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WO1995021260A1 - Vecteurs d'acide nucleique a expression intensifiee - Google Patents

Vecteurs d'acide nucleique a expression intensifiee

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Publication number
WO1995021260A1
WO1995021260A1 PCT/US1995/001471 US9501471W WO9521260A1 WO 1995021260 A1 WO1995021260 A1 WO 1995021260A1 US 9501471 W US9501471 W US 9501471W WO 9521260 A1 WO9521260 A1 WO 9521260A1
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WIPO (PCT)
Prior art keywords
phb
host cell
nucleic acid
promoter
plasmid
Prior art date
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Ceased
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PCT/US1995/001471
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English (en)
Inventor
John P. Kidwell
Douglas E. Dennis
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Virginia Innovation Partnership Corp
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Center for Innovative Technology
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Priority to EP95909483A priority Critical patent/EP0742832A1/fr
Publication of WO1995021260A1 publication Critical patent/WO1995021260A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the present invention relates generally to nucleic acid vectors for the expression of a desired product arising from a desired nucleic acid sequence, including downstream products made by enzymes encoded by the desired nucleic acid sequence.
  • nucleic acid vectors suitable for the cloning, sub- cloning and expression of nucleic acid sequences, or molecules, such as genes are well known in the fields of genetic engineering and biotechnology.
  • One such group of vectors are used largely for cloning and sub-cloning, such as pBR322, considered to be one of the workhorses in these fields, which contains numerous restriction endonuclease recognition sites suitable for the insertion of a desirable nucleic acid sequence (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; this reference, and all other references cited herein, are hereby expressly incorporated by reference in their entirety).
  • Another group of such vectors include multicopy expression vectors able to produce large amounts of a desired product encoded by, or resulting from, the desired nucleic acid sequence, such as a gene.
  • Desirable products include mRNA, proteins such as enzymes, and products produced by the action of an encoded enzyme.
  • expression vectors act by providing a regulable promoter that is induced to produce large amounts of mRNA encoded by the desired nucleic acid sequence in the presence of an inducer molecule.
  • lac promoter which can be derepressed by the addition of a chemical inducer, such as isopropyl- ⁇ -D- thiogalactoside (IPTG), resulting in increased production from the desired nucleic acid sequence (Jacob and Monod, J. Mol. Biol. 3: 318-356, 1961; Maniatis et al., supra).
  • a chemical inducer such as isopropyl- ⁇ -D- thiogalactoside (IPTG)
  • IPTG isopropyl- ⁇ -D- thiogalactoside
  • Such vectors permit close control of the production of the desired gene product and provide an ample amount of mRNA corresponding to the desired nucleic acid sequence, but the addition of the inducer molecule can be very expensive.
  • runaway replicon expression vectors produce large amounts of the operon itself by providing large quantities of the nucleic acid vector, as with pOU71 (Larsen, et al., Gene 28: 45-54, 1984) or pRA89 and pRA90 (Benzon Pharma A/S, Helseholmen 1, P.O. Box 1185, DK-2650 Hvidovre, Denmark).
  • pOU71 Lisen, et al., Gene 28: 45-54, 1984
  • pRA89 and pRA90 Benzon Pharma A/S, Helseholmen 1, P.O. Box 1185, DK-2650 Hvidovre, Denmark.
  • Such a vector may not need the addition of an inducer molecule (the vector copy number (i.e., the gene dosage) can be increased by an upshift in temperature, for example), but the vector may not provide a large quantity of mRNA, and may not provide as rapid a production of a the desired product.
  • the vectors when such vectors are grown (in a suitable host cell and growth medium), the vectors require a selective marker gene in order for the vector to be maintained in its host cell over a number of generations.
  • selective marker genes include, for example, the chloramphenicol resistance gene (cat) or the ampicillin resistance gene (bla), which genes in turn require the addition of a selective pressure agent (such as an antibiotic) in the growth medium.
  • PHAs poly- ⁇ - hydroxyalkanoates
  • PHAs can be, for example, "random" copolymers wherein the copolymer comprises poly- ⁇ -hydroxybutyrate (PHB) and poly- ⁇ -hydroxyvalerate (PHV) dispersed randomly in the polymer backbone, or "semi-random," or blocked, copolymers wherein the copolymer comprises long or short chains of one particular PHA, for example PHB, that is separated by long or short chains of other PHAs, for example, randomly dispersed PHB and PHV.
  • PHAs are synthesized by the action of three enzymes: ⁇ -ketothiolase, acetoacetyl-CoA reductase, and PHB synthase (Oeding and Schlegel, Biochem. J.
  • ⁇ -ketothiolase condenses two acetyl-CoA molecules to acetoacetyl-CoA.
  • Acetoacetyl-CoA reductase reduces this compound to ⁇ -hydroxybutyryl-CoA.
  • PHB synthase typically polymerizes ⁇ -hydroxybutyryl-CoA into PHB, although other PHAs are produced under particular conditions.
  • PHB synthase, ⁇ -ketothiolase, and acetoacetyl-CoA reductase are encoded in the phb operon, consists of three genes designated phbC, phb A, and phbB.
  • Clones that carry the phb operon on a multicopy plasmid can produce PHAs to levels as high as 80% (Janes et al., Novel Biodegradable Microbial Polymers, 175, 1990) and the cost of producing about 22,000 pounds of PHAs in a 100,000 liter fermentor would be about $22,000 if no antibiotic were necessary. But, the addition of the commonly used antibiotic chloramphenicol (at a concentration of 25 ⁇ g/ml) increases the cost by more than $14,000, and the addition of IPTG (at a concentration of 1 mM (0.238g/L)) increases the cost by more than $749,000.
  • One advantage in the expression of a desired product arising from a desired nucleic acid sequence would be a nucleic acid vector capable of being induced to rapidly produce large amounts of the desired gene product without the addition of an inducer molecule and capable of being maintained in a host cell without the addition of a selective pressure agent to the growth medium.
  • the present invention provides nucleic acid vector constructs capable of regulating the transcription and/or translation of a desired nucleic acid sequence, such as a gene or an operon.
  • the present invention also provides methods of using such constructs to produce a desired product from the desired nucleic acid sequence, host cells transformed with such constructs, and desired products from the desired nucleic acid sequence.
  • the present invention provides a runaway replicon nucleic acid vector construct, comprising a) a promoter that is negatively regulated by a repressor molecule, b) an operator region capable of binding the repressor molecule, c) a desired nucleic acid sequence, wherein the promoter is operably linked to the operator region and the desired nucleic acid sequence, and d) a stabilization locus.
  • the promoter comprises a -35 region of a trp promoter operably linked to a -10 region of a lac promoter, and an operator region of the lac promoter, such as a tac promoter;
  • the runaway replicon includes a ⁇ pR promoter operably linked to a repA gene; and/or the stabilization locus is parB.
  • the vector construct further comprises a consensus Shine-Dalgarno sequence operably linked to the desired nucleic acid sequence.
  • a Shine-Dalgarno sequence is a lac Shine-Dalgarno sequence.
  • the present invention provides methods of inducing a vector construct comprising, a) introducing into a host cell a runaway replicon vector construct, comprising i) a promoter that is negatively regulated by a repressor molecule, ii) an operator region capable of binding the repressor molecule, iii) a desired nucleic acid sequence, wherein the promoter is operably linked to the operator region and to the desired nucleic acid sequence, and iv) a stabilization locus; and b) increasing the temperature of the host cell, thereby expressing the desired nucleic acid sequence.
  • the methods further comprise culturing the host cell on an appropriate medium, then increasing the temperature of the host cell, and then further culturing the host cell for a time sufficient to produce a desired product from the desired nucleic acid sequence.
  • the methods comprise the step of isolating the desired product from the host cell.
  • the promoter of the vector construct comprises a -35 region of a trp promoter operably linked to a -10 region of a lac promoter, and an operator region of the lac promoter, such as a tac promoter;
  • the runaway replicon includes a ⁇ pR promoter operably linked to a repA gene; and/or the stabilization locus is parB.
  • the vector construct further comprises a consensus Shine-Dalgarno sequence operably linked to the desired nucleic acid sequence.
  • the host cell is an Enterobacteriaceae host cell, more preferably an E. coli or Klebsiella, particularly Klebsiella aerogenes.
  • the method is performed in a host cell containing an operable lacIQ gene, and/or during step (b), the methods include determining whether the culture of the host cells is growing rapidly or slowly; and then increasing the temperature early in a log phase of a growth cycle of the culture when the culture is fast-growing, or increasing the temperature late in a log phase of a growth cycle of the culture when the culture is slow-growing.
  • the temperature is increased to at least 33°C when the vectors are maintained in Klebsiella and at least 36°C when the vectors are maintained in E. coll
  • the methods further comprise growing the host cell for multiple generations and not adding a selective pressure agent , typically an antibiotic, to the medium.
  • a selective pressure agent typically an antibiotic
  • the present invention provides an Enterobacteriaceae host cell containing a vector construct as described above.
  • the Enterobacteriaceae is E. coli or Klebsiella, particularly Klebsiella aerogenes.
  • Figure 1 depicts the nucleotide sequence of a series of vector constructs having certain transcriptional and/or translational fusions to the phb operon.
  • the nucleotide sequence denoted “a” comprises a phb promoter operably linked to a putative phbC Shine-Dalgarno sequence (denoted “SD” in the figure) (Seq. ID No. ). This nucleotide sequence is present in the plasmids pJM9131 and pJM9117.
  • the nucleotide sequence denoted “b” comprises a tac promoter and a phbC Shine-Dalgarno sequence wherein there is an approximately 72 base pair leader sequence prior to the structural gene (Seq.
  • This nucleotide sequence is present in the pJM9229 and pJM9236 vector constructs.
  • the nucleotide sequence denoted “c” comprises a tac promoter and a putative phbC Shine-Dalgarno sequence wherein there is an approximately 355 base pair leader prior to the structural gene (Seq. ID No. ).
  • This nucleotide sequence is present in the pJM9232 and pJM9238 vector constructs.
  • the nucleotide sequence denoted” comprises a tac promoter and two lac Shine-Dalgarno sequences (Seq. ID No. ). This nucleotide sequence is present in the pJM9375 vector construct.
  • the nucleotide sequence denoted "e” comprises a tac promoter, two lac Shine-Dalgarno sequences and a phbC Shine-Dalgarno sequence (Seq. ID No. ).
