# Patent

## Title
Promoter and plasmid system for genetic engineering

## Abstract

This invention provides a series of low-copy number plasmids comprising restriction endonuclease recognition sites useful for cloning at least three different genes or operons, each flanked by a terminator sequence, the plasmids containing variants of glucose isomerase promoters for varying levels of protein expression. The materials and methods are useful for genetic engineering in microorganisms, especially where multiple genetic insertions are sought.

## Background
This application claims the benefit of U.S. Provisional Application No. 60/374,931, filed Apr. 22, 2002.


FIELD OF THE INVENTION
This invention is in the field of molecular biology. More specifically, this invention pertains to a series of low-copy-number plasmids comprising restriction endonuclease recognition sites useful for cloning at least three different genes or operons, each site flanked by a terminator sequence and a set of promoters for varying levels of protein expression. The invention is useful for genetic engineering in microorganisms, especially where multiple genetic insertions are sought.
BACKGROUND OF THE INVENTION
Molecular biotechnology is a discipline that is based on the ability of researchers to transfer specific units of genetic information from one organism to another. This process, known as cloning, relies on the techniques of recombinant DNA technology to produce a useful product or a commercial process (Glick, B. R.; Pasternak, J. J., Molecular Biotechnology Principles and Applications of Recombinant DNA, 2nd ed. American Society for Microbiology, Washington, D.C. (1998)).
Commercial processes often require that proteins encoded by the cloned gene are produced at high rates of expression. There is no single strategy for achieving maximal expression of every cloned gene. Most cloned genes have distinctive molecular properties that require the investment of considerable time and effort before a specific set of conditions that result in an acceptable level of expression is found.
Merely inserting a gene into a cloning vector does not ensure that it will be successfully expressed at the level needed. In response to the need for a high expression rate, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation, and secretion from the host cell. More specifically, the molecular features that have been manipulated to control gene expression include: (1) the nature of the relevant transcriptional promoter and terminator sequences, (2) the strength of the ribosome binding site, (3) the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell, (4) the final cellular location of the synthesized foreign protein, (5) the efficiency of translation in the host organism, and (6) the intrinsic stability of the cloned gene protein within the host cell.
Additionally, the introduction and expression of foreign DNA in a host organism often changes the metabolism of the organism in ways that may impair normal cellular functioning. This phenomenon is due to a metabolic load or burden imposed upon the host by the foreign DNA. The metabolic load may result from a variety of conditions including 1) increasing plasmid copy number, 2) overproduction of proteins, 3) saturation of export sites, and/or 4) interference of cellular function by the foreign protein itself.
Techniques to address some of the obstacles presented above are known. Several groups have used multiple promoters in tandem to express genes at different phases of cell growth (CN 1186856), from different RNA polymerases or in different phage species (U.S. Pat. No. 5,547,862; J. Biotechnol. 2(5):303-316 (1985); Biotechniques, 18(1):152-154, 156-157(1995)). Another group has used tandem repeated multiple cloning sites (MCS) (Gene, 139 (1):83-86 (1994)) to facilitate moving DNA in and out of the plasmid vector. One group has reported the use of a high-copy-number vector with three multiple cloning sites each behind a different promoter for expression of different genes in mammalian cells (Biotech. Bioeng., 57(1):1-10 (1998)).
Despite these techniques, the problem to be solved remains how to easily and quickly clone multiple genes or operons while minimizing the impact of metabolic load, controlling the yield of the recombinant protein to meet production needs, and enhancing the stability of the transformed host cell.
SUMMARY OF THE INVENTION
Applicants have created novel glucose isomerase promoter sequences that allow varying levels of gene expression in production organisms. Applicants incorporated expression cassettes containing the variant GI promoters into a low-copy-number plasmid derived from pCL1920 to construct a series of plasmids for genetic engineering. Transcription terminators isolate the associated promoter from transcription from other promoters located outside this construct.
Applicants also have constructed a unique nucleotide sequence containing cloning sites for multiple rare restriction enzymes, further facilitating cloning in this construct or transfer of this construct to alternate plasmid or vector backbones. The unique cloning sites allow introduction of genes or operons to be expressed under the control of suitable promoters of varying strengths.
The invention encompasses:
1. an isolated or recombinant nucleic acid molecule encoding a Streptomyces lividins glucose isomerase variant, the nucleic acid molecule selected from the group consisting of SEQ ID NOS:9-28;
2. an isolated or recombinant nucleic acid molecule encoding a Streptomyces lividins glucose isomerase variant, the nucleic acid molecule comprising a nucleotide sequence of any of SEQ ID NOs:9-28;
3. a library of isolated or recombinant nucleic acid molecules encoding a Streptomyces lividins glucose isomerase variant, the library comprising the nucleotide sequences of SEQ ID NOS:9-28;
4. an expression cassette comprising the nucleic acid molecule of the various GI variants set out above, and;
5. a kit comprising the nucleic acid molecules encoding the various Streptomyes lividins glucose isomerase variants set out above.
A further embodiment of the invention is a DNA construct comprising at least three transcriptional terminators and at least one cloning site situated between any two transcriptional terminators. A preferred embodiment of this DNA construct comprises the transcriptional terminators tonB, thrA, or aspA, and the cloning sites are selected from the group consisting of AvrIl, NheI, BfaI, Cac8I, BsaJI, and StyI. Preferred cloning sites are NheI or AvrIl. A library of these constructs is also encompassed in the invention.
The invention includes the following DNA constructs:

    
    
        the pSYCO109mcs plasmid consisting of SEQ ID NO:30,
        the short 1.5 GI promoter consisting of SEQ ID NO:31,
        the short 1.20 GI promoter consisting of SEQ ID NO:32,
        the pAH105 plasmid consisting of SEQ ID NO:70,
        the pSYCO101 plasmid consisting of SEQ ID NO:71,
        the pSYCO103 plasmid consisting of SEQ ID NO:72,
        the pSYCO106 plasmid consisting of SEQ ID NO:73,
        the pSYCO109 plasmid consisting of SEQ ID NO:74,
        the pSCYO106mcs plasmid consisting of SEQ ID NO:78, and
        the pRJ50 plasmid consisting of SEQ ID NO:79.
    
    


A further embodiment of the invention is a vector having a multiple cloning site containing restriction recognition site sequences specific for the restriction endonucleases AscI, NheI, PacI, RsrII, NsiI, SacII, Mlul, AgeI, SapI, and SnaBI. A particular embodiment of this vector is the nucleotide sequence of SEQ ID NO:77.
The genetic materials of this invention include transformed host cells containing the nucleic acid molecules described above and the polypeptides encoded by the polynucleotides.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND BIOLOGICAL DEPOSIT
Applicants have provided 83 sequences in conformity with Rules for the Standard Representation of Nucleotide and Amino Acid Sequences in Patent Applications (Annexes I and II to the Decision of the President of the EPO, published in Supplement No. 2 to OJ EPO, Dec. 1992), with 37 C.F.R. 1.821-1.825and Appendices A and B (Requirements for Application Disclosures Containing Nucleotides and/or Amino Acid Sequences) with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373 (1984) which are herein incorporated by reference.
SEQ ID NO:1 is the nucleotide sequence for the wildtype Streptomyces lividans glucose isomerase (GI) promoter.
SEQ ID NOs:2-8 are oligonucleotide primers used for saturation mutagenesis of the GI promoter. In SEQ ID NOs:3-8, “N” represents either A, T, C, or G.
SEQ ID NOs:9-28 are nucleotide sequences for the GI promoter variants.
SEQ ID NO:29 is the nucleotide sequence for the yqhD gene from E. coli. 
SEQ ID NO:30 is the nucleotide sequence for the pSYCO109mcs plasmid.
SEQ ID NO:31 is the nucleotide sequence for the short 1.5 GI promoter.
SEQ ID NO:32 is the nucleotide sequence for the short 1.20 GI promoter.
SEQ ID NO:33 is the nucleotide sequence for the short wild-type GI promoter.
SEQ ID NOs:34-37 are the oligonucleotide primers used for amplification of yqhD with incorporation of the short GI promoters.
SEQ ID NOs:38-39 are oligonucleotide primers used to construct the yqhD disruption.
SEQ ID NOs:40-43 are oligonucleotide primers used to confirm disruption of yqhD.
SEQ ID NOs:44-46 are oligonucleotide primers used for replacement of the chromosomal ppc promoter with the short wild-type GI promoter.
SEQ ID NO:47 is the nucleotide sequence for a multiple cloning site and terminator.
SEQ ID NO:48 is the nucleotide sequence for the pHK28-26 plasmid.
SEQ ID NOs:49-50 are oligonucleotide primers used to amplify dhaB3.
SEQ ID NOs:51-52 are oligonucleotide primers used to amplify dhaB1.
SEQ ID NOs:53-54 are oligonucleotide primers used to create the dhaT deletion.
SEQ ID NOs:55-56 are oligonucleotides used to create a linker.
SEQ ID NO:57 is a nucleotide sequence encoding three transcriptional terminators separated by restriction sites.
SEQ ID NOs:58-59 are oligonucleotides used to create SEQ ID NO:60.
SEQ ID NO:60 is the nucleotide sequence encoding three transcriptional terminators flanked by EcoRI and KpnI sites.
SEQ ID NOs:61-62 are oligonucleotide primers used to amplify SEQ ID NO:60.
SEQ ID NOs:63-66 are oligonucleotide primers used to amplify an expression cassette.
SEQ ID NO:67 is the nucleotide sequence of a double-stranded linker used to generate pCR-pCL1920.
SEQ ID NOs:68-69 are oligonucleotide primers used to amplify the rmBT1 T2 terminator from pTrc99A.
SEQ ID NO:70 is the nucleotide sequence for the pAH105 plasmid.
SEQ ID NO:71 is the nucleotide sequence for the pSYCO101 plasmid.
SEQ ID NO:72 is the nucleotide sequence for the pSYCO103 plasmid.
SEQ ID NO:73 is the nucleotide sequence for the pSYCO106 plasmid.
SEQ ID NO:74 is the nucleotide sequence for the pSYCO109 plasmid.
SEQ ID NOs:75-76 are oligonucleotide primers used to form SEQ ID NO:77.
SEQ ID NO:77 is the nucleotide sequence of a multiple cloning fragment containing restriction recognition sites for the following enzymes: NheI, RsrII, SacI, AgeI, SnaBI, AscI, PacI, NsiI, MluI, and SapI.
SEQ ID NO:78 is the nucleotide sequence for the pSCYO106mcs plasmid.
SEQ ID NO:79 is the nucleotide sequence for the pRJ50 plasmid.
SEQ ID NOs:80-81 are oligonucleotide primers used to amplify the orf operon.
SEQ ID NOs:82-83 are oligonucleotide primers to check transformants in Example 4.
Applicants have made the following biological deposit under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure:















