WO1998008954A2 - Production d'heme et d'hemoproteines de recombinaison - Google Patents

Production d'heme et d'hemoproteines de recombinaison Download PDF

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WO1998008954A2
WO1998008954A2 PCT/US1997/014165 US9714165W WO9808954A2 WO 1998008954 A2 WO1998008954 A2 WO 1998008954A2 US 9714165 W US9714165 W US 9714165W WO 9808954 A2 WO9808954 A2 WO 9808954A2
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Prior art keywords
heme
host cell
hema
ala
hemoglobin
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PCT/US1997/014165
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WO1998008954A3 (fr
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Elaine A. Best
Evie L. Verderber
Louise J. Lucast
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Somatogen, Inc.
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Priority to AU41495/97A priority Critical patent/AU4149597A/en
Publication of WO1998008954A2 publication Critical patent/WO1998008954A2/fr
Publication of WO1998008954A3 publication Critical patent/WO1998008954A3/fr

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • This invention generally relates to the production of heme and heme-containing proteins. More specifically, the invention relates to methods of enhancing heme production in host cells capable of expressing heterologous, heme-containing proteins.
  • Heme is an iron-containing porphyrin that serves as a prosthetic group in proteins such as hemoglobin, myoglobin and the cytochromes.
  • the biochemical pathway for heme biosynthesis is well known. Except for the initial steps in the formation of ⁇ -aminolevulinic acid (ALA), the pathway is fairly well conserved throughout plants, animals and bacteria.
  • ALA ⁇ -aminolevulinic acid
  • heme b which is also known as protoheme and ferrous protoporphyrin IX
  • Heme b serves as an essential cofactor for b-type cytochromes, catalase and peroxidase.
  • Biosynthesis of heme b occurs via a complex, branched pathway that involves up to twelve gene products (Fig. 1).
  • ALA the committed precursor in the heme b pathway, is formed from the 5-carbon skeleton of glutamate via the C5 pathway.
  • Production of ALA in E. coli occurs in three steps: (1) ligation of tRNA (GTR) synthetase, (2) reduction of the resulting GTR to glutamate 1-semialdehyde (GSA) by GTR reductase, and (3) transamination of GSA to ALA by GSA aminotransferase.
  • GTR tRNA
  • GSA glutamate 1-semialdehyde
  • E. coli cells In non-recombinant E. coli cells, accumulation of large pools of heme b pathway intermediates or free heme b is deleterious to the cells. For example, E. coli cells with mutations in the gene encoding hemH (vis A), are impaired in their ability to insert iron into protoporphyrin IX, and accumulate large pools of protoporphyrin IX, which is light sensitive. Because heme b has a propensity to cause oxidative damage to the lipid and protein components of cellular membranes, heme b is normally found associated with proteins in the cell, rather than as free heme,,. The biochemical and/or genetic mechanism by which E. coli and related bacteria regulate expression of the heme b pathway is poorly understood.
  • GTR reductases have been described in E. coli. Two GTR reductase activities of different molecular masses (85 kDa and 45 kDa) have been purified (Jahn et al., J. Biol. Chem.. 266:2542-2548 (1991)). The 45 kDa enzyme is the product of hemA and was thought to be a minor enzyme, and that the major GTR reductase is a 23 kDa enzyme encoded by hemM (Ikemi et al., Gene. 121: 127-132 (1992)).
  • Intracellular pools of free heme b and heme synthetic pathway intermediates are regulated in E. coli to prevent toxicity.
  • E. coli or other cells produce heme or hemoproteins, especially by recombinant DNA methods, such regulation may cause a problem because the limited availability of precursors can limit the amount of heme and or hemoprotein produced. Therefore, a need exists to overcome the normal regulatory mechanism that limits heme production when enhanced heme production is desired.
  • the present invention satisfies this need and provides related advantages as well.
  • the present invention relates to methods of enhancing the expression of heterologous heme-containing proteins by increasing the amount of endogenous heme available to host cells. Such methods are accomplished by exposing the hosts cells to increased amounts of ALA either by stimulating the production of ALA endogenously or by adding exogenous ALA to the culture medium containing the host cells.
  • the methods are accomplished by inserting at least one copy, preferably multiple copies, of the hemA gene into a host cell to stimulate the endogenous production of ALA in the heme biosynthesis pathway.