  • This nucleotide sequence is present in the pJM9376 vector construct.
  • Figure 2 depicts a map of the vector construct pJM8801 (formerly known as p4A), which contains the phb operon, and the construction of the vector construct pJM9002, which is an 8.10 kb plasmid produced by cleaving the Eco I/Hind III phb operon-containing fragment from p4A and ligating the fragment into the same sites of pBluescript SK+.
  • Figure 2 also depicts the construction of the pJM9131 vector construct, which is an 8.55 kb plasmid derived from pJM8801, but a kanamycin gene block is located at the Eco RI site, and ampicillin resistance has been removed by Dra I digestion.
  • Figure 2 further depicts the construction of the pJM9226 vector construct, which is a 3.10 kb plasmid comprising the tac promoter and a lac Shine-Dalgarno sequence ligated into the 3 kb Hind Ill-Bam HI fragment of pJM9002.
  • Figure 3 depicts a map of the vector construct pJM8703, which is an 8.50 kb plasmid comprising the phb operon cloned into pTZl ⁇ U from United States Biochemicals. The transcription pathway starts at the Kpn I site and ends at the Eco RI site.
  • Figure 3 also depicts the construction of the pTZ18U-4c vector construct, which is a 7.00 kb plasmid produced by digesting pJM8703 with Sph I and Bam HI, then deleting from the Bam HI to base 835 of the published sequence followed by religation.
  • Figure 3 further depicts the construction of the pSP72/PHB vector construct, which is a 8.50 kb plasmid comprising the Eco Rl/Pst I fragment from pJM8703 ligated into the same sites of pSP72 (Promega).
  • Figure 3 also depicts the construction of the pJM8905 vector construct, which is an 8.49 kb plasmid comprising a Pst l partial of Eco Rl-digested pJM8703 and ligated into pSP72, which was in turn digested with Xho I and Eco RI to provide a phb operon-containing fragment that was ligated into pGEM7Zf+.
  • Figure 3 further depicts the construction of the pJM9117 vector construct, which is a 10.13 kb plasmid that contains the phb operon from pGEM7-PHBr cloned into the Bam H I site of pRA89, which is a 5.13 kb plasmid that is inducible above 41°, and typically has a basal copy number of 1.
  • the pJM9117 vector construct was formerly known as pRA89/PHB/Fo.
  • Figure 4 depicts the construction of the pJM9227 vector construct, which is a 7.20 kb plasmid comprising the Bst BI-Bam HI fragment from pJM8905 inserted into the Bam HI site of pJM9226 to provide a vector construct having a tacr.phb fusion.
  • Figure 4 also depicts construction of the pJM9228 vector construct, which is an 8.50 kb plasmid comprised of a kanamycin gene block inserted into the Eco RI site downstream of the phb operon of pJM9227 (i.e., pJM9228 is similar to pJM9227 with kanamycin resistance).
  • Figure 4 further depicts the construction of the pJM9229 vector construct, which is a 7.80 kb plasmid comprising a 0.71 kb deletion in the bla gene of pJM9228 (i.e., the ampicillin resistance of pJM9228 was deleted).
  • Figure 5 depicts the construction of the pJM9230 vector construct, which is a 7.50 kb plasmid that includes the phb operon-containing Hind lll-Eco RI fragment from pTZ18U-4c ligated into the Bam HI site of pJM9226.
  • Figure 5 also depicts the construction of the pJM9231 vector construct, which comprises an 8.80 kb plasmid including a kanamycin gene block inserted into the Spe I site downstream of the phb operon in pJM9230 (i.e., pJM9231 is similar to pJM9230 with kanamycin resistance).
  • Figure 5 also depicts the construction of the pJM9232 vector construct, which is an 8.10 kb plasmid, including a 0.71 kb deletion in the bla gene of pJM9231 (i.e., the ampicillin resistance of pJM9231 was deleted).
  • Figure 6 depicts the construction of the pJM9233 vector construct, which is a 9.10 kb plasmid including the Hind ⁇ ll-Eco RI fragment from pJM9227 (which contains the tac:: phb fusion), ligated into the filled-in Bam HI site of pRA89, and the construction of the pJM9234 vector construct, which is a 9.10 kb plasmid similar to the pJM9233 vector construct, except that the phb operon is in the reverse orientation.
  • Figure 7 depicts the construction of the pJM9235 vector construct, which is a 9.40 kb plasmid that is similar to the pJM9233 vector construct, except that the insert was placed in the pRA90 vector construct, which is a 5.37 kb plasmid that is inducible at 41°, has a basal copy number of 1 and is resistant to chloramphenicol at 30 ⁇ g/ml.
  • Figure 7 also depicts the construction of the pJM9236 vector construct, which is a 9.40 kb plasmid that is similar to the pJM9235 vector construct, except that the phb operon is in the reverse orientation.
  • Figure 8 depicts the construction of the pJM9237 vector construct, which is a 9.70 kb plasmid comprising the Hind Wl-Spe I fragment from pJM9230 vector construct (which contains the tacAeader-phb operon fusion) ligated into the filled-in B ⁇ m HI site of pRA90, and the pJM9238 vector construct, which is a 9.70 kb plasmid similar to the pJM9237 vector construct except that the phb operon is in the reverse orientation.
  • Figure 9 depicts the pMS421 vector construct, which is a 5.5 kb plasmid containing the l ⁇ c I gene.
  • Figure 10 depicts a series of graphs indicating the synthesis of phb operon gene products in the E. coli strains HMS174 pJM9131 (panel a), HMS174 pJM9232 pMS421 (panel b), HMS174 pJM9117 (panel c), and HMS174 pJM9238 (panel d), as a function of percentage of total protein over time.
  • Figure 11 depicts the construction of the pJM9275 and pJM9376 vector constructs, which are 7.20 kb plasmids constructed by Exo III deletion from the Bst BI site in pJM9230 to potentially remove all or part of the native phb Shine-Dalgarno sequence, while inserting a consensus Shine-Dalgarno sequence.
  • Figure 12 depicts a series of graphs indicating the production of PHB in the E. coli strains HMS174 pJM9238 (panel a), HMS174 pJM9117 (panel b), HMS174 pJM9231 pMS421 (panel c), and HMS174 pJM9232 pMS421 (panel d), as a function of time and glucose consumption.
  • Figure 13 depicts a pair of graphs comparing PHB yield as a percentage of dry weight in clones containing runaway replicon vector constructs.
  • Figure 14 depicts a pair of graphs comparing plasmid copy number in clones containing a runaway replicon vector construct.
  • Figure 15 depicts a graph indicating a comparison of PHB production in clones containing a multicopy tac:: phb vector construct.
  • Figure 16 depicts a graph indicating PHB production in E. coli strain HMS 174 pJM9238 at different incubation temperatures.
  • Figure 17 depicts a graph indicating PHB yield in E. coli strain HMS 174 pJM9238 as a function of the optical density of a culture at the time of induction.
  • Figure 18 depicts a graph indicating PHB yield in E. coli strain HMS 174 pJM9238 with and without chloramphenicol.
  • Figure 19 depicts a graph indicating a comparison of PHB production in clones containing transcriptional and translational fusions (pJM9375 and pJM9376) versus a vector construct having only a transcriptional modification (pJM9232).
  • Figure 20 depicts a pair of graphs as follows: Panel a depicts the stability of plasmid pJM9238 Klebsiella strain KC2671 over approximately 120 generations when grown in media without chloramphenicol (or other antibiotics). Panel b depicts PHB production in KC2671 pJM9238 at 31°C and 33°C.
  • Figure 21 depicts a pair of graphs as follows: Panel a depicts the production of PHB in mg/ml in KC2671 pJM9238 over time. The graph also indicates the total dry cell weight of the Klebsiella host cells including the PHB. Panel b depicts a comparison of PHB yield in KC2671 for plasmids pJM9131 and pJM9238.
  • the present invention provides nucleic acid vector constructs suitable for introduction into an appropriate prokaryotic host wherein the vector constructs provide for stable, regulatable overproduction of a desired product encoded by, or resulting from, a desired nucleic acid sequence (typically a gene), but without the addition of a chemical inducer or a selective pressure agent to a growth medium suitable for host cells containing the inventive nucleic acid vector constructs.
  • a desired nucleic acid sequence typically a gene
  • the present invention provides a vector construct comprising (a) a negatively regulated promoter operably linked to the desired nucleic acid sequence, (b) a runaway replicon, providing multiple copies of the vector construct upon heat induction, and (c) a stabilization locus.
  • the vector construct will typically be a plasmid, but viral vectors, cosmids, and other nucleic acid vector constructs are also included within the scope of the present invention.
  • the vector construct further comprises a consensus Shine-Dalgarno sequence (preferably a lac Shine-Dalgarno sequence), operably linked to the desired nucleic acid sequence, thereby providing for increased translation of the desired nucleic acid sequence;
  • the consensus Shine- Dalgarno sequence may either replace the native Shine-Dalgarno sequence or it may be in addition to such native sequence.
  • Representative embodiments of suitable promoters and Shine-Dalgarno sequences are depicted in Figure 1 (Seq. ID Nos. to ).
  • the present invention also provides methods of producing a desired product encoded by, or resulting from, the desired nucleic acid sequence, bacterial host cells transformed with such vector constructs, and desired products produced according to the methods of the present invention.
  • the negatively regulated promoters of the present invention are strongly expressed and tightly regulated promoters, and are operably linked to the desired nucleic acid sequence.
  • Such promoters can be controllably “turned on” and “turned off by titrating out the effects of a repressor, or de-inducer, in the cell.
  • When “turned on,” such promoters permit substantially uninterrupted transcription of the desired nucleic acid sequence operably linked thereto (and are not repressed by substances found in the host cell).
  • When “turned off,” such promoters do not permit any substantial transcription of the gene.
  • the -35 region of such a negatively regulated promoter typically comprises an approximately 6- to 12-base sequence centered around the -35 nucleotide (plus or minus two or three nucleotides, measured from the transcription initiation site).
  • the -35 region of the trp promoter includes the nucleotide sequence TTGACA (Darnell, et al., Mol. Cell Biol, 270-
  • the -10 region also known as a Pribnow box, typically comprises an about 6-base sequence centered around the -10 nucleotide (plus or minus two or three nucleotides, also measured from the transcription initiation site). Also for example, the lac -10 region includes the nucleotide sequence TATAAT (Darnell, supra; Seq. ID No. ).