Depositor Identification
Int'l. Depository




Reference
Designation
Date of Deposit









Escherichia coli RJ8n
ATCC PTA-4216
9 Apr. 2002










As used herein, “ATCC” refers to the American Type Culture Collection International Depository located 10801 University Blvd., Manassas, Va. 20110-1109, U.S.A. The “ATCC No.” is the accession number to cultures on deposit with the ATCC.
The listed deposits will be maintained in the indicated international depository for at least thirty (30) years and will be made available to the public upon grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.


DETAILED DESCRIPTION OF THE INVENTION
The Applicants have solved the stated problem by creating a series of constructs containing at least three unique cloning sites, each cloning site operably separated from each other by transcription terminators and promoters of different strengths. The promoters of different strengths are variants of the Streptomyces lividans glucose isomerase (GI) promoter (SEQ ID NO:1). Combining the variant GI promoters in expression cassettes with a SYCO plasmid as the production platform provides a system useful for biocatalyst development in a wide variety of bioprocess projects.
The invention allows the facile and stable incorporation of endogenous or exogenous genes or operons in a vector controlling the levels of gene expression. The use of the single plasmid to express multiple genes or operons reduces the number of antibiotic markers needed to maintain the multiple plamids in the E. coli host that previous methods required to produce a gene product. Use of the invention can minimize the impact of metabolic load, optimize the yield of the recombinant protein, and enhance the stability of the transformed host cell. The invention is especially useful for genetic engineering in bioprocesses where expressing two or more genes or operons may be required for product formation.
Applicants have created novel GI promoter sequences that allow varying levels of gene expression. Applicants incorporated expression cassettes containing the variant GI promoters into a low-copy-number plasmid derived from pCL1920 to construct a series of plasmids for genetic engineering. The transcription terminators isolate the associated promoter from transcription from other promoters located outside this construct.
Applicants also have constructed a unique nucleotide sequence containing cloning sites for at least ten rare restriction enzymes, further facilitating cloning in this construct or transfer of this construct to alternate plasmid or vector backbones. The unique cloning sites allow introduction of genes or operons to be expressed under the control of suitable promoters of varying strengths. Further, a given construct may be flanked by unique cloning sites for facile integration into any number of plasmid backbones including pUC, pBR322, pACYC, pSC101, or others known and contemplated by those skilled in the art.
Applicants have demonstrated a specific utility of the invention in the biosynthesis of 1,3-propanediol (3G) from glucose in E. coli transformed with the claimed materials. Expression cassettes were constructed in a low-copy-number plasmid as described herein and genes for production of 1,3-propanediol were cloned into this vector. The invention may be used to vary gene expression in other expression systems.
Definitions
The following definitions and abbreviations are to be used to interpret the claims and specification.
“Open reading frame” is abbreviated ORF.
“Polymerase chain reaction” is abbreviated PCR.
The terms “host cell” or “host organism” refer to a microorganism capable of receiving foreign or heterologous genes or multiple copies of endogenous genes and of expressing those genes to produce an active gene product.
The terms “DNA construct” or “construct” refer to an artificially constructed fragment of DNA
“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign”, “exogenous”, or “heterologous” gene refers to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. A “gene construct” refers to a nucleic acid fragment that encodes for expression of one or more specific proteins. In the gene construct the gene may be native, chimeric, or foreign in nature.
The term “isolated nucleic acid” refers to a nucleic acid (e.g., an RNA, DNA, or a mixed polymer) which is substantially separated from other components that naturally accompany a native sequence (e.g., ribosomes, polymerases, and/or flanking genomic sequences from the originating species). The term includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
The terms “encoding” and “coding” refer to the process by which a gene, through the mechanisms of transcription and translation, produces an amino acid sequence. The process of encoding a specific amino acid sequence includes DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated. For example, alterations in the gene sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue (such as glycine), or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes that result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid), or one positively charged residue for another (such as lysine for arginine) can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity in the encoded products. Moreover, the skilled artisan recognizes that sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC , 0.1% SDS, 65° C.), with the sequences exemplified herein.
The term “expression” refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product.
The term “promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.
The terms “transcription terminator” or “terminator” refer to the genetic element that ends protein synthesis.
The term “operon” refers to a cluster of genes that are coordinately regulated.
The terms “polypeptide” and “protein” are used interchangeably to refer to the gene product.
The terms “plasmid”, “vector”, and “cassette” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences (linear or circular) of a single- or double-stranded DNA or RNA, derived from any source. Such elements contain a number of nucleotide sequences that have been joined or recombined into a unique construction capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in its host.
The term “restriction endonucleases” refers to a class of enzymes that cut a given length of DNA at a specific and unique internal location. By creating the cut in the DNA, restriction endonucleases enable the subsequent splicing or insertion of segments of DNA into the internal location. The terms “restriction site” or “restriction recognition site” refer to a nucleotide sequence (of base pairs) in a DNA molecule that is “recognized” and cut by a given restriction enzyme.
The term “rare”, as applied to restriction enzyme sites, refers to the low frequency of occurrence of a given sequence in a gene. A preferred group of rare restriction enzymes sites for purposes of this specification are AscI, NheI, PacI, RsrII, NsiI, SacII, MluI, AgeI, SapI, and SnaBI.
The term “cloning site” refers to a location on a vector into which DNA can be inserted. The term “multiple cloning site” or “mcs” refers to a synthetic DNA sequence that contains any one or a number of different restriction enzyme sites to permit insertion at a defined locus (the restriction site) on a vector. The term “unique cloning site” refers to a cloning site that appears one time with a given DNA sequence.
In describing the relative locations of the elements of a vector, a given site or locus of interest is “between” two others if it is situated in the intermediate length of DNA that separates the two others. In the case of a circular vector, the given site or locus of interest is “between” two others if it is situated within the shortest length of DNA that separates the two other sites on the vector. The given site or locus is said to be “flanked” by another situated either preceding or following the site or locus of interest.
The term “genetically altered” refers to the process of changing hereditary material by transformation or mutation. The terms “transformation” and “transfection” refer to the acquisition of new genes in a cell after the incorporation of nucleic acid. The acquired genes may be integrated into chromosomal DNA or introduced as extrachromosomal replicating sequences. The term “transformant” refers to the product of a transformation.
The terms “glycerol dehydratase” or “dehydratase enzyme” refer to the polypeptide(s) responsible for a coenzyme B12-dependent enzyme activity that is capable of isomerizing or converting a glycerol molecule to the product 3-hydroxypropionaldehyde. For the purposes of the present invention, the dehydratase enzymes include a glycerol dehydratase (GenBank U09771, U30903) and a diol dehydratase (GenBank D45071) having preferred substrates of glycerol and 1,2-propanediol, respectively. Glycerol dehydratase of K. pneumoniae ATCC 25955 is encoded by the genes dhaB1, dhaB2, and dhaB3 (GenBank U30903). The dhaB1, dhaB2 and dhaB3 genes code for the α, β, and γ subunits of the glycerol dehydratase enzyme, respectively. Glycerol dehydratase and diol dehydratase are complexes (with an α2β2γ2 subunit composition) that utilize coenzyme B12.
