in length. In a preferred embodiment the inter-promoter linker is a polynucleotide about 5 to about 50 nucleotides, or about 10 to about 50 nucleotides, or about 20 to about 50 nucleotides, or about 30 to about 50 nucleotides in length. In a preferred embodiment the inter-promoter linker is a polynucleotide having a length of 43 nucleotides. In a preferred embodiment the inter-promoter linker has the sequence of SEQ ID NO:14.
In a preferred embodiment, a tandem promoter comprises an anthranilate promoter sequence selected from the group consisting of nucleotides 1221-1365, 1221-1371, 1329-1365, and 1329-1371 of SEQ ID NO:13 attached upstream of a benzoate promoter sequence selected from the group consisting of nucleotides 1430-1503, 1430-1509, 1477-1503, and 1477-1509 of SEQ ID NO:13. In a preferred embodiment, a tandem anthranilate-benzoate promoter of the present invention comprises, for the benzoate promoter portion, both a “−35 to −10 region” and a benzoate promoter activator (or repressor) protein binding site, preferably an activator protein binding site. In a preferred embodiment, a tandem promoter comprises nucleotides 1329-1503 of SEQ ID NO:13. In a preferred embodiment, a tandem promoter comprises nucleotides 1329-1509 of SEQ ID NO:13. In a preferred embodiment, a tandem promoter comprises nucleotides 1221-1503 of SEQ ID NO:13. In a preferred embodiment, a tandem promoter comprises nucleotides 1221-1509 of SEQ ID NO:13. In a preferred embodiment, a tandem promoter comprises nucleotides 1329-1544 of SEQ ID NO:13. In a preferred embodiment, a tandem promoter comprises nucleotides 1221-1544 of SEQ ID NO:13.
In a preferred embodiment, an anthranilate activator protein coding sequence or a benzoate activator protein coding sequence is included in, and expressed within, a system using, respectively, a Pant-containing or Pben-containing tandem promoter of the present invention Where the tandem promoter contains both a Pant and a Pben, preferably an anthranilate promoter activator protein coding sequence is selected, for example, the anthranilate activator protein (AntR) described above in regard to Pant promoters. Even more preferred in any expression system is the presence of such an expressed coding sequence in multiple copies.
Sources of Native Promoters for Use in Constructing Tandem Promoters Tandem promoters according to the present invention may be constructed, e.g., by obtaining from prokaryotic cells, preferably bacterial cells, native promoters from genes or operons encoding enzyme(s) responsible for utilization of alternative carbon sources, i.e. carbon sources other than glucose. In a preferred embodiment, the bacterial cells will be chosen from among the bacterial cells belonging to the “Pseudomonads and closely related bacteria,” or any one of the 19 Subgroups thereof, as defined below.
Bacteria are known that are capable of utilizing a wide range of alternative carbon sources. In a preferred embodiment, a native promoter selected for use in constructing a tandem promoter will be obtained from a gene or operon from which is expressed an enzyme(s) having degradative activity toward at least one alternative carbon source chosen from among:

    
    
        Straight-chain, Branched-chain, Cyclic, and Alicyclic Homo- and Hetero-Hydrocarbons (saturated or unsaturated) and derivatives thereof;
        Aromatic and Alkylaryl Compounds and derivatives thereof, e.g., benzene, naphthalene, anthracene, phenanthrene, toluene, xylene, biphenyl;
        Heterocyclic compounds and derivatives thereof, e.g., steroids, sterols, allantoins, cyclic terpenes, yohimbines, indoles, imidazoles, oxazines, quinolines, phenazines, xanthenes;
        Alcohols & Polyols and derivatives thereof, e.g., ethanol, phenol, naphthol, cresol, catechol, glycerol, benzyl alcohol, menthanol;
        Acids, Esters, Anhydrides, and derivatives thereof, e.g., acetate, salicylate, benzoate, hydroxybenzoate, anthranilate, phthalate, benzylalkaioates, gentisate, amino acids (e.g., glutamate); mono-, di-, and tri-carboxylic acids; fatty acids, lipids, and related compounds;
        Aldehydes, Ketones, Ethers and derivatives thereof;
        Halogenated Organic Compounds and derivatives thereof, e.g., chlorobenzoate, chlorophenols, iodonaphthalene, bromoxylene, fluoropentane, trichloropropane;
        Organo-Phosphorus Compounds and derivatives thereof, e.g., organophosphonates, organophosphates, organophosphites, phospha-compounds, phospho-compounds;
        Organo-Sulphur Compounds and derivatives thereof, e.g., organosulfonates, organosulfates, organosulfites, thia-compounds, thio-compounds;
        Organo-Nitrogen Compounds and derivatives thereof, e.g., organonitrates, nitroorganics (e.g., nitrobenzoate, nitrophenol), cyano compounds, hydrazines, amines, imines, amides, imides, purines, pyrimidines, aza-compounds, azo-compounds;
        Other Hetero-Organic compounds, e.g., organo-boron compounds, organo-silicon compounds, organometallic compounds, multi-heteroatom-organic compounds; and
        Multi-functional organic compounds.
    
    


The genes and operons encoding these biodegradative activities may be either catabolite-repressed or non-catabolite-repressed, as described above. The native promoters thereof may be readily obtained by one of ordinary skill in the art by methods well known in the art, e.g., by isolating mRNA encoding such an enzyme and using the nucleic acid sequence of the mRNA or cDNA made therefrom, to probe the bacterial genome (or a record of the genomic sequence thereof) for occurrence(s) of the corresponding DNA gene. This is followed by identification of regulatory regions, including a transcription start site, located in the segment of DNA immediately upstream of (i.e. 5′ to) the coding sequence. Expression constructs containing such regulatory region nucleic acid sequences are then formed and the expression construct(s) tested for induction in bacterial host cells by one or more alternative carbon source compounds, both in the presence and absence of glucose. This provides catabolite-repressed and non-catabolite-repressed promoters that may be used in constructing a tandem promoter according to the present invention.
A variety of catabolite repressed and non-catabolite-repressed genes and operons are known that either (a) encode enzymes that utilize (e.g., that transport, anabolize, or catabolize) alternative carbon sources or (b) encode regulatory genes that control expression from such enzyme-encoding gene(s) and operon(s). The promoter of a typical gene or operon of this type is regulated in that transcription therefrom depends upon, le. the promoter is induced or derepressed by, the presence of a relevant alternative carbon source or an analog compound thereof.
Examples of such catabolite repressed genes and operons include, e.g.:

    
    
        the styABCD operon of Pseudomonas fluorescens ST, which encodes enzymes required for the conversion of styrene to phenylacetate [Ref. 8];
        the xylCMABN operon of P. putida mt-2, which encodes enzymes required for the transformation of toluene to benzoate and the transformation of xylenes to toluates [Ref. 9];
        the alkBFGHJKL operon of Burkholderia cepacia, which encodes enzymes, including alkane hydroxylase, required for metabolism of alkanes and alkenes [Ref. 10];
        the P. aeruginosa gene, oprD, which encodes a specific porin that facilitates the uptake of basic amino acids, and of the carbapenem antibiotic, imipenem, a thienamycin derivative [Ref. 11];
        the P. aeruginosa gene, aotJ, which is part of an operon encoding enzymes required for the transport of arginine and ornithine [Ref. 12]; and
        the P. putida and P. aeruginosa genes, bkdR, which encode a protein regulating expression of an operon encoding a branched-chain keto acid dehydrogenase complex that is required for the metabolism of the branched-chain amino acids [Ref 13].
    
    


Examples of such non-catabolite-repressed genes and operons include, e.g.:

    
    
        the ttgDEF operon of P. putida DOT-TlE, which encodes enzymes of a secondary toluene efflux system [Ref. 14];
        the P. aeruginosa gene, gdhb, which encodes an arginine-inducible NAD(+)-dependent glutamate dehydrogenase [Ref. 15]; and
        the putA and putP genes of P. putida mt-2, which encode enzymes necessary for proline utilization [Ref. 16].

Mutant and Closely Related Sequences of Promoters and Polypeptides

    
    


