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WO1993025697A1 - Renforcement de la croissance des cellules par expression de proteines clonees fixatrices d'oxygene - Google Patents

Renforcement de la croissance des cellules par expression de proteines clonees fixatrices d'oxygene Download PDF

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Publication number
WO1993025697A1
WO1993025697A1 PCT/US1993/005527 US9305527W WO9325697A1 WO 1993025697 A1 WO1993025697 A1 WO 1993025697A1 US 9305527 W US9305527 W US 9305527W WO 9325697 A1 WO9325697 A1 WO 9325697A1
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Prior art keywords
oxygen
host
cell
protein
expression
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PCT/US1993/005527
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English (en)
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James E. Bailey
Chaitan Khosla
Dallas E. Hughes
Jorge Galazzo
Fred C Sander, Jr.
J. David Rozzell
John Demodena
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California Institute Of Technology
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Publication of WO1993025697A1 publication Critical patent/WO1993025697A1/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
    • 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
    • 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
    • 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
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi

Definitions

  • This invention relates to the production of oxygen- binding proteins, particularly members of the globin family, and to enhancement of the growth and product synthesis characteristics of aerobic organisms in environments with sufficient as well as reduced or low levels of oxygen.
  • This invention relates generally to the use of recombinant DNA technology to direct or otherwise control gene expression in cultured cells, and more particularly, to methods and materials useful in subjecting the transcription and translation of DNA sequences to selective regulation by external control.
  • the present invention is directed toward the expression and use of oxygen-binding proteins, including, but not limited to, the evolutionary- related globin superfamily: hemoglobins (found in many eukaryotic organisms) ; leghemoglobins (found in many plants) ; myoglobins (found mainly in animal muscle tissue) ; and icrobial globins such as Vitreoscilla hemoglobin; _£____ coli hmp (a flavohemoglobin) , and yeast flavohemoglobin.
  • Other oxygen-binding hemoproteins include cytochrome C reductase, cytochrome c oxidase, and kinase in the oxygen sensor of Rhizobium meliloti.
  • Additional oxygen-binding proteins which function in the transport of oxygen in invertebrates include hemocyanins and hemerythrins.
  • globin-like oxygen-binding proteins may also be distributed in microrganisms.
  • Such endogenous, homologous globins may also be utilized in accordance with the present invention, providing the added advantage of expressing a globin from a gene homologous to the desired host organism.
  • the oxygen uptake rate is principally limited by the rate of transfer of dissolved oxygen in the environment or growth medium to the exterior cell surface.
  • closer examination of cell structure reveals several potential diffusional barriers between environmental oxygen and the cytochro es where the oxygen finally undergoes reaction.
  • the diffusing oxygen needs to cross transport barriers such as the cell wall, the outer membrane, the periplasmic space and the inner membrane before accepting electrons from metabolic reactions.
  • unicellular eucaryotes where oxidative phosphorylation takes place in the mitochondria, there are further diffusional resistances. Small neutral molecules like oxygen are assumed to passively diffuse across these barriers; however, these barriers make a non-trivial contribution to the overall resistance to mass transfer to the actual reaction site and thus could be of significance under conditions of low oxygen or oxygen limitation.
  • oxygen-binding proteins In addition to the respiratory oxygen requirement of aerobic organisms, oxygen-binding proteins have other potential applications as well, including, for example, the enhancement of particular oxidative transformations such as steroid conversions, vinegar production, biological waste treatment or enzymatic degradations, and in some steps in brewing or making distilled and fermented foods and beverages.
  • the filamentous bacterium Vitreoscilla. a member of the Beggiatoa family, is a strict aerobe that is found in oxygen-poor environments such as stagnant ponds and decaying vegetable matter. Growth of the bacterium under hypoxic conditions results in a several-fold induction of synthesis of a homodimeric soluble heme protein (subunit MW 15,775) (Boerman et al.. Control of heme content in Vitreoscilla by oxygen. Journal of General Applied Microbiology 28:35-42, 1982) which has a remarkable spectral (Webster, et al..
  • oxygen-binding proteins are useful in enhancing oxygen supply to cells or in other oxygen-utilizing processes, and for binding and separating oxygen from other fluids or gases. Furthermore, the oxygen- binding proteins are capable of increasing production of cells, or of proteins or metabolites normally made by a cell, or of natural or unnatural metabolites and proteins expressed in a cell via genetic manipulation. These proteins are also useful as selective markers in recombinant-DNA work, and have applications as diverse as enhancing certain oxygen- requiring steps in fermentation, enzymatic degradation, toxic chemical waste treatment, brewing, and particular oxidative reactions and transformations.
  • a preferred method of expressing these proteins in bacteria is to use the promoter/regulator sequence of V. hemoglobin of control expression of the homologous oxygen-binding protein.
  • the DNA sequences which usually precede a gene in a DNA polymer and which provide a site for initiation of the transcription of that gene into mRNA. These are referred to as
  • promoter sequences Other DNA or RNA sequences, also usually but not necessarily “upstream” of a structural gene, bind proteins that determine the frequency or rate of transcription and/or translation initiation. These other sequences, including attenuators, enhancers, operators and the like, are referred to as “regulator” sequences. Thus, sequences which operate to determine whether the transcription and eventual expression of a gene will take place are collectively referred to as “promoter/regulator” DNA sequences.