  • the negatively regulated promoter comprises a -35 region of a trp promoter operably linked to a -10 region of a lac promoter, the promoter operably linked to (and typically overlapping) an operator region of a lac promoter, such promoter being further operably linked to the desired nucleic acid sequence, thereby providing multiple copies of mRNA encoded by the desired nucleic acid sequence.
  • a promoter are the tac promoter (Russell and Bennett, Gene 20:231, 1982) and the trc promoter (Borel et al., FEBS 324: 162, 1993). Representative examples of a tac promoter are found in Figure 1 (Seq. ID Nos. to ).
  • promoters are also suitable for use in the present invention, provided that such promoters are repressed when present in lower numbers in a cell than the given promoter's repressor molecule, and that an increase in the number of operators (i.e., repressor binding sites) effectively titrates out the effects of the repressor molecules, thereby inducing transcription of the desired gene.
  • a promoter is an unaltered trp promoter (Yansura and Henner, Meth. Enz. 185: 54-61, 1990).
  • Yansura and Henner, Meth. Enz. 185: 54-61, 1990 A person having ordinary skill in the art in light of the present specification would be able to utilize other promoters in the vector constructs, methods and other aspects of the invention.
  • a promoter is operable can be readily determined by a person of ordinary skill in the art in light of the present specification, by screening for the presence or absence of a desired product arising from the desired nucleic acid sequence (for example, by examining cells under a light microscope for the presence of PHA and/or PHB), or for the presence or absence of mRNA produced from the desired nucleic acid sequence (for example by hybridization assay).
  • initiation of transcription may be repressed by binding a repressor, such as the lad gene product, to the operator, which is located between the promoter and the phb operon.
  • a repressor such as the lad gene product
  • IPTG isopropyl- ⁇ -D- thiogalactoside
  • Such inducers may include glucose- ⁇ -galactoside (lactose), glucose- ⁇ -galactoside (melibiose), and other lactose analogues such as methyl- ⁇ - galactoside and methyl- ⁇ -thiogalactoside (Jacob and Monod, J. Mol. Biol. 3: 318-356, 1961).
  • the runaway replicons of the present invention can be controllably induced and, upon induction, significantly increase the copy number of the vector construct in the cell.
  • the runaway replicon is controlled by temperature.
  • the runaway replicon includes the repA gene, which encodes a protein that is required for the initiation of plasmid replication, under the control of the ⁇ pR promoter (Nordstrom and Uhlin, Biotechnology 10:661, 1992).
  • the ⁇ cI857 gene encodes a heat-sensitive repressor that actively inhibits transcription from the ⁇ pR promoter at low temperature, but that is inactive at high temperatures.
  • the incorporation of the ⁇ cI857 gene in a host cell permits repression of the ⁇ pR promoter at a low temperature.
  • low temperatures such as 30°C
  • high temperatures such as 42°C
  • synthesis of repA mRNA increases, and the vector construct copy number is high.
  • the vector constructs of the present invention further comprise a stabilization locus.
  • Suitable stabilization loci include parB (Gerdes, K., Bio/Technology (5:1402-1405, 1988), ccd, which appears to operate by a mechanism that involves post-segregational mortality of cells that lose a plasmid carrying the ccd locus (Gerdes, supra), the pemK/peml system (Tsuchimoto, S. et al., J. Bact.
  • the vector constructs of the present invention comprise a novel expression system in which the copy number and the transcription of the desired nucleic acid sequence are both efficiently controlled by temperature, even when IPTG (or other derepressor or inducer) is not present.
  • the promoter includes a lac operator region
  • the copy number of the vector construct is lower than the number of lad repressor proteins present in the cell (such number is typically about 5-10 proteins per cell (Muller-Hill et al., Proc. Natl Acad. Scl 59:1259, 1968)).
  • Such a number of lad repressor molecules is sufficient to substantially repress transcription of the phb operon at low temperatures.
  • the vector constructs of the present invention provide the highly advantageous, and unexpected, result that the system is stable without the addition of antibiotics or other selective pressure agents to retain the vector construct. It is believed that the vector constructs of the present invention are stable without the provision of a selective pressure agent in the growth medium even without a stabilization locus, and thus a preferred embodiment of the present invention comprises a vector construct and method able to provide the above-discussed superior and unexpected results, comprising a negatively regulated promoter operably linked to a desired nucleic acid sequence, and comprising a runaway replicon, but without a stabilization locus.
  • the present invention provides vector constructs in which the native Shine-Dalgarno sequence (e.g., the native phbC Shine-Dalgarno sequence) is supplemented by or replaced with a consensus Shine-Dalgarno sequence, preferably the lac Shine-Dalgarno sequence.
  • a Shine-Dalgarno sequence is a sequence located about 10 bases to the 5' side of the start codon (typically AUG) of an mRNA sequence (Zubay, Biochemistry, 944-45, 1983).
  • the consensus Shine- Dalgarno sequence comprises AGGA, although other suitable Shine-Dalgarno sequences could be easily utilized by a person having ordinary skill in the art in light of the present specification. Determination of the effectiveness of a Shine- Dalgarno sequence is also well within the skill of the art in light of the present specification, for example by screening for mRNA copy number.
  • the present invention provides methods of producing large amounts of a desired product encoded by, or resulting from, the desired nucleic acid sequence, such as mRNA, proteins such as enzymes, or a product produced by the action of an encoded enzyme (such as PHAs), utilizing the vector constructs described above.
  • such methods include elevating the temperature of a culture at a certain time point in order to maximize production.
  • the temperature is preferably elevated at a later time in the log phase of the growth curve.
  • the temperature is preferably elevated earlier in the log phase of the growth curve.
  • Whether a culture is slow growing or fast growing will depend upon such factors as growth media, strain background, temperature, and aeration. In light of the present specification, determination of whether a culture is slow growing or fast growing and the preferred time at which to induce the culture involves routine experimentation well within the ordinary skill in the art.
  • the present invention provides prokaryotic host cells transformed by the vector constructs described above.
  • Various prokaryotic host cells may be utilized within the context of the present invention.
  • preferred prokaryotic host cells should have a well-characterized genetic system, including known cloning vectors and methods of genetic manipulation. They should also preferably grow well in minimal medium, ideally to a high cell density, without any special requirements (physical or physiological).
  • Representative examples of such host cells include members of the Bacillaceae, Nocardiaceae, Streptomycetaceae, Pseudomonadaceae, Corynebacteria, and Enterobacteriaceae.
  • Preferred host cells in the Family Enterobacteriaceae include Escherichia, Citrobacter, Klebsiella, Enterobacter, and Serratia, as well as Zymomonas and Flavobacterium, which are within the Enterobacteriaceae but of uncertain affiliation.
  • Particularly preferred host cells include E. coli, Klebsiella oxytoca, and Klebsiella aerogenes.
  • Preferred host cells in the Family Pseudomonaceae include P. aeruginosa.
  • the present invention provides an advantageous and unexpected result that the overproduction may be induced by a temperature increase to generally about 32°C to about 35°C, typically about 32.5°C to about 34°C, and preferably to about 33°C.
  • a temperature increase typically above 33°C, and generally above 34°C, results in smaller cell size and decreased yields.
  • prokaryotes may be readily obtained from a variety of commercial sources including, for example, the American Type Culture Collection (ATCC) (Rockville, Maryland). Alternatively, many of the above-described bacteria may be isolated from sources that are known by those of skill in the art to contain such prokaryotes, based upon techniques that are known in the art (See Bergy's Shorter Manual of Determinative Bacteriology, Williams & Wilkins (pub.), John G. Holt (ed.), 8th edition, 1977). Once the host cell has been cultured under conditions and for a time sufficient to generate large amounts of the desired product, the desired product is preferably isolated from the host cell. Isolation may be accomplished by a variety of methods.
  • the host cells may be lysed, and desired product assayed via hybridization or immunological methods, or where the desired product is PHA, the product may be agglomerated, essentially as described in U.S. Application Serial No. 07/528,549.
  • lysozyme plasmids may be introduced into the host cell to aid isolation of the desired product, as described in U.S. Application Serial No. 07/890,925.
  • Examples 1-10, 12 and 13 are directed toward the construction of desired nucleic acid vectors.
  • Examples 11 and 14-19 are directed toward assays for the effectiveness of various aspects of the present invention.
  • Example 1 is directed toward the construction of plasmid pJM9002 by inserting the phb operon-containing gene fragment from plasmid pJM8801 (previously designated p4A) into pBluescript SK + .
  • Example 2 is directed toward the construction of plasmid pTZ18U-4c by deleting a segment containing the phb genes from plasmid pJM8703, which is also known as pTZ-18U-PHB.
  • Example 3 is directed toward the construction of plasmid pJM8905 by transferring a phb operon-containing fragment from pJM8703 into pSP72 to create pSP72/PHB, followed by excision of the phb fragment from pSP72/PHB and inserting it into pGEM-7Zf t" .
  • Example 4 is directed toward the construction of plasmid pJM9131 by the insertion of kanamycin resistance into, and the deletion of ampicillin resistance from, pJM8801.
  • Example 5 is directed toward the construction of plasmid pJM9117 by the insertion of the phb operon-containing fragment from pJM8703 into pRA89.
  • Example 6 is directed toward the construction of plasmid pJM9226 by the deletion of the phb operon-containing fragment from pJM9002 and the insertion of the t ⁇ c promoter into pJM9002.
  • Example 7 is directed to the creation of t ⁇ cr.phb fusion plasmids pJM9227-pJM9229 by inserting the phb operon-containing fragment from pJM8905 into the t ⁇ c promoter-containing pJM9226.
  • pJM9227 has only ampicillin resistance
  • pJM9228 has both ampicillin resistance and kanamycin resistance
  • pJM9229 has only kanamycin resistance.
  • Example 8 is directed toward the construction of t ⁇ cr.phb fusion plasmids pJM9230-pJM9232 by the insertion of the phb operon-containing fragment from pJM8703 into pJM9226.
  • pJM9230 has only ampicillin resistance
  • pJM9231 has both ampicillin and kanamycin resistance
  • pJM9232 has only kanamycin resistance.
  • pJM9230-pJM9232 differ from pJM9227-pJM9229 in that pJM9230-pJM9232 have a phbC leader of approximately 355-bases that contains a cw-acting element, while pJM9227-pJM9229 have a phbC leader of approximately 72 base pairs without such an element.