Glycerol and diol hydratases are subject to mechanism-based suicide inactivation by glycerol and some other substrates (Daniel et al., FEMS Microbiol. Rev. 22:553 (1999)). The term “dehydratase reactivation factor” refers to those proteins responsible for reactivating the dehydratase activity. The terms “dehydratase reactivating activity”, “reactivating the dehydratase activity”, or “regenerating the dehydratase activity” refer to the phenomenon of converting a dehydratase not capable of catalysis of a substrate to one capable of catalysis of a substrate or to the phenomenon of inhibiting the inactivation of a dehydratase or the phenomenon of extending the useful half-life of the dehydratase enzyme in vivo. Two proteins have been identified as being involved as the dehydratase reactiviation factor (see WO 9821341 (U.S. Pat. No. 6,013,494 herein incorporated by reference) and references therein; Daniel et al., supra; Toraya and Mori, J. Biol. Chem. 274:3372 (1999); and Tobimatsu et al., J. Bacteriol. 181:4110 (1999)).
The terms “oxidoreductase” or “1,3-propanediol oxidoreductase” refer to the polypeptide(s) responsible for an enzyme activity that is capable of catalyszing the reduction of 3-hydroxypropionaldehyde to 1,3-propanediol. 1,3-Propanediol oxidoreductase includes, for example, the polypeptide encoded by the dhat gene (GeneBank U09771, U30903). Alternatively, yqhD, an E. coli open reading frame with 40% identity to the gene adhB in Clostridium (a probable NADH-dependent butanol dehydratase 2), encodes a polypeptide that functions as a 1,3-propanediol oxidoreductase (WO 0112833).
The enzymes expressed by the pSYCO plasmids (pSYCO101, pSYCO103, pSYCO106, pSYCO109, pSYCO106mcs, and pSYCO109mcs) can all be said to comprise genes required to express glycerol dehydratase, dehydratase reactiviation factor, glycerol-3-phosphate dehydrogenase, and glycerol-3-phosphatase.
The terms “fermentable carbon substrate” and “fermentable carbon source” refer to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, glycerol, dihydroxyacetone and one-carbon substrates or mixtures thereof.
Gene Expression System with GI Promoter Variants of Differing Strengths
The minimal requirement for an effective gene expression system is the presence of a promoter (a site on DNA where the RNA polymerase binds and begins transcription) upstream from a cloned gene. Often a strong promoter, one that has a high affinity for RNA polymerase, is used with the result that the adjacent downstream region is highly or frequently transcribed.
In the promoter, the main sequence determinant of promoter strength (the level at which the downstream gene is transcribed) is the most highly conserved base pairs. Promoters that have deviations from the conserved sequences have decreased transcription initiation frequency (Hawley, D. K.; McClure, W. R., Nucleic Acids Res., 11:2237-2255 (1983)).
Promoters for E. coli RNA polymerase have been shown to contain two regions of conserved DNA sequences, located about 10 and 35 base pairs upstream of the transcription start site. Twelve base pairs were determined to be the most highly conserved among promoters. These bases are TTGACA around 35 base pairs upstream, the so called −35 region, and TATMT around 10 base pairs upstream, the so called −10 region. Optimum spacing between the −10 and −35 regions is 17 base pairs. The promoter is stronger if the spacing is closer to 17 base pairs; however, promoters with interspacings of 15 and 20 base pairs retain partial function.
Applicants have created a series of constructs incorporating variants of the Streptomyces lividans glucose isomerase (GI) promoter. The constructs form a library or kit of promoter variants with a range of different strengths conferring the ability to tailor varying levels of gene expression as needed. The Streptomyces glucose isomerase (EC 5.3.1.9) catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate. Transcription of the gene encoding phosphoglucose isomerase (pgi) is controlled by a promoter that contains a characteristic −10 signature sequence (AATMT) and a characteristic −35 signature sequence (TTGACA). Although saturation mutagenesis was carried out in the −35 region of the promoter, changes to a SpeI restriction site approximately 122 bp upstream from the −35 region also had effects on expressed gene activity. Further, a 25 bp deletion between the −10 and the end of this promoter allowed retention of 86% of the enzyme's activity even with the changes to the SpeI restriction site. These particular results had not been previously reported.
Transcription termination of RNA synthesis occurs at specific base sequences on the DNA and regulates termination of transcription. A common termination sequence on the DNA is one containing an inverted repeat with a central non-repeating segment. When such a DNA sequence is transcribed, the RNA can form a stem-loop structure by intrastrand base pairing. When such stem-loop structures in the RNA are followed by runs of uridines, they are effective transcription terminators. Other termination sites are regions where a GC-rich sequence is followed by an AT-rich sequence. Such kinds of structures lead to termination of transcription without adding any extra factors and are sometimes termed intrinsic terminators or rho-independent terminators.
Other types of terminator sequences have been discovered that require protein factors like Rho from E. coli in addition to RNA polymerase to function. Rho does not bind to RNA polymerase or to DNA but binds tightly to RNA and moves down the chain towards the RNA polymerase-DNA complex. Once RNA polymerase has paused at a Rho-dependent termination site, Rho can then cause the RNA and polymerase to leave the DNA, thus terminating transcription. Other proteins involved in transcription termination are, like Rho, RNA-binding proteins. In all cases the sequences involved in termination operate at the level of RNA. However, RNA is transcribed from DNA, and so transcription termination is ultimately determined by specific nucleotide sequences on the DNA. (Madigan, M. T.; Martinko, J. M.; Parker, J.; Brock Biology of Microorganisms, 8th ed., Prentice Hall; Upper Saddle River, N.J. (1997)).
Applicants have constructed a termination region in which three different terminator sequences have been placed in tandem. These three terminators are flanked by unique restriction enzyme sites useful for the cloning of genes or operons. The tonB terminator is a bidirectional rho-independent transcriptional terminator found between the E. coli tonB gene and an opposing gene (Postle, K.; Good, R. F., Cell, 41, 577-585 (1985)). The thr attenuator, similar in structure to other rho-independent terminators facilitates transcriptional termination of the E. coli threonine operon (Yanget et al., J. BioL Chem., 270:23330-23336 (1995)). The aspA terminator with a structure characteristic of rho-independent terminators, facilitates transcriptional termination of the E. coli aspartase operon (Takagi et al., Nucleic Acid Res., 13:2063-2074 (1985)).
As autonomous, self-replicating genetic elements, plasmids have the basic attributes to make them potential vectors for carrying cloned DNA. Naturally-occurring plasmids often lack several important features required in a high-quality cloning vector. These features include (1) a small size (necessary for efficient transfer of exogenous DNA into a host), (2) unique restriction endonuclease recognition sites into which the insert DNA can be cloned, and (3) one or more selectable genetic markers for identifying recipient cells that carry the cloning vector-insert DNA construct. Consequently, plasmid cloning vectors have to be genetically engineered (Glick, B. R., Pasternak, J. J., Molecular Biotechnologqy Principles and Applications of Recombinant DNA, 2nd ed., American Society for Microbiology, Washington, D.C. (1998)).
pCL1920/21 vectors are a pair of low-copy-number plasmids that contain a 580 bp BstUI fragment carrying the lac promoter/operator, a multiple cloning site and lacZ fragment of pUC19 cloned in place of the polylinker region in pGB2, a pSC101-derived plasmid which confers spectinomycin and streptomycin resistance in E. coli. pCL1920/21 vectors (five copies per cell) have a 40-fold difference in plasmid copy number between pCL1920/21 vectors and pUC vectors (200 copies per cell). Thus, the pCL1920/21 vectors allow regulated low-level expression of genes inserted downstream of the promoter-operator when transformed into strains. They should also be useful for cloning genes that may be deleterious at high copy number. Since the pCL1920/21 vectors are compatible with ColE1-derived plasmids they can be used to form stable co-transformants together with pBR322 or pUC derived plasmids (Lerner et al., Nucleic Acids Res., 18:4631 (1990)).