  • lactose (“lac”) promoter/operator systems have also been commonly used, for they are very controllable through the mode of action of the operator. When the operator is repressed, the DNA dependent RNA polymerase is completely prevented from binding and initiating transcription, thus effectively blocking promoter operability.
  • This system can be derepressed by induction following the addition of a known inducer, such as isopropyl-beta- D-thiogalactoside (IPTG) .
  • IPTG isopropyl-beta- D-thiogalactoside
  • the inducer causes the repressor protein to fall aw-:. • so the RNA polymerase can function.
  • Cells transformed with plas ids carrying the lac promoter/operator system can be permitted to grow up to maximal density while in the repressed state through the omission of an inducer, such as IPTG, from the media.
  • an inducer such as IPTG
  • the system can be derepressed by addition of inducer.
  • the promoter is then free to initiate transcription and thus obtain expression of the gene products at yields commensurate with the promoter strength.
  • certain of these inducible promoter systems are relatively weak and commercial or research productions using such systems do not urge the cell to generate maximum output.
  • tryptophan (trp) promoter/operator system
  • This system is one of several known systems with at least three times the strength of the lac promoter. However, it has the disadvantage of less promoter control.
  • the trp promoter is not inducible in the way the lac promoter is, namely, the bound repressor is not removed by induction. Instead, the system operates on a sort of feedback loop as described above.
  • a system was devised whereby the attenuator region of the trp promoter/operator system was removed, with the resultant transformed cells being grown in tryptophan-rich media. This provided sufficient tryptophan to essentially completely repress the operator so that cell growth could proceed uninhibited by premature expression of any desired foreign proteins.
  • a hybrid system has been developed from the tryptophan and lactose promoter, wherein both promoters can be repressed by the lac repressor and both can be derepressed with IPTG. See De Boer et al..
  • the tac promoter A functional hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. USA, 80: 21-25, 1983.
  • This system shares a disadvantage with the two discussed above, namely the required introduction of additional agents to a normal growth medium.
  • Another regulator/promoter system commonly used for expression of cloned proteins in E. coli is based on the P L promoter system from phage lambda. See Bernard and Helsinki, Methods in Enzymology, 68:482- 492, 1979; Use of Lambda Phage Promoter P L to Promote Gene Expression In Hybrid Plasmid Cloning Vehicles. Induction of this promoter requires increase of culture temperature from 30°C to 42°C. This system has the disadvantages of suboptimal growth rates at 30°C prior to induction and upsetting of cell metabolism by the temperature shift. Temperature shift effects on metabolism are discussed, for example, by Neidhart, et. al.. The Genetics And Regulation Of Heat-Shock Proteins. Annual Reviews of Genetics, 18:295-329, 1984. The promoter systems described above are thus useful for expression of proteins of the globin family.
  • the present invention relates to the expression and use of oxygen-binding proteins, particularly hemoglobins, recombinant-DNA methods of producing same, and to portable DNA sequences capable of directing intracellular production of these oxygen- binding proteins.
  • the present invention provides novel methods and materials for subjecting DNA sequences of living microorganisms to external regulation which is dependent upon availability of oxygen in the environment. Particularly, it relates 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 provide for the control of expression of any selected chromosomal or extrachromosomal gene or DNA sequence through the incorporation of a promoter/regulator DNA sequence which is functionally responsive to environmental variations in the concentration of oxygen.
  • oxygen-binding proteins are set forth which are capable of stoichiometric reaction with oxygen.
  • portable DNA sequences coding for hemoglobin proteins are provided.
  • portable sequences which code for the hemoglobin of the filamentous bacterium Vitreoscilla. These sequences comprise nucleotide sequences capable of directing intracellular production of oxygen- binding proteins.
  • the portable sequences may be either synthetic sequences or restriction fragments ("natural" DNA sequences) .
  • Vitreoscilla genomic library contains the genetic information capable of directing a cell to synthesize the hemoglobin of the present invention.
  • Other natural DNA sequences which may be used in the recombinant DNA methods set forth herein may be isolated from other genomic libraries.
  • 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 which results in manufacture by a host cell or microorganism of the instant oxygen-binding proteins using the portable DNA sequences referred to above.
  • This recombinant-DNA method comprises:
  • recombinant-DNA methods which subject to external control the translation and transcription of gene products by a host cell or microorganism using the portable DNA sequences referred to above.
  • Processes of the invention include methods for subjecting the expression of a selected DNA sequence in a living cell or virus to regulation by oxygen level through the site-specific insertion of promoter/regulator DNA sequences responsive thereto. Also disclosed are improvements in prior methods for securing expression of a selected "foreign" or exogenous sequence in a host microorganism wherein the DNA sequence is stably incorporated as chromosomal or extrachromosomal constituent of the host. Such improvements comprise fusing to the selected DNA sequence a promoter/regulator DNA sequence capable of selectively promoting or inhibiting expression of the selected DNA in response to variations in environmental concentration of oxygen.
  • cloning vectors comprising at least one portable DNA sequence.
  • plasmid pUC19/pRED2 is disclosed.
  • Fig. 1 is a partial restriction map of the plasmid pUC19/pRED2.