  • ALA can be supplemented directly in a culture of host cells to increase heme production.
  • the host cells of the present invention can be prokaryotic or eukaryotic, such as bacteria, yeast, plant, or animal (vertebrate and invertebrate) cells.
  • the host cells are bacteria, for example, E.coli.
  • E.coli E.coli and certain other bacteria, the increased production of heme, results in the enhanced expression of the heterologous heme-containing protein.
  • Heme-containing proteins include, for example, hemoglobin, myoglobin, chlorophyll, siroheme, factor F430 and heme-containing enzymes.
  • Such enzymes include, for example, vitamin B12 catalase and nitric oxide synthetase.
  • Various hemoglobins are also contemplated, including wild-type human hemoglobin and variants thereof, including mutant human hemoglobins such as rHbl.l.
  • the present invention further provides methods for enhancing heme b production in a host cell, particularly in E.coli.
  • the methods are accomplished by inserting one or more copies of the hemA gene into the host cell and culturing the transformed host cell to allow production of an enhanced amount of heme b .
  • Figure 1 shows, schematically, the pathway for heme synthesis.
  • ALA production is boxed. Genes and enzymes relevant to this study are indicated.
  • Figure 2 shows the effect of ALA supplementation and rHbl. l production, on heme pools. Cultures contained no IPTG (D) or 300 mM IPTG ( ⁇ ). Each data point represents the average of three independent trials. Experimental variation (standard deviation) is shown by error bars.
  • Figure 3 shows the features of DNA fragments used to identify promoters of hemA.
  • the start of the hemA coding sequence is indicated by the ATG codon. Arrows mark the transcription initiation sites identified by Verkamp & Chel , J. Bacterio 171 :4728-4735 (1989).
  • the triangle below pSGE864 DNA fragment represents the 72bp DNA segment containing the Al transcription start site that was deleted in the PCR amplification.
  • Figure 4 shows the effect of ALA concentration on hemA-lacZ expression.
  • Cells were grown in 5 ml cultures and assayed as described in Example 1. Data points represent the average of four independent trials.
  • the present invention generally relates to the enhanced production of heterologous hemoproteins and heme in host cells.
  • the invention is based on the results of the studies described in the Examples below. The results show:
  • heme b is a feedback inhibitor of the heme b pathway; (2) overexpression of rHbl .l (a genetically fused version of human hemoglobin) and
  • HemA glutamyl tRNA (GTR) reductase
  • GTR glutamyl tRNA reductase
  • HemA is rate-limiting
  • heme b does not repress ALA formation, while ALA formation limits heme b synthesis.
  • the present invention is based on the surprising results of the studies. Therefore, in one aspect of the invention, methods of enhancing the expression of a heterologous hemoprotein are provided. Such methods can be accomplished by first exposing host cells to an increased amount of ALA effective to enhance or increase heme production in the host cell.
  • the host cells can then be cultured to allow enhanced production of the target hemoprotein.
  • the host cells can be exposed to enhanced amounts of ALA either endogenously or exogenously.
  • ALA is added as a supplement to culture medium in which the host cells are grown as described in the Examples below.
  • hemA refers to the gene
  • HemA refers to the glutamyl tRNA (GTR) reductase.
  • heterologous when referring to a gene, indicates that the gene has been inserted into a host cell that does not naturally carry the gene, either by way of a stable plasmid or through integration into the genome.
  • protein when referring to a protein, indicates that the protein is the product of a heterologous gene.
  • Heterologous proteins are proteins that are normally not produced by a host cell.
  • the terms “enhanced” and “increased” are used interchangeably and mean a measurably greater amount of expression (i.e., overexpression) or production of a gene or target protein compared to the amount of expression or production of the same gene or protein prior to any manipulation of the host cell or culture medium.
  • Recombinant systems for producing heterologous proteins or polypeptides, including hemoproteins are well known in the art.
  • the genes encoding the target protein can be placed in a suitable expression vector and inserted into a microorganism, animal, plant, insect or other organism, or inserted into cultured animal or plant cells or tissues.
  • These host cells, organisms or tissues may be produced using standard recombinant DNA techniques following the teachings of the present invention, and may be grown in cell culture or in fermentations.