Mutant promoters made from a promoter(s) of a preferred embodiment hereof may also be created using any of the various random and/or directed, mutagenesis techniques known in the art. In a preferred embodiment, site-specific mutagenesis will be performed (e.g. via mutagenic oligonucleotide-directed mutagenesis). In a preferred embodiment, an improved mutant promoter will be selected from a library of mutants made by an error-prone polymerase chain reaction (EP-PCR) performed on a promoter polynucleotide. Multiple rounds of mutagenesis may be performed either upon the pool of polynucleotides resulting from a previous round or upon one or more mutant promoters selected therefrom. Advantageous mutations identified in improved promoters may also be combined to obtain further increases in improvement (e.g., cumulative improvements).
In addition to generating mutant tandem promoters by performing one or more of the techniques described above upon a non-mutant tandem promoter (i.e. a tandem promoter in which the individual promoter elements are themselves of native sequence), individual mutant promoters may be used in forming a tandem promoter(s) according to the present invention. For example, two mutant promoters may be linked together, or a mutant promoter and a non-mutant promoter may be linked together, to form a tandem promoter according to the present invention. In addition, directed mutagenesis and/or recombination may be performed (e.g., using a technique such as is described in WO 91/16427) in order to create multiple promoter-promoter combinations in a given round.
Closely related promoters may be obtained by use of polynucleotides containing tandem and/or native promoter constructs and/or elements as hybridization probes, under stringent hybridization conditions, according to any of the various protocols known in the art. An exemplary stringent hybridization protocol is set forth below. Alternatively, a peptide nucleic acid (PNA), or other nucleic acid analog, having a base sequence of such a promoter may be used as a hybridization probe. Preferably the probe will contain a base sequence of at least about 6, at least about 8, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 bases in length. In a preferred embodiment, the probe will contain a base sequence of not more than about 100, about 80, about 60, or about 50 bases in length. In a preferred embodiment, the probe will contain a base sequence of about 20 to about 50, or about 25 to about 45, or about 30 to about 40 bases in length.
In order to perform hybridization probing of target nucleic acids, e.g., target DNA at least suspected of containing a promoter, the target DNA to be probed is denatured, blotted crosslinked onto a nitrocellulose or nylon membrane according to standard protocols (see Sambrook et al. [Cold Spring Harbor Press], Current Protocols in Molecular Biology [John Wiley and Sons, Inc.]). The blot is then pre-hybridized using standard buffers as described in Sambrook et al. or Current Protocols or using a commercially available hybridization buffer such as EXPRESSHYB (BD Biosciences Clontech, Palo Alto, Calif.). Pre-hybridization may be performed at temperatures ranging from 50-65° C.
The probe to be used (e.g., DNA representing or containing an, e.g., anthranilate or benzoate, promoter fragment) may be labeled to identify specifically bound target DNA and/or the probe nucleic acid may be used as a primer to enzymatically copy specifically bound target DNA. The probe may be labeled according to any of the techniques known in the art. For example, the probe may be labeled with any detectable label, including, but not limited to a: peptide tag, an immunogenic moiety, avidin, biotin, a fluorescent or colored moiety, a detectable chelate, or a radionuclide moiety. In a preferred embodiment, a nucleic acid, preferably DNA, is used as the probe. In a preferred embodiment, the DNA of the probe is labeled. In a preferred embodiment, the label is a radioactive moiety, e.g., a radionuclide-containing compound such as γ-32P dATP. Kits for performing such labeling are commercially available: for example, the HIGH PRIME DNA labeling kit (Roche Molecular Biochemicals, Indianapolis, Ind.), in conjunction with a radionuclide-containing nucleoside-5′-triphosphate, such as γ-32P dATP, may be used. The probe or labeled probe is then boiled and added to the pre-hybridization buffer. The blot is incubated with the probe at 50-65° C. overnight, then washed twice with 2×SSC/0.5%SDS for 5 minutes per wash at room temperature. Then the blot is washed twice with 0.1×SSC/0.1%SDS for 15 minutes per wash at 50-65° C. The blot is then developed as appropriate for viewing the specifically bound labeled probe. For example, if a radionuclide moiety is used as the label on the probe, the blot is used to expose a film or a phosphor screen for viewing.
Alternately, an oligo or set of oligos may be designed that hybridize to known promoter elements (i.e., the −35 and −10 sequences with intervening sequence), or to known activator protein binding sites; a set of degenerate oligos can be designed, at least one of which can hybridize to the target sequence of interest. These may be used as probes for Southern blot analysis as described above, or may be used to initiate synthesis of single (one oligo) or double (two oligos) stranded DNA that may be homologous to the promoter of interest. DNA synthesis may be carried out with, e.g., Taq polymerase (with extension carried out at 72° C. or as indicated in the manufacturer's protocol), or other polymerase, with buffers supplied by the manufacturer, 1-5 mM concentration of primer, and 0.2-1 mM final concentration dNTP mix. Annealing temperature can be varied to attain optimal amplification. Extension times for the polymerase may be 20-60 seconds, depending upon length of desired product. A linker could be added to the single-stranded fragment to allow for synthesis of a second strand and amplification, if necessary. Double-stranded fragments may then be sequenced using primers designed for extension/amplification. If restriction sites are also designed onto the oligo, this fragment could subsequently be directly cloned into a standard vector, such as pUC18, e.g., for sequence analysis;
The target nucleic acid to which the probe has specifically bound is then selected by means of selecting probe-target hybrids that have been viewed. In a preferred embodiment, a selected target nucleic acid, will be at least 90% homologous, i.e. at least 90% identical in base sequence to the probe or the complementary base sequence thereof (wherein T and U are considered equivalent bases for these purposes). In a preferred embodiment, a selected target nucleic acid will be at least about 95% homologous thereto. In a preferred embodiment, a selected target nucleic acid will be at least about 98% homologous thereto. Where such a target nucleic acid is situated as a portion within a larger polynucleotide molecule, the target nucleic acid, or a fragment containing said target nucleic acid, may be recovered therefrom by any means known in the art, including, e.g., endonuclease digestion and exonuclease digestion.
Alternatively, the base sequence of the probe may be used, in the form of an information string, to perform searching of a nucleotide sequence record, such as a paper or electronic database record of nucleotide sequences present in polynucleotides containing with a polynucleotide source. The search parameters may specify that a successful match must be 100% identical (100% homologous) or less than 100% identical (heterologous) to the probe information string. Preferably, the search parameters will be selected so that a successful match must be at least 90% homologous, at least 95% homologous, or at least 98% homologous to the probe information string. Preferably, the search parameters will be selected so that a successful match must be heterologous to the probe information string. Once a successful match has been identified, the polynucleotide source corresponding thereto is selected.
Alternatively, a probe information string may be created by altering a first information string representing the nucleobase sequence of a given promoter from a modified information string representing a heterologous nucleobase sequence at least 90% homologous to that of said given promoter. This modified information string may then be used to synthesize a polynucleotide molecule containing the base sequence thereof or may be used to perform searching of a nucleotide sequence record for an identical information string as described above. Upon a successful match, the polynucleotide source corresponding thereto is selected.
Once a polynucleotide at least 90% homologous to the probe sequence is obtained, it is then tested, by forming an expression construct therewith, inserting the expression construct into a transcription system (or transcription and translation system), such as a prokaryotic host cell, and screening the resulting system, e.g., the transformed cell, for the ability of the polynucleotide to direct transcription. Preferably, the screening also involves identifying at least one promoter property improved relative to that of the original promoter.
Alignments and searches for homologous sequences can be performed using the U.S. National Center for Biotechnology Information (NCBI) program, MegaBLAST (currently available at http://www.ncbi.nlm.nih.gov/BLAST/). Use of this program with options for percent identity set at 90% will identify those sequences with 90% or greater homology to the query sequence. Other software known in the art is also available for aligning and/or searching for homologous sequences, e.g., sequences at least 90% homologous to an information string containing a promoter base sequence or activator-protein-encoding base sequence according to the present invention. For example, sequence alignments for comparison to identify sequences at least 90% homologous to a query sequence can be performed by use of, e.g., the GAP, BESTFIT, BLAST, FASTA, and TFASTA programs available in the GCG Sequence Analysis Software Package (available from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), with the default parameters as specified therein, plus a parameter for the extent of homology set at 90%. Also, for example, the CLUSTAL program (available in the PC/Gene software package from Intelligenetics, Mountain View, Calif.) may be used.
These and other sequence alignment methods are well known in the art and may be conducted by manual alignment, by visual inspection, or by manual or automatic application of a sequence alignment algorithm, such as any of those embodied by the above-described programs. Various useful algorithms include, e.g.: the similarity search method described in W. R. Pearson & D. J. Lipman, Proc. Nat'l Acad. Sci. USA 85:244448 (April 1988); the local homology method described in T. F. Smith & M. S. Waterman, in Adv. Appl. Math. 2:482-89 (1981) and in J. Molec. Biol. 147:195-97 (1981); the homology alignment method described in S. B. Needleman & C. D. Wunsch, J. Molec. Biol. 48(3):443-53 (March 1970); and the various methods described, e.g., by W. R. Pearson, in Genomics 11(3):635-50 (November 1991); by W. R. Pearson, in Methods Molec. Biol. 24:307-31 and 25:365-89 (1994); and by D. G. Higgins & P. M. Sharp, in Comp. Appl'ns in Biosci. 5:151-53 (1989) and in Gene 73(1):237-44 (15 Dec. 1988).
In a preferred embodiment, a nucleobase polymer (e.g., a polynucleotide or polynucleotide analog) that is heterologous to, i.e. whose base sequence is heterologous to, the base sequence of a given promoter, promoter region, or other non-codon- or non-anti-codon-containing polynucleotide segment, will be at least 90% homologous thereto; preferably about or at least 93% homologous thereto; preferably about or at least 95% homologous thereto; preferably about or at least 96% homologous thereto; preferably about or at least 97% homologous thereto; preferably about or at least 98% homologous thereto; preferably about or at least 99% thereto.
In a preferred embodiment, a polypeptide (or segment thereof) that is heterologous to, i.e. whose amino acid sequence is heterologous to, the amino acid sequence of a given polypeptide (or segment thereof) will be at least 90% homologous thereto; preferably about or at least 93% homologous thereto; preferably about or at least 95% homologous thereto; preferably about or at least 96% homologous thereto; preferably about or at least 97% homologous thereto; preferably about or at least 98% homologous thereto; preferably about or at least 99% thereto.
In a preferred embodiment, a nucleobase polymer (or segment thereof) that is heterologous to, i.e. whose base sequence is heterologous to, the base sequence of a given codon- or anti-codon-containing polynucleotide (or segment thereof), will be at least 90% homologous thereto; preferably about or at least 93% homologous thereto; even more preferably about or at least 95% homologous thereto; still more preferably about or at least 96% homologous thereto. In a preferred embodiment, such a nucleobase polymer has such a degree of homology to the given codon- or anti-codon-containing polynucleotide that the amino acid sequence encoded by the nucleobase polymer will be at least 90% homologous to the amino acid sequence of the given polynucleotide; preferably about or at least 93% homologous thereto; preferably about or at least 95% homologous thereto; preferably about or at least 96% homologous thereto; preferably about or at least 97% homologous thereto; preferably about or at least 98% homologous thereto; preferably about or at least 99% thereto.
In a preferred embodiment, a nucleobase polymer (or segment thereof) that is heterologous to, i.e. whose base sequence is heterologous to, the base sequence of a given codon- or anti-codon-containing polynucleotide (or segment thereof), is about or at least 97% homologous thereto; preferably about or at least 98% homologous thereto; preferably about or at least 99% thereto.
Expression Constructs
In an expression construct, e.g., a gene or operon, according to the present invention, a nucleic acid containing a transcription product-encoding sequence will be operably linked to a promoter according to the present invention, spacer. Where the transcription product is an mRNA or a precursor molecule thereto, the spacer will be a ribosome-binding-site-containing spacer (“RBS spacer”).
A “transcription product-encoding polynucleotide” is any polynucleotide that contains a transcription product-encoding sequence, wherein the transcription product is any functional or structural RNA molecule, including, but not limited to, e.g., mRNA, rRNA, tRNA, cRNA, gRNA, hnRNA, miRNA, mtRNA, nRNA, ncRNA, pRNA, satRNA, scRNA, siRNA, snRNA, snoRNA, srpRNA, stRNA, tmRNA, vRNA, anti-sense RNA (also called “aRNA”), aptamer RNA, chromosomal RNA, enzyme-inhibitor RNA, genetic-control-element RNA, plastid RNA, ribozyme RNA, self-cleaving RNA, self-splicing RNA, telomerase RNA (TER or TERC), X-chromosome-inactivator RNA (XIST RNA), or a precursor RNA of any such RNA molecule. In a preferred embodiment, the transcription product will be an mRNA or a precursor RNA molecule thereto.
Other elements may be included in an expression construct. Such elements include, but are not limited to, e.g.: transcriptional enhancer sequences; translational enhancer sequences; leader peptide-encoding sequences, e.g., for intra-cellular-targeting-peptides or secretion signal peptides; pro-peptide-, pre-peptide-, and pre-pro-peptide-coding sequences; other promoters; translational start and stop signals; polyadenylation signals; transcription terminators; introns; and tag sequences, such nucleotide sequence “tags” and “tag” peptide coding sequences (a “tag” facilitates identification, separation, purification, or isolation of an expressed polynucleotide, for which a nucleotide sequence tag is used, or of an expressed polypeptide, for which a “tag” peptide coding sequence is used).
At a minimum, an expression construct according to the present invention will include (in addition to a promoter and either a spacer or an RBS-spacer, operably linked to a transcription product-encoding sequence), a transcriptional terminator. Where the transcription product is an mRNA or pre-mRNA, the expression construct will, at minimum, further include translational start and stop signals operably linked to the transcription product-encoding sequence. The term “operably linked,” as used herein, refers to any configuration in which the transcriptional and any translational regulatory elements are covalently attached to the encoding sequence in such disposition(s), relative to the encoding sequence, that in and by action of the host cell, the regulatory elements can direct the expression of the coding sequence. Every regulatory element in the expression construct must be “operably linked” to the transcription product-encoding sequence. In cases wherein the cell processes the expression construct before transcription or processes a precursor RNA transcribed from the expression construct, the regulatory element(s) must be so positioned that the cell's processing systems can manipulate the expression construct or the pre-RNA to operably link the regulatory element(s) therein. Likewise, in cases wherein the expression construct is present in the cell in distinct segments of polynucleotide(s), the segments, i.e. the polynucleotide molecules or regions collectively containing the regulatory element(s) and transcription product-encoding sequence(s), must be so positioned that the cell can manipulate the segments to create or to re-connect the expression construct wherein the regulatory elements are operatively linked to the transcription product-encoding sequence, and/or are so positioned that the cell's processing systems can manipulate a to-be-transcribed pre-RNA to operably link the regulatory element(s) thereto.
Any prokaryotic ribosome binding site (RBS) may be utilized in such an expression construct. Preferably a bacterial RBS is utilized. In preferred embodiment, an RBS operative in Gram positive bacteria is used; even more preferably an RBS operative in Gram negative bacteria is used. Many specific and a variety of consensus RBSs are known, e.g., those described in and referenced by D. Frishman et al., Starts of bacterial genes: estimating the reliability of computer predictions, Gene 234(2):257-65 (8 Jul. 1999); and B. E. Suzek et al., A probabilistic method for identifying start codons in bacterial genomes, Bioinformatics 17(12):1123-30 (December 2001). In addition, either native or synthetic RBSs may be used, e.g., those described in: EP 0207459 (synthetic RBSs); O. Ikehata et al., Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli, Eur. J. Biochem. 181(3):563-70 (1989) (native RBS sequence of AAGGAAG, SEQ ID NO:42); or J. A. Wells et al., Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis, Nucl. Acids Res. 11(22):7911-25 (1983) (native RBS sequence of GAGAGG, SEQ ID NO:43).
Furthermore, one or more marker genes or reporter genes may be used in an expression system to verify expression. Many such useful marker or reporter genes are known in the art. See, e.g., U.S. Pat. No. 4,753,876 to Hemming et al., and DL Day et al., in J. Bact. 157(3):937-39 (March 1984). In a preferred embodiment, the marker gene is selected from among the antibiotic resistance-conferring marker genes. In a preferred embodiment, the marker gene is selected from among the tetracycline and kanamycin resistance genes. In a preferred embodiment, a reporter gene is selected from among those encoding: (1) fluorescent proteins (e.g., GFP); (2) colored proteins; and (3) fluorescence- or color-facilitating or -inducing proteins, the latter class (3) including, e.g., luminases, alkaline phosphatases, and beta-galactosidases. Alkaline phosphatases hydrolyze BCIP to produce a blue color, and hydrolyze PNPP to produce a yellow color. Beta-galactosidases hydrolyze X-gal to create a blue-colored derivative, and hydrolyze ONPG to produce a yellow color. Fluorescent substrates are also available for alkaline phosphatase and β-galactosidase.
Further examples of methods, vectors, and translation and transcription elements, and other elements useful in the present invention are described in, e.g.: U.S. Pat. No. 5,055,294 to Gilroy and U.S. Pat. No. 5,128,130 to Gilroy et al.; U.S. Pat. No. 5,281,532 to Rammler et al; U.S. Pat. Nos. 4,695,455 and 4,861,595 to Barnes et al.; U.S. Pat. No. 4,755,465 to Gray et al.; and U.S. Pat. No. 5,169,760 to Wilcox.
Vectors
A great many bacterial vectors are known in the art as useful for expressing proteins in bacteria, and any of these may be used for expressing the genes according to the present invention. Such vectors include, e.g., plasmids, cosmids, and phage expression vectors. Examples of useful plasmid vectors include, but are not limited to, the expression plasmids pMB9, pBR312, pBR322, pML122, RK2, RK6, and RSF1010. Other examples of such useful vectors include those described by, e.g.: N. Hayase, in Appl. Envir. Microbiol. 60(9):3336-42 (September 1994); A. A. Lushnikov et al, in Basic Life Sci. 30:657-62 (1985); S. Graupner & W. Wackernagel, in Biomolec. Eng. 17(1): 11-16. (October 2000); H. P. Schweizer, in Curr. Opin. Biotech. 12(5):439-45 (October 2001); M. Bagdasarian & K. N. Timmis, in Curr. Topics Microbiol. Immunol. 96:47-67 (1982); T. Ishii et al., in FEMS Microbiol. Lett. 116(3):307-13 (Mar. 1, 1994); I. N. Olekhnovich & Y. K. Fomichev, in Gene 140(1):63-65 (Mar. 11, 1994); M. Tsuda & T. Nakazawa, in Gene 136(1-2):257-62 (Dec. 22, 1993); C. Nieto et al., in Gene 87(1):14549 (Mar. 1, 1990); J. D. Jones & N. Gutterson, in Gene 61(3):299-306 (1987); M. Bagdasarian et al., in Gene 16(1-3):237-47 (Dec 1981); H. P. Schweizer et al., in Genet. Eng. (NY) 23:69-81 (2001); P. Mukhopadhyay et al., in J. Bact. 172(1):477-80 (January 1990); D. O. Wood et al., in J. Bact. 145(3):1448-51 (March 1981); and R. Holtwick et al., in Microbiology 147(Pt 2):337-44 (February 2001).
Further examples of useful Pseudomonas expression vectors include those listed in Table 3.