The plamids of the inventions may be used in a variety of hosts for the controlled bioproduction of materials.
Replacing Chromosomally-Located Native Promoters of any Endogenous Gene or Operon to Alter Transcriptional Level.
Claimed promoter variants (constructs comprising SEQ ID NOs:31 and 32) may be used in a method to replace chromosomally-located native promoters associated with any endogenous gene or operon in order to alter the transcription level of the gene or operon. The result is changed protein production levels. The promoter to be replaced can be any gene in any microorganism where the Llambda red method of Datsenko and Wanner [(2000) PNAS 97:6640-6645] or an equivalent method is operable.
In the method, a chimeric DNA molecule comprising a selectable marker operably linked to a divergently-arranged non-native promoter operably linked to the 5′ coding region of a target gene is synthesized by polymerase chain reaction (PCR). The synthesis is accomplished using: (1) a pair of chemically synthesized primers, (a) the first primer comprising: (i) a DNA region distal to the target natural promoter to be replaced, (ii) a non-native promoter, and (iii) a DNA region from either the 3′ or 5′ end of the selectable marker; and (b) the second primer comprising: (i) a DNA region proximal to the targeted insertion site, and (ii) a DNA region from the opposite end of the selectable marker than was used in the first primer; and (2) a DNA template encoding a selectable marker. This product is integrated in the DNA product synthesized above at the chromosomal target site of any host cell using the method of Datsenko and Wanner (supra). The result of this protocol is that the target native promoter(s) are replaced with the PCR-synthesized chimeric molecule that carries the non-native promoter.
An extension of the method may be used to assess the effect of the varying gene expression level on biocatalyst performance.
Biosynthesis of 1,3-propanediol (3G) from Glucose in E. coli 
The plasmids of the invention may be used in E. coli for the biosynthesis of 1,3-propanediol (3G) from glucose. The examples herein include the construction of a production organism that incorporates the claimed invention and the genetic machinery necessary to convert a fermentable carbon substrate to 1,3-propanediol.
The genes involved in 1,3-propanediol production include a dehydratase gene (typically a glycerol or diol dehydratase) and an oxidoreductase as well as other proteins expected to aid in the assembly or in maintaining the stability of the dehydratase enzyme. These genes may be transgenes introduced into the host cell, or may be endogenous. At least one of these genes will be a transgene and introduced into the production cell. Recombinant organisms containing the necessary genes that encode the enzymatic pathway to convert a carbon substrate to 1,3-propanediol may be constructed using techniques well known in the art. The transformed production cell is then grown under appropriate conditions for the production of 1,3-propanediol.
Production of 1,3-propanediol in E. coli has been previously described (U.S. Pat. No. 5,633,362; U.S. Pat. No. 5,821,092; U.S. Pat. No. 5,686,276; U.S. Pat. No. 6,025,184; U.S. Pat. No. 6,013,494; U.S. Pat. No. 5,599,689; U.S. Pat. No. 6,136,576). Expression of many different genes are involved in the production from glucose of 1,3-propanediol by a recombinant E.coli. Genes encoding glycerol dehydratase (dhaB) and 1,3-propanediol oxidoreductase (dhaT) were isolated from a native host such as Klebsiella and used to transform host strains such as E.coli strain DH5α or FM5; K. pneumoniae strain ATCC 25955; K. oxytoca strain ATCC 8724 or M5a1, S. cerevisiae strain YPH499, P. pastoris strain GTS115, and A. niger strain FS1.
In Klebsiella pneumonia, Citrobacter freundii, and Clostridium pasteurianum, the genes encoding the three structural subunits of glycerol dehydratase (dhaB1-3 or dhaB, C, and E) are located adjacent to a gene encoding a specific 1,3-propanediol oxidoreductase (dhaT). Although the genetic organization differs somewhat among these microorganisms, these genes are clustered in a group that also includes orfX and orfZ (genes encoding a dehydratase reactivation factor for glycerol dehydratase), as well as orfY and orfW (genes of unknown function). The specific 1,3-propanediol oxidoreductases (dhaT's) of these microorganisms are known to belong to the family of type III alcohol dehydrogenases; each exhibits a conserved iron-binding motif and has a preference for the NAD+/NADH linked interconversion of 1,3-propanediol and 3-HPA. However, the NAD+/NADH linked interconversion of 1,3-propanediol and 3-HPA is also catalyzed by alcohol dehydrogenases which are not specifically linked to dehydratase enzymes (for example, horse liver and baker's yeast alcohol dehydrogenases (E.C. 1.1.1.1)), albeit with less efficient kinetic parameters. Glycerol dehydratase (E.C. 4.2.1.30) and diol [1,2-propanediol] dehydratase (E.C. 4.2.1.28) are related but distinct enzymes that are encoded by distinct genes. Diol dehydratase genes from Klebsiella oxytoca and Salmonella typhimurium are similar to glycerol dehydratase genes and are clustered in a group which comprises genes analogous to orfX and orfZ (Daniel et al., FEMS Microbiol. Rev. 22:553 (1999); Toraya and Mori, J. Biol. Chem. 274:3372 (1999); GenBank AF026270).
The gene encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) has been cloned and sequenced from S. diastaticus (Wang et al., J. Bact. 176:7091-7095 (1994)). The DAR1 gene was cloned into a shuttle vector and used to transform E. coli where expression produced active enzyme. Wang et al. (supra) recognize that DAR1 is regulated by the cellular osmotic environment but do not suggest how the gene might be used to enhance 1,3-propanediol production in a recombinant microorganism.
Other glycerol-3-phosphate dehydrogenase enzymes have been isolated. For example, sn-glycerol-3-phosphate dehydrogenase has been cloned and sequenced from Saccharomyces cerevisiae (Larason et al., Mol. Microbiol. 10: 1101 (1993)). Albertyn et al. (Mol. Cell. Biol. 14:4135 (1994)) teach the cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae. Like Wang et al. (supra), both Albertyn et al. and Larason et al. recognize the osmo-sensitivity of the regulation of this gene but do not suggest how the gene might be used in the production of 1,3-propanediol in a recombinant microorganism.
As with G3PDH, glycerol-3-phosphatase has been isolated from Saccharomyces cerevisiae and the protein identified as being encoded by the GPP1 and GPP2 genes (Norbeck et al., J. Biol. Chem. 271:13875 (1996)). Like the genes encoding G3PDH, it appears that GPP2 is osmosensitive.
EXAMPLES
The present invention is further defined in the following Examples that indicate preferred embodiments of the invention. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
General Methods
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et a., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).
Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg, and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis., DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), New England Biolabs (Beverly, Mass.) or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified.
The meaning of abbreviations is as follows: “h” means hour(s), “min” means minute(s), “sec” means second(s), “d” means day(s), “mL” means milliliters, “L” means liters, “mm” means millimeters, “nm” means nanometers, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” mean micromole”, “g” means gram, “μg” means microgram.
Example 1
Construction of Glucose Isomarase Promoter Variants
The Streptomyces lividans glucose isomerase (GI) promoter (SEQ ID NO:1) contains a characteristic −10 signature sequence (AATAAT) and a characteristic −35 signature sequence (−35 T, −34 T, −33 G, −32 A, −31 C, −30 A). Using mixed base oligonucleotides, saturation mutagenesis of the −35 region of the GI promoter in pMP38 (as described in Example 6 following) was performed by standard PCR. In six individual PCR reactions, an upstream primer (SEQ ID NO:2) was paired with one of six downstream primers (SEQ ID NOs:3-8), each of which contains an equal mixture of all four possible bases at a single position in the −35 region, designated as N. The upstream primer also incorporates two single base pair changes that change a SpeI restriction site (ACTAGT) immediately following the EcoRI site to an AvrIl restriction site (CCTAGG). The six PCR products were digested with EcoRI and HindIII, and individually ligated to EcoRI/HindIII digested pMP38. Ligations were transformed into E. coli, and recombinant plasmids were identified through restriction analysis by the SpeI to AvrIl conversion, and subjected to nucleotide sequencing. Only recombinant plasmids would be expected to harbor possible −35 region changes. Of the twenty-four possible recombinant outcomes (4 bases in 6 positions), 18 were obtained, of which 13 represent changes in the −35 region (Table 1).