  • human alpha and beta globin genes have been cloned and sequenced by Liebhaver et al. fProc. Natl. Acad. Sci. USA. 77:7054-58, 1980) and Marotta et al. fj.
  • Methods for incorporating the desired mutations are well known in the art and include, for example, site-directed mutagenesis. Random mutagenesis is also useful for generating a number of mutants at a particular site. Other recombinant techniques are also known, such as those described in U.S. Patent No. 5,028,588, U.S. Patent No. 5,545,727, U.S. Patent No. 5,599,907, PCT Publications WO 96/40920 and WO 97/04110, all incorporated herein by reference.
  • the genes can be used to construct plasmids to be inserted into appropriate host cells according to conventional methods or as described in WO 96/40920, incorporated herein by reference.
  • Any suitable host cell can be used to express the novel polypeptides.
  • Suitable host cells include, for example, bacteria, yeast, plant, vertebrate and invertebrate animal cells, including mammalian and insect cells. Host cells in transgenic animals are also contemplated. E. coli cells are particularly useful for expressing desired recombinant hemoprotein.
  • the transformed host cell is then cultured or fermented until soluble hemoglobin is harvested.
  • the protein generally should be released from the cell to create a crude protein solution. This can usually be done by breaking open the cells, e.g., by sonication, homogenization, enzymatic lysis or any other cell breakage technique known in the art.
  • the proteins can also be released from cells by dilution at a controlled rate with a hypotonic buffer so that some contamination with cellular components can be avoided (U.S. Patent No. 5,264,555).
  • Cells also may be engineered to secrete the protein of interest by methods known in the art
  • the target protein is contained in a crude cell lysate or crude cell broth or solution.
  • the protein may be purified according to methods well known in the art. For example, methods for purifying hemoglobin-like proteins are taught in PCT publication WO 95/14038, incorporated herein by reference. The hemoproteins, so-produced, can be used for their known purposes.
  • Heme- containing compounds known in the art include, for example, nitric oxide synthase, myoglobin, chlorophylls (from e.g., plants and bacteria), vitamin B12, catalase, siroheme, factor F430 and various hemoglobins, including those from human, yeast, bacteria, worms, crocodiles, and other sources.
  • Heme-containing proteins for example the various types of hemoglobins, have many uses, including, for example, for delivery of oxygen or therapeutic uses.
  • Other hemoproteins for example, P-450 enzymes which can be used for oxidation of drugs, alkaloids, terpenes, pesticides, carcinogens, and other xenobiotic chemicals are also important (Porter & Coo.
  • Cytochrome P-450 enzymes can be used for detoxification of various chemicals.
  • recombinant hemoglobin can be used for a number of in vitro or in vivo applications.
  • in vitro applications include, for example, the delivery of oxygen by compositions of the instant invention for the enhancement of cell growth in cell culture by maintaining oxygen levels in vitro (DiSorbo and Reeves, PCT publication WO 94/22482, herein incorporated by reference).
  • hemoglobins of the instant invention can be used to remove oxygen from solutions requiring the removal of oxygen (Bonaventura and Bonaventura, US Patent 4,343,715, incorporated herein by reference) and as reference standards for analytical assays and instrumentation (Chiang, US Patent 5,320,965, incorporated herein by reference) and other such in vitro applications known to those of skill in the art.
  • recombinant hemoglobin can be formulated for use in various therapeutic applications.
  • Example formulations suitable for the recombinant hemoglobin of the instant invention are described in Milne, et al., WO 95/14038 and Gerber et al., WO 96/27388, both herein incorporated by reference.
  • Pharmaceutical compositions can be administered by, for example, subcutaneous, intravenous, or intramuscular injection, topical or oral administration, large volume parenteral solutions, aerosol, transdermal or mucus membrane adsorption and the like.
  • the recombinant hemoglobins of the present invention can be used in compositions useful as tissue oxygenating therapeutics, as substitutes for red blood cells in any application that red blood cells are used or for any application in which oxygen delivery is desired.
  • the recombinant hemoglobin formulated as oxygen therapeutics can be used for the treatment of hemorrhages, traumas and surgeries where blood volume is lost and either fluid volume or oxygen carrying capacity or both must be replaced.
  • the recombinant hemoglobins of the instant invention can be made pharmaceutically acceptable, they can be used not only as blood substitutes that deliver oxygen but also as simple volume expanders that provide oncotic pressure due to the presence of the large hemoglobin protein molecule.