TABLE 3







Some Examples of Useful


Expression Vectors










Replicon
Vector(s)







pPS10
PCN39, pCN51



RSF1010
PKT261-3




PMMB66EH




PEB8




PPLGN1



RK2/RP1
PRK415




PJB653



pRO1600
PUCP




PBSP










The expression plasmid, RSF1010, is described, e.g., by F. Heffron et al., in Proc. Nat'l Acad. Sci. USA 72(9):3623-27 (September 1975), and by K. Nagahari & K. Sakaguchi, in J. Bact. 133(3):1527-29 (March 1978). Plasmid RSF1010 and derivatives thereof are particularly useful vectors in the present invention. Exemplary, useful derivatives of RSF1010, which are known in the art, include, e.g., pKT212, pKT214, pKT231 and related plasmids, and pMYC1050 and related plasmids (see, e.g., U.S. Pat. Nos. 5,527,883 and 5,840,554 to Thompson et al.), such a, e.g., pMYC1803. Other exemplary useful vectors include those described in U.S. Pat. No. 4,680,264 to Puhler et al.
In a preferred embodiment, an expression plasmid is used as the expression vector. In a preferred embodiment, RSF1010 or a derivative thereof is used as the expression vector. In apreferred embodiment, pMYC1050 or a derivative thereof, or pMYC1803 or a derivative thereof, is used as the expression vector.
A vector can then be transformed into a bacterial host cell.
Transformation
Transformation of the host cells with the vector(s) may be performed using any transformation methodology known in the art, and the bacterial host cells may be transformed as intact cells or as protoplasts (i.e. including cytoplasts). Exemplary transformation methodologies include poration methodologies, e.g., electroporation, protoplast fusion, bacterial conjugation, and divalent cation treatment, e.g., calcium chloride treatment or CaCl/Mg2+ treatment.
In addition to the above elements of an expression construct, the bacterial host cell will also contain at least one, and preferably more than one, copy of a gene containing a coding sequence of an activator or repressor protein of the promoter. This gene may be attached to the expression construct, or it may be part of a separate nucleic acid. In a preferred embodiment, an anthranilate activator protein having the amino acid sequence encoded by the complement of the coding sequence shown at nucleotides 4-993 of SEQ ID NO:7 will be utilized; and a benzoate activator protein encoded by nucleotides 225-1229 or nucleotides 285-1229 of SEQ ID NO:1 will be utilized. Preferably, the activator- (or repressor-) encoding gene will be constitutively expressed in the bacterial host cell. When expression of the (e.g., exogenous) coding sequence is desired, the host cell will be contacted with an activator (or de-repressor) compound to induce expression. In a preferred embodiment, the bacterial host cell will be contacted with anthranilic or benzoic acid or a biologically acceptable salt (preferably a sodium salt) thereof, in the case of anthranilate and benzoate promoters, respectively. In a preferred embodiment for a tandem promoter, the bacterial host cell will be contacted with an inducer compound that induces either the natively catabolite-repressed promoter element or the (natively) non-catabolite-repressed promoter element thereof. In a preferred embodiment of a tandem promoter, benzoic acid, anthranilic acid, or biologically acceptable salt(s) (preferably a sodium salt) thereof, will be used as the inducer (activator) compound.
Host Cells
In a preferred embodiment, the host cell in which the promoter is used will be selected from the prokaryotes. In a preferred embodiment, the host cell is selected from the bacteria. In a preferred embodiment, the host cell is selected from the Proteobacteria. In a preferred embodiment, the host cell is selected from the “Pseudomonads and closely related bacteria” or from a Subgroup thereof, as defined below. In a preferred embodiment, the host cell is selected from the genus Pseudomonas. A particularly preferred species of Pseudomonas is P. fluorescens; even more preferred is Pseudomonas fluorescens biotype A.
In a preferred embodiment, both the organism from which the native promoter(s) are obtained and the host cells in which a promoter according to the present invention is utilized, will be selected from the prokaryotes. In a preferred embodiment, both the organism from which the native promoter(s) are obtained and the host cells in which a promoter according to the present invention is utilized, will be selected from the bacteria. In a preferred embodiment, both the bacteria from which the native promoter(s) are obtained and the bacterial host cells in which a promoter according to the present invention is utilized, will be selected from the Proteobacteria. In a preferred embodiment, both the bacteria from which the native promoter(s) are obtained and the bacterial host cells in which a promoter according to the present invention is utilized, will be selected from the Pseudomonads and closely related bacteria or from a Subgroup thereof, as defined below.
In a preferred embodiment, both the promoter source organism and the host cell will be selected from the same species. Preferably, the species will be a prokaryote; more preferably a bacterium, still more preferably a Proteobacterium. In a particularly preferred embodiment, both the promoter source organism and the host cell will be selected from the same species in a genus selected from the Pseudomonads and closely related bacteria or from a Subgroup thereof, as defined below; more preferably from the genus Pseudomonas. Especially preferred is the species Pseudomonas fluorescens; even more preferably, Pseudomonas fluorescens biotype A.
In a preferred embodiment, the host cells in which the promoter is used will lack biocatalyst(s) effective to degrade the inducer compound: e.g., benzoate or anthranilate or an analog thereof, and/or the degradation product(s) thereof, if any, that is directly responsible for induction; and/or gratuitous inducer compounds. Such host cells are readily obtained as knock-out mutants. For example, the present inventors have found that, in the case of an anthranilate promoter, inactivation of at least the antA portion of the host cell's antABC operon does inhibit the consumption of an anthranilate inducer and thereby permits the inducer to effect lasting induction. The antA open reading frame encodes the large subunit of the first enzyme utilized in the pathway for degradation of anthranilate. Similarly, in the case of a benzoate promoter, the inventors have found that inactivation of the benAB portion of the host cell's benABCD operon, e.g., by deletion or mutation, does inhibit the consumption of a benzoate inducer, thereby improving the level of induction; inactivation of at least the beta portion would work similarly, as this encodes the large subunit of the first enzyme utilized in the pathway for degradation of benzoate.
Gene knock-outs may be constructed according to any method known effective in the art. Gene inactivation by insertion of a gene has been previously described. See, e.g., D L Roeder & A Collmer, Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi, J Bacteriol. 164(1):51-56 (1985). Briefly, a portion of the gene to be disrupted in amplified and cloned into a vector containing a selectable marker, such as an antibiotic resistance gene, that is not able to replicate in the target host. Homologous recombination between the chromosomal copy of the gene and the portion of the target gene contained on the plasmid results in the disruption of the chomosomal copy of the gene and incorporation of the antibiotic resistance marker. Alternatively, transposon mutagenesis and selection for desired phenotype (such as the inability to metabolize benzoate or anthranilate) may be used to isolate bacterial strains in which target genes have been insertionally inactivated. See, e.g., K Nida & P P Cleary, Insertional inactivation of streptolysin S expression in Streptococcus pyogenes, J Bacteriol. 155(3):1156-61 (1983). Specific mutations or deletions in a particular gene can be constructed using cassette mutagenesis, for example, a described in J A Wells et al., Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, Gene 34(2-3):315-23 (1985); whereby direct or random mutations are made in a selected portion of a gene, and then incorporated into the chromosomal copy of the gene by homologous recombination.
Pseudomonads and Closely Related Bacteria
The “Pseudomonads and closely related bacteria,” as used herein, is co-extensive with the group defined herein as “Gram(−) Proteobacteria Subgroup 1.” “Gram(−) Proteobacteria Subgroup 1” is more specifically defined as the group of Proteobacteria belonging to the families and/or genera described as falling within that taxonomic “Part” named “Gram-Negative Aerobic Rods and Cocci” by R. E. Buchanan and N. E. Gibbons (eds.), Bergey's Manual of Determinative Bacteriology, pp. 217-289 (8th ed., 1974) (The Williams & Wilkins Co., Baltimore, Md., USA) (hereinafter “Bergey (1974)”), and the genus, Acinetobacter. Table 4 presents the families and genera of organisms listed in the Bergey taxonomic “Part.”







TABLE 4





Families and Genera Listed in the Part,


“Gram-Negative Aerobic Rods and Cocci” (in


Bergey (1974))


