  • Example 9 is directed to the construction of runaway replicon tacrphb fusion plasmids pJM9233- ⁇ JM9236 by the insertion into the runaway replicon vectors pRA89 and pRA90 the tacrphb fusion from pJM9227.
  • pJM9233-pJM9236 have both a tac promoter and a heat inducible promoter ( ⁇ pR). These plasmids differ from each other in the orientation and precise placement of t & phb gene fragment within the vector.
  • Example 10 is directed to the construction of runaway replicon tacrphb fusion plasmids pJM9237 and pJM9238, which have an approximately 355-base leader.
  • Example 11 is directed to a graphic analysis of phb operon gene products, which analysis indicates that the phbC gene product (PHB synthase) is subject to post-translational regulation, and therefore is not overproduced by the plasmids constructed pursuant to Examples 1-10.
  • PHB synthase phbC gene product
  • Examples 12 and 13 are directed to the construction of plasmids pJM9375 and pJM9376, which were created by the addition of a consensus Shine-Dalgarno sequence operably linked to the phbC gene.
  • pJM9375 the consensus (lac) Shine-Dalgarno sequence replaced the native phbC Shine- Dalgamo sequence.
  • pJM9376 the consensus (lac) Shine-Dalgarno sequence was added to the native phbC Shine-Dalgarno sequence.
  • Example 14 is directed to a graphic comparison of PHB production in native versus tac promoter clones.
  • Example 15 is directed to a graphic comparison of PHB production in the approximately 72 base pair leader and 355 base pair leader phbC tacrphb fusion constructs.
  • Example 16 is directed to the optimization of PHB production at different temperatures using the heat- inducible plasmid pJM9238.
  • Example 17 is directed to the determination of the optimal cell density during the cell growth cycle for initiation of PHB production using the plasmid pJM9238.
  • Example 18 is directed to a comparison of PHB production using the plasmid pJM9238 with or without chloramphenicol.
  • Example 19 is directed to the quantitation of PHB production in plasmids pJM9375 and pJM9376, each of which contain a tac promoter and a consensus (lac) Shine-Dalgarno sequence.
  • Example 20 is directed to the determination of the stability of PHB-producing plasmids in Klebsiella.
  • Example 21 is directed to the production of PHB in Klebsiella at varying temperatures.
  • Example 22 is directed to the production of PHB in Klebsiella using plasmid pJM9238 based on fed-batch fermentation.
  • Example 22 is also directed to a comparison of PHB production using plasmid pJM9238 versus plasmid pJM9131.
  • phb operon fragment was cloned into pBluescript SK + (Stratagene) as follows.
  • plasmid pJM8801 previously designated p4A in U.S. Application Serial No. 07/890,925
  • the vector pBluescript SK + were digested with the restriction endonucleases -EcoR I and Hind III (Gibco BRL) as described (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982.).
  • the released fragment containing the phb genes from plasmid pJM8801 was ligated into the EcoR l-Hind III digested pBluescript SK + fragment using T4 DNA Ligase (Gibco BRL) as described in Gibco Focus Technical Bulletin 5224-1.
  • the resulting recombinant plasmid was designated pJM9002 ( Figure 2).
  • Plasmid pTZ-18U-PHB (deposited with American Type Culture Collection and assigned ATCC Deposit No. 299006, currently designated as pJM8703 ( Figure 3), was digested with Sph I (which yields a 3' overhang) and Bam HI (which creates a 5' overhang).
  • the resulting linearized fragment containing the phb genes was deleted from the Bam HI end using the procedure of Henikoff (Henikoff, S., Gene 28, 351, 1984) to approximately base 835 in the phb operon sequence previously disclosed (U.S. Application Serial No. 07/705,806).
  • the fragment was then religated using T4 DNA Ligase and the resulting circularized plasmid was designated pTZ18U-4c ( Figure 3).
  • Example 3 Construction of plasmid p JM8905
  • Plasmid pJM8905 was constructed as follows.
  • the linearized plasmid DNA was then partially digested with Pst I as follows. From a lOO ⁇ l digestion reaction, performed as described (Maniatis, supra), 10 ⁇ l aliquots were removed every 2.0 minutes to microplate wells containing 2 ⁇ l of 150 mM EDTA on ice. A total of 12 time-points were taken. Three microliters of 6X loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% (w/v) sucrose) were added to each well.
  • 6X loading buffer 0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% (w/v) sucrose
  • coli strain DH5 ⁇ endAl hsdRll (rk", mk + ) supE44 thi-1 recAl gyrA (Nal r ) relAl A(lacZYA-argF)U169 ( ⁇ 80dlacd(lacZ)M15)).
  • the resulting plasmid was pSP72 containing the Pst I- EcoR I phb fragment in the multiple cloning site.
  • This plasmid, designated pSP72/PHB ( Figure 3) was next digested with EcoR I and oXho I.
  • Kanamycin Resistance GENBLOCK® (Eco RI) (Pharmacia) restriction fragment was ligated into the Eco RI site of plasmid pJM8801 (p4A)
  • the phb fragment was cloned into pRA89 (Benzon Pharma A/S, Helseholmen 1, P.O. Box 1185, DK-2650 Hvidovre, Denmark; Figure 3) as follows. Plasmid p8703 (pTZ-18U-PHB) was digested with Eco RI. The resulting DNA fragment was partially digested with Pst I and the approximately 5 kb fragment containing the entire phb fragment was ligated using T4 DNA Ligase (Gibco BRL) into plasmid pSP73 (Promega) that had been digested with Eco RI and Pst I. The recombinant plasmid was transformed into E. coli strain DH5 ⁇ .
  • This plasmid was purified by the alkaline-lysis method (Maniatis et al., supra). The plasmid was then digested with oXho I and EcoR I to release the DNA fragment containing the phb operon. This fragment was ligated using T4 DNA Ligase into plasmid pGEM-7Zf l" (Promega) that had been digested with Xho I and EcoR I. The resulting plasmid was designated pGEM7f-PHB reverse. Plasmid pGEM7f-PHB reverse was digested with BamR I.
  • the released phb fragment was ligated using T4 DNA Ligase into the Bam HI site of Bam Hi- digested pRA89, which had been treated with Calf Intestinal Alkaline Phosphatase (Boehringer Mannheim).
  • the resulting plasmid designated pJM9117 ( Figure 3), was introduced into E. coli strain HMS 174 recAl hsdR Rif by electroporation as previously described (see U.S. Application Serial No. 08/035,433).
  • the tac promoter was cloned into the plasmid pBluescript SK + (Stratagene) as follows.
  • the vector pJM9002 (described above) was digested with the restriction endonucleases BamR I and Hind III (Gibco BRL) as described (Maniatis et al., supra). This released the phb fragment from the pBluescript plasmid.
  • the tac promoter GENBLOCK® (Hind ILl/BamH I) (Pharmacia) restriction fragment was ligated into the Hind Ul-BamH I-digested pBluescript fragment using T4 DNA Ligase (Gibco BRL) as described in Gibco BRL Focus Technical Bulletin 5224-1.
  • the recombinant plasmid was introduced into E. coli strain DH5 ⁇ by electroporation (Miller, Bacterial Electroporation, Molecular Biology Reports No. 5, Bio-Rad Laboratories, Richmond, CA, 1988) using the GENE PULSER® (Bio-Rad). Electroporation was performed as follows. An isolated colony of E. coli strain DH5 ⁇ was inoculated into a 13 x 100 mm S/P diSPo culture tube (Baxter) containing 3 ml of Luria-Bertani (LB) medium (Maniatis et al., supra).
  • LB Luria-Bertani
  • This culture was grown overnight in a Lab-Line Incubator-Shaker (Lab-Line Instruments, Inc.) at 37°C, 225 rpm. The next morning 1 ml of the culture was inoculated in 50 ml of LB medium in a baffled 250 ml Erlenmeyer flask (Wheaton). Growth of the culture was followed by withdrawing aliquots at regular time intervals and measuring the optical density at 600 nanometers using a Shimadzu UV 160 U Spectrophotometer. The culture was incubated at 37°C, 225 rpm, until the optical density at 600 nanometers reached approximately 0.5. At this time, the culture was placed on ice for 10 minutes.
  • the mixture was subjected to a pulse of 2.5KV, at 200 Ohms and 25 ⁇ farads using a GENE PULSER® (Bio-Rad Laboratories).
  • the bacterial suspension was then transferred to a sterile 13 x 100 mm culture tube containing 3 ml of LB broth.
  • the culture was incubated for 1 hour in a Lab-Line Incubator-Shaker (Lab-Line Instruments, Inc.) at 37°C, 225 rpm.
  • Transformants were selected by spreading the cells on LB agar plates containing 200 ⁇ g/ml ampicillin (Sigma). The plates were incubated at 37°C in a Fisher IsoTemp Oven Model 350D until colonies were visible.
  • Transformants were purified by picking well-isolated colonies and streaking for single colony isolation on LB agar plates containing 200 ⁇ g/ml ampicillin. Purified clones were inoculated into 3 ml of LB media containing 200 ⁇ g/ml ampicillin in 13 xlOO mm culture tubes (Baxter) and incubated overnight at 37°C in a Lab-Line Incubator-Shaker (Lab-Line Instruments, Inc.) at 200 rpm.
  • the plasmid DNA was purified by a modification of the alkaline lysis method as follows: A single colony was inoculated into a 13 x 100 mm culture tube containing 3 ml of LB broth + 200 ⁇ g/ml ampicillin and grown overnight. The culture was pipetted into two sterile 1.5 ml microfuge tubes (West Coast Scientific, Inc.) and pelleted in an Eppendorf Centrifuge 5415C microcentrifuge for two minutes. The supernatant was decanted. Each pellet was resuspended in 1 ml of ice-cold SET butter (20% sucrose, 50 mM EDTA, 50 mM Tris, pH 8.0) and centrifuged two minutes as before.
  • the supernatant was withdrawn with a pipette.
  • the cells were resuspended in a total of 150 ⁇ l of SET buffer and placed on ice.
  • Five microliters of RNase (Boehringer Mannheim) (10 ⁇ g/ml, boiled for 2 minutes) was added and the tube was vortexed.
  • Three hundred-fifty microliters of 0.2 N NaOH/1.0% SDS was added and the tube was inverted several times. The tube was incubated on ice for 20 minutes.
  • the tube was centrifuged for 5 minutes and the supernatant was decanted into a sterile 1.5 ml microfuge tube.