  • the recombinant hemoglobins of the instant invention can be crosslinked by methods known in the art and used in situations where it is desirable to limit the extravasation or reduce the colloid osmotic pressure of the hemoglobin-based blood substitute.
  • the recombinant hemoglobins can act to transport oxygen as a red blood cell substitute, while reducing the adverse effects that can be associated with excessive extravasation.
  • a typical dose of recombinant hemoglobin as an oxygen delivery agent can be from 2 mg to 5 grams of hemoglobin per kilogram of patient body weight.
  • a typical dose for a human patient might be from a few grams to over 350 grams.
  • the unit content of active ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount could be reached by administration of a number of administrations. The selection of dosage depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of those skilled in the art.
  • Administration of recombinant hemoglobin can occur for a period of seconds to hours depending on the purpose of the hemoglobin usage.
  • an oxygen carrier the usual time course of administration is as rapid as possible.
  • Typical infusion rates for hemoglobin solutions as oxygen therapeutics can be from about 100 ml to 3000 ml/hour.
  • the hemoglobins of the instant invention can be used to treat anemia, both by providing additional oxygen carrying capacity in a patient that is suffering from anemia, and/or by stimulating hematopoiesis as described in PCT publication WO 95/24213, incorporated herein by reference.
  • the recombinant hemoglobins of the instant invention can be used for applications requiring administration to a patient of high volumes of hemoglobin as well as in situations where only a small volume of the hemoglobin of the instant invention is administered.
  • the hemoglobins of the present invention can be used to deliver oxygen to areas that red blood cells cannot penetrate. These areas can include any tissue areas that are located downstream of obstructions to red blood cell flow, such as areas downstream of thrombi, sickle cell occlusions, arterial occlusions, angioplasty balloons, surgical instrumentation, any tissues that are suffering from oxygen starvation or are hypoxic, and the like. Additionally, all types of tissue ischemia can be treated using the hemoglobins of the instant invention.
  • tissue ischemias include, for example, stroke, emerging stroke, transient ischemic attacks, myocardial stunning and hibernation, acute or unstable angina, emerging angina, infarct, and the like.
  • Recombinant hemoglobin can also be used as an adjunct with radiation or chemotherapy for the treatment of cancer. Because of the broad distribution in the body, the recombinant hemoglobins of the instant invention can also be used to deliver drugs and for in vivo imaging as described in WO 93/08842, incorporated herein by reference.
  • Recombinant hemoglobins can also be used as replacement for blood that is removed during surgical procedures where the patient's blood is removed and saved for reinfusion at the end of surgery or during recovery (acute normovolemic hemodilution or hemoaugmentation).
  • the recombinant hemoglobins of the instant invention can be used to increase the amount of blood that can be predonated prior to surgery, by acting to replace some of the oxygen carrying capacity that is donated.
  • the present invention further provides methods for enhancing the production of heme b . Such methods can be accomplished by inserting one or more copies of the hemA gene into a host cell capable of expressing heme b . The transformed host cell is then cultured to allow production and accumulation of heme,, Heme, usually in the form of heme-arginate as described in U.S. Patent No.
  • 5,008,388 is a clinically important compound which is administered as treatment for hepatic porphyrias which result from a defect in the human heme synthetic pathway. It is also used to treat myelodysplastic syndrome and has potential for treatment of sickle cell disease, ⁇ - thalassemia, and prevention of myelosuppression associated with chemotherapy and AZT treatment (Mustajoki, Br. Med. J. 293:538-39 (1986); Volin, Leukemia Res. 12:423-31 (1988)). Therefore, heme and particularly heme-arginate can be used for hematopoiesis, myelodysplastic syndrome, porphyrias, as well as a natural coloring agent.
  • ALA formation is a rate-limiting step in E.coli heme b biosynthesis, and that hemA, not hemM, encodes the major GTR reductase in the pathway.
  • Three enzymes are necessary for conversion of glutamate to ALA by the C5 pathway (Fig. 1 ).
  • GLTX and HemL are well known, the roles of HemA and HemM in the reaction catalyzed by GTR reductase are not.