Family I. Pseudomonadaceae
Gluconobacter




Pseudomonas




Xanthomonas




Zoogloea



Family II. Azotobacteraceae
Azomonas




Azotobacter




Beijerinckia




Derxia



Family III. Rhizobiaceae
Agrobacterium




Rhizobium



Family IV. Methylomonadaceae
Methylococcus




Methylomonas



Family V. Halobacteriaceae
Halobacterium




Halococcus



Other Genera
Acetobacter




Alcaligenes




Bordetella




Brucella




Francisella




Thermus










“Gram(−) Proteobacteria Subgroup 1” contains all Proteobacteria classified thereunder, as well as all Proteobacteria that would be classified thereunder according to the criteria used in forming that taxonomic “Part.” As a result, “Gram(−) Proteobacteria Subgroup 1” excludes, e.g.: all Gram-positive bacteria; those Gram-negative bacteria, such as the Enterobacteriaceae, which fall under others of the 19 “Parts” of this Bergey (1974) taxonomy; the entire “Family V. Halobacteriaceae” of this Bergey (1974) “Part,” which family has since been recognized as being a non-bacterial family of Archaea; and the genus, Thermus, listed within this Bergey (1974) “Part,” which genus which has since been recognized as being a non-Proteobacterial genus of bacteria.
Also in accordance with this definition, “Gram(−) Proteobacteria Subgroup 1” further includes those Proteobacteria belonging to (and previously called species of) the genera and families defined in this Bergey (1974) “Part,” and which have since been given other Proteobacterial taxonomnic names. In some cases, these re-namings resulted in the creation of entirely new Proteobacterial genera. For example, the genera Acidovorax, Brevundimonas, Burkholderia, Hydrogenophaga, Oceanimonas, Ralstonia, and Stenotrophomonas, were created by regrouping organisms belonging to (and previously called species of) the genus Pseudomonas as defined in Bergey (1974). Likewise, e.g. the genus Sphingomonas (and the genus Blastomonas, derived therefrom) was created by regrouping organisms belonging to (and previously called species of) the genus Xanthomonas as defined in Bergey (1974). Similarly, e.g., the genus Acidomonas was created by regrouping organisms belonging to (and previously called species of) the genus Acetobacter as defined in Bergey (1974). Such subsequently reassigned species are also included within “Gram(−) Proteobacteria Subgroup 1” as defined herein.
In other cases, Proteobacterial species falling within the genera and families defined in this Bergey (1974) “Part” were simply reclassified under other, existing genera of Proteobacteria. For example, in the case of the genus Pseudomonas, Pseudomonas enalia (ATCC 14393), Pseudomonas nigrifaciens (ATCC 19375), and Pseudomonas putrefaciens (ATCC 8071) have since been reclassified respectively as Alteromonas haloplanktis, Alteromonas nigrifaciens, and Alteromonas putrefaciens. Similarly, e.g., Pseudomonas acidovorans (ATCC 15668) and Pseudomonas testosteroni (ATCC 11996) have since been reclassified as Comamonas acidovorans and Comamonas testosteroni, respectively; and Pseudomonas nigrifaciens (ATCC 19375) and Pseudomonas piscicida (ATCC 15057) have since been reclassified respectively as Pseudoalteromonas nigrifaciens and Pseudoalteromonas piscicida. Such subsequently reassigned Proteobacterial species are also included within “Gram(−) Proteobacteria Subgroup 1” as defined herein.
Likewise in accordance with this definition, “Gram(−) Proteobacteria Subgroup 1I” fiurter includes Proteobacterial species that have since been discovered, or that have since been reclassified as belonging, within the Proteobacterial families and/or genera of this Bergey (1974) “Part.” In regard to Proteobacterial families, “Gram(−) Proteobacteria Subgroup 1” also includes Proteobacteria classified as belonging to any of the families: Pseudomonadaceae, Azotobacteraceae (now often called by the synonym, the “Azotobacter group” of Pseudomonadaceae), Rhizobiaceae, and Methylomonadaceae (now often called by the synonym, “Methylococcaceae”). Consequently, in addition to those genera otherwise described herein, further Proteobacterial genera falling within “Gram(−) Proteobacteria Subgroup1” include: 1) Azotobacter group bacteria of the genus Azorhizophilus; 2) Pseudomonadaceae family bacteria of the genera Cellvibrio, Oligella, and Teredinibacter; 3) Rhizobiaceae family bacteria of the genera Chelatobacter, Ensifer, Liberibacter (also called “Candidatus Liberibacter”), and Sinorhizobium; and 4)Methylococcaceae family bacteria of the genera Methylobacter, Methylocaldum, Methylomicrobium, Methylosarcina, and Methylosphaera. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 1,” as defined above.
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 2.” “Gram(−) Proteobacteria Subgroup 2” is defined as the group of Proteobacteria of the following genera (with the total numbers of catalog-listed, publicly-available, deposited strains thereof indicated in parenthesis, all deposited at ATCC, except as otherwise indicated): Acidomonas (2); Acetobacter (93); Gluconobacter (37); Brevundimonas (23); Beijerinckia (13); Derxia (2); Brucella (4); Agrobacterium (79); Chelatobacter (2); Ensifer (3); Rhizobium (144); Sinorhizobium (24); Blastomonas (1); Sphingomonas (27); Alcaligenes (88); Bordetella (43); Burkholderia (73); Ralstonia (33); Acidovorax (20); Hydrogenophaga (9); Zoogloea (9); Methylobacter (2); Methylocaldum (1 at NCIMB); Methylococcus (2); Methylomicrobium (2); Methylomonas (9); Methylosarcina (1); Methylosphaera; Azomonas (9); Azorhizophilus (5); Azotobacter (64); Cellvibrio (3); Oligella (5); Pseudomonas (1139); Francisella (4); Xanthomonas (229); Stenotrophomonas (50); Oceanimonas (4); and Acinetobacter (160).
Exemplary species of “Gram(−) Proteobacteria Subgroup 2” include, but are not limited to the following bacteria (with the ATCC or other deposit numbers of exemplary strain(s) thereof shown in parenthesis): Acidomonas methanolica (ATCC 43581); Acetobacter aceti (ATCC 15973); Gluconobacter oxydans (ATCC 19357); Brevundimonas diminuta (ATCC 11568); Beijerinckia indica (ATCC 9039 and ATCC 19361); Derxia gummosa (ATCC 15994); Brucella melitensis (ATCC 23456), Brucella abortus (ATCC 23448); Agrobacterium tumefaciens (ATCC 23308), Agrobacterium radiobacter (ATCC 19358), Agrobacterium rhizogenes (ATCC 11325); Chelatobacter heintzii (ATCC 29600); Ensifer adhaerens (ATCC 33212); Rhizobium leguminosarum (ATCC 10004); Sinorhizobium fredii (ATCC 35423); Blastomonas natatoria (ATCC 35951); Sphingomonas paucimobilis (ATCC 29837); Alcaligenes faecalis (ATCC 8750); Bordetella pertussis (ATCC 9797); Burkholderia cepacia (ATCC 25416); Ralstonia pickettii (ATCC 27511); Acidovorax facilis (ATCC 11228); Hydrogenophaga flava (ATCC 33667); Zoogloea ramigera (ATCC 19544); Methylobacter luteus (ATCC 49878); Methylocaldum gracile (NCIMB 11912); Methylococcus capsulatus (ATCC 19069); Methylomicrobium agile (ATCC 35068); Methylomonas methanica (ATCC 35067); Methylosarcina fibrata (ATCC 700909); Methylosphaera hansonii (ACAM 549); Azomonas agilis (ATCC 7494); Azorhizophilus paspali (ATCC 23833); Azotobacter chroococcum (ATCC 9043); Cellvibrio mixtus (UQM 2601); Oligella urethralis (ATCC 17960); Pseudomonas aeruginosa (ATCC 10145), Pseudomonas fluorescens (ATCC 35858); Francisella tularensis (ATCC 6223); Stenotrophomonas maltophilia (ATCC 13637); Xanthomonas campestris (ATCC 33913); Oceanimonas doudoroffii (ATCC 27123); and Acinetobacter calcoaceticus (ATCC 23055).
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 3.” “Gram(−) Proteobacteria Subgroup 3” is defined as the group of Proteobacteria of the following genera: Brevundimonas; Agrobacterium; Rhizobium; Sinorhizobium; Blastomonas; Sphingomonas; Alcaligenes; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Methylobacter; Methylocaldum; Methylococcus; Methylomicrobium; Methylomonas; Methylosarcina; Methylosphaera; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter; Francisella; Stenotrophomonas; Xanthomonas; Oceanimonas; and Acinetobacter. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 4.” “Gram(−) Proteobacteria Subgroup 4” is defined as the group of Proteobacteria of the following genera: Brevundimonas; Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Methylobacter; Methylocaldum; Methylococcus; Methylomicrobium; Methylomonas; Methylosarcina; Methylosphaera; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter; Francisella; Stenotrophomonas; Xanthomonas; Oceanimonas; and Acinetobacter. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 5.” “Gram(−) Proteobacteria Subgroup 5” is defined as the group of Proteobacteria of the following genera: Methylobacter; Methylocaldum; Methylococcus; Methylomicrobium; Methylomonas; Methylosarcina; Methylosphaera; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter; Francisella; Stenotrophomonas; Xanthomonas; Oceanimonas; and Acinetobacter. 
In a preferred embodiment, the host cell is selected from “Gram(−) Proteobacteria Subgroup 6.” “Gram(−) Proteobacteria Subgroup 6” is defined as the group of Proteobacteria of the following genera: Brevundimonas; Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter; Stenotrophomonas; Xanthomonas; Oceanimonas; and Acinetobacter. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 7.” “Gram(−) Proteobacteria Subgroup 7” is defined as the group of Proteobacteria of the following genera: Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter; Stenotrophomonas; Xanthomonas; Oceanimonas; and Acinetobacter. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 8.” “Gram(−) Proteobacteria Subgroup 8” is defined as the group of Proteobacteria of the following genera: Brevundimonas; Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Pseudomonas; Stenotrophomonas; Xanthomonas; Oceanimonas; and Acinetobacter. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 9.” “Gram(−) Proteobacteria Subgroup 9” is defined as the group of Proteobacteria of the following genera: Brevundimonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Pseudomonas; Stenotrophomonas; Oceanimonas; and Acinetobacter. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 10.” “Gram(−) Proteobacteria Subgroup 10” is defined as the group of Proteobacteria of the following genera: Burkholderia; Ralstonia; Pseudomonas; Stenotrophomonas; Xanthomonas; and Acinetobacter. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 11.” “Gram(−) Proteobacteria Subgroup 11” is defined as the group of Proteobacteria of the genera: Pseudomonas; Stenotrophomonas; Xanthomonas; and Acinetobacter. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 12.” “Gram(−) Proteobacteria Subgroup 12” is defined as the group of Proteobacteria of the following genera: Burkholderia; Ralstonia; Pseudomonas. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 13.” “Gram(−) Proteobacteria Subgroup 13” is defined as the group of Proteobacteria of the following genera: Burkholderia; Raistonia; Pseudomonas; Xanthomonas; and Acinetobacter. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 14.” “Gram(−) Proteobacteria Subgroup 14” is defined as the group of Proteobacteria of the following genera: Pseudomonas and Xanthomonas. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 15.” “Gram(−) Proteobacteria Subgroup 15” is defined as the group of Proteobacteria of the genus Pseudomonas. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 16.” “Gram(−) Proteobacteria Subgroup 16” is defined as the group of Proteobacteria of the following Pseudomonas species (with the ATCC or other deposit numbers of exemplary strain(s) shown in parenthesis): Pseudomonas abietaniphila (ATCC 700689); Pseudomonas aeruginosa (ATCC 10145); Pseudomonas alcaligenes (ATCC 14909); Pseudomonas anguilliseptica (ATCC 33660); Pseudomonas citronellolis (ATCC 13674); Pseudomonas flavescens (ATCC 51555); Pseudomonas mendocina (ATCC 25411); Pseudomonas nitroreducens (ATCC 33634); Pseudomnonas oleovorans (ATCC 8062); Pseudomonas pseudoalcaligenes (ATCC 17440); Pseudomonas resinovorans (ATCC 14235); Pseudomonas straminea (ATCC 33636); Pseudomonas agarici (ATCC 25941); Pseudomonas alcaliphila; Pseudomonas alginovora; Pseudomonas andersonii; Pseudomonas asplenii (ATCC 23835); Pseudomonas azelaica (ATCC 27162); Pseudomonas beijerinckii (ATCC 19372); Pseudomonas borealis; Pseudomonas boreopolis (ATCC 33662); Pseudomonas brassicacearum; Pseudomonas butanovora (ATCC 43655); Pseudomonas cellulosa (ATCC 55703); Pseudomonas aurantiaca (ATCC 33663); Pseudomonas chlororaphis (ATCC 9446, ATCC 13985, ATCC 17418, ATCC 17461); Pseudomonas fragi (ATCC 4973); Pseudomonas lundensis (ATCC 49968); Pseudomonas taetrolens (ATCC 4683); Pseudomonas cissicola (ATCC 33616); Pseudomonas coronafaciens; Pseudomonas diterpeniphila; Pseudomonas elongata (ATCC 10144); Pseudomonas flectens (ATCC 12775); Pseudomonas azotoformans; Pseudomonas brenneri; Pseudomonas cedrella; Pseudomonas corrugata (ATCC 29736); Pseudomonas extremorientalis; Pseudomonas fluorescens (ATCC 35858); Pseudomonas gessardii; Pseudomonas libanensis; Pseudomonas mandelii (ATCC 700871); Pseudomonas marginalis (ATCC 10844); Pseudomonas migulae; Pseudomonas mucidolens (ATCC 4685); Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas synxantha (ATCC 9890); Pseudomonas tolaasii (ATCC 33618); Pseudomonas veronti (ATCC 700474); Pseudomonas frederiksbergensis; Pseudomonas geniculata (ATCC 19374); Pseudomonas gingeri; Pseudomonas graminis; Pseudomonas grimontii; Pseudomonas halodenitrificans; Pseudomonas halophila; Pseudomonas hibiscicola (ATCC 19867); Pseudomonas huttiensis (ATCC 14670); Pseudomonas hydrogenovora; Pseudomonas jessenii (ATCC 700870); Pseudomonas kilonensis; Pseudomonas lanceolata (ATCC 14669); Pseudomonas lini; Pseudomonas marginata (ATCC 25417); Pseudomonas mephitica (ATCC 33665); Pseudomonas denitrificans (ATCC 19244); Pseudomonas pertucinogena (ATCC 190); Pseudomonas pictorum (ATCC 23328); Pseudomonas psychrophila; Pseudomonas fulva (ATCC 31418); Pseudomonas monteilli (ATCC 700476); Pseudomonas mosselii; Pseudomonas oryzihabitans (ATCC 43272); Pseudomonas plecoglossicida (ATCC 700383); Pseudomonas putida (ATCC 12633); Pseudomonas reactans; Pseudomonas spinosa (ATCC 14606); Pseudomonas balearica; Pseudomonas luteola (ATCC 43273); Pseudomonas stutzeri (ATCC 17588); Pseudomonas amygdali (ATCC 33614); Pseudomonas avellanae (ATCC 700331); Pseudomonas caricapapayae (ATCC 33615); Pseudomonas cichorii (ATCC 10857); Pseudomonas ficuserectae (ATCC 35104); Pseudomonas fuscovaginae; Pseudomonas meliae (ATCC 33050); Pseudomonas syringae (ATCC 19310); Pseudomonas viridiflava (ATCC 13223); Pseudomonas thermocarboxydovorans (ATCC 35961); Pseudomonas thermotolerans; Pseudomonas thivervalensis; Pseudomonas vancouverensis (ATCC 700688); Pseudomonas wisconsinensis; and Pseudomonas xiamenensis. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 17.” “Gram(−) Proteobacteria Subgroup 17” is defined as the group of Proteobacteria known in the art as the “fluorescent Pseudomonads” including those belonging, e.g. to the following Pseudomonas species: Pseudomonas azotoformans; Pseudomonas brenneri; Pseudomonas cedrella; Pseudomonas corrugata; Pseudomonas extremorientalis; Pseudomonas fluorescens; Pseudomonas gessardii; Pseudomonas libanensis; Pseudomonas mandelii; Pseudomonas marginalis; Pseudomonas migulae; Pseudomonas mucidolens; Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas synxantha; Pseudomonas tolaasii; and Pseudomonas veronii. 
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 18.” “Gram(−) Proteobacteria Subgroup 18” is defined as the group of all subspecies, varieties, strains, and other sub-special units of the species Pseudomonas fluorescens, including those belonging, e.g., to the following (with the ATCC or other deposit numbers of exemplary strain(s) shown in parenthesis): Pseudomonas fluorescens biotype A, also called biovar 1 or biovar I (ATCC 13525); Pseudomonas fluorescens biotype B, also called biovar 2 or biovar II (ATCC 17816); Pseudomonas fluorescens biotype C, also called biovar 3 or biovar III (ATCC 17400); Pseudomonas fluorescens biotype F, also called biovar 4 or biovar IV (ATCC 12983); Pseudomonas fluorescens biotype G, also called biovar 5 or biovar V (ATCC 17518); and Pseudomonas fluorescens subsp. cellulosa (NCMB 10462).
In a preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 19.” “Gram(−) Proteobacteria Subgroup 19” is defined as the group of all strains of Pseudomonas fluorescens biotype A. A particularly preferred strain of this biotype is P. fluorescens strain MB101 (see U.S. Pat. No. 5,169,760 to Wilcox), and derivatives thereof.
In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 1.” In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 2.” In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 3.” In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 5.” In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 7.” In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 12.” In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 15.” In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 17.” In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 18.” In a particularly preferred embodiment, the bacteria are selected from “Gram(−) Proteobacteria Subgroup 19.”
An expression system according to the present invention can be cultured in any fermentation format. For example, batch, fed-batch, semi-continuous, and continuous fermentation modes of any volume may be employed herein.
In the present invention, growth, culturing, and/or fermentation of the host cells is performed within a temperature range permitting survival of the host cells, preferably a temperature within the range of about 4° C. to about 55° C., inclusive. Thus, e.g., the terms “growth” (and “grow,” “growing”), “culturing” (and “culture”), and “fermentation” (and “ferment,” “fermenting”), as used herein in regard to the host cells of the present invention, inherently and necessarily means “growth,” “culturing,” and “fermentation,” within a temperature range of about 4° C. to about 55° C., inclusive. In addition, “growth” is used to indicate both biological states of active cell division and/or enlargement, as well as biological states in which a non-dividing and/or non-enlarging cell is being metabolically sustained, the latter use of the term “growth” being synonymous with the term “maintenance.”
In addition, growth “under conditions permitting expression” when used in regard to the bacterial host cells and expression systems of the present invention, is defined herein to mean: (1) growth of the recombinant bacterial host cells per se, where the promoter used in the control sequence operably linked to the coding sequence is a constitutive promoter; and (2) where the promoter used in the control sequence operably linked to the coding sequence is a regulated promoter, (a) growth of the recombinant bacterial host cells in the presence of (i.e. in contact with) an inducer thereof, and (b) growth of the recombinant bacterial host cells in the absence of an inducer thereof, followed by addition of such an inducer to the system, thereby causing contact between the cell and the inducer.
Biocatalyst Preparation
Once the coding sequence(s) under control of the promoter is expressed, the resulting gene product(s) and/or secondary products (e.g., metabolites) resulting from expression of the gene product(s) can be separated, isolated, and/or purified using any recovery and/or purification methods known in the art as useful for such a product, e.g., a protein, nucleic acid, or other molecule. Alternatively, the host cells themselves can be used, e.g., in whole cell bioreactors or in other applications.
EXAMPLES
Materials & Methods
Promoters and Promoter-Plasmid Constructs
The following promoter nucleotide sequences are referred to herein,

    
    
        Pben509: nucleotides c994-c1502 of SEQ ID NO:1.
        Pben278: nucleotides g1228-c1502 of SEQ ID NO:1.
        Pben88: nucleotides g1228-c1316 of SEQ ID NO:1 with deletion of g1306.
        Pant713: nucleotides c592-c1304 of SEQ ID NO:7.
        Pant705: nucleotides c592-g1296 of SEQ ID NO:7.
        Pant311: nucleotides c994-c1304 of SEQ ID NO:7.
        Pant289: nucleotides 994-1283 of SEQ ID NO:7 with deletion of g1269 and t1278.
        AntR+Pant (also Pant+AntR): a1-c1395 of SEQ ID NO:13.
        Ptandem: SEQ ID NO:13.
    