  • the tube was centrifuged for 5 minutes in a Eppendorf Centrifuge 5415C microcentrifuge at room temperature. The top aqueous phase was transferred to a sterile microfuge tube (West Coast Scientific, Inc.).
  • the DNA was digested with BamR I (Gibco BRL) and Hind III (Gibco BRL).
  • the restriction fragments were separated by gel electrophoresis on a 1.0% SeaPlaque (FMC BioProducts) agarose gel in TAE buffer (40 mM Tris- acetate, 1 mM EDTA, pH 8.0) using a BRL Horizon Model 58 Horizontal Gel Electrophoresis System and a BRL Model 200 Power Supply.
  • the DNA was stained by placing the gel in a 0.5 ⁇ g/ml solution of ethidium bromide for 20 minutes, followed by destaining the gel in water for 30 minutes.
  • pJM9226 This plasmid, designated pJM9226 ( Figure 2), was subsequently used to construct tac promoter fusions to the phb operon. The construction of these fusions is described below.
  • Example 7 Construction of the multicopy tacr.phb fusion plasmids pJM9227 and pJM9229 containing a phbC leader of approximately 72 base pairs
  • Plasmid pJM8905 ( Figures 3 and 4) was digested with BstB I (New
  • T4 DNA Polymerase (Gibco BRL) was used to remove the single stranded DNA from the ends.
  • Phosphorylated BamR I linkers (New England Biolabs) were ligated to the blunt ends using T4 DNA Ligase (Gibco BRL). The resulting DNA was digested with BamR. This released a BamR I fragment that contained the phb structural genes but did not contain the promoter/regulatory region.
  • the DNA restriction fragments were separated by preparative gel electrophoresis on a 1.0% SeaPlaque (FMC BioProducts) agarose gel in TAE buffer using a BRL Horizon Model 58 Horizontal Gel Electrophoresis System and a BRL Model 200 Power Supply. The DNA was visualized by staining with ethidum bromide. The restriction fragment containing the phb genes was excised from the gel and purified using GENECLEAN® (BIO101) according to the manufacturer's protocol. This fragment was then ligated into the BamR I site of plasmid pJM9226 ( Figures 2 and 4). The recombinant plasmid was then introduced into E.
  • GENECLEAN® BIO101
  • coli strain DH5 ⁇ by electroporation and the transformants were spread on LB agar plates containing 100 ⁇ g/ml ampicillin and 1.0% glucose. The plates were incubated at 37°C in a Fisher IsoTemp Oven Model 350D until colonies were visible. White (PHB+) colonies were picked and streaked for single isolates. Purified clones were inoculated into 3 ml of LB media containing 200 ⁇ g/ml ampicillin and grown to saturation. Plasmid DNA was prepared from the cultures as described above and digested with BamR I and Hind III to confirm the tacrphb fusion construct. The resulting plasmid was designated pJM9227 ( Figure 4).
  • This pJM9227 vector construct contains the tac promoter fused 78 bp upstream of the phbC structural gene.
  • the native phbC Shine-Dalgarno sequence and ribosome-binding site is retained in this fusion.
  • expression of the phb genes is transcriptionally regulated by the tac promoter and translationally regulated by the native phbC Shine-Dalgarno sequence.
  • the Kanamycin Resistance GENBLOCK® (Eco RI) (Pharmacia) restriction fragment was ligated into the EcoR I site located downstream of the phb genes.
  • the resulting plasmid, pJM9228 ( Figure 4) was introduced into E. coli strain DH5 ⁇ by electroporation as previously described.
  • the transformants were spread on LB agar plates containing 50 ⁇ g/ml kanamycin (Sigma). The plates were incubated at 37°C in a Fisher IsoTemp Oven Model 350D until colonies were visible. Plasmid DNA was isolated from purified clones.
  • the plasmid-encoded ampicillin resistance gene was inactivated by Dra I (Gibco BRL) digestion to delete a 0.71 kb fragment from the bla structural gene, followed by religation.
  • the resulting plasmid was designated pJM9229 ( Figure 4).
  • This plasmid construct contains the tac promoter fused 78 bp upstream of the phbC structural gene, and results in a leader of approximately 72 bases (see Figure 1, panel b; Seq. ID No. ).
  • the native phbC Shine- Dalgarno sequence is retained in this fusion.
  • expression of the phb genes is transcriptionally regulated by the tac promoter and translationally regulated by the native phb ribosome binding sites.
  • Example 8 Construction of multicopy tacr.phb fusion plasmids pJM9230, pJM9231 and pJM9231 containing a phbC leader of approximately 355 base pairs
  • Plasmid pJM9226 ( Figures 2 and 5) was digested with BamR I and the 5' recessed ends were filled in with T4 DNA Polymerase to form blunt ends.
  • a restriction fragment containing the phb operon was fused to the tac promoter as follows:
  • the plasmid pTZ18U-4c was digested with EcoR I and Hind III. This released a restriction fragment containing the phb structural genes and 290 bp of the upstream leader sequence.
  • the ends of the fragment were filled in using T4 DNA Polymerase (Gibco BRL). The fragment was then excised from an agarose gel and purified using GENECLEAN®.
  • the phb fragment was ligated into the plasmid containing the tac promoter fragment at the filled-in BamR I site.
  • the resulting plasmid designated pJM9230 ( Figure 5) was introduced into E. coli strain DH5 ⁇ by electroporation as previously described.
  • the transformed cells were plated on LB agar plates containing 200 ⁇ g/ml ampicillin.
  • the presence of the tacrphb operon fusion was confirmed by screening for the PHB + phenotype (i.e., white colonies) on LB plates containing 200 ⁇ g/ml ampicillin and 1% glucose.
  • a kanamycin resistance gene was cloned into the plasmid as follows: The plasmid was digested with Spe I (New England Biolabs) and the recessed 5'-ends were filled-in with T4 DNA Polymerase to create blunt ends. The 5' recessed ends of a Kanamycin Resistance GENBLOCK® (Eco RI) (Pharmacia) restriction fragment were filled in with T4 DNA Polymerase. The Kanamycin Resistance GENBLOCK® was ligated into the filled-in Spe I site downstream of the phb operon. The recombinant plasmid, designated pJM9231 ( Figure 5), was introduced into E.
  • pJM9231 Figure 5
  • coli strain DH5 ⁇ by electroporation and transformants were selected on LB agar plates containing 50 ⁇ g/ml kanamycin.
  • Single clones were isolated and plasmid DNA isolated from these clones was purified as described above.
  • the ampicillin resistance gene (bla) was inactivated by digestion with Dra I, followed by religation, which removed a 0.71 kb fragment from the structural gene.
  • the resulting plasmid was designated pJM9232 ( Figure 5). This construct contains the tac promoter fused 361 bp upstream of the phbC structural gene, resulting in a leader of approximately 355 bases (see Figure 1, panel c; Seq. ID No. ).
  • Example 9 Construction of runaway replicon tacr.phb fusion plasmids pJM9233, pJM9234, pJM9235 and pJM9236 containing a phbC leader of approximately 72 base pairs
  • runaway replicon vector pRA89 (Benzon Pharma A/S,
  • restriction fragments were separated by preparative gel electrophoresis on a 1.0% SeaPlaque (FMC BioProducts) agarose gel in TAE buffer using a BRL Horizon Model 58 Horizontal Gel Electrophoresis System and a BRL Model 200 Power Supply.
  • the DNA was stained with 0.5 ⁇ g/ml ethidum bromide for 20 minutes and destained in water for 20 minutes. The bands were visualized using a UV transilluminator.
  • the restriction fragment containing the tacr.phb fusion was excised from the gel and purified using GENECLEAN® (BIO101) according to the manufacturer's protocol.
  • Phosphorylated BamR I linkers (New England Biolabs) were ligated to the ends of the fragment using T4 DNA Ligase and the DNA fragment was purified using GENECLEAN® (BIO 101). This DNA fragment was then ligated into the BamR I-digested vector pRA89. The recombinant plasmid was introduced into E.
  • Transformants were purified by picking well-isolated colonies and streaking for single colony isolation on LB agar plates containing 25 ⁇ g/ml chloramphenicol. The plates were incubated at 30°C to prevent runaway replication. The purified isolates were screened for the presence of the phb operon by streaking the isolates onto LB agar plates containing 25 ⁇ g/ml chloramphenicol + 1.0% glucose. The plates were incubated in a Fisher IsoTemp Oven Model 350D at 37°C to induce runaway replication. White colonies indicated the production of PHB, and thus the presence of the phb genes on the plasmid.
  • Recombinants that exhibited a PHB + phenotype at 37°C were inoculated into LB media containing 25 ⁇ g/ml chloramphenicol. The culture was incubated at 30°C for 6 hours, then 100 ⁇ l of the culture was used to inoculate 3 ml of LB media containing 50 ⁇ g/ml chloramphenicol. The cultures were incubated at 37°C overnight. Plasmid DNA was isolated from the cultures and digested with Kpn I (New England Biolabs) to determine the orientation of the tacrphb insert.
  • the plasmid containing the tacrphb operon fusion with the tac promoter proximal to the chloramphenicol resistance gene (cat) was designated pJM9233 ( Figure 6).
  • the plasmid containing the tacrphb operon fusion with the tac promoter proximal to the cI857 gene was designated pJM9234 ( Figure 6).
  • These tacrphb fusions were also cloned into the vector plasmid pRA90 (Benzon Pharma A/S, Helseholmen 1, P.O. Box 1185, DK-2650 Hvidovre, Denmark) ( Figure 7) by the same procedure.
  • the plasmid containing the tac promoter proximal to the parB locus was designated pJM9235 ( Figure 7).
  • the plasmid containing the tac promoter proximal to the chloramphenicol resistance gene (cat) was designated pJM9236 ( Figure 7).
  • these plasmid constructs contain the tac promoter fused 78 bp upstream of the phbC structural gene, resulting in an approximately 72 bp leader (see Figure 1, panel b; Seq. ID No. ). These plasmids retain the native phbC Shine-Dalgarno sequence and ribosome binding site.
  • Example 10 Construction of runaway replicon tacrphb fusion plasmids pJM9237 and pJM9238 containing a phbC leader of approximately 355 base pairs
  • the runaway replicon vector pRA90 (Benzon Pharma A/S, Helseholmen 1, P.O. Box 1185, DK-2650 Hvidovre, Denmark) ( Figures 7 and 8) was digested with BamR I and the 5' recessed ends were filled in with T4 DNA Polymerase.