  • the hemA gene allowed an E.coli hemM mutant to form only small colonies on medium lacking ALA, and that hemM allowed that mutant to form large colonies, Ikemi et al.
  • hemM encodes the major GTR reductase in E.coli, and that the GTR reductase encoded by hemA is involved in "an alternative minor pathway for ALA formation.” (Ikemi, et al., supra). The existence of two pathways for ALA formation is supported by the fact that there are two GTR reductase proteins in E.coli: a 45-kDa protein encoded by hemA, and an 85-kDa protein whose gene product has not yet been identified. Two facts suggest that hemM may not encode GTR reductase.
  • the E.coli hemM protein encodes a 23-kDa protein in E.coli maxicells, and the deduced amino acid sequence of HemM bears no obvious homology to GTR reductase, or to any proteins whose functions have been described in Ikemi et al., supra.
  • hemM encodes the predominant GTR reductase for the C5 pathway of ALA synthesis or that both hemA and hemM are required for maximal accumulation of ALA.
  • a GTR reductase (hemA) mutant was not complemented with a plasmid containing hemM (pSGE1 104). ALA accumulation was enhanced when cells were provided with multiple copies of hemA, but not further enhanced when both hemA+M were provided.
  • a wild-type strain containing this hemM-lacZ fusion produced 6118+ 492 Miller units of beta-gal actosidase activity, while a control strain, containing the pMLB1034 vector, produced less than one unit of activity.
  • Both the hemM (pSGE1 104) and ⁇ e -4+M(pSGE1103) containing plasmids used herein contain the complete hemM coding sequence as well as 200 bp of upstream sequence.
  • hemA and hemA RC Two different systems for increasing ALA pools— hemA and hemA RC — were evaluated in the present studies. Both systems allowed for an increase in cellular heme content, but the two systems had different effects on rHbl .1 accumulation.
  • the strong tac promoter that drives the hemA RC gene in pSGEl 1 10 appears to be more efficient than either the hemA, or hemA 2 promoters, neither of which has ideal -10 or -35 consensus elements (Verkamp et al., 1989, supra.). While not wishing to be bound by any theory, it is possible that the level of HemA RC protein produced by pSGEl l lO is so high that the cell's capacity to produce rHbl.
  • Bacterial strains and plasmids Bacterial strains and plasmids. Bacterial strains and plasmids used in this study are listed in Table 1.
  • pSGE715 is a pUC-derived plasmid containing a synthetic, to -controlled operon composed of two genetically fused alpha subunits and one beta subunit of human hemoglobin and the lad gene for repression of the tac promoter as described in WO 97/04110.
  • pSGE518 was derived from pRS415 (Simon et al., Gene.
  • capsulatus hemA RC pSGE715 high copy plasmid for IPTG-con trolled expression of rHb 1.1 ; Tc pSGE494 pACYC184 with 1.2 kb Bamlll-Hindlll PCR fragment containing hemA pSGE862 pRS518 with 0.5 kb Hindlll-Bglll fragment of pSGE494 containing hemA) and hemA 2 promoters pSGE863 pRS518 containing 0.3 kb Hindlll-Bglll fragment containing hemA / promoter pSGE864 pRS518 containing 0.4 kb Hindlll-Bglll fragment containing hemA 2 promoter
  • All PCR amplification reactions contained the following reagents: -100 ng of template DNA, 20-50 pmol of each primer, 20 mM Tris-HCl, 10 M KC 1 , 6 mM (NH 4 ) 2 SO 4 , 1.5 mM MgCl 2 , 0.1% Triton X-100, 0.2 mM of each of the four deoxyribonucleotide triphosphate
  • All three hemA promoter fragments (Fig. 3) were amplified using the following cycle conditions (program "J"): one cycle of 5 min. at 95°C, 5 min. at 50°C, 1 min at 72°C; 28 cycles of 1 min. at 94°C, 1 min 50°C, and 30 sec. at 72°C; one cycle of 10 min. at 72°C.
  • the hemA promoter fragment in pSGE863 (any other E. coli having a hemA promoter can be used) was amplified using primers EV50 and EV39.
  • the hemA promoter fragment in pSGE864 (any E.
  • coli having a hemA promoter was amplified using two sets of primers: EV45 an EV57, and EV53 and EV54.