TABLE 1







GI promoter variants obtained by saturation mutagenesis PCR

















SEQ







ID



Position
Base
Name
Comments
NO.

















−30A
A
P1.6
No change in −35 region
9




T
P1.5

10




G
P1.20

11




C
P1.10

12



−31C
C
P2.8
No change in −35 region
9




A
P2.39

13



−32A
A
P3.4
No change in −35 region;
14






25 bp deletion between






−10 and -HindIII site




C
P3.5

15



−33G
G
P4.49
No change in −35 region
9




A
P4.15

16




C
P4.1

17



−34T
T
P5.10
No change in −35 region
9




C
P5.12

18




A
P5.17

19




G
P5.19

20



−35T
T
NA
No change in −35 region
9




G
P6.5

21




A
P6.14

22




C
P6.20

23







*NA = Not Applicable






Although five of the possible 18 changes in the −35 region were not isolated, these may also be useful for varying expression levels of cloned or chromosomally-encoded native and non-native genes or operons. These five additional GI promoter variants are described in Table 2.







TABLE 2







Other Potential GI Promoter Variants











Position
Base
SEQ ID NO.







−31C
G
24




T
25



−32A
G
26




T
27



−33G
T
28










Example 2
Analysis of Glucose Isomerase Promoter Variants by Measuring Glycerol Dehydratase Activity
Glycerol dehydratase (GDH; encoded by dhaB1-3) activity was used as a reporter to measure the effect of the GI promoter mutations (Table 3). It was observed that even in the absence of a change in the −35 region, GDH activity dropped significantly due to the two base pair changes which converted SpeI to AvrIl (for example, P1.6). It was also determined that P3.4 did not have a −35 mutation, but did have a 25 base pair deletion immediately following the −10 region, and had nearly wild-type (86%) promoter strength.
Dehydratase activity in cell-free extracts was determined using either glycerol or 1,2-propanediol as substrate. Cell-free extracts were prepared by cell disruption using a French press followed by centrifugation of the cellular debris. The assay, based on the reaction of aldehydes with methylbenzo-2-thiazolone hydrazone, has been described by Forage and Foster (Biochim. Biophys. Acta 569:249 (1979)).







TABLE 3







Measure of Relative GDH Activity In GI Promoter Variants











relative GDH



Plasmid
activity














pMP38
100



pMP38/1.6
13



pMP38/1.5
3



pMP38/1.20
1



pMP38/1.10
1



pMP38/2.39
0



pMP38/3.4
86



pMP38/3.5
1



pMP38/4.1
0



pMP38/4.15
0



pMP38/5.12
0



pMP38/5.17
0



pMP38/5.19
0



pMP38/6.5
0



pMP38/6.14
1



pMP38/6.20
2










Example 3
Analysis of GI Promoter Variants Using LUX Assays
A second type of reporter was used to measure levels of expression driven from the GI promoter variants. Bacterial bioluminescence is a phenomenon in which the products of 5 structural genes (luxA, luxB, luxC, luxD, and luxE) work in concert to produce light. The luxD product generates a C14 fatty acid from a precursor. The C14 fatty acid is activated in an ATP dependent reaction to an acyl-enzyme conjugate through the action of the luxE product, which couples bacterial bioluminescence to the cellular energetic state. The acyl-enzyme (luxE product) serves as a transfer agent, donating the acyl group to the luxC product. The acyl-LuxC binary complex is then reduced in a reaction in which NADPH serves as an electron pair and proton donor reducing the acyl conjugate to the C14 aldehyde. This reaction couples the reducing power of the cell to bacterial light emission. The light production reaction, catalyzed by luciferase (the product of luxA and luxB), generates light. The energy for light emission is provided by the aldehyde to fatty acid conversion and FMNH2 oxidation, providing another couple between light production and the cellular energy state.
The Photorabdus luminenscens luxAB genes were used as reporters for GI promoter variant strength (Van Dyk et al., Appl. Environ. Microbiol., 180:785-792 (1995)). A PCR fragment carrying the P. luminenscens luxAB genes and containing SpeI sites at the 3′ and 5′ ends and an NcoI site engineered at the initiation codon of luxA was subcloned into the SpeI site in pMCS5 (MobiTec, Göttingen, Germany) yielding pJT13. Then a gene SOEing PCR-based kanamycin cassette with SwaI/Ncol ends was cloned into SwaI/NcoI-digested pJT13 to make pJT14.HIGHCOPY, the high copy luxAB promoter probe. pJT14.HIGHCOPY was then digested with SpeI producing the luxAB::kanamycin cassette, which was subcloned into the unique NheI site (compatible with SpeI), in pRJ50 (SEQ ID NO:79) to make pJT14.LOWCOPY.1, the low copy luxAB promoter probe. The GI promoters 1.6, 1.5, 1.20, and native were cloned into pJT14.HIGHCOPY and pJT14.LOWCOPY as NotI/NcoI fragments to make the high-copy constructs pJT18, pJT19, pJT20, and pJT25, respectively, and low-copy constructs pJT21.1, pJT22.1, pJT23.1, and pJT26.1, respectively. The plasmids were then transformed into select E. coli strains for in vivo bioluminescence measurements.
Promoter strengths were measured by luminometry using broth cultures of E. coli reporter strains, n-decanal as the aldehyde substrate, and a luminometer, as described by Van Dyk and Rosson (Methods in Molecular Biology, Vol. 102: Bioluminescence Methods and Protocols, 85 (1998)). E. coli clones were inoculated from a fresh agar plate into test tubes containing standard Luria-Bertani liquid growth medium with the appropriate antibiotic and grown aerobically (with shaking) at 37° C. for approximately 16 h. Cells were then subcultured into 100-mL flasks containing 25 mL of fresh medium and grown under the same conditions for approximately 8-10 h. Aliquots (200 μL) were then taken from each culture and placed into 96-well clear and white plates for optical density measurements at 600 nm (SpectraMax 190 Plater Reader, Molecular Devices Corporation, Sunnyvale, Calif.) and luminometer measurements (Luminoscan Ascent TAype 392, LabSystems, Helsinki, Finland), respectively. For the luminometry readings, 2 μL of exogenous aldehyde (n-decanal) was added to each well and measurements made. Results from these assays are listed in Table 4. These luminometry measurements indicated a level of promoter strength similar to that indicated by glycerol dehydratase assays.







TABLE 4







Bioluminescence Measurements











GI Promoter
Plasmid construct
Relative Bioluminescence







GI wildtype
pJT26.1
 100%



GI 1.6
pJT21.1
12.9%



GI 1.5
pJT22.1
 3.0%



GI 1.20
pJT23.1
 1.3%










Example 4
Use of Shortened GI Promoter Sequences to Achieve Different Levels of Gene Expression
A subset of the GI promoter sequences described and used in Examples 1-3 were used to vary levels of expression of E. coli yqhD (SEQ ID NO:29) from the pSYCO109mcs plasmid (as described in Example 8 and SEQ ID NO:30) in strain RJ8n in which the yqhD gene was disrupted on the chromosome to create strain RJ8n (yqhD−).
Three expression cassettes for yqhD were constructed. These cassettes contain (i) one of the shortened GI promoters designated short 1.5 GI (SEQ ID NO:31), short 1.20 GI (SEQ ID NO:32), or short wild-type GI (SEQ ID NO:33); (ii) yqhD from E. coli KLP23 (WO9928480); and (iii) the threonine terminator (Lynn et al., J. Mol. Biol., 183:529-541 (1985)). The yqhD gene was isolated by PCR amplification from genomic KLP23 DNA using forward synthetic primers for short 1.5 GI (SEQ ID NO:34), short 1.20 GI (SEQ ID NO:35), or short wild-type GI (SEQ ID NO:36) which contain one of the shortened GI promoters and also incorporate a RsrIl restriction site, and the reverse primer for yqhD (SEQ ID NO:37) that contained the threonine terminator and included a SacI site. Plasmid pSYCO109mcs was digested with RsrIl/SacI and the RsrIl/SacI digested PCR products were ligated into the plasmid. The ligation mixture was transformed into the RJ8n (yqhD−) strain by electroporation and the enzyme activity levels in each of the strains were compared (Table 5.)
The enzyme activity expressed by yqhD will reduce the aldehydes 3-hydroxypropionaldehyde (3-HPA) and butanal with similar rates using NADPH as the source of reducing equivalents. Since 3-HPA is not commercially available, butanal is generally used. The assay mixture contained in 1 mL total volume: 200 mM potassium phosphate buffer (pH 7.5), 10 mM butanal, 0.2 mM NADPH, and approximately 0.01 mg protein from cell-free extracts to be assayed. The initial rate of oxidation of NADPH after addition of protein sample was followed by measuring the change in absorbance at 340 nm (Δε=6.22 mM−1). A unit of activity is defined as that required to oxidize 1 micromole of NADPH in 1 minute in the presence of 10 mM butanal at 35° C. The activities of various strains are given in Table 5 below and were consistent with levels of expression allowed by the longer GI promoter variants.