  • An EcoR l-Hind III restriction fragment from plasmid pJM9230 ( Figures 5 and 8) containing the tac promoter and the phb structural genes was gel purified as previously described and the single-stranded ends were filled using T4 DNA Polymerase. This fragment was ligated into the filled-in BamR I site of pRA90.
  • the recombinant plasmid was introduced into E.
  • coli strain XL1- Blue (Stratagene) by electroporation as previously described except the transformed cells were incubated at 30°C to allow expression of the plasmid- encoded antibiotic resistance factors.
  • the transformants were spread on LB agar plates containing 25 ⁇ g/ml chloramphenicol. The plates were incubated in a Fisher IsoTemp Oven Model 350D at 30°C to prevent runaway replication. Transformants were purified by single colony isolation and screened for the presence of the phb operon by streaking the isolates onto LB agar plates containing 25 ⁇ g/ml chloramphenicol and 1.0% glucose. The plates were incubated at 37°C to induce runaway replication.
  • Recombinants that exhibited a PHB + phenotype at 37°C were inoculated into LB media containing 25 ⁇ g/ml chloramphenicol. The culture was incubated at 30°C for 6 hours, then 100 ⁇ l of the culture was used to inoculate 2.5 ml of LB media containing 50 ⁇ g/ml chloramphenicol. The cultures were incubated at 37°C overnight. Plasmid DNA was isolated from the cultures and digested with Kpn I to determine the orientation of the tacr.phb insert. The plasmid containing the tacr.phb operon fusion with the tac promoter proximal to the chloramphenicol resistance gene (cat) was designated pJM9237 ( Figure 8). The plasmid containing the tacr.phb operon fusion with the tac promoter proximal to the cI857 gene was designated pJM9238 ( Figure 8).
  • these plasmid constructs contain the tac promoter fused 361 bp upstream of the phbC structural gene, resulting in a leader of approximately 355 bases (see Figure 1, panel c; Seq. ID
  • the purpose of this experiment was to quantitate induction of the phb operon gene products in the tacrphb promoter constructs and to compare induction of the gene products to that observed in the native phb promoter clones.
  • all of the plasmids were introduced into E. coli strain HMS 174 by electroporation as previously described.
  • the plasmid pMS421 obtained from G. Weinstock ( Figure 9), was introduced into the strains containing tacr.phb fusions on multicopy plasmids by electroporation.
  • the plasmid pMS421 is a low copy number vector that confers streptomycin resistance and contains the lacN gene, which overproduces the Lac repressor protein (Muller- Hill and Gilbert, Proc. Natl Acad. Scl 59:1259, 1968).
  • Five E. coli strains were used in this experiment: HMS 174, HMS 174 pJM9131, HMS 174 pJM9232 pMS421, HMS 174 pJM9117 , and HMS 174 pJM9238.
  • E. coli strain HMS 174 was inoculated into 3 ml of LB medium.
  • E. coli strain HMS 174 pJM9131 was inoculated into 3 ml of LB medium containing 50 ⁇ g/ml kanamycin.
  • E. coli strain HMS 174 pJM9232 pMS421 was inoculated into 3 ml LB medium containing 50 ⁇ g/ml kanamycin and 10 ⁇ g/ml streptomycin. These cultures were shaken at 200 rpm at 37°C in a Lab-Line Incubator- Shaker (Lab-Line Instruments, Inc.) for approximately 15 hours.
  • coli strain HMS 174 pJM9238 were each inoculated into 3 ml of LB medium containing 25 ⁇ g/ml chloramphenicol. These cultures were shaken at 200 rpm at 30°C in a Lab-Line Orbital Environ- Shaker (Lab-Line Instruments, Inc.) for approximately 15 hours. The cultures were diluted 1 :100 into 50 ml of the same media in a 250 ml baffled Erlenmeyer flask (Wheaton), except glucose was added to a final concentration of 1.0%, and the cultures were incubated at the same temperature and agitation as previously described.
  • Wheaton baffled Erlenmeyer flask
  • the growth of the culture was followed by withdrawing aliquots at regular time intervals and measuring the optical density at 600 nanometers using a Shimadzu UV 160U Specfrophotometer.
  • the phb operon was induced in E. coli strain HMS 174 pJM9232 pMS421 by the addition of IPTG to a final concentration of 10 mM at an OD600 of 2.75.
  • the phb operon was induced in E. coli strain HMS 174 pJM9117 and E. coli strain HMS 174 pJM9238 by transferring the cultures to a 41°C waterbath for 30 minutes when an OD600 of 0.7 was reached.
  • a sterile stir bar was added and the cultures were mixed at 200 rpm using a Fisher Scientific Electronic Stirrer 2008. Following the heat pulse, the cultures were incubated in a Lab-Line Incubator-Shaker (Lab-Line Instruments, Inc.) at 37°C and shaken at 200 rpm. One milliliter aliquots were withdrawn at time intervals, centrifuged in an Eppendorf Centrifuge 5415C and frozen at -70°C. These samples were subsequently analyzed by 1-D SDS-PAGE analysis. Proteins were separated by gel electrophoresis using precast Mini-PROTEAN II Ready Gels (Bio-Rad). A 12% polyacrylamide gel was used to resolve the thiolase and reductase proteins.
  • the proteins were electrophoresed for approximately 45 minutes using the Mini- PROTEAN II® Electrophoresis Cell (Bio-Rad) and BRL Model 200 Power Supply. Prestained SDS-PAGE Standards (Bio-Rad) were used as molecular weight markers to monitor protein migration through the gels during electrophoresis. The proteins were visualized by silver staining using the Bio- Rad Silver Stain Plus kit according to the manufacturer's protocol. The gels were dried between cellophane sheets using drying frames (Integrated Separation Systems). The phb gene products were quantitated as a relative percentage of total protein by densitometry using a Ultrascan XL Enhanced Laser Densitometer (LKB).
  • LLB Ultrascan XL Enhanced Laser Densitometer
  • synthase production appears to be primarily regulated at the post-transcriptional level.
  • Synthesis of thiolase is regulated at the level of transcription.
  • Synthesis of reductase appears to be a function of plasmid type or copy number, and may be regulated by a gene dosage effect.
  • Example 12 Replacement of the native phbC ribosome-binding site with the lac ribosome-binding site to create plasmids pJM9375 and pJM9376
  • the phbC Shine-Dalgarno sequence was replaced as follows.
  • a 50 ml culture of E. coli strain XL-1 Blue (Stratagene) pJM9230 was grown to saturation in LB + 50 ⁇ g/ml kanamycin in a 250 ml Erlenmeyer flask (Wheaton).
  • Plasmid pJM9230 was purified from the culture using the QIAGEN Plasmid Kit (QIAGEN Inc.) according to the manufacturer's protocol. This procedure is based on a modification of the alkaline lysis method (Bimboim and Doly, Nucl Acids. Res. 7:1513-1522, 1979.).
  • the plasmid was digested the BstB I, which cleaves at a site approximately 30 bp upstream of the phbC structural gene. Small deletions were made from this site into the phbC ribosome-binding region with Exonuclease III using the double-stranded Nested Deletion Kit (Pharmacia) according to the manufacturer's protocol, with the following modifications:
  • the incubation temperature was 30°C and the digestion buffer contained 150 mM NaCl.
  • Phosphorylated BamR I linkers were ligated to the blunt ends of the deletion endpoints using T4 DNA Ligase and the DNA was introduced into E. coli strain XL-1 Blue cells by electroporation as previously described. Transformants were spread onto LB agar plates containing 200 ⁇ g/ml ampicillin and 1.0% glucose. Light-brown translucent (PHB”) colonies were picked and purified by single colony isolation. These mutants were defective in PHB production, presumably because they were unable to synthesize the phbC gene product.
  • Colonies were replica plated onto LB agar plates containing 200 ⁇ g/ml ampicillin + 1.0% glucose + 1 mM IPTG using an Accutran Replica Plater (Schleicher & Schuell). Deletion derivatives that yielded large white colonies were isolated from the original selection plates and purified.
  • the plasmids were designated pJM9375 and pJM9376 ( Figure 11).
  • Example 13 Determination of the tac GeneBlock-/?/rbC leader fusion joint in plasmids pJM9375 and pJM9376 by sequence analysis.
  • the precise endpoints of the Exo III deletions were determined by sequence analysis.
  • the plasmid DNA used as template was isolated from 50 ml cultures of E. coli strain HMS174 pJM9375 pMS421 and HMS174 pJM9376 pMS421 grown to saturation in LB media containing 200 ⁇ g/ml ampicillin + 10 ⁇ g/ml streptomycin.
  • the plasmid DNA was purified using a QIAGEN Plasmid Kit (QIAGEN Inc.) as previously described.
  • the DNA was sequenced using a Li-Cor DNA Sequencer Model 4000.
  • the primer used was an infrared dye- labeled M13 17-mer -20 Sequencing Primer (3TGACCGGCAGCAAAATG5')
  • the ATG start codon was replaced with a GTG start codon. Although some mRNAs exhibit the same translational yield with ATG and GTG, the ATG codon usually results in higher translation (Gold, supra).
  • Example 14 Comparison of PHB production in native and tac promoter phb multicopy clones
  • E. coli strain HMS 174 pJM9131 was inoculated into 50 ml of LB medium containing 50 ⁇ g/ml kanamycin in a 250 ml Erlenmeyer flask.
  • E. coli strain HMS 174 ⁇ JM9232 pMS421 was inoculated into 50 ml LB medium containing 50 ⁇ g/ml kanamycin and 10 ⁇ g/ml streptomycin in a 250 ml Erlenmeyer flask. These cultures were shaken at 225 rpm at 37°C in a G24 Environmental Incubator Shaker (New Brunswick Scientific) for approximately 15 hours.
  • E. coli strain HMS 174 pJM9232 and E. coli strain HMS 174 pJM9238 were each inoculated into 50 ml of LB medium containing 25 ⁇ g/ml chloramphenicol in a 250 ml Erlenmeyer flask.
  • the phb operon was induced in E. coli strain HMS 174 pJM9232 pMS421 by the addition of IPTG to a final concentration of 10 mM at an OD600 of 2.75.
  • the phb operon was induced in E. coli strain HMS 174 pJM9117 and E. coli strain HMS 174 pJM9238 by transferring the cultures to a 41°C waterbath for 30 minutes when an OD600 of 0.7 was reached.
