EP0497916A1 - Expression d'hemoglobine bacterienne et amelioration de l'expression de produits clones et natifs dans des streptomycetes - Google Patents

Expression d'hemoglobine bacterienne et amelioration de l'expression de produits clones et natifs dans des streptomycetes

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Publication number
EP0497916A1
EP0497916A1 EP91900091A EP91900091A EP0497916A1 EP 0497916 A1 EP0497916 A1 EP 0497916A1 EP 91900091 A EP91900091 A EP 91900091A EP 91900091 A EP91900091 A EP 91900091A EP 0497916 A1 EP0497916 A1 EP 0497916A1
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Prior art keywords
streptomyces
host
expression
protein
vector
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German (de)
English (en)
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Dallas E. Hughes
Sharon K. Magnolo
John Anthony Demodena
Joseph Edward Curtis
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Exogene Corp
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Exogene Corp
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    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • TECHNICAL FIELD This invention relates to the expression of Vitreoscilla hemoglobin in Streptomyces s . to enhance growth characteristics and antibiotic yields at low or reduced oxygen levels.
  • This invention relates to the use of Vitreoscilla hemoglobin gene promoter to obtain high level expression of cloned proteins in Streptomyces.
  • the bacteria of the genus Streptomyces are used for the production of approximately 60% of the commercially available antibiotics (Atkinson and Mavituna, Biochemical Engineering and Biotechnology Handbook, Macmillan, England, 1987) .
  • Examples of widely-used antibiotic compounds produced in Streptomyces fermentations include the spriamycins, neomycins, tetracycline ⁇ , and streptomycins (Demain and Solomon, Manual of Industrial Microbiology and Biotechnology. American Society for Microbiology, 1986) .
  • many compounds produced by Streptomyces have antineoplastic (the bleomycins, mithramycins, and daunomycins) and antihelminthic (the avermectins) activity.
  • Streptomyces are obligate aerobes that reguire high levels of oxygen for optimal growth. Providing sufficient oxygen to a high cell density culture represents a major obstacle due to the tendency for Streptomyces to form long filamentous strands that results in a highly viscous culture. Viscosity dramatically reduces the oxygen transfer rate to the culture medium.
  • a typical Streptomyces fermentation begins with the growth of cells to high densities (growth phase) . There is little antibiotic production during growth phase. The final cell densities achieved are usually limited by the oxygen supply. After cessation of growth, antibiotic synthesis begins (production phase) . The antibiotic production phase is maintained as long as possible by supplying the appropriate nutrients. Eventually, acidic waste products accumulate and the cells die.
  • the effect of bacterial hemoglobin expression on growth of a unicellular organism was investigated by Khosla and Bailey (Khosla and Bailey, ibid.).
  • the bacterial hemoglobin was originally discovered in the obligate aerobic bacterium, Vitreoscilla (Tyree and Webster, J. Biol. Chem. , 253: 6988, 1978).
  • the hemoglobin is a soluble, di eric protein that combines with oxygen and displays a spectral response to carbon monoxide binding characteristic of eukaryotic hemoglobins (Wakabayashi, et al. , Nature, 332: 481, 1986). It was conjectured that the hemoglobin protein functioned to facilitate oxygen transfer to Vitreoscilla and thus allowed it to propagate under oxygen-poor conditions.
  • the gene for the Vitreoscilla hemoglobin has been isolated along with its native transcriptional regulatory sequences. (Khosla and Bailey, Mol. Gen. Genet, 214: 158, 1988). Interestingly, this gene was expressed from its native promoter when introduced into E. coli. Of particular interest was that expression of hemoglobin was regulated by the culture oxygen content such that maximal induction occurred under microaerobic conditions. Under fed-batch fermentation conditions, E. coli cells expressing hemoglobin displayed significantly higher specific growth rates and achieved 2-3 fold the final cell densities as non-expressing strains (Khosla and Bailey, Nature. 331:633, 1988).
  • the present invention relates to oxygen-binding proteins, particularly hemoglobins, a reco binant-DNA method of producing same, and to portable DNA sequences capable of directing intracellular production of these oxygen-binding proteins in Streptomyces.
  • the present invention also relates to vectors containing these portable DNA sequences.
  • One object of the present invention is to provide a recombinant-DNA method for the production of these oxygen-bindingproteins.