  • Figs. 2a and 2b are partial restriction maps of plas ids pWLD 5 and pWLD 10, respectively.
  • Fig. 3 is a partial restriction map of plasmid pBHb3.
  • Figs. 4A, B, and C and 5A, B, and C show the total cell number, total t-PA and t-PA produced/10 6 cells according to Example 18.
  • Fig. 6 is a partial restriction map of plasmid pMSG.
  • Fig. 7 is a graph of results described in Example 37.
  • the present invention relates in part to portable DNA sequences capable of directing intracellular production of oxygen-binding proteins in a variety of host cells and host microorganisms.
  • "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 amino 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 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 intracellular expression 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 intracellular production of the corresponding amino acid sequence, 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 foreign proteins include oxygen-binding proteins such as hemoglobins, leghemoglobins, myoglobins, hemoproteins, hemocyanias, hemerythrins and the like.
  • the promoter/regulator contains transcription and translation initiation and control sequences substantially equivalent to those for directing intracellular production of a hemoglobin protein biologically equivalent to that previously isolated from the filamentous bacterium, Vitreoscilla.
  • substantially equivalent is meant that a promoter/regulator operates to express a downstream gene product upon reduction of the level of oxygen available to the host cell below some critical value.
  • promoter/regulators of the present invention may control and initiate transcription and translation of an unlimited number of endogenous and/or exogenous foreign proteins.
  • a first preferred portable DNA sequence for the promoter/regulators of the present invention contains at least a portion of SEQUENCE ID No. 1, a nucleotide sequence, which reads 5* to 3' and includes the translation initiation sequence ATG and some of the nucleotide sequence of the Vitreoscilla structural gene
  • the SEQUENCE ID No. 1 exhibits homology with certain sequences which are highly conserved in a variety of promoter/regulators. Using conventional numbering, with the underlining showing the homology in the SEQUENCE ID No. 1 to the consensus sequence, 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) TG.
  • the SEQUENCE ID No. 1 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 amino acid sequence of SEQUENCE ID No. 2
  • the SEQUENCE ID No. 2 is disclosed in Wakabayashi et al.. supra. Nature 322:483, 1986. It is presently believed that the protein purified and prepared through the practice of this invention will exhibit a homology of over 80% with this sequence. The protein of this invention has been observed to enhance functioning of a cell in low oxygen environments.
  • 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 Cloning—A Laboratory Manual. Cold Spring Harbor Laboratory (1984) , and Horvath et al. An
  • the portable DNA sequence may be a fragment of a natural sequence, i.e., a fragment of a polynucleotide which occurred in nature and which has been cloned and expressed for the first time by the present inventors.
  • 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. While it is envisioned that the portable DNA sequences of this invention may desirably be inserted directly into the host chromosome, the present invention also provides a series of vectors, each containing at least one of the portable DNA sequences described herein.
  • 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.
  • 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.
  • supplemental sequences are those that will 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, pUC19/pRED2 contains the preferred nucleotide sequence which codes for the amino acids set forth above.
  • Vector pUC19/pRED2 cells are on deposit in the American Type Culture Collection ("ATCC") in Rockville, Maryland under Accession No. 67536.
  • a preferred nucleotide sequence encoding the Vitreoscilla hemoglobin protein and adjacent Vitreoscilla sequences described above is identified in Figure 1 as region A. The above nucleotide sequence reads counter-clockwise through region A of Figure 1.
  • Plasmid pUC19/pRED2 may also contain supplemental nucleotide sequences preceding and subsequent to the preferred DNA sequence in region A, such as terminators, enhancers, attenuators and the like.
  • supplemental nucleotide sequences preceding and subsequent to the preferred DNA sequence in region A, such as terminators, enhancers, attenuators and the like.
  • at least one leader sequence and any other DNA sequences necessary or preferred 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 or preferred 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.
  • an E. coli vector system is a preferred embodiment.
  • Various cloning vehicles are required for the range of host cells and organisms suitable for insertion of the portable DNA sequences of the present invention, as set forth below. In light of the available literature, choice of such a cloning vehicle, if necessary, is within the ordinary skill in the art.
  • Additional bacterial hosts are suitable, including, without limitation: bacteria such as members of the genera Bacillus. Pseudomonas. Alcaligenes. Streptococcus. Lactobacillus. Methylophilus. Xanthomonas. Corynebacterium. Brevibacterium. Acetobacter. and Strepotomyces.
  • eucaryotic host microorganisms examples include fungi, yeasts such as Saccharomvces and Candida, and molds such as Aspergillus. Pennicillium. Trichederma and Cephalosporium (Acremonium) . It is envisioned that the scope of this invention would cover expression systems in eucaryotic microorganisms and host cultured cells derived from multicellular organisms, including animals, insects and plants, which are grown in the presence of oxygen. The promoter/regulator of the present invention is especially useful in a host which switches from low to very high expression activity upon reduction of dissolved oxygen concentration in the medium. Such expression systems need not be derived from Vitreoscilla.
  • vector systems will be suitable for these and other desirable hosts, including plasmids, viruses 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.
  • telomeres For example, many selectable cloning vectors have been characterized for use in E. coli. including pUC8, pUC9, pBR322, pGW7, placl q , and pDP8, Maniatis et al.. supra.