    


The following promoterless plasmid constructs are referred to herein.

    
    
        pDOW1017: No Promoter, but carrying a promoterless lacZ reporter gene.
        pDOW1033: No Promoter, but carrying a promoterless phoA reporter gene.
    
    


The plasmid promoter constructs listed in Tables 5 and 6 are referred to herein.







TABLE 5







Plasmid Individual Promoter Constructs











Promoter
Plasmid
Promoter or Activator-Promoter




Type
Designation
Sequence Identity
Construct Description





Benzoate
pDOW1028
SEQ ID NO:1 N1228–N1502
Pben278::lacZ




pDOW1041
SEQ ID NO:1 N1228–N1502
Pben278::phoA



pDOW1019
SEQ ID NO:1 N994–N1502
Pben509::lacZ



pDOW1102
SEQ ID NO:1 N1228–N1316 with
Pben88::lacZ




deletion of g1306



pDOW1081
SEQ ID NO:1 N1228–N1316 with
Pben88::phoA




deletion of g1306



pDOW1083
SEQ ID NO:1 N1228–N1316 with
Pben88(−10con)::phoA




deletion of g1306 and substitution of




native −10 ‘tacggt’ 1296–1301 by




‘tataat’



pDOW1126
SEQ ID NO:1 N1–N1316 with
BenR-Pben88(−10con)::lacZ




deletion of g1306



pDOW1090
SEQ ID NO:1 N1–N1316 with
BenR-Pben88(−10con)::phoA




deletion of g1306



pDOW1100
SEQ ID NO:1 N1228–N1316 with
Pben88(—10benAc)::lacZ




deletion of g1306 and substitution of




native −10 ‘tacggt’ 1296–1301 by




‘taaggt’



pDOW1084
SEQ ID NO:1 N1228–N1316 with
Pben88(−10benAc)::phoA




deletion of g1306 and substitution of




native −10 ‘tacggt’ 1296–1301 by




‘taaggt’


 


Anthranilate
pDOW1039
SEQ ID NO:7 N1–N1304
AntR-Pant



pDOW1101
SEQ ID NO:7 N994–N1304
Pant311::lacZ



pDOW1035
SEQ ID NO:7 N1–N1304
AntR-Pant::lacZ



pDOW1056
SEQ ID NO:7 N1–N1304
AntR-Pant::phoA



pDOW1029
SEQ ID NO:7 N592–N1304
Pant713::lacZ



pDOW1095
SEQ ID NO:7 N1–N1283 with
AntR-Pant(−10wt)::phoA




deletion of t1278



pDOW1082
SEQ ID NO:7 N994–N1283 with
Pant289(−10con)::phoA




deletion of g1269 and t1278 and




substitution of native −10 region




‘ttaat’ 1264–1268 by consensus −10




region ‘tataat’



pDOW1098
SEQ ID NO:7 N1–N1283 with
AntR-Pant289(−10con)::phoA




deletion of g1269 and t1278 and




substitution of native −10 region




‘ttaat’ 1264–1268 by consensus −10




region ‘tataat’


 


Mosaic
pDOW1099
SEQ ID NO:7 N1–N1283 with
AntR-Pant289(−10ben)::phoA




deletion of g1269 and t1278 and




substitution of native Pant −10 region




‘ttaat’ 1264–1268 by native Pben −10




region ‘tacggt’
















TABLE 6







Plasmid Tandem Promoter Constructs










Plasmid
Promoter or Activator-Promoter




Designation
Sequence Identity
Construct Description





pDOW1057
SEQ ID NO:13
AntR-Ptandem::lacZ



 


pDOW1111
SEQ ID NO:13 N1085–N1541
Pant311-Pben278::lacZ


 


pDOW1107
SEQ ID NO:13 N1–N1518 with
AntR-Ptandem[Pben88(−10wt)]::lacZ



deletion of g1508


 


pDOW1108
SEQ ID NO:13 N1–N1518 with
AntR-Ptandem[Pben88(−10con)]::lacZ



deletion of g1508 and substitution of



Pben native −10 ‘tacggt’ 1498–1503 by



‘tataat’


 


pDOW1109
SEQ ID NO:13 N1–N1518 with
AntR-Ptandem[Pben88(−10benAc)]::lacZ



deletion of g1508 and substitution of



Pben native −10 ‘tacggt’ 1498–1503 by



‘taaggt’









The oligonucleotides listed in Table 7 are utilized in the following examples.







TABLE 7







Oligonucleotides Used Herein








Primer Name
Sequence (all listed 5′→3′)













AntAKO5
GGAATTCTTCGTGACGATGCG
(SEQ ID NO:16)



 


AntAKO3
CGGGATCCGCTCGCGATGCTGC
(SEQ ID NO:17)


 


lacZPE
GGATGTGCTGCAAGGC
(SEQ ID NO:18)


 


lacZPE2
GTAACCATGGTCATCGC
(SEQ ID NO:19)


 


M13forward
GTAAAACGACGGCCAGT
(SEQ ID NO:20)


 


M13reverse
AACAGCTATGACCATG
(SEQ ID NO:21)


 


Bambenwtshort
CGGGATCCGTATCAGGCGCCTCACCGTACGTGCTC
(SEQ ID NO:22)


 


Bambenconshort
CGGGATCCGTATCAGGCGCCTCATTATACGTGCTC
(SEQ ID NO:23)


 


BambenAcshort
CGGGATCCGTATCAGGCGCCTCACCTTACGTGCTC
(SEQ ID NO:24)


 


Bamantwtshort
CGGGATCCGCTAACGGTGAGCCATTAAGCGGCTGC
(SEQ ID NO:25)


 


Bamantconshort
CGGGATCCGCTAACGGTGAGCATTATAGCGGCTGC
(SEQ ID NO:26)


 


BenactKO-for
CGCGACACATTGCTGCCCAG
(SEQ ID NO:27)


 


BenactKO-rev
AGTATCAGCCATCGCACCTT
(SEQ ID NO:28)


 


1803H3seq
GTCCTGCAATTTCAGCCGA
(SEQ ID NO:29)


 


BenL278
CCTTAATTAAGTTAAGCGACGTGCGC
(SEQ ID NO:30)


 


3′Antactiv
CCCAAGCTTCTATCGAGGCAAGCCAG
(SEQ ID NO:31)


 


Benact5′
AGCTTTGTTTAAACGCATGACGTTGTTGATTC
(SEQ ID NO:32)


 


H3_5′BenAKOclean
CCCAAGCTTGCCATGAGGCGGAAAACGCTGC
(SEQ ID NO:33)


 


H3_3′BenBKOclean
CCCAAGCTTCGGTGATCGCCACGCTGTCGC
(SEQ ID NO:34)


 


BenKOmega
CATACGTCATGGCCCTCCGTTGTTC
(SEQ ID NO:35)


 


InvbenKOmega
GAACAACGGAGGGCCATGACGTATG
(SEQ ID NO:36)


 


5′BenA_Seq
CTGCTGGAAAACGCCTGCCTGGAG
(SEQ ID NO:37)


 


Seq_3′BenB
GAGCACTTCAAGCATCGACAGGAAC
(SEQ ID NO:38)


 


1261-8378F
CTTCAGATCCAGACTCACCAG
(SEQ ID NO:39)


 


1261-103R
GACCATGATTACGCCAAGCGC
(SEQ ID NO:40)


 


M13R21
CACACAGGAAACAGCTATGAC
(SEQ ID NO:41)









Host Cells:

E. coli JM109 (obtained from Promega Corp.), E. coli TOP10 (obtained from Invitrogen Corp.), and Pseudomonas fluorescens biotype A (strains MB 101 and MB214). P. fluorescens MB214 is a derivative of strain MB101 (a wild-type prototrophic P. fluorescens biovar A). MB214 had been prepared by integrating the E. coli laclZYA operon (deleted of the lacZ promoter region) into the chromosome of strain MB 101 to provide a host cell wherein the lac promoter and its derivatives can be regulated by lactose or IPTG to drive inducible expression of transgenes of interest. The MB101 strain is Lac(−) whereas the MB214 strain is Lac(+).
Inducer Compounds
As used in the Examples below, an “anthranilate” inducer means sodium anthranilate, and a “benzoate” inducer means sodium benzoate.
Transformation Protocols
E. coli: Transformations of E. coli were performed as per the manufacturer's protocol, using strain JM109 chemically competent cells from Promega (Madison, Wis.).
P. fluorescens: Electroporation of P. fluorescens was performed by subculturing 1 mL of an overnight culture (grown in rich medium; the present examples used Luria-Bertni Broth, Miller (ie. LB Broth, Miller) (available from Difco, Detroit, Mich.) into 50 mL LB Broth, Miller and incubating at 30° C. with shaking until an A600 measurement falls within the range of 0.4-0.6. The resulting cells were washed twice with 50 mL cold ddH20 and resuspend in 1 mL cold ddH20. To 100 μL aliquots of competent cells were added approximately 10 ng of a plasmid of interest, in a 0.2 cm gap electroporation cuvette (Bio-Rad Laboratories, Inc., Hercules, Calif.). Electroporation was performed under the following conditions: 200 Ohms, 25 μF, 2.25 kV. This was followed by the addition of 1 mL cold LB broth. Cells were permitted to recover on ice for 2 minutes, then incubated at 30° C., with no shaking, for 2 hours to overnight. Cells were then plated on selective medium; the present examples used LB agar Miller (Luria-Bertani) (available from Difco, Detroit, Mich.), supplemented with 15 μg/mL tetracycline (Fisher Scientific, Pittsburgh, Pa.) as the selective medium.
Cell Growth Protocols:
Cell growth for induction: Strains of interest were grown overnight (at 30° C. with shaking at 250 rpm) in 1×M9 minimal salts medium (diluted from a 5× preparation purchased from Fisher Scientific, Pittsburgh, Pa.) supplemented with 0.5% or 1% (w/v) glucose, 1 mM MgSO4, and trace elements (for trace elements, the present examples used a solution containing salts of sodium, magnesium, manganese, iron, and cobalt, all at less than 0.5 mg/mL final concentration). Strains were then subcultured 1:4 in the same medium to a volume of 10 or 20 mL and then induced with 0-10 mM concentrations of anthranilate or benzoate, as indicated.
Cell growthforplasmidpropagation: E. coli cells containing a plasmid of interest were grown overnight in 50-200 mL of LB Broth, Miller, supplemented with 15 μg/ML tetracycline or 100 μg/mL ampicillin (depending on the plasmid to be isolated) at 37° C., with shaking at 250 rpm. Plasmids preparations were performed using the NUCLEOSPIN kit (plasmid DNA purification “miniprep” kit for use with culture volumes up to 5 mL; available from BD Biosciences Clontech, Palo Alto, Calif.) or the NUCLEOBOND kit (plasmid DNA purification “midiprep” kit for use with culture volumes up to 200 ml; available from BD Biosciences Clontech, Palo Alto, Calif.).
Induction Protocols:
Strains of interest were grown overnight at 30° C. in 1×M9 medium supplemented with 0.5% or 1% (w/v) glucose, 1 mM MgSO4, and 5L/L trace elements (as described above), and optionally tetracycline at 15 ug/mL. These were then subcultured 1:4 or 1:5 in the same medium and then induced with indicated concentrations of anthranilate, benzoate, or other inducer, for a desired amount of time (e.g., for 2, 4, 6, 8, 12, or 24 hours, or overnight). Samples were taken at indicated times and those samples were assayed for reporter gene activity. Results are reported at time points taken at a given number of hours post-induction; time points are indicated by either a numeral for the number of hours, and in some cases this number is immediately preceded by the letter “I” indicating post-induction.
EP-PCR Protocol
The following protocol was used for error-prone PCR mutagenesis (see “Mutagenesis of Cloned DNA,” in F. M. Ausubel, Current Protocols in Molecular Biology on CD-ROM (2002) (John Wiley & Sons, New York, N.Y.)). The following reagents were combined: 63 μL water, 10 μL 0.1M Tris (pH 8.3), 5 μL 1M KCl, 0.7 μL 1M MgCl2, 4 μL dNTP mix (either mix #1 [25 mM dCTP, 25 mM dTTP, 5 mM dATP, 5 mM dGTP] or mix #2 [20 mM DCTP, 20 mM dTTP; 2 mM dATP, 2 mM dGTP]), 2 μL 100 μM M13forward primer (GTAAAACGACGGCCAGT) (SEQ ID NO:16), 2 μL 100 μM M13reverse primer (AACAGCTATGACCATG) (SEQ ID NO:17), 1 μL ( ̃5 ng) template (consisting of a Pant or Pben promoter polynucleotide cloned into pNEB193, a plasmid available from New England BioLabs, Beverly, Mass.), 2 μL 25 mM MnCl2, and 1 μL Taq polymerase (5 Units/μL, obtained from Invitrogen Corp.). PCR conditions were as follows: 94° C. for 3 min.; 30 cycles of 30 sec. at 94° C., 30 sec. at 50° C., and 90 sec. at 72° C.; hold at 4° C. PCR products were purified using MICROCON YM-100 or MICROCON-PCR columns (nucleic acid purification columns, Millipore Corp., Bedford, Mass.) according to the manufacturer's instructions for AMICON devices. Products were digested with BamHI and HindIII (New England BioLabs) in 1×NEBUFFER BAMH I+BSA (BamH I restriction endonuclease buffer, available from New England BioLabs) and purified by gel extraction using either QIAEX II (gel extraction kit, from Qiagen, Valencia, Calif.), for fragments of 300 bp and smaller, or PREP-A-GENE (DNA purification kit, from Bio-Rad Laboratories), for fragments larger than 300 bp. The fragments were then cloned upstream of the lacZ or phoA reporter gene of pDOW1017 or pDOW1033, respectively.
Knock-Out Protocols
Construction of AntA Knock-Out. An internal fragment of the antA gene was amplified using primers AntAKO5 (GGAATTCTTCGTGACGATGCG) (SEQ ID NO:16) and AntAKO3 (CGGGATCCGCTCGCGATGCTGC) (SEQ ID NO:17) from P. fluorescens genomic DNA (EcoRI and BamHI sites, respectively, shown in italics). The reaction mixture was formed by combining: 5 μL 10.times.buffer (i.e. the buffer supplied by Invitrogen with the Taq polymerase, which buffer contained 200 mM Tris-HCl (pH 8.4) and 500 mM KCl), 2.5 μL 50 mM MgCl2, 1 μL 10 mM dNTPs, 0.5 μL 100 μM AntKO3, 0.5 μL 100 μM AntKO5, 1 μL (5 Units/μL) Taq polymerase (Invitrogen Corp., Carlsbad, Calif.), 0.5 μL P. fluorescens MB214 genomic DNA ( ̃50 ng), and 39 μL ddH2O. The PCR cycle conditions used were: 2 min. at 96° C.; 30 cycles of 30 sec. at 96° C., 30 sec. at 52° C., and 30 sec. at 72° C. The resulting PCR product was cloned into a plasmid unable to replicate in P. fluorescens (a pUC type plasmid was used, though, e.g., pBR type plasmids will also work). The resulting plasmid was transformed into electrocompetent P. fluorescens cells, and the transformants were selected with the appropriate antibiotic. Since the plasmid cannot replicate in P. fluorescens, only those bacteria which have the plasmid integrated at the antA locus, resulting in two truncated antA ORFs separated by the plasmid backbone, can be selected. Several transformants were cultured in M9 medium+1.0% glucose, 5 mM anthranilate, at 30° C. with shaking for 24 hours, and culture supernatants were analyzed by HPLC for anthranilate concentration.
Construction of BenAB Knock-Out. Generally, following the above-described method for deletion of the AntA gene in P. fluorescens, the plasmid pDOW1139 was constructed to facilitate deletion of the benAB genes as follows. The 3′ portion of the benR gene and the 5′ portion of the benC gene were amplified using P. fluorescens MB214 genomic DNA as template. The benR region was amplified using primers H3—5′benAKOclean and BenKOmega. The benC region was amplified using primers H3—3′BenBKOclean and InvbenKOmega. For both reactions, the cycling conditions were 95° C. for 5 minutes; (94° C., 30 seconds; 55° C., 30 seconds; 72° C., 1 minute) for 35 times; then 72° C. for 5 minutes. This reaction was performed using Taq polymerase (Invitrogen) according to the manufacturer's protocol. The benR and benC fragments were fused using primers H3—5′benAKOclean and H3—3′benBKOclean, with both fragments as template. This fusion reaction employed KOD HOTSTART DNA polymerase (Novagen) under conditions of 94° C. for 2 minutes; (94° C., 30 seconds; 50° C., 30 seconds; 68° C., 1.5 minutes) for 35 times; then 68° C. for 5 minutes. The expected 1.1 kb fragment was gel purified using QIAEX II (Qiagen) and cloned into SrfI-digested plasmid DNA to form plasmid, pDOW1139. pDOW1139 was then transformed into P. fluorescens). Transformants were selected by plating on LB medium with tetracycline for selection. Since the plasmid could not replicate in P. fluorescens, colonies resistant to tetracycline arose from the plasmid being integrated into the chromosome. The site of integration of the plasmid was analyzed by PCR. To obtain strains that lost the integrated plasmid by recombination between the homologous regions, single colonies of the first transformants were inoculated into liquid LB medium, grown overnight, and then plated onto selective medium to counterselect for loss of the plasmid (data not shown). Isolates having the expected phenotype were selected. DNA from the resulting strains was analyzed by PCR to confirm removal of the benAB region using primers 5′BenA_seq, Seq—3′BenB, M13R21, 1261-8378F and 1261-103R.
Inactivation of the P. fluorescens Chromosomal BenR Gene. The open reading frame (ORF) upstream of the benA gene e FIG. ). A DNA fragment containing a portion of the ORF was amplified by PCR using the BenactKOfor and BenactKOrev primers, and P. fluorescens MB214 genomic DNA as template. Recombinant Taq polymerase (from Invitrogen Corp.) was used according to the manufacturer's protocol. The cycling profile [94° C. for 2 min.; (94° C. for 30 sec, 62° C. for 30 sec, 72° C. for 30 sec) for 30 cycles; then 72° C. for 7 min] was used. The resulting products were cloned into the pCR2.1 vector (form Invitrogen Corp.) and transformed into E. coli Top10 cells. Transformants were screened for insert by colony PCR using the above primers/conditions, and the positive clones were further confirmed by DNA sequencing. The resulting plasmids were then used to insertionally inactivate the corresponding chromosomal ORFs. DNA samples were prepared using a NucLEoBoND plasmid midiprep kit (from Clontech Corp.) and 4 μg of plasmid DNA was transformed into P. fluorescens strain MB101. The resulting transformants were screened again by colony PCR. To do this, putative knockout clones were picked into 20 μl H2O and incubated at 100° C. for 10 min. PCR was performed on the DNA of the resulting lysed cells, using PCR reaction conditions of: 20 μl pre-incubated clone, 5 μl 10×buffer, 3 μl 25 mM MgCl2, 1 μl 10 mM dNTP, 5 μl 5 μM BenactKO-for, 5 μl 5 μM M13F (−40) and M13R (−21), 0.5 μl Taq polymerase (5U/μl; from Promega Corp.), and 5.5 μl H2O. PCR reaction cycle conditions used were: 94° C. for 1 min; (94° C., 1 min; 50° C., 30 sec; 72° C. 2 min.) for 30 times; then 72° C. for 10 min, followed by 4° C. hold. MB101 genomic DNA and pDOW1125 were used as controls. Inactivation of this BenR gene resulted in inability of the knock-out host cells to activate transgenic Pben-reporter gene constructs, as well as inability to metabolize benzoate.
Site-Directed Mutagenesis Protocol
Oligonucleotides used for site directed mutagenesis are found listed among SEQ ID NOs:16-41. Construction of the Pben −10 promoter mutants was conducted as follows. The plasmid pDOW1022 was used as template for polymerase chain reaction (PCR) with 1 uM primer benL278 and 1 μM of bambenconshort, bambenwtshort, or bambenAcshort. Recombinant Taq polymerase (from Invitrogen Corp.) was used according to the manufacturer's instructions. The reaction cycling protocol was 94° C. for 2 min.; (30 sec at 94° C., 30 sec at 62° C., and 30 sec at 72° C.) for 25 times; then 72° C. for 7 min. The resulting products were cloned into the pCR2.1 vector (Invitrogen Corp.) and transformed into E. coli TOP10. The insert containing the mutated promoter was digested with BamHI and PacI and subsequently ligated to pDOW1033 digested with the same restriction enzymes yielding plasmids pDOW1081 and 1083-1084, which have a promoter::phoA transcriptional fusion. These plasmids were used as templates to re-amplify the mutant promoters using the primer 1803H3seq and either bambenconshort, bambenwtshort or bambenAcshort and using a recombinant Taq polymerase (from Promega Corp.), according to the manufacturer's instructions. Reaction cycling conditions were 94° C. for 1 min., (1 min at 94° C., 30 sec at 50° C., and 1 min. at 72° C.) for 30 times; then 72 ° C. for 7min. The resulting products were digested with HindIII and BamHI, and subsequently ligated to pDOW1017 that had been digested with the same restriction enzymes. This resulted in formation of promoter::lacZ fusions pDOW1102, 1106 and 1100.
Construction of Pant-10 promoter mutants was conducted as follows. The plasmid pDOW1039 was used as template for PCR with 1 uM primer 3′ Antactiv and 1 uM of primer bamantwtshort or bamantconshort. Recombinant Taq polymerase (from Invitrogen Corp.) was used according to the manufacturer's instructions. The reaction cycling protocol was 94° C. for 2 min.; (30 sec at 94° C., 30 sec at 60° C., and 30 sec at 72° C.) for 25 times; then 72° C. for 7 min. The resulting products were digested with HindHI and BamHI, and cloned into the same sites of pDOW1033: plasmids pDOW1095 and 1098 contain antR-Pant::phoA fusions, with variations of the −10 region of the promoter.
DNA Sequencing Protocol
Cloned inserts were sequenced using ABI PRISM BIGDYE V2.0 or V3.0 DNA sequencing kit from (Applied Biosystems, Inc., Foster City, Calif.) as follows: 4 μL of premix (containing buffer, Taq polymerase, and dye terminators, as supplied in the Applied Biosystems kit), 50 fmol of plasmid template, 3.2-5 pmol of desired sequencing primer, and 2 μL of 5×buffer (as supplied in the Applied Biosystems kit) were combined (to a fiaal volume of 20 μL). The PCR cycling profile used was: 45 cycles of 30 sec. at 95° C., 20 sec. at 50° C., and 4 min. at 60° C. Samples were purified using SEPHADEX G-50 (a bead-form, dextran gel for chromatographic purification of nucleic acids, from Sigma Chemical Company, St. Louis, Mo.), dried, resuspended in formamide, and then run on an AB13100 automated DNA sequencer (a 16 capillary array, automated DNA sequencer, from Applied Biosystems, Inc.).
Primer Extension Protocol
RNA Isolation. An RNA isolation procedure was followed in order to identify the transcription start sites under the control of the P. fluorescens Pant and Pben promoters. The procedure used is as follows. An overnight culture of P. fluorescens MB101 carrying the appropriate plasmid was grown in 1×M9 medium supplemented with 1% glucose (w/v), 1 mM MgSO4, and trace elements (as described above) was subcultured 1:4 (v/v) in the same medium to a final volume of 50 mL. The culture was induced with 5 mM benzoate or anthranilate as appropriate for 8 or 24 hours. Cells were pelleted and total RNA isolated using an RNEAsY kit (a “maxi” bacterial RNA isolation kit from Qiagen, Valencia, Cal.). The RNA was resuspended to a final volume of 200 μL and treated with 10 Units of DNAse I (ribonuclease-free, from Ambion, Inc., Austin, Tex.) according to manufacturer's protocol. Following DNAseI treatment, the RNA was purified using an RNEASY column (a “midi” or “mini” RNA purification column, from Qiagen) as appropriate (the RNEASY “midi” column was used for RNA amounts up to 1 mg; the RNEASY “mini” column was used for RNA amounts up to 100 μg). Once purified, the RNA concentration was determined using RIBOGREEN (RNA quantitation kit, from Molecular Probes, Inc., Eugene, Oreg.), following the manufacturer's protocol.
Primer Labeling: This was performed by mixing 1 μL 10 μM primer (either lacZPE, GGATGTGCTGCAAGGC (SEQ ID NO:18), or lacZPE2, GTAACCATGGTCATCGC (SEQ ID NO:19)), 1 μL 10×T4 polynucleotide kinase buffer (700 mM Tris-HCl (pH 7.6), 100 mM MgCl2, 50 mM dithiothreitol (DTT)), 5 μL 32P-γATP (50 μCi, Amersham-Pharmacia), 1 μL T4 kinase (New England BioLabs), and 2 μL ddH2O; and incubating the resulting reaction mixture at 37° C. for 30-60 min. Following incubation, 5 μL of the reaction mixture was reserved to use for a “sequencing ladder” analysis. 20 μL TE (10 mM Tris, 1 mM EDTA (pH8.0)) was added to the other 5 μL and mixed and the result was spun through a MICROSPIN G-25 column (Amersham-Pharmacia, Piscataway, N.J.) to remove unincorporated nucleotides, thereby yielding a final concentration of 0.2 μM labeled primer.
Sequencing ladder: This was performed according to the protocol that came with the FMOL kit (DNA sequencing kit from Promega Corp.), using 1 picomole (pmol) of the labeled primer described above. Plasmid template used corresponds with that contained in the strain from which RNA was isolated for the extension reaction.
Primer Extension reaction: Primer extension reactions were performed by mixing 10-20 μg of total RNA with 0.2 pmol primer to yield a final volume of 12 μL, followed by incubation at 70° C. 10 min. Then, the following were added: 4 μL 5×SUPERSCRIPT II buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, available from Life Technologies, now Invitrogen Corp., Carlsbad, Calif.), 2 μL 1M DTT, 1 μL 10 mM dNTPs, and 1 μL SUPERSCRIPT II (reverse transcriptase, from Life Technologies, now Invitrogen), followed by incubation at 42° C. for 1 hour. Then the resulting mixture was treated by either an addition of 5 μL sequencing stop solution (containing formamide and tracking dye, as supplied in the Promega FMOL kit) or, in those cases where the signal was weak, by: precipitation with 2 μL 3M sodium acetate/40 μL 100% ethanol, followed by centrifugation for 10 minutes to pellet suspended matter, drying of the pellet, and resuspension in 4 μL H2O+2 μL sequencing stop solution. The product mixture resulting from the primer extension reaction was then electrophoresed on a LONG RANGER gel (made from 6% pre-mixed gel solution, from Biowhittaker Molecular Applications, Rockland, Me.) containing 8M Urea and 1.2×TBE (ie. Tris-Borate-EDTA, as diluted from 10×TBE obtained from Fisher Scientific, Pittsburgh, Pa.) next to the sequencing ladder, with 0.6×TBE as an electrophoretic “running” buffer. The gel was dried and exposed to a phosphor screen (from Molecular Dynamics, now Amersham Biosciences, Inc., Piscataway, N.J.) to detect radiolabeled DNA fragment, and imaged on the TYPHOON PHOSPHORIMAGER (Molecular Dynamics, now Amersham Biosciences, Inc., Piscataway, N.J.).
Primer extension using Thermoscript reverse transcriptase: 30 ng total RNA, 1 μL 0.2 μM primer, and ddH2O to a final volume of 12 μL were mixed and then incubated at 70° C. for 10 min. To this mixture were added 4 μL 5×cDNA synthesis buffer (250 mM Tris acetate (pH 8.4), 375 mM potassium acetate, 40 mM magnesium acetate), 1 μL 0.1M DTT, 2 μL 10 mM dNTPs, 1 μL THERMOSCRIPT RT (reverse transcriptase from Invitrogen Corp.), and the resulting mixture was incubated at 55° C. for 1 hour. The reaction product was precipitated, dried, and resuspended in 4 μL ddH2O+2 μL stop solution (described above). All reactions were heated at 70° C. for 2 minutes immediately before being loaded onto the gel as described above. The gel was run as described above.
Microtiter β-Galactosidase Assay
We prepared enough of the following assay medium to provide for each sample well of a 96-well plate (i.e. for all those wells used, with at least one well being used for each time point measured during the reaction course for each sample): 152 μL Z buffer (0.06M Na2HPO4.7H2O, 0.04M NaH2PO4.H2O, 0.01M KCl, 0.001M MgSO4.7H2O)+8 μL 1M β-mercaptoethanol. For each 900 μL of the resulting mix, we added one drop of 0.1%SDS and two drops of CHCl3, mixed (using a vortex-type mixer), and then added 144 μL thereof to each well. 16 μL of cells were then added to each well and the plate sealed with a plastic plate sealer. The plate was then mixed (by vortex) for 10 seconds, and then equilibrated to incubation temperature (room temperature) for 5 minutes. 50 μL 4 mg/mL ONPG was then added. When a significant yellow color developed, 90 μL stop solution (1M Na2CO3) was added and the reaction time recorded. The resulting color intensity for each sample was then read at A420 and A550. In addition, the cell density of each culture providing the 16 μL of cells used in each sample was read at A600. Miller Units were calculated as follows:

    
    
        1000 * ((A420−(1.75*A550))/(time(in minutes) *0.1 * A600)).

Alkaline Phosphatase Assas

    
    


For this assay we prepared SIGMA FAST (p-nitrophenyl phosphate (PNPP) substrate, from Sigma-Aldrich Corp., St. Louis, Mo.) by adding one of each tablet provided by the manufacturer (one table each PNPP and Tris; stored at −20° C.) to 20 mL ddH2O, giving a final concentration of 1 mg/mL PNPP and 0.2M Tris. At each time point, for each sample, 50 μL SIGMA FAST substrate was combined with 5 μL of cells. The result was then incubated at room temperature for 30 minutes. The resulting color intensity for each sample was then read at A410. In addition, the cell density of each culture providing the 5 μL of cells used in each tested sample was read at A600 (i.e. the cell cultures were read in a 96 well plate). The value of A410/(0.1 * A600) was then calculated to express alkaline phosphatase activity/cell.
Example 1
Cloning and Analysis of a Benzoate-Inducible Promoter
Benzoate is an inexpensive, essentially nontoxic compound, making it an ideal candidate for an inducer. A 509 bp region of P. fluorescens DNA was cloned. This region was found located upstream of a putative benA translational start site (FIG. 5), which was part of the coding sequence of a subunit of benzoate dioxygenase. The cloned region was found to contain a benzoate-inducible promoter (Pben), and was named “Pben509”.
Benzoate-inducible promoter activity was tested by fusing the DNA fragment containing the putative promoter sequence of Pben509, or of Pben278 (described below), upstream of an easily assayable reporter gene (i.e. either lacZ, which encodes β-galactosidase and was used as the chief reporter gene, orphoA). The resulting plasmid was transformed into P. fluorescens MB101. Following addition of sodium benzoate, induction of β-galactosidase activity was measured using the chromogenic substrate o-nitrophenol-β-D-galactopyranoside (ONPG) (see FIG. 6). Similar experiments were carried out using the phoA reporter gene and the chromogenic substrate p-nitrophenyl phosphate (PNPP). P. fluorescens strains carrying these constructs show β-galactosidase or alkaline phosphatase activity, respectively, upon addition of 1-10 mM sodium benzoate. Varying the concentration of the inducer and/or the time of induction resulted in varying levels of reporter gene expression (data not shown).
A truncated version of the promoter-plus-reporter gene construct, containing a 275 bp portion upstream of the predicted translational start site (FIG. 5), which portion was named “Pben278”, was found to retain activity similar to that of Pben509 (see FIG. 6).
Both Pben 509 and Pben278 promoter activity was found to be inhibited during fermentation, due to the presence of a small, but significant concentration of glucose. Thus, these promoters are catabolite-repressed.
Northern analysis indicated that expression from Pben occurred only upon addition of the inducer compound (e.g., sodium benzoate), demonstrating that inducible expression of Pben is not leaky like that of the lac family of promoters (data not shown). Primer extension analysis of total RNA isolated from induced cultures of MB101 carrying either Pben509 or Pben278 fused to lacZ indicated that the transcriptional start site was 196 nucleotides (nt) upstream of the predicted benA translational start site. This indicates that the promoter sequence and the positive regulatory cis acting elements are contained within 82 bp upstream of the transcriptional start in the Pben278 clone.
The literature teaches that cis, cis-muconate, a benzoate metabolite, acts to induce the benABCD operon of other bacteria such as Acinetobacter sp. and P. putida. However, both cis, cis-muconate and the presumed preceding compound in the known metabolic pathway for benzoate degradation, i.e. catechol, fail to induce activity of either Pben509 or Pben278 (data not shown). As a result, either benzoate or an initial benzoate derivative, e.g., 2-hydro-1,2-dihydroxybenzoate, may be directly responsible for inducing the benzoate promoter.
Example 2
Cloning and Analysis of an Anthranilate-Inducible Promoter
Anthranilate, like benzoate, is an inexpensive low toxic compound that can be utilized by P. fluorescens, making it an ideal compound to investigate as an inducer. Four promoter constructs have been cloned upstream of either a lacZ or phoA reporter and have been found to possess similar activity upon induction with anthranilate: Pant713, Pant705, Pant311, and Pant+antR coding sequence (CDS) (FIG. 7). Pant713 and Pant705 have the same 5′ end, but Pant713 contains the predicted ribosome binding site of the antA gene, whereas Pant705 does not (see the underlined CCTCC, nucleotides 1299 through 1303 of SEQ ID NO:7, in the final octamer shown for Pant713). In an effort to determine the minimal region of DNA necessary for anthranilate -induced activation, the Pben713 construct was truncated on the 5′ terminus to 311 base pairs (bp) The Pant311 construct was found to retain activity similar to that of Pant713 (data not shown). Expansion of the promoter clone to include the transcriptional activator gene 5′ of the antA open reading frame (ORF) increased expression levels of the lacZ fusion. The transcriptional start site was mapped to 31 nucleotides upstream of the predicted antA translational start site, for both Pant713 and the expanded clone that includes the transcriptional activator AntR (data not shown; also see FIG. 7). The presence of antR in multi-copy with the lacZ fusion was found to enable faster and stronger induction (see FIG. 8).
In addition, further increasing expression of the AntR has been found to result in more improved anthranilate-inducible expression by Pant promoters. For example, as shown in FIG. 8, both pDOW1029- and pDOW1035-constructs were induced in P. fluorescens host cells. FIG. 8 demonstrates a substantial difference in the rates of induction and the maximum levels of induction achieved for each of these promoters during the 24 hour time course of the study. pDOW1029 contains the Pant713 promoter, which lacks the antR coding sequence; pDOW1035 contains the full activator CDS. The P fluorescens host cell used contains an actively expressed, chromosomal copy of the antR CDS. Thus, the results shown in FIG. 8 for pDOW1029 are for a system in which the antR gene is present in a single copy, while the results for pDOW1035 represent a two-copy system. These results demonstrate that the presence of an extra copy of the antR gene dramatically improves both the rate and level of response of the Pant promoter. Such improved expression can alternatively be obtained, or further enhanced, by driving antR expression with a very strong promoter. Improved induction/expression can also be obtained, as described below, by using a host cell in which a key gene (e.g., antA) responsible for degradation of the inducer compound (e.g., anthranilate) has been inactivated. Moreover, mutating the activator and/or promoter sequences (and selecting for mutants with increased activity) could also enhance the activator/promoter interaction and thereby allow for more improved anthranilate-inducible expression by Pant.
The anthranilate promoter was also found to be inducible by anthranilate analogs, including the halo-substituted anthranilic acid derivatives: 3-chloro-, 4-chloro-, 5-chloro- and 6-chloro-anthranilate. 6-chloroanthranilate is found to act as a gratuitous inducer of anthranilate metabolism, i.e. it is not metabolized by P. fluorescens yet induces expression from the anthranilate promoter. For example, 6-chloroanthranilate was found to induce the Pant713 and antR/Pant constructs (FIG. 8). Taken together, these results indicate that anthranilate itself induces the metabolic pathway; and that it is possible to utilize substituted anthranilate compounds as gratuitous inducers as an alternative to inactivating the anthranilate metabolic pathway of the host organism.
Example 3
Construction and Testing of a Fused, Pant-Pben Tandem Promoter
The relative strength of the Pant promoter with multi-copy antR was found to be approximately 1⁄5 that of the Pben278 promoter. However, unlike the catabolite-repressed Pben promoter, the activity of the Pant promoter was not inhibited during fermentation A fusion of these two promoters was created by linking them together, as shown in SEQ ID NO:3, i.e. by cloning a fragment antR and Pant, upstream of the Pben278 promoter fused to lacZ.
The strength of the tandem “‘antR/Pant’-‘Pben278’” construct, induced with anthranilate, was surprisingly found to be improved over that of “antR/Panf” alone upon induction with anthralate. The strength of the tandem promoter upon induction with benzoate was found to be similar to that of Pben278 alone (FIG. 9). The induction of greater β-galactosidase activity from the tandem promoter, upon addition of anthranilate in the presence of glucose, indicates that transcription from the tandem promoter, in which a natively catabolite-repressed Pben is located proximal to the coding sequence, surprisingly is not blocked by the catabolite repression of Pben. This is even more surprising in light of the fact that both (1) the bacterial source of the Pben and Pant elements in the tandem promoter and (2) the host cell in which induction was tested are the same: Pseudomonas fluorescens biotype A. Thus, even though the Pben element is native to the host cell, the upstream presence of the natively non-catabolite-repressed promoter (Pant) is able to overcome the catabolite repression of the natively catabolite-repressed promoter (Pben). Moreover, the presence of antR and Pant upstream of the Pben promoter appears to relieve the catabolite repression of Pben since the tandem promoter is active during fermentation, in the presence of glucose (see FIGS. 12 and 13).
Example 4
Improved Mutants of Pben509
In an effort to improve the Pben promoter, the Pben509 promoter was subjected to mutagenesis by error prone PCR. Mutants were screened for improved activity following induction with 10 mM benzoate at the shake flask scale. The mutants identified showed approximately 2-fold improvement over the wild type promoter (FIG. 10). Positive hits were re-transformed into P. fluorescens and re-tested to ensure that the improved activity was in fact due to the new construct.
Sequence analysis (FIG. 11) revealed one change in mutant 2d3 and two changes in mutant 21b5. As illustrated in the attached figure, the mutation in isolate 2d3 and one of the mutations in 21b5 fall within the coding region of the upstream ORF. This region is not contained within in the Pben278 construct. The fact that these mutations were isolated in improved promoter mutants indicates that upstream regions may affect transcription, although they are not necessary for activated transcription. The second mutation identified in 21b5 is located five base pairs upstream of the transcriptional start site.
Example 5
Rationally Mutated Pben and Pant Promoters
Construction and Analysis ofPben—10 Mutants. The native Pben predicted −10 region was mutated in an attempt to improve promoter activity. The promoter itself was truncated to 88 bp, and three derivatives of the −10 were constructed: wild type (TACGGTT, SEQ ID NO:44, consensus (TATAAT, SEQ ID NO:45), and Acinetobacter (Ac) Pben−10 (TAAGGT, SEQ ID NO:46), as described in Materials and Methods. The primers were constructed such that one bp (G:C) upstream of the previously identified transcriptional start site was removed and 9 bp downstream of the previously identified transcriptional start site are included. These promoters were fused to the phoA reporter gene and tested for activity in P. fluorescens MB 101. FIG. 14 shows that the truncation of Pben promoter to 88 bp is sufficient to confer benzoate-activated expression, although altering the −10 region either to the consensus TATAAT or to the −10 sequence of the Acinetobacter Pben promoter did not appear to significantly improve benzoate induced promoter activity.
Construction and Analysis of Pant—10 Mutants. As described above for Pben, the predicted −10 region of the Pant promoter was mutated in an attempt to improve promoter activity. In the construction of two Pant −10 mutants, the promoter was truncated to 289 bp. DNA and fragments containing the anthranilate transcriptional activator and the mutant promoter were fused to the phoA reporter gene. The resulting plasmids were transformed into a derivative of MB 101 in which the antA gene has been insertionally inactivated. FIG. 15 shows that, following induction with anthranilate, the 3′ truncation of the promoter to 289 bp did not affect activity (pDOW1095). Altering the putative −10 region to consensus −10 (pDOW1098) resulted in the promoter becoming capable of expression even in the absence of inducing compound. Addition of antlranilate did result in higher expression, indicating that the promoter was still inducible.
Example 6
Mutant Ptandem Promoters
The tandem promoter having the sequence as shown in pDOW1057 (SEQ ID NO:13) was mutated in the Pben−10 region as follows to construct mutant Ptandem promoters. A 1.6 kb DNA fragment containing antR and Pant, obtained by digestion of pDOW1039 with HindIII and SmaI, was gel purified (using QIAEX II gel column, from Qiagen Corp.) and ligated into each of pDOW1102 (Pben88wt-10), pDOW1106 (Pben88con−10), and pDOW1100 (Pben88Ac-10), each of which had been digested with HindIII and PmeI. Following transformation into host cells, positive clones were identified for each plasmid by colony PCR and then confirmed by DNA sequencing. The resulting plasmids were named pDOW1107, pDOW1108, and pDOW1109, respectively.