  • the present invention also provides novel methods and materials for expression of cloned genes in Streptomyces. Particularly, it related to promoter/regulators, a recombinant-DNA method of producing same, and to portable DNA sequences capable of directing the translation and transcription initiation and control of the expression of desired gene products.
  • another object of the present invention is to provide for the expression in Streptomyces of any selected chromosomal or extrachromosomal gene or DNA sequence through the incorporation of a promoter/regulator DNA sequence.
  • Such expression may thus provide native or heterologous enzyme activities which increase antibiotic production or which enable synthesis of modified or novel antibiotics.
  • promoter/ regulators are also set forth.
  • portable DNA sequences for these promoter/regulators are provided.
  • Particularly preferred promoter/regulator DNA sequences for use in the practice of the present invention are derived from the filamentous bacterium Vitreoscilla.
  • Portable nucleotide sequences are provided for these promoter/regulators.
  • the portable sequences may be either synthetic sequences or restriction fragments ("natural" DNA sequences) .
  • portable DNA sequences useful in the processes of the present invention may be synthetically created. These synthetic DNA sequences may be prepared by polynucleotide synthesis and sequencing techniques known to those of ordinary skill in the art.
  • a recombinant-DNA method is disclosed which results in manufacture by cells of the genus Streptomyces of the instant oxygen-binding proteins using the portable DNA sequences referred to above.
  • recombinant- DNA methods which provide transcription and translation of gene products by a host Streptomyces using the portable DNA sequences referred to above.
  • cloning vectors comprising at least one portable DNA sequence.
  • plasmids pWLD5 and pWLDlO are disclosed.
  • Figure 1 is a partial restriction map of plasmids pWLD 10 and pWLD 5.
  • one objective of this invention is metabolically improved Streptomyces cells which have preferred functional characteristics in aerobic manufacturing processes.
  • the present invention relates in part to portable DNA sequences capable of directing intracellularproduction of oxygen- binding proteins in a variety of Streptomyces species.
  • "Portable DNA sequence” in this context is intended to refer either to a synthetically produced nucleotide sequence or to a restriction fragment of a naturally occurring DNA sequence.
  • oxygen-binding protein is intended to mean a protein with a primary structure as defined by the codons present in the deoxyribonucleic acid sequence which directs intracellular production of the a ino acid sequence, and which may or may not include post- translational modifications. It is contemplated that such post-translational modifications include, for example, association with a heme prosthetic group. It is further intended that the term “oxygen-binding protein” refers to either the form of the protein as would be excreted from a cell or as it may be present in the cell from which it was not excreted.
  • the intracellular presence of cloned hemoglobin may provide a modified form(s) of the antibiotic molecule(s) normally produced by the host strain of Streptomyces.
  • the portable DNA sequences are capable of directing intracellular production of hemoglobin.
  • the portable DNA sequences are capable of directing intracellular production of a hemoglobin biologically equivalent to that previously isolated from the filamentous bacterium, Vitreoscilla.
  • biologically equivalent as used herein, it is meant that a protein, produced using a portable DNA sequence of the present invention, is capable of binding oxygen in the same fashion, but not necessarily to the same degree, as the homodimeric soluble heme protein (subunit MW 15,775) isolable from Vitreoscilla.
  • the present invention also relates in part to portable DNA sequences which contain promoter/regulators which are capable of directing intracellularexpression of endogenous or exogenous gene products, in a variety of host cells and host microorganisms.
  • "Portable DNA sequence” and “promoter/regulator” in this context are intended to refer either to a synthetically produced nucleotide sequence or to a restriction fragment of a naturally occurring DNA sequence.
  • the portable DNA sequences of the present invention may also include DNA sequences downstream from a promoter/regulator which code for at least one foreign protein.
  • "foreign protein” is intended to mean a protein with a primary structure as defined by the codons present in the deoxyribonucleic acid sequence which directs intracellularproduction of the corresponding a ino acid seguence, and which may or may not include post- translational modifications. It is further intended that the term “foreign protein” refers to either the form of the protein as it would be excreted from a cell or as it may be present in the cell from which it was not excreted.