  • the use of two primer sets allowed removal of a small DNA segment containing the hemA, transcription start site described by Verkamp and Chelm (J. Bacteriol.. 171 :4728-4735 (1989)).
  • the hemA gene fragment contained in pSGE494 (any other E. coli having a hemA gene fragment can be used) was amplified using program "J" and primers TG40 and TG41. The function of the hemA gene in pSGE494 was verified by complementation assay using an E. coli hemA mutant.
  • a DNA fragment containing the coding sequence of hemA C was obtained by PCR using chromosomal DNA isolated from Rhodobacter strain SB 1003 (Hornberger, Mol. Gen- Genet. 221 :371-78 (1990); any other Rhodobacter strain containing the hemA RC promoter can be used), primers EV98 and EV99, and program "J" described above.
  • Rhodobacter strain SB 1003 Rhodobacter strain SB 1003 (Hornberger, Mol. Gen- Genet. 221 :371-78 (1990); any other Rhodobacter strain containing the hemA RC promoter can be used
  • primers EV98 and EV99 primers EV98 and EV99
  • program "J" described above.
  • Primers and cycle conditions used for amplification of the BamHI-HindlH fragment in pSGEl 104 were as follows: EV1 13 and TG224; one cycle of 5 min. at 95°C, 5 min. at 60°C, 3 min. at 72°C,
  • Plasmid DNA for cloning and DNA sequencing was isolated using the Wizard plasmid isolation kit (Promega, Madison, WI) according to the manufacturer's instructions. Restriction digests, gel electrophoresis, DNA ligations and transformations were performed according to standard methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Springs Harbor, 1989). PCR fragments for ligation reactions were purified using a GENECLEAN II kit (Bio 101, Vista, CA). Both strands of DNA fragments generated by PCR were sequenced using the Prism DYE-terminator cycle sequencing system (Applied Biosystems, Foster City, CA) and an Applied Biosystems model 373 automated sequencer (Applied Biosystems, Foster City, CA). DNA sequences were analyzed using programs contained in the MacVector (version 4.5.2.) software package (International Biotechnologies, Inc. New Haven, CT).
  • Beta-galactosidase assays Strains were grown overnight in 5 ml of M63 salts (Miller, Experiments in Molecular Genetics. (Cold Springs Harbor, 1972)) supplemented with 0.4% glucose, 0.1% casamino acids, 50 ⁇ g/ml proline, 40 ⁇ g/ml thiamine, 1 mM MgSO 4 , and appropriate antibiotics. Cells were diluted 1 :50 into test tubes containing 5 ml of the same medium, and grown at 37°C with shaking to OD ⁇ 0.4-0.8. Cultures were assayed for ⁇ - galactosidase according to the method described by Miller, supra.
  • Fermentations were performed at 30°C in 15 liter fermentors (LSL Biolafitte, Inc.
  • the seed inoculum for 15 liter fermentors was prepared in a two stage process.
  • the primary seed stage was 500 ml of DM59 medium in a 2.5 liter shake flask inoculated with 0.5 ml of a stock culture preserved in 7% (v/v) dimethyl sulfoxide at ⁇ 80°C.
  • the salts used in DM59 medium were as follows: 33 mM KH 2 PO 4 , 46 mM K 2 HPO 4 , 13 mM NaH 2 PO 4 , 18 mM Na-HPO 4 , 19 mM (NH 4 ) 2 SO 4 , 5.4 mM K 3 citrate, 2.2 mM Na 3 citrate, 4.2 mM MgSO 4 , 7.2 mM H 3 PO 4 .
  • Trace metals were added to DM59 salts to the final concentrations indicated: 0.91 mM FeCl 3 , 0.14 mM ZnCl 2 , 12 ⁇ M CoCl 2 , 10 ⁇ M Na 2 Mo0 4 , 0.13 mM MnCl 2 , 0.41 mM CaCl 2 and 54 ⁇ M CuSO 4 .
  • Glucose was added to a final concentration of 1% (w/v).
  • Thiamine was added to a final concentration of 0.32 mg/ml.
  • Antibiotics (see below) were added as needed to maintain plasmids. The primary seed stage . ,.
  • the pH of the secondary seed stage culture was maintained at 20% in 2 liter fermentors.