TABLE 5







YqhD Activity










Activity
%


Strain and construct
(U/mg)
Activity












RJ8n(yqhD-)
0.015
0.8


RJ8n(yqhD-)/pSYCO109mcs
0.010
0.5


RJ8n(yqhD-)/pSYCO109mcs-short 1.20 GI yqhD
0.14
7.3


RJ8n(yqhD-)/pSYCO109mcs-short 1.5 GI yqhD
0.29
15


RJ8n(yqhD-)/pSYCO109mcs-short wild-type GI yqhD
1.92
100









To create RJ8n (yqhD−) the yqhD gene was disrupted in E. coli MG1655 using the procedure as described by Wanner and Datsenko (PNAS, 97(12):6640-6645 (2000)) for Red-mediated homologous recombination. The forward PCR primer H1::6574 (SEQ ID NO:38) (containing 42 bp of sequence homologous to yqhD and the primer binding site P1 to pKD13) and the reverse PCR primer H2::6706 (SEQ ID NO:39) (containing 47 bp of homologous yqhD sequence and the primer binding site P4 to pKD13) were prepared. PCR amplification with pKD13 as the template produced a PCR product that had yqhD sequence on each end followed by FRT (FLP recognition target) sites that flank a kanamycin resistance (kanR) marker. The PCR product was electrotransformed into E. coli MG1655 cells and kanamycin-resistant transformants were selected. Correct insertion in the transformants was confirmed by PCR using primers yqhDUP (SEQ ID NO:82) and yqhDDN (SEQ ID NO:83) flanking the yqhD gene. The temperature-sensitive plasmid containing the Lambda Red system was cured by growth of the strains at 42° C.
The yqhD::kan disruption was moved into RJ8n by P1 transduction and confirmed by PCR using the yqhDUP2 (SEQ ID NO:40) and yqhDDN2 (SEQ ID NO:41) primers paired with primers internal to the kanR gene (Vec 61; SEQ ID NO:42 and Vec 60; SEQ ID NO:43). To remove the kanamycin marker, integrants were transformed with the temperature-sensitive replicon, pCP20, which contains the gene for the FLP recombinase. FLP recombinase excises the kanamycin marker at the flanking FRT (FLP recognition target) sites. Kanamycin-sensitive cells were then grown at 42° C. to cure pCP20. The resultant strain was RJ8n (yqhD−).
Example 5
Replacement of the E. Coli Phosphoenolpyruvate Carboxylase Chromosomal Promoter with a GI Promoter
Example 5 describes the replacement in the Escherichia coli genome of the natural ppc (encoding the phosphoenolpyruvate carboxylase or PEP carboxylase) promoter by the short wild-type GI promoter (SEQ ID NO:33).
Design of the Oligonucleotides for the ppc Promoter Replacement
Two oligonucleotides (ppcF, SEQ ID NO:44 and ppcR, SEQ ID NO:45) were designed to amplify by PCR a cassette containing an 80-bp sequence homologous to the upstream region of the natural ppc promoter, a chloramphenicol-resistance encoding gene (cat) flanked by baker yeast FRT sites, the short wild-type GI promoter sequence (SEQ ID NO:33), and a 40-bp sequence homologous to the downstream region of the +1 transcription start site of the natural ppc promoter.
The ppcR primer (SEQ ID NO:45) is 100 nucleotides long and includes: the entire sequence from the +1 of P1 (natural ppc promoter) transcription start to 41 bp upstream the ATG of ppc, the short wild-type GI promoter sequence (SEQ ID NO. 33) from 4 bp upstream of the −35 to 9 bp downstream of the −10, and the priming site for pKD3 (Wanner and Datsenko, supra), an R6K plasmid containing the cat gene flanked by two FRT sites. The ppcF primer (SEQ ID NO:44) is 100 nucleotides long and includes 80 bp of sequence upstream of the natural ppc promoter and the priming site for pKD3.
Primers ppcF and ppcR (SEQ ID NOs:44 and 45) were used to amplify the promoter replacement cassette using plasmid pKD3 as a template. The 1.15-kb PCR product was purified by agarose gel electrophoresis followed by QIAquick gel extraction Kit (Qiagen, Inc., Valencia, Calif.).
Replacement of the Natural ppc Promoter into Escherichia coli Genome by Homologous Recombination Using Linear DNA
Competent Escherichia coli MG1655 cells containing pKD46 (Datsenko and Wanner, supra), a Red-recombinase plasmid expressing γ, β, and exo under the control of the arabinose promoter, were electrotransformed with 0.5 μg of the above 1.15-kb linear DNA and the resulting transformants were screened for chloramphenicol resistance (15 μg/mL). The recombinant strains were checked by PCR using primers ppcF and seqppcR (SEQ ID NO:46). Non-specific integration of the cassette gives no PCR products while true recombinants give a 1.25-kb PCR product. The sequence of the short wild-type GI promoter was confirmed by sequencing the 1.25-kb PCR product with the seqppcR primer (SEQ ID NO:46).
Measurement of Enzymatic Activity
The PEP carboxylase activities in MG1655 and in MG1655 (short wild-type GI-ppc) were measured on ultra-centrifuged cell-free extract using the following assay and are indicated in Table 6. The activity of PPC under control of the short wild-type GI promoter was over three times higher than under control of the natural promoter.
The decrease at 340 nm (due to consumption of NADH) was measured in a mixture containing: 0.11 M Tris buffer (pH 8.5), NADH (0.22 mM), Magnesium sulfate (11.1 mM), Sodium bicarbonate (11.1 mM), Acetyl-CoA (0.25 mM), MalateDH (Sigma), 50 μL of 6 U cell extracts and 0.03 Phosphoenolpyruvate (1.11 mM). The following formula was used to determine activity:




  
    Units
    ⁢
    
      /
    
    ⁢
    mg
  
  =
  
    
      
        Δ
        ⁢
        
          
        
        ⁢
        A
        ⁢
        
          
        
        ⁢
        
          340
          /
          min
        
        ⁢
        
          
        
        ⁢
        
          (
          test
          )
        
      
      -
      
        Δ
        ⁢
        
          
        
        ⁢
        A
        ⁢
        
          
        
        ⁢
        
          340
          /
          min
        
        ⁢
        
          
        
        ⁢
        
          (
          blank
          )
        
      
    
    
      6.22
      ×
      mg
      ⁢
      
        
      
      ⁢
      protein
      ⁢
      
        /
      
      ⁢
      mL
      ⁢
      
        
      
      ⁢
      reaction
      ⁢
      
        
      
      ⁢
      mixture
    
  











TABLE 6







Activity of PPC from natural and GI1.6 promoters










Strain
Activity (U/mg)