  • the promoter/regulator contains transcription and translation initiation and control ⁇ eguences substantially equivalent to those for directing intracellular production of a hemoglobin protein biologically equivalent tothatpreviously isolated from the filamentous bacterium, Vitreoscilla.
  • a preferred portable DNA sequence for the promoter/regulators of the present invention contains at least a portion of the following nucleotide sequence, which reads 5 ' to 3 ' and includes the translation initiation sequence ATG (underlined) and some of the nucleotide sequence of the Vitreoscilla structural gene (also underlined) :
  • the above sequence exhibits homology with certain sequences which are highly conserved in a variety of promoter/regulators.
  • the -10 consensus sequence or Pribnow box sequence is TATAAT(A/G) .
  • the -35 consensus sequence is TTGACA, and the consensus Shine-Dalgarno sequence is AGGAGGTXXX(XX)ATG.
  • the above sequence is operatively fused with at least a portion of a downstream sequence of nucleotides which code for at least a portion of the Vitreoscilla hemoglobin protein which contains at least a portion of the following amino acid sequence: 5 10
  • amino acids represented by the foregoing abbreviations are as follows:
  • substantially homology is meant a degree of homology to native Vitreoscilla hemoglobin in excess of 50%, preferably in excess of 80%.
  • the portable DNA sequences of the present invention may be synthetically created, by hand or with automated apparatus. It is believed that the means for synthetic creation of these polynucleotide sequences are generally known to one of ordinary skill in the art, particularly in light of the teachings contained herein. As examples of the current state of the art relating to polynucleotide synthesis, one is directed to Maniatis et al. , Molecular Clonin — LaboratoryManual, Cold SpringHarbor Laboratory (1984) , and Horvath et al. , An Automated DNA Synthesizer Employing Deoxynucleoside 3'-Phosphoramidites. Methods in Enzymology 154:313-326, 1987, hereby incorporated by reference.
  • the portable DNA sequence may be a fragment of a natural sequence, i.e. , a fragment of a polynucleotide which occurred in nature.
  • the portable DNA sequence is a restriction fragment isolated from a genomic library.
  • the genomic library is created from the bacterium Vitreoscilla.
  • the portable DNA sequence is isolated from other genomic and cDNA libraries.
  • the present invention also provides a series of vectors, each containing at least one of the portable DNA sequences described herein. It is contemplated that additional copies of the portable DNA sequence may be included in a single vector to increase a host cell's ability to produce large quantities of the desired oxygen-binding protein. It is also envisioned that other desirable DNA sequences may also be included in the vectors of this invention. Further, the invention may be practiced through the use of multiple vectors, with additional copies of at least one of the portable DNA sequences of this invention and perhaps -other desirable DNA sequences.
  • the cloning vectors within the scope of the present invention may contain supplemental nucleotide sequences preceding or subsequent to the portable promoter/regulator and/or DNA sequence. These supplemental sequences are those thatwill not adversely interfere with transcription of the portable promoter/regulator and/or any fused DNA sequence and will, in some instances, enhance transcription, translation, posttranslational processing, or the ability of the primary amino acid structure of the resultant gene product to assume an active form.
  • a preferred vector of the present invention is set forth in Figure 1.
  • This vector, pWLDIO contains the preferred nucleotide sequence which codes for the amino acids set forth above.
  • Plasmid pWLDIO (and pWLD5) may also contain supplemental nucleotide sequences such as terminators, enhancers, attenuators and the like.
  • supplemental nucleotide sequences such as terminators, enhancers, attenuators and the like.
  • at least one leader sequence and any other DNA sequences necessary orpreferred for appropriate transcription and subsequent translation of the vector DNA may be included within the scope of this invention.
  • cloning vectors containing and capable of expressing the portable DNA sequence of the present invention contain various operational elements in addition to or instead of the promoter/regulator disclosed and claimed herein.
  • These "operational elements" may include at least one promoter, at least one sequence that acts as expression regulator, and at least one terminator codon, at least one leader sequence, and any other DNA sequences necessary orpreferred for appropriate transcription and subsequent translation of the vector DNA.
  • Additional embodiments of the present invention are envisioned as employing other known or currently undiscovered vectors which would contain one or more of the portable DNA sequences described herein.