  • IPTG isopropyl B-D-thiogalactopyranside
  • Heme b obtained as bovine hemin (Amesresco, Solon, OH), was dissolved in IN NaOH to a final concentration of 50 mg/ml. Where specified, heme b was added to fermentors at the time of IPTG induction, and at 3 and 6 hrs. post-induction, as 10 ml, 13 ml, and 17 ml aliquots.
  • Antibiotics Sigma Chemical Co., St. Louis, MO were added to shake flasks and fermentors in the following concentrations: tetracycline, 15 ⁇ g/ml; chloramphenicol, 25 ⁇ g/ml, and ampicillin, 100 ⁇ g/ml.
  • ALA and PBG assays One ml fermentation samples were resuspended in 10 mM
  • MES pH 6.0
  • Lysozyme, NaCl, and DNAse were added to final concentrations of 500 ⁇ g/ml (lysozyme), 100 mM (NaCl) and 60 ⁇ g/ml (DNAse), and samples were incubated on ice for 20 min., and then at 37°C for 2 min.
  • Proteinase K Sigma Chemical Co., St. Louis, MO was added to a final concentration of 150 ⁇ g/ml, and samples were incubated for an additional 20 min. on ice. The sample pH was lowered to 5.5-5.9 by addition of 10% acetic acid.
  • ALA and PBG were separated using a two-column chromatography system (ALA and PBG Column Test Kit) obtained from BioRad (Hercules, CA). ALA and PBG levels were quantified by their reactivity with Ehrlich's reagent, essentially as described in the BioRad test kit.
  • Hemoglobin assay Samples (1 ml of fermentation broth at) were pelleted and resuspended in 25 mM Na ⁇ O ? . Lysozyme and NaCl were added to final concentrations of 1 mM NaCl and 0.75 mg/ml lysozyme, and the samples were incubated first at 4°C (30-40 min), and then at 37°C (3 min). DNAse (60 ⁇ g/ml) was added, and the samples were incubated 15 min. at room temperature. The samples were freeze-thawed, treated for 10 sec. with CO gas, and then diluted into a solution of 80 mM Tris-HCl, 2 M NaCl (pH8.0).
  • the crude lysates were heated at 65°C for 4 min., and centrifuged at 13,000 x g for 2 min to remove cellular debris and contaminating proteins.
  • the supernatant fraction containing partially purified hemoglobin was saved, and the hemoglobin was quantified by immobilized metal chelate chromatography using a Biocad Perfusion Chromatography Workstation (PerSeptive Biosystems, Cambridge, MA).
  • the capture column was charged with a solution of 20 mM Zn(OAc) 2 , 200 mM NaCl, and equilibrated with 8 mM Tris-HCl, 200 mM NaCl (pH 8.0).
  • Plasmid pSGE715 is a high-copy number plasmid for IPTG-controIled expression of the alpha and beta subunits of human hemoglobin. Following IPTG induction, E. coli strains containing pSGE715 produce large quantities of fully functional tetrameric hemoglobin variant (rHbl.l; WO 97/041 10). Because each hemoglobin tetramer has the capacity to bind four heme b groups, induction of rHbl.l is expected to rapidly deplete cellular pools of heme b . Accordingly, if heme b is a repressor of E.coli heme b synthesis, removal of heme b by rHbl .
  • heme b is a negative regulator of the heme b pathway.
  • shake flask cultures of SGE1453 Prior to induction of rHbl.l, shake flask cultures of SGE1453 contain approximately 25 pmoles/OD 600 *ml of heme b in heme b -containing proteins and as free heme b (Fig. 2, no ALA, zero hour time point).
  • Fig. 2, no ALA, zero hour time point Prior to induction of rHbl.l, shake flask cultures of SGE1453 contain approximately 25 pmoles/OD 600 *ml of heme b in heme b -containing proteins and as free heme b (Fig. 2, no ALA, zero hour time point).
  • Fig. 2, no ALA, zero hour time point Four hours following induction of rHbl . l, cultures of SGE1453 have approximately 75 pmoles/OD 600 *ml heme b (Fig. 2, no ALA, zero time point).
  • This divergent transcript was shown to encode a 23-kDa protein, hemM, and a role for HemM in ALA synthesis was proposed (Chen et al., J. Bacteriol.. 176:2743-2746 (1994); Ikemi, supra.) .