  • a bifunctional vector that replicates in E. coli and can also be used in Streptomyces is pKC462a.
  • Suitable vectors for use in Bacillus include: pUBHO, pSA0501, pSA2100, pBD6, pBD8, and pT127, Ganesan and Hock, eds.. Genetics and Biotechnology of Bacilli. Academic Press 1984. In Pseudomonas.
  • RSF1010, Pmsl49, pXT209, and RK2 are suitable; some of these vectors are useful in a wide range of gram-negative bacteria including Agrobacterium and Xanthomonas.
  • Saccharomyces it is possible to use YEp24, YIp5, and YRpl7, Botstein and Davis, Molecular Biology of the Yeast Saccharo yces (Strathern et al.. eds) , Cold Spring Harbor Laboratory, 1982. In mammalian systems retrovirus vectors such as those derived from SV40 are typically used.
  • the host organism would produce greater amounts per vector of the desired oxygen-binding protein.
  • the number of multiple copies of the DNA sequence which may be inserted into the vector is limited only by the ability of the resultant vector, due to its size, to be transferred into and replicated and expressed in an appropriate host.
  • 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 ampicillin resistance is included in vector pUC19/pRED2.
  • 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.
  • 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, and also engender an easily visible reddish tint in the host cells.
  • 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 narural or unnatural metabolites and proteins expressed in a cell via genetic manipulation. This would include heterologous proteins—either intracellular or extracellular—as well as biopolymers such as polysaccharide materials, simpler metabolites such as amino acids and nucleotides, antibiotics and other chemicals produced by living cells or cellular biocatalysts.
  • the oxygen-binding proteins of the present invention prepared by the recombinant-DNA methods set forth herein, will enable increased research into the growth of organisms in oxygen-poor environments.
  • the oxygen-binding proteins of the present invention are useful in enhancing oxygen supply to cells or in other oxygen-utilizing processes (Adlercreutz et al.. Biocatalvst in Organic Synthesis. Symposium of the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology, Abstracts, p.18, 1985) , and for binding and separating oxygen from other fluids or gases (Bonaventura et al.. Underwater Life Support Based on Immobilized Oxygen Carriers. Applied Biochemistry and Biotechnology 9:65-80, 1984) .
  • oxygen-binding proteins of this invention are capable of increasing production ⁇ cells, or of proteins or metabolites normally by a cell, or of natural or unnatural metabolites b.. ⁇ proteins expressed in a cell via genetic manipulation. This would, as described above, include heterologous proteins, biopolymers, simpler metabolites, antibiotics, and other chemicals produced by living cells or cellular biocatalysts.
  • the protein products of this invention also have applications as diverse as enhancing certain oxygen- requiring steps in fermentation, enzymatic degradation, toxic chemical waste treatment, brewing and particular oxidative reactions and transformations such as steroid conversions.
  • This invention also relates to a recombinant-DNA method for the production of oxygen-binding proteins.
  • this method includes:
  • the portable DNA sequences are those synthetic or naturally-occurring polynucleotides described above.
  • the portable DNA sequence codes for at least a portion of the Vitreoscilla hemoglobin protein are described above.
  • This invention also relates to a recombinant-DNA method for the use of these promoter/regulators.
  • this method provides a process for subjecting the expression of a selected DNA sequence to external control under given environmental conditions which comprises the steps of:
  • 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 vector pUC19/pRED2 is used in the disclosed method.
  • a vector thus obtained may then be transferred into the appropriate host cell or organism. It is believed that any microorganism 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 certain microorganisms such as yeast, 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 yeast for ultimate expression of the oxygen-binding protein.
  • the host cells or microorganisms are cultured under 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, in light of the published literature regarding the growth conditions for such organisms.
  • conditions necessary for the regulation of the expression of the DNA sequence dependent upon any operational elements inserted into or present in the vector, would be in effect at the transformation and culturing stages.
  • the cells are grown to a high density in the presence of appropriate regulatory conditions which inhibit the expression of the DNA sequence. When optimal cell density is approached, the environmental conditions are altered to those appropriate for the expression of the portable DNA sequence.
  • cloned protein will occur in a time span subsequent to the growth of the host cells to near optimal density, and that the resultant cloned protein product would be harvested, if desired, at some time after the regulatory conditions necessary for its expression were induced.
  • the operational elements used are in the promoter/regulator sequence of this invention, these conditions are as follows.
  • the cells are grown to a high density in the presence of appropriate levels of oxygen which inhibit the expression of the DNA sequence.
  • the environmental oxygen level is altered to a lower value appropriate for the expression of the portable DNA sequence.
  • Levels from less than about 1% oxygen- saturation to an oxygen saturated solution are within the scope of this invention. It is thus contemplated that the production of any desired fused product will occur in a time span subsequent to the growth of the host cells to near optimal density, and that the resultant product would be harvested, if desired, at some time after the oxygen level necessary for its expression were reached. If harvesting of the oxygen-binding protein products of the present invention is desired, it may be done prior or subsequent to purification and prior or subsequent to assumption of an active structure.