  • these vectors have some or all of the following characteristics: (1) possess a minimal number of host-organism sequences; (2) be stable in the desired host; (3) be capable of being present in a high copy number in the desired host; (4) possess a regulatable promoter; and (5) have at least one DNA sequence coding for a selectable trait present on a portion of the plasmid separate from that where the portable DNA sequence will be inserted.
  • Alteration of vectors to meet the above criteria are easily performed by those of ordinary skill in the art in light of the available literature and the teachings herein. It is to be understood that additional cloning vectors may now exist or will be discovered which have the above- identified properties and are therefore suitable for use in the present invention and these vectors are also contemplated as being within the scope of this invention.
  • Streptomyces Any strain of Streptomyces which admits stable insertion of cloned DNA can serve as a host for the practice of this invention.
  • Examples of Streptomyces strains which can be transformed or transduced are:
  • Streptomyces lividans 66 - Hopwood, et aJL. Genetic Manipulation of Streptomyces: A Laboratory Manual. The John Innes Foundation, Norwich, 1985.
  • Streptomyces fradiae - Chung J. Bacteriol. , 169: 4436, 1987. Streptomyces ambofaciens - Matsushima and
  • Various vector systems will be suitable for Streptomyces species, including plasmids, and bacteriophages.
  • the following, noninclusive list of cloning vectors is believed to set forth vectors which can easily be altered to meet the above criteria and are therefore preferred for use in the present invention. Such alterations are easily performed by those of ordinary skill in the art in light of the available literature and the teaching herein.
  • Streptomyces plasmids have been used as vectors: pIJ699 - Kieser and Melton, Gene. 65:83, 1988, pIJ702 - Katz, et al. , J. Gen. Microbiol., 129: 2703, 1983), pHJL400 - Larson and Hershberger, Plasmid, 15: 199, 1986) , pKC505 - Richardson, et aJL. , Gene, 61:231, 1987,
  • Phages used as Streptomyces vectors include derivatives of ⁇ C31 (Hopwood, et. al. , Methods Enzvmol.. 153:116, 1987) . See, for example, phage KC515 - Rodicio, et al. , Gene, 34:283, 1985.
  • the cloning vector contain a selectable marker, such as a drug resistance marker or other marker which causes expression of a selectable trait by the host.
  • a selectable marker such as a drug resistance marker or other marker which causes expression of a selectable trait by the host.
  • the gene for thiostrepton resistance is included in vector pWLDIO.
  • Such a drug resistance or other selectable marker is intended in part to facilitate in the selection of transformants.
  • the presence of such a selectable marker on the cloning vector may be of use in keeping contaminating microorganisms from multiplying in the culture medium. In this embodiment, such a pure culture of the transformed host organisms would be obtained by culturing the organisms under conditions which require the induced phenotype for survival.
  • the portable DNA sequence of the present invention may themselves be used as a selectable marker, in that they provide enhanced growth characteristics in low oxygen circumstances.
  • the promoter/regulators of this invention are capable of controlling expression of proteins or, thereby, of controlling synthesis of metabolites normally made by a cell, or of natural or unnatural metabolites and proteins expressed in a cell via genetic manipulation. This would include heterologous proteins—either intracellular or extracellular—as well as antibiotics and other chemicals produced by Streptomyces cells.
  • This invention also relates to a recombinant-DNA method for the production of oxygen-binding proteins.
  • this method includes:
  • the portable DNA sequences may be inserted directly into the host chromosome, or alternatively may utilize a vector cloning system.
  • the vectors contemplated as being useful in the present method are those described above.
  • the cloning vectors pWLDIO -and pWLD5 are used in the disclosed method.
  • a vector thus obtained may then be transferred into the appropriate Streptomyces species. It is believed that any Streptomyces species having the ability to take up exogenous DNA and express those genes and attendant operational elements may be chosen. Particular hosts which may be preferable for use in this invention include those described above. Methods for transfer of vectors into hosts are within the ordinary skill in the art. For ultimate expression in Streptomyces . it may be desirable that the cloning vector be first transferred into another microorganism such as Escherichia coli, where the vector would be allowed to replicate and, from which the vector would be obtained and purified after amplification, and then transferred into the Streptomyces for ultimate expression of the oxygen-binding protein.