MG1655
0.05



MG1655 (1.6GI ppc)
0.164










Example 6
Construction of an Expression Plasmid for Use in Transformation of Escherichia Coli with Genes from the Klebsiella Pneumoniae dha Regulon
Construction of the Expression Vector pTacIQ:
The E. coli expression vector pTacIQ was prepared by inserting the lacIQ gene (Farabaugh, Nature, 274(5673):765-769 (1978)) and tac promoter (Amann et al., Gene 25:167-178 (1983)) into the EcoRI site of pBR322 (Sutcliffe, Cold Spring Harb. Symp. Quant. Biol. 43:77-90 (1979)). A multiple cloning site and terminator sequence (SEQ ID NO:47) replaced the pBR322 sequence from EcoRI to SphI.
Subcloning the Glycerol Dehydratase Genes (dhaB1, 2, 3, X):
The open reading frame for the dhaB3 gene was amplified from pHK28-26 (SEQ ID NO:48) by PCR using primers (SEQ ID NOs:49-50) incorporating an EcoRI site at the 5′ end and a XbaI site at the 3′ end. The product was subcloned into pLitmus29 (New England Biolabs) to generate the plasmid pDHAB3 containing dhaB3.
The region (containing the entire coding region for dhaB1, dhaB2, dhaB3, and dhaBX of the dhaB operon from pHK28-26) was cloned into pBluescriptIlKS+ (Stratagene, La Jolla, Calif.) using the restriction enzymes KpnI and EcoRI to create the plasmid pM7.
The dhaBX gene was removed by digesting plasmid pM7 with ApaI and XbaI, purifying the 5.9-kb fragment and ligating it with the 325-bp ApaI-XbaI fragment from plasmid pDHAB3 to create pM11 (containing dhaB1, dhaB2, and dhaB3).
The open reading frame for the dhaB1 gene was amplified from pHK28-26 by PCR using primers (SEQ ID NOs:51-52) incorporating a HindIII site and a consensus ribosome-binding site (RBS) at the 5′ end and a XbaI site at the 3′ end. The product was subcloned into pLitmus28 (New England Biolabs) to generate the plasmid pDT1 containing dhaB1.
A NotI-XbaI fragment from pM11 (containing part of the dhaB1gene, the dhaB2 gene, and the dhaB3 gene) was inserted into pDT1 to create the dhaB expression plasmid, pDT2. The HindIII-XbaI fragment (containing the dhaB(1,2,3) genes from pDT2) was inserted into pTacIQ to create pDT3.
Subcloning the 1.3-Propanediol Dehydrogenase Gene (dhaT):
The KpnI-SacI fragment of pHK28-26 (containing the 1,3-propanediol dehydrogenase (dhaT) gene) was subcloned into pBluescriptIl KS+ creating plasmid pAH1. The dhat gene was amplified by PCR using pAH1 as template DNA and the synthetic primers (SEQ ID NOs:53-54) which incorporated an XbaI site at the 5′ end and a BamHI site at the 3′ end. The product was subcloned into pCR-Script (Stratagene) at the SrfI site to generate the plasmids pAH4 and pAH5 containing dhaT. The plasmid pAH4 contains the dhat gene in the correct orientation for expression from the lac promoter in pCR-Script and pAH5 contains dhaT gene in the opposite orientation. The XbaI-BamHI fragment from pAH4 (containing the dhaT gene) was inserted into pTacIQ to generate plasmid pAH8. The HindIl-BamHl fragment from pAH8 (containing the RBS and dhat gene) was inserted into pBluescriptIlKS+ to create pAH11.
Construction of an Expression Cassette for dhaT and dhaB(1.2.3):
An expression cassette for dhaT and dhaB(1,2,3) was assembled from the individual dhaB(1,2,3) and dhaT subclones described previously using standard molecular biology methods. A SpeI-SacI fragment (containing the dhaB(1,2,3) genes from pDT3) was inserted into pAH11 at the SpeI-SacI sites to create pAH24. A SaIl-XbaI linker created from SEQ ID NOs:55-56 was inserted into pAH5 that was digested with the restriction enzymes SaIl-XbaI to create pDT16. The linker destroys the XbaI site. The 1-kb SaIl-MluI fragment from pDT16 was then inserted into pAH24 replacing the existing SaIl-MluI fragment to create pDT18. pDT21 was constructed by inserting the SaIl-NotI fragment from pDT18 and the NotI-XbaI fragment from pM7 into pCL1920 (GenBank AX085428). The glucose isomerase promoter sequence from Streptomyces lividans (SEQ ID NO:1) was cloned by PCR and inserted into EcoRI-HindIII sites of pLitmus28 to construct pDT5. pCL1925 was constructed by inserting the EcoRI-PvuII fragment of pDT5 (containing the GI promoter) into the EcoRI-PvuII site of pCL1920 (GenBank AX085428).
Construction of Expression Vector for Glycerol Dehydratase under the Control of the Streptomyces Glucose Isomerase Promoter:
The HindIII restriction fragment (containing dhaT) was deleted from pDT24 to generate pRN105. The pDT24 plasmid was constructed by cloning the HindIII-MluI fragment of pDT21 and the MluI-XbaI fragment of pDT21 into the HindIII-XbaI sites of pCL1925. A PCR product (comprising the 3′ region of dhaX, from a unique Hpal restriction site to the end of dhaX, and incorporating the HpaI restriction site at the 5′ end and an XbaI restriction site at the 3′ end) was generated from pRN105 template and used to replace the existing HpaI/XbaI restriction fragment in pRN105, generating pMP37. A PCR product (comprising the 5′ region of dhaB1, from a unique HindIII restriction site just upstream of the start codon to a unique NotI restriction site within dhaB1, and incorporating the HindIII restriction site at the 5′ end and the NotI restriction site at the 3′ end) was generated from pDT29 template and used to replace the small HindIII/NotI restriction fragment in pRN105, generating pRJ25. The pDT29 had been constructed by inserting the SacI-EcoRI fragment of pHK28-26 into SacI-EcoRI sites of pCL1925. The small HpaI/XbaI restriction fragment (containing the 5′ region of dhaX from pMP37) was ligated to the large XbaI/HpaI restriction fragment from pRJ25 to generate pMP38, in which the Streptomyces lividans glucose isomerase promoter (SEQ ID NO:1) drives expression of the K. pneumoniae dhaB1-3,X operon using the native ribosome-binding site.
Example 7
Construction of SYCO Plasmids for Production of 1,3-Propanediol
To produce 1,3-propanediol from glucose in an E. coli host, several operons from different sources can be expressed. These include genes coding for a glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate phosphatase, and glycerol dehydratase activity. These genes may come from sources such as the dha operon from Klebsiella pnuemoniae (containing dhaR, dhaT, dhaX, and dhaB1-3), and the orf operon also from Klebsiella pnuemoniae (containing orfYXW), and an operon containing DARI and GPP2 from Saccharomyces. In order to maintain strain stability in fermentation it is preferable to maintain as few plasmids as possible in the E. coli host. To this end a series of plasmids were constructed to enable cloning of at least three different operons on a single plasmid. Three transcriptional terminators were used to flank unique cloning sites in order to prevent read-through of the RNA polymerase. These transcriptional terminators included the tonB terminator, thr attenuator and aspA terminator. The tonB terminator is a bi-directional rho-independent terminator located between the E. coli tonB gene and an opposing gene (Postle, K. and Good, R. F., Cell, 41:577-585 (1985)). The thr attenuator facilitates transcriptional termination of the E. coli threonine operon (Lynn et al., J. Mol. Biol., 183:529-541 (1985)). The aspA terminator facilitates transcriptional termination of the E. coli aspartase operon (Takagi et al., Nucleic Acid Research. 13(6):2063-2072 (1985)).
Construction of pRJ50 Comprising Three Transcriptional Terminators Flanked by Unique Cloning Sites:
A synthetic DNA fragment (comprising the tonB, thr, and aspA transcriptional terminators (SEQ ID NO:57) and several restriction sites) was assembled using PCR-mediated overlap extension (Horton et al., BioTechniques, 8:528-535, (1990)). Two 100 base oligonucleotides (SEQ ID NOs:58-59) that complement each other for a span of 25 base pairs at the 3′ ends were annealed to generate a 175-base DNA fragment (SEQ ID NO:60). Two additional oligonucleotide primers (SEQ ID NOs:61-62) were used to further amplify the 175-base fragment, which is flanked by EcoRI and KpnI restriction sites. The 175-base pair PCR product was digested with EcoRI and KpnI and subcloned into EcoRI/KpnI digested plasmid pCL1925 to generate pRJ50 (SEQ ID NO:79).
Construction of an Expression Cassette for dhaR, orfY, orfX, orfW and dhaB(1.2.3,X):
A derivative of plasmid pDT29 was constructed in which all except the first five and the last five codons (plus stop codon) of the gene dhaT were deleted by a technique known as PCR-mediated overlap extension. Using pDT29 as the template, two primary PCR products were generated using the following primers:









SEQ ID NO:63 =
5′GAC GCA ACA GTA TTC CGT CGC3′;



 


SEQ ID NO:64 =
5′ATG AGC TAT CGT ATG TTC CGC CAG



GCA TTC TGA GTG TTA ACG3′;


 


SEQ ID NO:65 =
5′GCC TGG CGG AAC ATA CGA TAG CTC



ATA ATA TAC3′;


 


SEQ ID NO:66 =
5′CGG GGC GCT GGG CCA GTA CTG3′.