  • the oxygen-binding proteins of the present invention will assume their proper, active structure upon expression in the host cell or organism. If desired, the oxygen-binding protein may be transported across a cell membrane. This will generally occur if DNA coding for an appropriate leader sequence has been linked to the DNA coding for the recombinant protein. The structures of numerous signal peptides have been published. It is envisioned that these leader sequences, included in or added to at least some portion of the portable DNA as necessary, will direct intracellular production of a fusion protein which will be transported through the cell membrane and will have the leader sequence cleaved upon release from the cell.
  • oxygen-binding proteins of the present invention are envisioned.
  • the purified proteins and/or the whole cells and/or extracts of the cells of the present invention themselves may be used to bind to oxygen or proteins and thus could function somewhat as ery hrocytes.
  • the present invention may also be used as a method for transporting and enhancing oxygen supply to cells or in other oxygen-utilizing processes by delivering the oxygen-binding proteins—isolated in lysates and crude cell preparations, purified from extracts, in synthetic sequences, or in whole cells containing the proteins—where desired. It is envisioned that the protein products of the present invention could valuably be added to media for culturing cells and thereby enhance the transport of oxygen.
  • proteins of the present invention may be used for binding and separating of oxygen from fluids such as seawater and from other gases.
  • the products and processes of the present invention find usefulness in a range of medical, laboratory and industrial applications.
  • the invention provides metabolically engineered cells with enhanced growth characteristics for increasing production of various proteins or metabolites by those cells.
  • the invention further provides a method for subjecting expression of a certain DNA sequence to external control under given environmental conditions.
  • recombinant-DNA fusion gene products, expression vectors, and nucleotide base sequences for the practice of the invention.
  • the products and processes of the present invention find applications in a range of aerobic processes, such as manufacture of cloned proteins and synthesis of metabolites, chemical production by fermentation, enzymatic degradation, waste treatment, brewing and a range of oxidative reactions.
  • Vitreoscilla sp. (Murray strain no. 389) was obtained from Dr. Webster (Department of Biology, Illinois institute of Technology, Chicago, Illinois 60616, USA) , and grown in a medium containing 1.5% yeast extract, 1.5% peptone, and 0.02% sodium acetate (pH 8.0 with NaOH) . This strain is also available from ATCC, accession number 13981.
  • E. coli JM101 were obtained from the laboratory of Dr. Simon (Division of Biology, California Institute of Technology, Pasadena, California 91125, USA) , and grown in L broth containing 1% Bactotryptone, 0.5% yeast extract and 1% sodium chloride. This strain is also available from ATCC, accession number 33876.
  • Plasmid pUC19 (Yanisch-Perron et al.. Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of ml3mpl8 and PUC19 vectors. Gene 33:103- 109, 1985) packaging kits were purchased from Pharmacia. All restriction enzymes, T4 polynucleotide kinase and T4 ligase were from New England Biolabs or Bethesda Research Laboratories. Calf intestine alkaline phosphatase was from Pharmacia. Mixed oligonucleotide probes were synthesized with an Applied Biosystems synthesizer. Kodak XAR5 x-ray film was used for autoradiography. Geneclean kits were purchased from BiolOl. All other chemicals were of analytical grade.
  • Vitreoscilla genomic DNA was isolated according to the protocol of Silhavy et al.. Experiments with gene fusions. Cold Spring Harbor Laboratory (1984) , specifically incorporated herein. Hindlll-digested Vitreoscilla DNA was ligated into the phosphatased Hindlll site of pUC19 and transformed into JM101. Recombinant colonies and plaques were transferred on nitrocellulose filters as described in Maniatis, et al.. Molecular clonin —a laboratory manual. Cold Spring Harbor Laboratory (1982) and specifically incorporated herein. Rapid plasmid isolation from recombinant colonies were done according to Silhavy et al.. supra.
  • Digested fragments of plasmid DNA or fractions of genomic DNA were isolated from agarose gels using Geneclean kits. E. coli cells were transformed by the CaCl 2 method of Silhavy et al.. supra. Plasmid uptake was induced by heat-shocking chilled competent cells at 37°C for 5 minutes.
  • SDS-polyacrylamide gel electrophoresis was done according to standard protocols, Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:580-685, 1970, specifically incorporated herein, with a 12.5% resolving gel. Protein in the gel was visualized by the silver staining method or Merril et al.. Ultrasensitive stain for proteins in polvacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211:1437-1438, 1983.
  • Three sets of mixed oligonucleotide probes were synthesized which had a predicted homology to two domains in the hemoglobin gene, one N-terminal and one C-terminal.
  • a pUC19-HindIII library of Vitreoscilla DNA was test-plated on rich plates with ampicillin, X-gal and IPTG. More than 70% of the colonies were probable recombinants , as estimated by visual inspection. About 10,000 colonies were then screened. Three positives were identified. Because of the high density of colonies on the plate, these, along with their immediate clones from each group, were assayed by rapid isolation and Hindlll digestion. One of these, pREDl, had three inserted fragments including a 2.2kb one.
  • the Hindlll fragment from pREDl that contained the hemoglobin structural gene was purified and reinserted by standard protocols into pUC19 in both orientations (pRED2 and pRED3) .
  • E. coli cells containing plasmids pREDl, pRED2, pRED3 and pUC19 as well as Vitreoscilla cells were grown to stationary phase and cell extracts were assayed on an SDS- polyacrylamide gel for the existence of the hemo ⁇ globin polypeptide.