  • another microorganism such as Escherichia coli
  • Thehost cells are culturedunder conditions appropriate for the expression of the oxygen-binding protein. These conditions are generally specific for the host organism, and are readily determined by one of ordinary skill in the art. It is understood that application of the teachings of the present invention to a specific problem or environment will be within the capabilities of one having ordinary skill in the art in light of teachings contained herein. Examples of the products of the present invention and representative processes for their isolation, use and manufacture appear below.
  • the products and processes of the present invention find usefulness in the production of antibiotics and the expression of any cloned proteins using Streptomyces in laboratory and industrial applications.
  • the invention provides metabolically engineered cells with enhanced growth characteristics for increasing production of proteins, antibiotics, or other metabolites in Streptomyces.
  • the invention also provides a DNA sequence that acts as a strong transcriptional initiation sequence for the expression of cloned proteins in Streptomyces.
  • a plasmid was constructed for the expression of a bacterial hemoglobin in Streptomyces.
  • This plasmid, pWLD5 contains the Vitreoscilla hemoglobin gene and its native transcriptional regulatory sequences [Khosla and Bailey (1988) Mol. Gen. Genet.. 214:158] cloned into a common Streptomyces plasmid, plJ699 [Keiser and Melton (1988) Gene, 65:83].
  • the 1.2 kilobase Hind III/SphI Vitreoscilla DNA fragment containing the hemoglobin gene was first inserted into the Hindlll/Sphl site of the Escherichia coli plasmid pUC19. This construct was then linearized with Hindiii and ligated into Hindlll-cut pIJ699.
  • the resulting plasmid, pWLD5 was stably maintained in both E. coli and Streptomyces lividans.
  • TK64:pWLD5 A single thiostrepton-resistant colony, designated TK64:pWLD5
  • TK64:pWLD5 A single thiostrepton-resistant colony
  • Hemoglobin expression in TK464:pWLD5 was confirmed by Western analysis of total cell protein.
  • a crude cell extract was generated by sonication and the proteins separated by SDS-polyacrylamide gel electrophoresis. The proteins were then electrotransferred to nitrocellulose membrane and screenedwith polyclonal antiserumgenerated against pure Vitreoscilla hemoglobin.
  • a hemoglobin band of identical molecular weight as pure hemoglobin was detected in the cell extracts.
  • Hemoglobin expression appeared to be constitutive as the levels were similar in cells sampled from any stage of growth. Expression of functional hemoglobin was demonstrated by a carbon monoxide difference spectrum technique [Webster and Liu (1974) J. Biol. Chem. 249:4257].
  • TK64:pWLD5 was compared with the plasmid-free strain (TK64) under two culture conditions corresponding to high and low aeration.
  • the culture medium used for the experiment was as follows: 3% dextrose, 2% N-Z a ine Type I, 1% yeast extract, and 1% v/v .
  • the first condition was a 50 mL culture volume in a 250 mL unbaffled erlenmeyer flask shaken at 250 rpm at 300C;
  • the second condition was a 75 culture volume in a 250 L unbaffled erlenmeyer flask shaken at 150 rpm at 30°C.
  • the maximum specific growth rates of the two strains were similar (0.10-O.11 h..,) under reduced aeration. Hemoglobin expression levels in the two strains were similar throughout the experiment as demonstrated by Western analysis.
  • Oxygen uptake rates were compared between TK64:pWLD5 and the plasmid-free strain throughout this experiment. Cells were removed at various times, washed, and resuspended in fresh medium at an OD 590 of 0.10. The OUR's were then measured using a Yellow
  • TK64:pWLD5 reached a final OD 590 of 4.5, while TK64 reached an OD 590 of only 3.3, a difference of 41%.
  • This experiment indicates that hemoglobin expression benefits Streptomyces cell growth under two additional conditions of reduced culture oxygen.
  • a plasmid similar to pWLD5 was constructed by inserting BamHI-linearized pRED2 [Khosla and Bailey (1988) Mol. Gen. Genet. 214:158] into Bglll-digested plJ699.
  • pRED2 contains the identical hemoglobin sequence as pWLD5 but contains an additional 1.5 kb of non-essential DNA.
  • the resultant plasmid, PWLDIO was transformed into Streptomyces coelicolor strain M145 (SCP1 " ' SCP2- obtained from Dr. David Hopwood, John Innes Institute, Norwich, England) and a single thiostrepton-resistant transformant, designated M145:pWLD10, was selected for further experiments.