  • SGE1857 included the hemA l promoter
  • SGE1858 included the hemA 2 promoter
  • SGE1859 included both the hemA x and hemA 2 promoters.
  • Beta- galactosidase activity assays indicated that both the hemA x and hemA 2 promoters are biologically active, and that the hemA, promoter is the stronger promoter (Table 2). Similar results were obtained when the three hemA-l ⁇ cZ fusions were integrated into the genome of a hemA mutant and grown with 0.2 ⁇ g/ml ALA.
  • Cells were grown in five ml cultures and assayed as described in Materials and Methods. b Values reported are the average of four independent experiments. Variation is indicated in parentheses as standard error. A control strain containing pSGE518 produced 40
  • heme b does not regulate hemA gene expression or the hemA promoters tested.
  • ALA which is structurally similar to the dipeptide glycyl-glycine, is taken up by E.coli using a dipeptide permease. Because ALA is normally synthesized by E.coli cells rather than taken up by cells, transport of ALA by the dipeptide permease may be a relatively inefficient method for increasing intracellular ALA pools. Therefore, increasing ALA pools by genetically manipulating enzymes involved in ALA formation, especially GTR reductase, was examined to determine if they have a greater effect on cellular heme b content than supplementation with exogenous ALA. Accordingly, plasmids for overexpression of the hemA and hemM genes from their native promoters were constructed and evaluated in fermentation cultures (Table 3).
  • Rhodobacter synthesize ALA by an alternate route, the C4 or Shemin pathway (Fig. 1).
  • the C4 pathway requires only a single enzymatic step, and is catalyzed by the enzyme ALA synthase (E.C. 2.3.1.37)(Fig. 1).
  • the Rhodobacter gene that encodes ALA synthase has been designated hemA. To avoid confusion with the E. coli hemA, which encodes a different activity, the Rhodobacter hemA gene is referred to herein as hemA RC .
  • E.coli hemA mutants which require ALA supplementation for growth, can be complemented by the Rhodobacter / ⁇ em ⁇ fiC (Hornberger et al., Mol. Gen. Genet.. 221:371-378 (1990)).
  • Rhodobacter / ⁇ em ⁇ fiC Rhodobacter / ⁇ em ⁇ fiC
  • E.coli can produce heme b using enzymes from either the C4 or C5 pathways.
  • hem RC was placed under control of the tac promoter to create an inducible system for studying the effect of high-level expression of ALA synthase on heme b pathway regulation.
  • ALA and PBG pools in cells expressing hemA RC were significantly higher than in a control strain (SGE1453) following induction of HemA RC and rHbl . l expression (Tables 4 and 5). Although strains expressing HemA RC and rHbl. l (SGE2681) produced more heme b , the rates of rHbl. l accumulation for strains SGE1453 and SGE2681 were indistinguishable (Tables 4 and 5). The ratios of ALA and PBG in cells expressing hemA (SGE2644), hemA+M (SGE2658) and hemA RC (SGE2681) were considerably different.

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Abstract

Procédés permettant d'améliorer la production de protéines contenant de l'hème par augmentation de l'accumulation de l'hème dans des cellules hôtes hétérologues. Lesdits procédés consistent à exposer les cellules hôtes à des quantités accrues d'acide δ-aminolévunilique (ALA) endogène ou exogène, ce qui entraîne une augmentation de la production d'hème. On peut augmenter la production de ALA en ajoutant des copies multiples du gène hemA dans la cellule hôte. Des procédés de production de quantités améliorées d'hème sont également décrits.
PCT/US1997/014165 1996-08-30 1997-08-29 Production d'heme et d'hemoproteines de recombinaison WO1998008954A2 (fr)

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WO2018046512A1 (fr) * 2016-09-08 2018-03-15 Universitaet Bielefeld Procédés et agents de production microbienne d'acide aminolévulinique
WO2022269550A1 (fr) * 2021-06-24 2022-12-29 Reliance Industries Limited Micro-organisme génétiquement modifié, procédé(s) et application(s) associés

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018046512A1 (fr) * 2016-09-08 2018-03-15 Universitaet Bielefeld Procédés et agents de production microbienne d'acide aminolévulinique
WO2022269550A1 (fr) * 2021-06-24 2022-12-29 Reliance Industries Limited Micro-organisme génétiquement modifié, procédé(s) et application(s) associés

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