  • the hemoglobin was expressed as a major cellular protein in all recombinant cells. Since both plasmids PRED2 and PRED3 express about equal amounts of this polypeptide, it is presently believed that the gene is probably expressed from its natural promoter in E. coli.
  • Hindlll-Sphl fragment from plasmid pRED2 which contains the structural gene and adjacent sequences was subcloned into pUC19 (purchased from Bethesda Research Labs) to obtain plasmid pRED4.
  • An Mlul site was identified, by restriction mapping the resulting plasmid, which breaks up the Hindlll-Sphl insert into two fragments which were individually sequenced using conventional protocols (Maxam and Gilbert, Seguencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology 65:499-560, 1980; Iverson and Dervan, Adenine specific DNA chemical sequencing reaction. Nuclear Acids Research 15:7823- 7830, 1987).
  • the nucleotide sequence of the important portion of the Hindlll-Sphl fragment includes a putative E. coli promoter, ribosome binding site, the complete VHb structural gene (start and stop codons are underlined) and a putative E.- coli transcription terminator (Khosla and Bailey, The Vitreoscilla hemoglobin gene: molecular cloning nucleotide sequence and genetic expression in Escherichia coli.
  • Plasmids pUC9 and pUC19 are essentially identical except for a difference in one restriction site unrelated to the insert or to any of the functional properties of the plasmid.
  • the shake-flask was inoculated with a 1% (V/V) dose of concentrated nutrient broth containing 430 g/L glucose, 5 g/L yeast extract, 110 g/L (NH 4 ) 2 S0 4 , 8 g/L MgS0 4 -7H 2 0, 0.27 g/L FeCl 3 -6H 2 0, 0.02 g/L ZnCl 2 -4H 2 0, 0.02 g/L CaCl 2 -2H 2 0, 0.02 g/L NaMo0 4 -2H 2 0, 0.01 g/L CuS0 4 -5H 2 0, 0.005 g/L H 3 B0 31 0.1% (V/V) cone.
  • V/V concentrated nutrient broth containing 430 g/L glucose, 5 g/L yeast extract, 110 g/L (NH 4 ) 2 S0 4 , 8 g/L MgS0 4 -7H 2 0, 0.27 g/L FeCl 3 -6H 2 0, 0.02 g
  • HC1 4.2 mg/L riboflavin, 54 mg/L pantothenic acid, 60 mg/L folic acid.
  • This formulation has been successfully used on a previous occasion to grow stationary cells to a high density in a fedbatch mode. The cells were then allowed to grow further until stationary phase was reached again. Optical density was measured at 600 nm on a Bausch & Lomb Spectronic 21 spectrophotometer. Dry weights were measured by spinning 10 mL samples at 4°C, washing once with distilled water and subsequently drying the resuspended sample at 100°C overnight.
  • the heme content of the cells was assayed according to the method of Lamba & Webster (Lamba & Webster, Effect of growth conditions on yield and heme content of Vitreoscilla. Journal of Bacteriology 142:169-173, 1980), and the hemoglobin activity was measured by the method of Webster & Liu (Webster and Liu, Reduced nicotinamide adenine dinucleotide cytochrome o reductase associated with cytochromic o purified from Vitreoscilla. Journal of Biological Chemistry, 249:4257-4260, 1974.).
  • EXAMPLE 3 GROWTH ENHANCEMENT OF E. COLI WITH PRED2.
  • a typical high-cell density fermentation is of a fedbatch type.
  • the optimal rate of nutrient addition, and consequently the productivity, is ultimately limited by the rate at which cells can aerobically catabolize the carbon source without generating growth-inhibitory metabolites such as acetate and lactate (Zabriskie and Arcuri, Factors influencing productivity of fermentations employing recombinant microorganisms.
  • Batch stationary phase refers to conditions before continuous feeding was started.
  • JM101:pRED2 was improved compared to the control strains at low DO levels, as observed in a Gilson respirometer.
  • hemoglobin is under the regulation of its native oxygen-regulated promoter. Hence, it is not possible to modulate independently the dissolved oxygen concentration (DO) and the intracellular VHb level.
  • E. coli such as trp (Russell and Bennett,
  • Plasmid PRED4 was linearized with Hindlll and treated with exonuclease Bal31 to generate 5' end deletions in the Hindlll-Sphl VHb fragment (Maniatis et al, supra) . After digestion with SphI, the resulting VHb fragments were cloned into Hindlll-Sphl digested pUC19. The positions of the deleted end-points were identified by sequencing.
  • trp and tac promoters and the chloramphenicol acetyl transferase gene (CAT) were purchased from Pharmacia, Inc. Oligonucleotides were synthesized at California Institute of Technology using an Applied Biosystems DNA synthesizer.
  • the functional assay for the VHb gene product is as described in Webster and Liu, supra.
  • the CAT gene (Alton and Vapnek, Nucleotide sequence analysis of the chlora phenicol resistance transposon Tn9. Nature 282:864-869, 1979) was inserted downstream and under the control of the lac promoter available on this PUC19-based plasmid. This gene product can be conveniently assayed (Neumann et al, Novel rapid assay for chloramphenicol acetyltransferase gene expression. BioTechniques 5:444-447, 1987) and hence serves as a useful reporter.