  • M145:pWLD10 cells were grown in liquid culture to exponential phase in 50 mL YEME medium (0.3% yeast extract, 0.5% peptone, 0.3% malt extract, 1% glucose,
  • Vitreoscilla hemoglobin promoter element functions in S. coelicolor to express a heterologous protein.
  • this promoter functions in different strains of Streptomyces.
  • Antibiotic production in Streptomyces coelicolor strains M145 and M145:pWLD10 was compared in a shake flask culture experiment.
  • One mL of exponential phase cells were inoculated into 50 mL of YEME medium (5 ug/ml thiostrepton was added to the M145:pWLD10 culture) in 250 mL unbaffled flasks.
  • the cells were grown at 250 rpm at 30°C.
  • Ten days later the cultures were analysed for the production of the pigmented antibiotic, undecylprodigiosin.
  • the assay was performed by mixing equal volumes of the culture and 0.1 M NaOH followed by a 30" sonication (50 Watt output) on ice.
  • the sonicate was then filtered through a 0.2 uM nitrocellulose membrane.
  • the OD 46g of the filtrate which is a measure of undecylprodigiosin, was then determined. While the hemoglobin-expressing strain had an OD 468 of 1.4, thenon- expressing strain had an OD 46g of only 0.6. This indicated that greater than twice as much antibiotic is produced in a hemoglobin-expressing strain of Streptomyces.

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Abstract

Procédé pour exprimer de l'hémoglobine bactérienne dans des Streptomycètes, et, par ce moyen, pour améliorer le métabolisme aérobie et la production d'antibiotiques dans des Streptomycètes. Sont également décrits des vecteurs d'expression permettant l'expression de gènes natifs et hétérologues dans des Streptomycètes. Des vecteurs pWLD5 et pWLD10 sont représentés à titre d'exemple dans les figures.
EP91900091A 1989-10-30 1990-10-26 Expression d'hemoglobine bacterienne et amelioration de l'expression de produits clones et natifs dans des streptomycetes Withdrawn EP0497916A1 (fr)

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EP (1) EP0497916A1 (fr)
AU (1) AU6891291A (fr)
CA (1) CA2072115A1 (fr)
IL (1) IL96142A0 (fr)
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ZA (1) ZA908687B (fr)

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EP0500652A4 (en) * 1989-10-30 1992-12-02 California Institute Of Technology Enhancement of cell growth by expression of a cloned hemoglobin gene
WO1993025697A1 (fr) * 1992-06-15 1993-12-23 California Institute Of Technology Renforcement de la croissance des cellules par expression de proteines clonees fixatrices d'oxygene
US5908764A (en) * 1997-05-22 1999-06-01 Solidago Ag Methods and compositions for increasing production of erythromycin
US8265357B2 (en) 2005-10-14 2012-09-11 Unisense Fertilitech A/S Determination of a change in a cell population
DK2173853T3 (da) 2007-06-29 2011-07-04 Unisense Fertilitech As Indretning, system og fremgangsmåde til at overvåge og/eller dyrke mikroskopiske objekter
WO2015091967A1 (fr) * 2013-12-19 2015-06-25 Basf Se Mélanges comprenant un polymère superabsorbant (sap) et un biopesticide
CA3007635A1 (fr) 2015-12-07 2017-06-15 Zymergen Inc. Promoteurs de corynebacterium glutamicum
US11208649B2 (en) 2015-12-07 2021-12-28 Zymergen Inc. HTP genomic engineering platform
US9988624B2 (en) 2015-12-07 2018-06-05 Zymergen Inc. Microbial strain improvement by a HTP genomic engineering platform
US10544390B2 (en) 2016-06-30 2020-01-28 Zymergen Inc. Methods for generating a bacterial hemoglobin library and uses thereof
JP2019519241A (ja) 2016-06-30 2019-07-11 ザイマージェン インコーポレイテッド グルコース透過酵素ライブラリーを生成するための方法およびその使用

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CA2072115A1 (fr) 1991-05-01
ZA908687B (en) 1991-11-27
AU6891291A (en) 1991-05-31
WO1991006628A1 (fr) 1991-05-16
IL96142A0 (en) 1991-07-18

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