  • the supernatant was aspirated and discarded, and the tubes containing the cell pellets were placed at -70°C for at least one hour. Uncapped screw-capped tubes containing the frozen pellets were then placed in a Labconco lyophilizer for approximately 2 hours until samples were freeze-dried.
  • Samples were then subjected to methanolysis as follows. To each tube was added 1.7 ml ACS grade methanol (Mallinckrodt), 2 ml ACS grade chloroform (Mallinckrodt), 0.3 ml concentrated sulfuric acid (added while vortexing tube) and 0.1 ml benzoic acid solution (2 mg/ml). Samples were capped tightly, placed in a heat-block adjusted to 100°C and incubated for 140 minutes. Samples were then removed from the heat block and allowed to cool to room temperature.
  • the gas chromatography system consisted of a Shimadzu GC-14A, connected to a CR-4A data processing unit, an AOC-14 autoinjector, and an AOC-1400 autosampler.
  • the carrier gas was UPC grade helium and detection was through a flame ionization detector.
  • the flow rate of the carrier was approximately 5 ml/min.
  • the column used for detection was a Supelcowax 10 column (Supelco Separation Technologies).
  • the column is a 15 meter column, 0.53 mm inner diameter, with a 1 ⁇ m thick coating. Samples (1 to 3 ⁇ l) were injected into the injection port (temperature 200°C) and carried into the column.
  • Test tubes were labeled blank, standard, and test. To each blank tube, 0.1 ml water was added. To the standard tubes, 0.1 ml of diluted Glucose Standard solution (Catalog No. 635-100) were added at concentrations of 1-20 mM. One milliliter of each culture sample to be tested was centrifuged in a Eppendorf Centrifuge 5415C microcentrifuge for two minutes to pellet the cells. To each test tube, 0.1 ml of culture supernatant was added, then 5.0 ml of o-Toluidine Reagent (Catalog No. 635-6) was added.
  • the tubes were mixed by vortexing and placed into a 100°C heat block for 10 minutes. The tubes were removed and cooled to room temperature. The contents of tubes were transferred to cuvettes and the absorbance at 635 nm was read using a Shimadzu UV 160U Specfrophotometer with the blank as reference.
  • the plasmid copy number was determined for each culture as follows. Two hundred microliters of cell suspension was centrifuged in a Eppendorf Centrifuge 5415C microcentrifuge for one minute. The supernatant was aspirated off and discarded. The cell pellet was resuspended in 50 ml of 10 mM Tris (pH 8.0), 10 mM EDTA, 100 mM NaCl, 20% sucrose, 1.5 mg/ml lysozyme (Sigma), 2 units/ml RNase. The solution was incubated for 30 minutes at 37°C. Fifty microliters of 2% SDS was added and the solution was mixed by vortexing at the maximum setting for two minutes.
  • the solution was frozen at -70°C and thawed for two cycles.
  • Five microliters of a 400 mg/ml proteinase K (BRL) stock solution was added and the tube was incubated for 30 minutes at 37°C.
  • Twenty-five microliters of loading buffer (50% glycerol, 1 mM EDTA, pH 8.0, 0.1% bromophenol blue) was added and 5-15 ⁇ l of the sample was loaded on a 0.9% agarose gel in TBE (89 mM Tris-borate, 2 mM EDTA, pH 8.0) buffer. The gel was run for three hours at 75 volts. The gel was stained for 40 minutes in 1 ⁇ g/ ml ethidum bromide solution.
  • the gel was then destained for 20 minutes in water, rinsed, and destained an additional 20 minutes.
  • the gel was placed on a UV transilluminator (Fotodyne) and photographed using a Polaroid MP-4 Land Camera with Polaroid Type 665 film. The lowest F-stop was used and the shutter was opened for 45 seconds.
  • the negative was placed in fixer solution and agitated gently for approximately 30 seconds (in the dark). The negative was then washed under 65°C running water for 5 minutes and dried.
  • LLB Ultrascan XL Enhanced Laser Densitometer
  • E. coli strain HMS 174 pJM9131 and HMS 174 pJM9232 pMS421, respectively ( Figure 12, panels c and d).
  • E. coli strain HMS 174 pJM9238 also retained a higher PHB yield as a percentage of dry weight than strain HMS 174 pJM9117 throughout the post-induction period ( Figure 13, panel a). This difference was not due to a higher gene dosage for the tac promoter clone. In fact, the pJM9117 copy number was slightly higher than the pJM9238 copy number ( Figure 14, panel a).
  • tacr.phb fusions were constructed. In one type of fusion the tac promoter was inserted 78 bp upstream of the phb C structural gene. In the other type of fusion the tac promoter was inserted 361 bp upstream of the phbC structural gene. Each type of fusion was cloned into a multicopy vector and runaway replicon vectors. To determine if the leader sequence contained cis- acting elements that regulated the expression of the phb genes, PHB production was quantitated in tacrphb fusion multicopy clones containing each type of fusion. The E.
  • coli strains used in this study were HMS 174 pJM9229 pMS421 and HMS 174 pJM9232 pMS421.
  • the strains were inoculated into 50 ml of LB media containing 50 ⁇ g/ml kanamycin and 10 ⁇ g/ml streptomycin in a 250 ml baffled Erlenmeyer flask (Wheaton).
  • the culture was incubated at 200 rpm at 37 °C in a Lab-Line Orbital Environ- Shaker (Lab-Line Instruments, Inc.) for approximately 15 hours.
  • One ml of the stationary phase culture was added to 250 ml of LB media containing 1.0% glucose + 50 ⁇ g/ml kanamycin + 10 ⁇ g/ml streptomycin in a 1 liter baffled Erlenmeyer flask (Bellco) and incubated at 200 rpm at 37°C.
  • the growth of the culture was followed by withdrawing aliquots at regular time intervals and measuring the optical density at 600 nanometers using a Shimadzu UV 160U Specfrophotometer.
  • IPTG United State Biochemical Corp.
  • Samples were withdrawn at regular time intervals for GC analysis.
  • Example 17 Determination of the optimal cell density to initiate PHB production in E. coli strain HMS174 pJM9238
  • the optimal cell density at which to induce the PHB biosynthetic pathway was determined.
  • a culture of E. coli strain HMS 174 pJM9238 was induced at various cell densities as follows: The strain was inoculated into 50 ml of LB + 25 ⁇ g/ml chloramphenicol and the culture was incubated overnight at 30°C. The next morning, 250 ml of LB media containing 2% glucose and 25 ⁇ g/ml chloramphenicol in a 1 liter baffled Erlenmeyer flask (Wheaton) was equilibrated to 36°C by placing the flask in an incubator. The optical density at 600 nm of the overnight culture was determined and enough volume of the culture was added to the medium to obtain an initial optical density at 600 nm of 0.10. The culture was incubated at a constant temperature of 36°C in a Innova 4000
  • results indicate tha- PHB production is highest when the culture is induced at low cell density.
  • the 24 hour culture contained 5.5 mg/ml PHB (71% of dry weight).
  • PHB production was lowest when the culture was induced at mid- log phase.
  • the 24 hour culture contained only 2.2 mg/ml PHB (48% of dry weight).
  • PHB production increased as the cells entered late log phase.
  • the 24 hour culture contained 3.7 mg/ml PHB (66% of dry weight) ( Figure 17).
  • Example 18 Comparison of PHB production in E. coli strain HMS174 pJM9238 grown in media with and without chloramphenicol
  • E. coli strain HMS 174 pJM9238 was inoculated into 50 ml of LB medium containing 25 ⁇ g/ml chloramphenicol in a 250 ml baffled Erlenmeyer flask and incubated at 30°C, 175 rpm overnight. Two 1 liter baffled Erlenmeyer flasks containing 250 ml of LB broth + 2% glucose were prepared.
  • E. coli strains HMS174 pJM9375 pMS421 and HMS174 pJM9376 pMS421 were tested for PHB production in liquid media.
  • the cultures were inoculated into 3 ml of LB media containing 200 ⁇ g/ml ampicillin and 10 ⁇ g/ml streptomycin in 16x100 mm culture tubes and grown to saturation in a Lab-Line Incubator-Shaker at 37°C with shaking at 200 rpm.
  • the cultures were diluted to an optical density at 600 nm of 0.10 into 50 ml of LB media containing 200 ⁇ g/ml ampicillin + 10 ⁇ g/ml streptomycin + 2% glucose.
  • IPTG was added to the culture to a final concentration of 10 mM. Aliquots of the cultures were withdrawn during growth for measuring optical density, dry weight analysis, and to quantitate PHB production as described above.
  • strain KC2671 The bacterial strain used in this study was Klebsiella aerogenes strain KC2671 hutC515 recA3011 A[bla]-2. Strain KC2671 was streaked onto an LB plate and strain KC2671 pMS421 was streaked onto an LB + 10 ⁇ g/ml streptomycin plate from frozen permanents. Single colonies were picked from the plates and patched onto the same medium. These were used as stock plates. Strain KC2671 was inoculated into 3 ml of LB and strain KC2671 pMS421 was inoculated into 3 ml LB + 10 ⁇ g/ml streptomycin and grown to saturation.
  • the supernatant was aspirated off, then the pellets were resuspended in 20 ml of 10% glycerol.
  • the cells were centrifuged as before, then resuspended in 10 ml of 10% glycerol in a 15 ml Falcon tube.
  • the cultures were centrifuged as before, then resuspended in 200 ⁇ l 10% glycerol. 40 ⁇ l of the cell suspension were pipetted into chilled microfuge tubes and stored at -70°C.
  • plasmid DNA was introduced into the cells by electroporation as previously described.
  • strain KC2671 pJM9238 was plated onto LB + 25 ⁇ g/ml chloramphenicol and strain KC2671 pMS421 pJM9232 was plated onto LB + 10 ⁇ g/ml streptomycin + 50 ⁇ g/ml kanamycin. The plates were incubated overnight at 30°C.
  • strain KC2671 pMS421 pJM9232 was patched onto a) LB + 10 ⁇ g/ml streptomycin + 50 ⁇ g/ml kanamycin, and b) LB + 10 ⁇ g/ml streptomycin + 50 ⁇ g/ml kanamycin + 1% glucose + 5 mM IPTG. Both plates were incubated at 30°C overnight.