  • the ⁇ - lactamase gene on this PUC19-based plasmid was deleted by digestion and religation with Pvul.
  • the purpose of this step is to eliminate the presence of a plasmid-encoded periplasmic protein.
  • the plasmid thus obtained was called pHbCAT and was transformed into JM101.
  • the CAT gene was cloned downstream and under the control of the lac promoter in pUC19.
  • the ⁇ -lactamase gene was identically deleted. This plasmid was called pCAT.
  • the restriction maps and the anticipated sequence of relevant regions of these two plasmids are shown below. pHbCAT (3.6 kb)
  • the sequence of the region spanning between EcoRI and the start of the VHb structural gene is shown above. It includes the trp promoter and a synthetic riboso e binding site.
  • an expression plasmid plasmid was made using a Hindlll-BamHI tac promoter cartridge, the BamHI/SphI fragment from pHbCAT, and the Hindlll-Sphl digested fragment of the vector pBR322 (Bolivar et al, Construction and characterization of new cloning vehicles. II. A multipurpose cloning system Gene 2:95-113, 1977).
  • VHb is expressed using promoters different from the native VHb oxygen-regulated promoter.
  • the strains:plasmids used are:
  • the two hosts have nearly identical genotypes, the major difference being the presence of an F 1 factor in JMlOl which harbors the lad 8 gene. This gene is necessary to keep a strong promoter like tac under control.
  • IX LB 10 g/L Bactotryptone, 5 g/L Yeast Extract, 5 g/L NaCl, 3 g/L K 2 HP0 4 1 g/L KH 2 P0 4 100 mg/L Ampicillin
  • 2X LB 20 g/L Bactotryptone, 10 g/L Yeast Extract, 5 g/L NaCl, 3 g/L K 2 HP0 4 , 1 g/L KH 2 P0 4 100 mg/L Ampicillin
  • 5X LB 50 g/L Bactotryptone, 25 g/L Yeast Extract,
  • VHb expression may be a function of specific growth properties of each cell line and/or plasmid construct as well as of the environmental conditions of growth. The optimum may thus have to be determined for different applications of this technology on a case-by-case basis; however, such determination does not require undue experimentation.
  • JMlOl:PCAT 2. JMlOl:PHbCAT.
  • Example 5 The construction of these plasmids is described in Example 5. The following media compositions were used:
  • LB 10 g/L Bactotryptone, 5 g/L Yeast Extract, 5 g/L NaCl, 3 g/L K 2 HP0 41 1 g/L KH 2 P0 4 , 30 mg/L Chloramphenicol
  • 10X feed 100 g/L Bactotryptone, 100 g/L Yeast Extract, 150 mg/L Chloramphenicol.
  • VHb may also enhance the specific activity (activity per unit amount of totally soluble protein) of the cloned gene product.
  • the Hindlll-Sphl fragment containing the VHb gene and flanking sequences was cloned into the corresponding sites of the vector pBR322, thereby creating the plasmid pOXl. This was then transformed into the E. coli hold, HB101.
  • the fermentation was conducted in a New Brunswick Bioflo II fermentor with a 2.5 L working volume using LB (10 g/L Bactotryptone, 5 g/L yeast extract, 5 g/L NaCl, 3 g/L K 2 H-P0 4 , 1 g/L KH 2 P0 4 ) plus 8 mg/L silicone antifoam as medium at 37°C, pH 7.0 with a constant agitation speed of 300 rpm. All other methods involve conventional protocols (Maniatis, et al .. supra) .
  • VHb mRNA The level of VHb mRNA increased about ten-fold as DO dropped from 70% to 1% air saturation.
  • VHb This may occur because of the requirement of additional heme biosynthesis in the host cell in order to produce active VHb.
  • VHb promoter was switched on to significant levels only below 40% air saturation and attains maximum induction levels below 5% air saturation.
  • the recombinant strain D603:pYREDl was significantly redder than the control D603:pBM150 when grown in the presence of galactose (2% peptone, 1% yeast extract, 2% galactose) . Inocula were grown in minimal galactose medium. Significant hemoglobin activity was determined in sonicates from D603:pYREDl compared to D603:pBM150 controls based on the difference spectrum hemoglobin analysis referenced in Example 2.
  • VHb expression was studied.
  • the VHb gene was cloned into a yeast expression plasmid, AAH5, that is stably maintained as an extrachromosomal plasmid in yeast cells.
  • Plasmid pEX-2 was constructed as follows. The BamHI/SphI fragment described in Example 4 was cloned by blunt-end ligation into the Hindlll site of the yeast expression vector AAH5 (Ammerer, Expression of genes in yeast using the ADC1 promoter. Methods in Enzymology 101:192-201, 1983).
  • AAH5 contains the selectable yeast marker Leu 2, the 2 micron circle origin of replication, and a unique Hindlll site flanked by the transcriptional promoter and terminator regions of the yeast alcohol dehydrogenase-1 (ADH-1) gene.
  • the ADH-1 promoter will support high levels of transcription of any sequence cloned into the Hindlll site.
  • the ADH-1 gene is constitutively expressed in yeast.