The effect of the Pben mutants on Ptandem activity was assessed at the shake flask scale. As shown in FIG. 16, the mutations did not have a significant effect on benzoate- or anthranilate-induced Ptandem activity. pDOW1107-1109 all showed β-galactosidase activity within 2 fold of that shown by the original construct, pDOW1057. One interesting finding was that the Pben88-10 consensus mutant alone or as part of a tandem promoter appeared to be expressed prior to induction with either benzoate or anthranilate. Addition of benzoate as an inducer resulted in an increased expression from Pben88−10 consensus alone (pDOW1 106), or as part of a tandem (pDOW1108) (FIG. 16A). However, addition of anthranilate did not result in an increase in lacZ expression from the Pben88-10 consensus tandem construct pDOW1108 (FIG. 16B).
Analysis of pDOW1108 at the 20 L scale revealed that MB101 carrying pDOW1108 induced with 2 mM or 5 mM benzoate pulses over a 24-hour period was not only active, but also was able to metabolize benzoate (data not shown). A relatively high level of β-galactosidase activity was detected at IQ, most likely a result of “leaky” expression, as had been detected at the shake flask scale (see FIG. 16A). An initial decrease in activity was consistently detected upon induction with benzoate, but activity then rose to a level greater than that detected at I0. Induction of pDOW1108 at the 20 L scale with 2 mM anthranilate every 4 hours for a 24-hour period showed that although anthranilate was metabolized efficiently, cloned tandem promoter expression as measured by β-galactosidase activity was leaky, as observed at shake flask scale, but actually declined after addition of anthranilate (see FIG. 18). A comparative induction of P. fluorescens carrying the original tandem promoter construct pDOW1057 with benzoate at the 20 L scale shows that benzoate is metabolized, as with pDOW1108. However, induction of β-galactosidase activity seems to be delayed compared to similar 2 mM dose inductions of the pDOW1108 construct, where increased activity was detected between 4 and 24 hours as opposed to between 30 and 48 hours post induction (data not shown).
Example 7
Use of Benzoate- and Anthranilate-Induced Promoters for Controlled Gene Expression during Fermentation
Testing of the Pben509 lacZ fusion at the 20 L scale revealed transcriptional regulation issues not detected at the shake flask scale. Induction of the fusion with 5 or 10 mM benzoate was not consistently observed (data not shown). A correlation between benzoate consumption and activation of Pben509 was also observed. The presence of glucose is thought to be responsible for the inhibition of reporter gene expression. Subsequent to these experirnents, it has been observed in shake flask experiments that metabolism of benzoate follows the depletion of glucose. The benzoate-inducible system may be useful in fermentation processes that utilize carbon sources other than glucose. Shake flask experirnents reveal that the highest levels of induction are observed when citrate is used as a carbon source. This observation should hold true for fermentation scale.
Testing of the antR Pant construct and of the tandem promoter construct at the 20 L scale showed activity similar to that observed at the shake flask scale. Because the inducer is consumed by the culture, anthranilate was fed during the course of induction. Activity was observed to increase over time. It is likely that higher activity will be observed in strains that are unable to metabolize the inducer. As observed in shake flask and 20 L fermentation experiments, the tandem promoter construct is more active than the antR Pant construct (FIG. 12). Inactivation of anthranilate metabolism by insertional inactivation of the antA gene allowed for greater expression of the tandem promoter:lacZ fusion at the 20 L scale. As shown in FIG. 13, anthranilate is not metabolized during the course of induction, and the level of β-galactosidase activity observe is much higher that that observed in a strain that does metabolize anthranilate.
Example 8
Characterization of the BenAB Knock-Out Strain
To verify whether benzoate is in fact the inducer of the Pben promoter, and not a downstream metabolite thereof, the benAB gene knock-out strain of P. fluorescens was further characterized. The benA and benB genes code for the large and small subunits of benzoate 1, 2 dioxygenase, respectively. Two isolated of the benAB knock-out strain were further tested for the ability to metabolize benzoate as follows. Cells were grown in LB-proline-uracil to high density; benzoate was then added to the cultures to a final concentration of  ̃5 mM before they were returned to incubate for 24 hr. The concentration of benzoate remaining in the cell-free broth, as measured by HPLC, showed that the benAB deletion mutants were unable to metabolize benzoate, while the parent, non-knock-out strain did metabolize benzoate efficiently. To assess whether the Pben promoter is still active in the benAB knockout strain, a plasmid containing a Pben278::lacZ construct was transformed into one of the strains, and transformants were grown in LB medium. Transformants were induced with 0 or 5 mM benzoate and lacZ activity demonstrated that benzoate was indeed the inducer for Pben, rather than a downstream metabolite. See FIG. 19.
Example 9
Effect of Multi-Copy Expression of BenR
A DNA fragment containing the BenR ORF upstream of benA along with Pben promoter was amplified from P. fluorescens MB214 genomic DNA using primers Benact5′ and Bambenconshort under the following conditions: 94° C. for 1 min; (94° C., 1 min; 50° C., 30 sec; 72° C., 90 sec) for 30 cycles; then 72° C. for 10 min, and 4° C. hold. The PCR product was ligated into the pCR2.1 vector, and the sequence verified. The insert fragment was digested with PmeI and BamHI, and ligated to pDOW1033. The resulting plasmid was stocked as pDOW1090. The same promoter construct was fused to the lacZ reporter by digesting pDOW1090 with BamHI and XhoI to remove the phoA reporter, and replacing it with the 3 Kb BamI-HXhoI fragment of pDOW1035, containing the lacZ reporter gene.
The benR ORF was cloned together with the Pben promoter upstream of the phoA reporter gene to determine whether expression of the transcriptional activator gene in multicopy would improve benzoate activated gene expression. At the shake flask scale, there was observed no significant difference in promoter activity with benR in multicopy. Since it has been shown in the literature that overexpressing the transcriptional activator can overcome catabolite repression, we tested 20 L fermentations of P. fluorscens MB 101 carrying pDOW1090. Previous studies showed that MB101 carrying a Pben::lacZ fusion was unable to metabolize benzoate during fermentation with a corn syrup feed. We found that MB101 carrying pDOW1090 is able to metabolize benzoate at the 20 L scale. Benzoate was found to be consistently metabolized in triplicate 20 L fermentations, indicating that the chromosomal Pben promoter was active. Thus, the presence of multi-copy expression of BenR overcame catabolite repression. See FIG. 17.
As a result, we have found that overexpression of benR allows P. fluorescens to overcome catabolite repression observed for benzoate metabolism at the 20 L scale when constructs containing Pben alone were tested. Demonstration of benzoate-induced promoter activity at the 20 L scale is an important improvement, since benzoate-induced activation of tandem promoters is greater that that of anthranilate-induced activity at the shake flask scale, even though anthranilate-induced activity under control of Ptandem is already stronger than anthranilate-induced activity of Pant. Both pDOW1057 and pDOW1108 were found to be benzoate-inducible at the 20 L scale. Although the pDOW1108 construct is “leaky”, in that significant expression occurs prior to addition of the inducer, this should not present a large problem for its use in protein expression. In addition, because it has been found that Pben is active in the benAB knock-out strain, use of such a knock-out strain will improve benzoate-induced promoter activity for Pben, as well as Ptandem. Likewise, because it has now been shown that induction of the tandem promoter construct pDOW1057 with anthranilate is improved in a strain carrying and insertionally inactivated chromosomal ant4 gene, improved anthranilate-induced promoter activity will be enhanced for Pant, as well as Ptandem.
Consequently, anthranilate- and benzoate-inducible promoters have now been developed for use in bacterial expression systems. These promoters have been found to permit tight regulation of transcription and are inducible with low-cost compounds such as benzoate and anthranilate; the presence of antR in multi-copy also now has been found to significantly improve the activity of the Pant promoter. In addition a new type of tandem promoters has now been developed for use in bacterial expression systems, exemplified by Pant-Pben tandem promoters that have been found to exhibit increased levels of anthranilate-induced gene expression, over Pant itself; were found to be benzoate-inducible, i.e. to the same level as Pben itself; and were found to surprisingly overcome the catabolite repression to which Pben alone was subject. Further, the present work has demonstrated that both the Pant promoter (with antR) and the tandem promoter constructs exhibit anthnnilate-inducible gene expression under fermentation-scale conditions (e.g., at the 20 L scale); the tandem promoter constructs also exhibits benzoate inducible gene expresion under fermentations-scale condiitons.
It is to be understood that the preferred embodiments described above are merely exemplary of the present invention and that the terminology used therein is employed solely for the purpose of illustrating these preferred embodiments; thus, the preferred embodiments selected for the above description are not intended to limit the scope of the present invention. The invention being thus described, other embodiments, alternatives, variations, and obvious alterations will be apparent to those skilled in the art, using no more than routine experimentation, as equivalents to those preferred embodiments, methodologies, protocols, vectors, reagents, elements, and combinations particularly described herein. Such equivalents are to be considered within the scope of the present invention and are not to be regarded as a departure from the spirit and scope of the present invention. AlR such equivalents are intended to be included within the scope of the following claims, the true scope of the invention thus being defined by the following claims.
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## Claims

1. A nucleic acid construct comprising two non-identical promoters in a tandem arrangement, wherein each promoter is responsive to an exogenously supplied compound, wherein the upstream promoter is set forth in nucleotides 1329-1369 of SEQ ID NO:13, wherein the downstream promoter in the tandem arrangement is the catabolite-repressed promoter set forth in nucleotides 1477-1509 of SEQ ID NO:13, and wherein induction of the upstream promoter in the tandem arrangement overcomes the catabolite repression of the downstream promoter.

2. The construct of claim 1 further comprising at least one activator protein binding site.

3. The construct of claim 2 wherein said at least one activator protein binding site is a benzoate activator protein binding site or an anthranilate activator protein binding site.

4. The construct of claim 1 further comprising a nucleotide sequence encoding a transgene of interest.

5. The construct of claim 4 wherein the transgene of interest is operably linked to the catabolite-repressed promoter.

6. A host cell comprising a nucleic acid construct comprising two non-identical promoters in a tandem arrangement, wherein each promoter is responsive to an exogenously supplied compound, wherein the upstream promoter is set forth in nucleotides 1329-1369 of SEQ ID NO:13, wherein the downstream promoter in the tandem arrangement is the catabolite-repressed promoter set forth in nucleotides 1477-1509 of SEQ ID NO:13, and wherein induction of the upstream promoter in the tandem arrangement overcomes the catabolite repression of the downstream promoter.

7. The host cell of claim 6, wherein the construct further comprises a transgene of interest.

8. The host cell of claim 6, wherein the construct further comprises at least one benzoate activator protein binding site and at least one anthranilate activator protein binding site.

9. A process for protein expression comprising growing in the presence of an inducing condition a cell comprising a nucleic acid construct comprising two non-identical promoters in a tandem arrangement, wherein the upstream promoter is set forth in nucleotides 1329-1369 of SEQ ID NO:13, wherein the downstream promoter in the tandem arrangement is the catabolite-repressed promoter set forth in nucleotides 1477-1509 of SEQ ID NO:13, wherein induction of the upstream promoter in the tandem arrangement overcomes the catabolite repression of the downstream promoter, and wherein said construct further comprises a transgene of interest.

10. The process of claim 9 wherein the presence of an inducing condition results in the production of a recombinant protein or peptide encoded by said transgene of interest.

11. The process of claim 9 wherein the inducing condition comprises the presence of anthranilate.

12. The process of claim 11 wherein the inducing condition further comprises the presence of benzoate.

13. The process of claim 9 wherein induction of the upstream promoter overcomes the catabolite repression of the downstream catabolite repressed promoter.

14. The process of claim 10 further comprising isolating the recombinant protein or peptide.