  • Strain KC2671 pJM9238 was patched onto a) LB + 25 ⁇ g/ml chloramphenicol and incubated at 30°C, and b) LB + 25 ⁇ g/ml chloramphenicol + 1% glucose and incubated at 37°C.
  • the glucose-containing plates were inspected and the apparent best PHB producer of each strain was chosen.
  • the strains were then picked from the plates that did not contain glucose (that is, the best-producing cultures were then grown under conditions in which they did not make PHB and were then picked) and inoculated into 50 ml of LB containing the appropriate antibiotics.
  • the cultures were incubated for 6-8 hours at 30°C (until the cultures were in early stationary phase), then frozen permanents of each strain were made as described above and stored at -70°C.
  • Plasmid Stability Four 50 ml cultures in 250 ml Erlenmeyer flasks were started from 500 ⁇ l of frozen permanents as follows: Strain KC2671 pJMS421 pJM9232 was inoculated into a) LB medium and b) LB + 10 ⁇ g/ml streptomycin + 50 ⁇ g/ml kanamycin. Strain KC2671 pJM9238 was inoculated into a) LB medium and b) LB + 25 ⁇ g/ml chloramphenicol. The cultures were incubated at a temperature of 30°C in an Innova shaker incubator at 200 rpm. The next morning each culture was diluted into sterile 0.85% saline solution and the IO' 6 , IO -7 , and IO" 8 dilutions were spread onto LB plates.
  • KC2671 pMS421 pJM9232 culture was pipetted into 1.5 ml microfuge tubes and centrifuged. The pellet was resuspended in 150 ⁇ l of SET buffer and stored at -20°C.
  • strain KC2671 pJM9238 45 ml of culture was centrifuged in a 50 ml Falcon tube, resuspended in 4 ml of PI buffer (QIAGEN kit), and stored at -20°C.
  • PI buffer QIAGEN kit
  • strain KC2672 pMS421 pJM9232 After 100 generations 100% (100/100) of the isolates tested from the LB culture and 100% (102/102) of the isolates tested from the LB + 10 ⁇ g/ml streptomycin + 50 ⁇ g/ml kanamycin culture retained streptomycin and kanamycin resistance. All isolates also produced PHB on plates containing glucose.
  • strain KC2671 pJM9238 after 100 generations, 99.4% (310/312) of the isolates tested from the culture grown in LB + 25 ⁇ g/ml chloramphenicol retained chloramphenicol resistance and produced PHB on plates containing glucose.
  • Klebsiella strain KC2671 pJM9238 was grown to saturation overnight in 50 ml of LB + 25 ⁇ g/ml chloramphenicol in a 250 ml Erlenmeyer baffled flask at 30°C with shaking at 175 rpm.
  • An aliquot of the culture was inoculated into 250 ml of LB media containing 2% glucose and 25 ⁇ g/ml chloramphenicol in a 1 liter Erlenmeyer baffled flask to yield an initial optical density at 600 nm of 0.10.
  • the culture was incubated at a given temperature in the range of 30°C to 40°C with shaking at 175 rpm.
  • the growth of the culture was followed by measuring the optical density at 600 nm. During exponential growth, samples of the culture were harvested for analysis of PHB production.
  • PHB could be detected in all of the cultures 2 to 4 hours after inoculation.
  • the results for two temperature induction experiments are shown in Figure 20, panel b.
  • PHB production rose from 1.054 ⁇ g/ml (0.405% of dry weight) at 3.0 hours after inoculation to 234.4 ⁇ g/ml (19.2% of dry weight) at 6.7 hours, then to 716.7 ⁇ g/ml (33.8% of dry weight) at 24 hours.
  • Klebsiella aerogenes strain KC2671 pJM9238 was tested for PHB production during fed-batch fermentation.
  • the fermentor used in this study was a B. Braun Type ES10 Biostat E 15 liter fermentor.
  • the parameters were controlled using the Micro-MFC S computer control system (B. Braun Melsungen AG) with a Hyundai Super-386C computer.
  • the strain was inoculated from a frozen permanent into 50 ml of LB medium containing 25 ⁇ g/ml chloramphenicol and grown at 31°C to saturation.
  • the cultures were inoculated into 5 liters of media containing the following components: 6 g/L Na2HP ⁇ 4 anhydrous, 6 g/L KH 2 P0 4 anhydrous, 5 g/L (NH 4 ) 2 S0 4 , 0.35 g/L MgS0 4 - 7H2O, 3 ml/L trace elements, 5 g/L yeast extract. Chloramphenicol was added to the medium at a final concentration of 25 ⁇ g/ml.
  • the feed media was composed of the following components: 33 g/L (NH 4 )2S0 4 , 400 g/L glucose, 7 g/L MgS0 4 -7H2 ⁇ , 5 ml/L trace elements, 5 g/L yeast extract.
  • the culture was incubated at 31°C to an optical density at 600 nm of approximately 3.0, at which time the temperature was shifted to 33°C. Aliquots were harvested at approximately 1 hour intervals for determination of dry weight, PHB content, and glucose concentrations. PHB content and dry weight were determined as previously described in Example 11.
  • Glucose was quantitated using the Sigma Diagnostics Glucose Assay Kit (Sigma), Procedure No. 635, p. 5, as previously described in Example 15.
  • results The results are depicted in Figure 21, panel a. PHB production was effectively repressed in KC2671 p.TM9238 when the culture was grown at 31°C. Prior to thermal induction, PHB levels were at or below 0.0441 mg/ml. After the incubation temperature was increased to 33°C, PHB synthesis was rapidly induced. At the 12 hour time point (approximately 6 hours after the temperature shift to 33°C) the PHB concentration was 7.017 mg/ml, an increase of over 150-fold. At the 24 hour time point (approximately 18 hours after the temperature shift) the PHB concentration was 27.4 mg/ml, an increase of over 600-fold.
  • the rate of PHB synthesis observed in strain KC2671 pJM9238 was significantly higher than that previously observed in strain KC2671 pJM9131, as shown in Figure 21, panel b.
  • the KC2671 pJM9238 culture contained 23 mg/ml PHB, while KC2671 pJM9131 contained only 10 mg/ml PHB.

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Abstract

Des vecteurs de synthèse comprenant (a) un promoteur à régulation négative et relié activement à la séquence d'acide nucléique désirée, (b) un réplicon fugitif permettant l'obtention de copies multiples du vecteur de synthèse lors de l'induction thermique, et (c) un locus de stabilisation. Les vecteurs de synthèse sont typiquement des plasmides, toutefois des vecteurs viraux, des cosmides et d'autres vecteurs de synthèse d'acide nucléique entrent également dans le cadre de la présente invention. Selon un mode de réalisation préféré, le vecteur de synthèse comprend de plus une séquence consensus Shine-Dalgarno (de préférence une séquence Shine-Dalgarno lac), reliée activement à la séquence d'acide nucléique désirée, permettant ainsi une translation intensifiée de la séquence d'acide nucléique désirée, la séquence consensus Shine-Dalgarno pouvant soit remplacer la séquence Shine-Dalgarno native, soit être utilisée en complément d'une telle séquence native. L'invention concerne également des procédés permettant d'obtenir un produit requis codé par la séquence d'acide nucléique désirée ou résultant de celle-ci, des cellules hôtes bactériennes transformées à l'aide de tels vecteurs de synthèse, et les produits désirés obtenus selon les procédés de la présente invention.
PCT/US1995/001471 1994-02-03 1995-02-02 Vecteurs d'acide nucleique a expression intensifiee Ceased WO1995021260A1 (fr)

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WO1998004713A1 (fr) * 1996-07-26 1998-02-05 Massachusetts Institute Of Technology Procede de regulation du poids moleculaire de polyhydroxyalcanoates
EP0881293A1 (fr) * 1997-05-28 1998-12-02 Eidgenössische Technische Hochschule Zürich Institut für Biotechnologie Préparation de poly-3-hydroxyalkanoates à chaíne moyenne en E. coli et monomères dérivés
RU2422504C2 (ru) * 2008-11-07 2011-06-27 Учреждение Российской академии наук Институт микробиологии им. С.Н. Виноградского РАН СПОСОБ ПОЛУЧЕНИЯ ЭКСПРЕССИОННОГО ПЛАЗМИДНОГО ВЕКТОРА, ОБЛАДАЮЩЕГО ПОВЫШЕННОЙ СТАБИЛЬНОСТЬЮ НАСЛЕДОВАНИЯ В КЛЕТКАХ Escherichia coli
CN112313336A (zh) * 2018-05-01 2021-02-02 Ambrx公司 一种用于优化抗体表达的方法

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D. KLEINER ET AL.;: "Construction of multicopy expression vectors for regulated over-production of proteins in Klebsiella pneumoniae and other enteric bacteria", J. GEN. MICROBIOL., vol. 134, pages 1779 - 1784 *
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998004713A1 (fr) * 1996-07-26 1998-02-05 Massachusetts Institute Of Technology Procede de regulation du poids moleculaire de polyhydroxyalcanoates
US5811272A (en) * 1996-07-26 1998-09-22 Massachusetts Institute Of Technology Method for controlling molecular weight of polyhydroxyalkanoates
EP0881293A1 (fr) * 1997-05-28 1998-12-02 Eidgenössische Technische Hochschule Zürich Institut für Biotechnologie Préparation de poly-3-hydroxyalkanoates à chaíne moyenne en E. coli et monomères dérivés
WO1998054329A1 (fr) * 1997-05-28 1998-12-03 Eidgenössische Technische Hochschule Zürich Institut Für Biotechnologie Production de poly-3-hydroxyalcanoates a longueur de chaine moyenne dans escherichia coli, et de monomeres derives
RU2422504C2 (ru) * 2008-11-07 2011-06-27 Учреждение Российской академии наук Институт микробиологии им. С.Н. Виноградского РАН СПОСОБ ПОЛУЧЕНИЯ ЭКСПРЕССИОННОГО ПЛАЗМИДНОГО ВЕКТОРА, ОБЛАДАЮЩЕГО ПОВЫШЕННОЙ СТАБИЛЬНОСТЬЮ НАСЛЕДОВАНИЯ В КЛЕТКАХ Escherichia coli
CN112313336A (zh) * 2018-05-01 2021-02-02 Ambrx公司 一种用于优化抗体表达的方法
US12404512B2 (en) 2018-05-01 2025-09-02 Ambrx, Inc. Method for optimizing antibody expression

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