SEQ ID NO:65 was paired with SEQ ID NO:66 to generate a product of 931 bps and encompassing nucleic acid including 5′ dhaB1 (to unique Scal site), all of orfY, and the first five codons of dhaT. SEQ ID NO:63 was paired with SEQ ID NO:64 to generate a product of 1348 bps and encompassing nucleic acid including the last five codons (plus stop codon) of dhaT, all of orfX, all of orfW, and 5′ dhaR (to unique SapI site). The 15 bases at the 5′ end of SEQ ID NO:64 constitute a tail that is the inverse complement of a 15-base portion of SEQ ID NO:65. Similarly, the 11 bases at the 5′ end of SEQ ID NO:65 constitute a tail that is the inverse complement of an 11-base portion of SEQ ID NO:64. Thus, the two primary PCR products were joined together after annealing (via 26-bp tail overlap) and extending by PCR, to generate a third nucleic acid product of 2253 bps. This third PCR product was digested with SapI and ScaI and ligated into pDT29 which was also digested with SapI and ScaI, to generate the plasmid pKP32, which is identical to pDT29, except for the large, in-frame deletion within dhaT.
Construction of Plasmids for Expression of orfWXY and dhaB1-3 Containing Different GI Promoter Variants:
The orf operon from pKP32 was PCR-amplified (SEQ ID NOs:80-81) with HindIII at the 5′ end and AvrtIl at the 3′ end, and subcloned between HindIII and AvrIl in pLitmus28 (New England Biolabs) to generate pKP38. The EcoRI/HindIII restriction fragment (containing the GI mutant promoter P1.6 (SEQ ID NO:9) from pMP38/1.6) was subcloned between EcoRI and HindIII in pKP38 to generate pKP39. The AvrIl/XbaI restriction fragment (containing the dhaB expression cassette from pMP38/1.6) was subcloned between AvrIl and XbaI in pLitmus28 (New England Biolabs) to generate pMP39. The AvrIl/XbaI. restriction fragment (containing the dhaB expression cassette from pMP39) was subcloned into the AvrIl site of pRJ50 to generate pSYCO11. The AvrIl restriction fragment (containing the orf expression cassette from pKP39) was subcloned into the NheI site of pSYCO11 to generate pSYCO12. The plasmids pSYCO11 and pSYCO12 are identical except that pSYCO11 does not contain the orf operon.
The EcoRI/HindIII restriction fragment (containing the GI mutant promoter P1.5 (SEQ ID NO:10) from pMP38/1.5) was subcloned between EcoRI and HindIII in pKP38 to generate pKP40. The AvrIl restriction fragment (containing the orf operon driven by P1.5 from pKP40) was subcloned into the NheI site of pSYCO11 to generate pSYCO13. The AvrIl/NotI restriction fragment (containing the P1.6 and 5′ end of dhaB1in pSYCO13) was replaced with the corresponding AvrIl/NotI restriction fragment from pMP38/1.5 to generate pSYCO19.
Construction of pSYCO101, pSYCO103, pSYCO106 and pSYCO109 Vectors with Three Operons each Isolated by Transcriptional Terminators:
A double-stranded nucleic acid linker (SEQ ID NO:67) was subcloned between the XbaI and SmaI restriction sites in pCL1920 (GenBank AX085428) to generate pCR-pCL1920. The glycerol pathway expression cassette in pAH48 comprising the trc promoter which was derived from pTrc99A (Amersham Pharmacia Biotech, Piscataway, N.J.), the coding sequences for DAR1 and GPP2, of S. cerevisiae, and the terminator rrnBT1T2 (from pTrc99A) was PCR- amplified (SEQ ID NOs:68-69) and subcloned into the SrfI restriction site of pCR-pCL1920 to generate pAH105 (SEQ ID NO:70).
The PvuIl(2)/PvuIl(4) restriction fragment (containing the DAR1/GPP2 expression cassette from pAH105) was subcloned into the Bst1107I site of pSYCO12 to generate pSYCO101 (SEQ ID NO:71). The DAR1/GPP2 operon is in the opposite orientation relative to the orf operon and the dhaB operon. The NheI restriction fragment (containing the DAR1/GPP2 expression cassette from pAH105) was subcloned into the XbaI site of pSYCO19 to generate pSYCO103 (SEQ ID NO:72).
The plasmid pSYCO103 comprises (a) a set of two exogenous genes obtained from Saccharomyces cerevisiae (DAR1 (a gene encoding glycerol-3-phosphate dehydrogenase) and GPP2 (a gene encoding glycerol-3-phosphatase)); (b) a set of three exogenous genes obtained from Klebsiella pneumoniae (dhaB1(a gene encoding the “α” subunit of glycerol dehydratase), dhaB2 (a gene encoding the “β” subunit of glycerol dehydratase), and dhaB3 (a gene encoding the “γ” subunit of glycerol dehydratase)); and (c) a set of two exogenous genes obtained from Klebsiella pneumoniae (dhaBX (a gene encoding the “α” subunit of dehydratase reactivation factor) and orfX (a gene encoding the “β” subunit of dehydratase reactivation factor)). In pSYCO103 the DAR1/GPP2 operon is in the same orientation relative to the orf operon and the dhaB operon.
The NheI restriction fragment (containing the DAR1/GPP2 expression cassette from pAH105) was subcloned into the XbaI site of pSYCO12 to generate pSYCO106 (SEQ ID NO:73). The DAR1/GPP2 operon is in the same orientation relative to the orfoperon and the dhaB operon. The PmIl/NotI restriction fragment in pSYCO106 was removed and replaced with the overlapping StuI/NotI restriction fragment from pSYCO106, resulting in a 141 base pairs deletion near the 3′ end of orfW to generate pSYCO109 (SEQ ID NO:74).
Example 8
A Novel Nucleotide Sequence with Ten Rare Restriction Enzyme Sites Useful for Cloning
A novel nucleotide sequence was designed to encode ten rare restriction endonuclease sites useful for cloning of additional genes, operons, or cassettes and as sites for transferring cassettes from this plasmid to another. The plasmid pSCYCO106deltaS was constructed by restricting pSYCO106 with SpeI, filling in the ends with Klenow and religating. The pSYCO106deltaS was digested with EcoRI to isolate the vector backbone and then recircularized by ligation to form pSpREPds. Oligonucleotides (SEQ ID NOs:75-76) were annealed at 60° C. and digested with KpnI/StuI. The multiple cloning fragment (SEQ ID NO:77) contains recognition sites for the following enzymes: NheI, RsrII, SacI, AgeI, SnaBI, AscI, PacI, NsiI, MluI, and SapI. The fragments were gel-purified and cloned to pSpREPds to form pSpREPmcs. The pSpREPmcs was linearized with EcoRI and the EcoRI fragments (containing the pathway genes from pSYCO106deltaS and pSYCO109) were ligated to pSpREPmcs to form pSYCO106mcs (SEQ ID NO:78) and pSYCO109mcs (SEQ ID NO: 30), respectively.
Example 9
Production of 1.3-Propanediol Using E. Coli Strain RJ8N/PSYCO101
The plasmid pSYCO101 (SEQ ID NO:71) was used to transform electrocompetent E coli RJ8n cells, resulting in the E. coli strain, RJ8n/pSYCO101.
RJ8n/pSYCO101 was pre-cultured for seeding a fermenter in 2YT medium (10 g/L yeast extract, 16 g/L tryptone, and 10 g/L NaCl) containing 50 mg/L spectinomycin. Cultures were started from frozen stocks (10% glycerol as cryoprotectant) in 500 mL of medium in a 2-L Erlenmeyer flask, grown at 35° C. in a shaker at 250 rpm until an OD550 of approximately 1.0 was reached and used to seed the fermenter.
The following components were sterilized together in the fermenter vessel: 45 g KH2PO4, 12 g citric acid monohydrate, 12 g MgSO4.7H2O, 30 g yeast extract, 1.8 g ferric ammonium citrate, 5 mL Mazu DF204 as antifoam, 1.2 g CaCl2.2H2O, 7.2 mL sulfuric acid and 60 mL of a trace element solution. After sterilization, the pH was raised to 6.8 with 20-28% NH4OH and the following components were added: 0.30 g spectinomycin, and glucose (from a 67 weight % feed). The solution of trace elements contained (g/L): citric acid. H2O (4.0), MnSO4.H2O (3.0), NaCl (1.0), FeSO4.7H2O (0.10), CoCl2.6H2O (0.10), ZnSO4.7H2O (0.10), CuSO4.5H2O (0.010), H3BO3 (0.010), and Na2MoO4.2H2O (0.010). After inoculation, the volume was 6.0 L and the glucose concentration was 10 g/L.
A 15-L stirred tank fermenter was prepared with the, medium described above. The temperature was controlled at 34° C. and aqueous ammonia (20-28 weight %) was used to control pH at 6.8. Dissolved oxygen (DO) control was set at 10% and back pressure was controlled at 0.5 bar. Except for minor excursions, glucose was maintained at between 10 g/L and 25 g/L with a 67% (wt) feed. An addition of 10 mg vitamin B12 was made at 10 h elapsed fermentation time and a co-feed (2.64 mg/h of a 0.0167 mg/mL solution) begun one hour later. A titer of 99 g/L 1,3-propanediol was obtained after 64 h.

## Claims

1. A pSYCO109mcs plasmid consisting of SEQ ID NO:30.