  • S. cerevisiae strain 488-0 (leu2, ura3, his 1-7) was transformed with plasmids AAH5 and PEX-2 by the rapid colony transformation procedure (Keszenman-Pereyra and Heida, A colony procedure for transformation of Saccharom ⁇ ces cerevisiae. Curr. Genet. 13:21-23, 1988) , and plated on synthetic dextrose (SD) medium (Rose, Isolation of genes by complementation in yeast. Methods in Enzymology, 152: 481-504, 1987) without leucine. A representative clonal cell line from each transformation was established after colony purification of a primary transformant.
  • single yeast colonies were inoculated into 2 mL of SD -leu (+leu for 488-0) and cultured for 24 hr at 260 rpm at 30°C in a Labline Model 3258 Orbit Enviro-shaker. 0.5 mL of this inoculum was added to 50 mL of the same medium in a 250 mL flask and cultured at 260 rpm at 30°C. Cell growth was measured by turbidity (A600nm) using a Perkin-Elmer Lambda 4A Spectrophotometer.
  • the cultures were pulsed with 1/40 volume of a concentrated medium containing 20 x SD (40% glucose, 13.3% Difco yeast nitrogen base without amino acids, and 1.6 mg/mL of all the amino acids except leucine.
  • 20 x SD 50% glucose, 13.3% Difco yeast nitrogen base without amino acids, and 1.6 mg/mL of all the amino acids except leucine.
  • Glucose concentration was estimated using Ames Glucostix test strips.
  • the VHb-containing strain 488-0:pEX-2 grew to a final optical density of 13.0, while strains 488- 0:AAH5 and 488-0 grew to optical densities of only 10.0 and 9.5, respectively.
  • this represents a 32.6% increase in the final cell density of 488-0:pEX-2 over the strain containing no AAH5- derived plasmid (488-0) .
  • TnlO transposon (Foster, et al.. Three TnlO-associated excision events: Relationship to transposition and role of direct and inverted repeats. Cell, 23:215-227, 1981) was constructed as follows. A kanamycin resistance gene (Pharmacia Inc.) was cloned into the Sail site of PINT1 (Example 5) .
  • the EcoRI/EagI fragment from the resulting plasmid which contains the entire tac-VHb fusion and Kan R gene, was cloned between the inverted repeats (bases 1-66 on the right end and bases 9234-9300 on the left end) of a TnlO derivative which lacks the transposase gene (obtained from Cold Spring Harbor Laboratory, NY) .
  • the resulting element, TnlOdKan- tac-VHb was cloned into a multicopy plasmid containing a tac-TnlO rightward transposase (obtained from Cold Spring Harbor Laboratory, NY) .
  • E . coli HB101 was transformed with pMSG using a standard CaCl 2 protocol (1) . After culturing transformants overnight, pMSG plasmid DNA was isolated by standard miniprep technique (1) . The authenticity of this plasmid was confirmed by performing several restriction digestions of the original and the HBlOl-derived pMSG plasmid DNA. A frozen stock of HB101-pRED302 (2) was plated out on a agar plate. The following day, a 10 ml overnight culture was inoculated from a single colony.
  • Miniprep plasmid isolation was carried out on this overnight culture for pRED302 isolation.
  • Plasmid pRED302 was digested with Xbal and Sspl (Boerhringer Mannheim, Indianapolis, IN) and the digested sample was run on a 0.7% agarose gel. This resulted in 3 fragments of approximate sizes of 2.5 kb, 0.56 kb and 0.54 kb.
  • Test digestions were carried out with the two smaller fragments using the restriction enzymes Mlul and Bsu361 since these sites are present in the VHb structural gene. Electrophoresis of these digested samples on a 2% agarose gel confirmed that the lower band contains the VHb structural gene.
  • the lower band was eluted and purified from the gel using the GeneClean Kit (Bio 101, La Jolla, CA) resuspended in 30 ml TE (pH 8.0) and stored at -20°C. Part of this purified fragment was used for construction of pMSG-VHb and remaining sample was frozen down at - 20°C for use in Southern hybridization experiments.
  • the construction of pMSG-VHb was done by utilizing the multicloning site of pMSG. pMSG was sequentially digested with Smal and Nhel with an intermediate phenol:chloroform extraction and ethanol precipitation step.
  • the digested vector was run on a 0.7% agarose gel and the larger fragment was purified using a GeneClean Kit, resuspended in 20 ml TE (pH 8.0) and stored at -20°C. An overnight blunt-sticky end ligation reaction was carried out at 8-15°C for cloning the Xbal/Sspl VHb fragment into the Nhel/Smal digested pMSG.
  • This vector was named pMSG-VHb.
  • competent HB101 were transformed separately with no plasmid, pMSG and pMSG-VHb respectively and spread on LB-ampicillin plates. Nine colonies were picked from pMSG-VHb plate as potential transformants for further analysis.
  • Minipreps were carried out on overnight cultures of all these potential transformants for pMSG-VHb plasmid DNA isolation.
  • Vectors pMSG and pMSG-VHb were digested with the differed restriction enzymes BamHI, Hinglll, Sail and Mlul for verifying the authenticity of the new construct. When run on a 0.7% agarose gel, those samples confirmed the presence of the VHb insert in the multicloning site of pMSG.
  • a maxiprep was carried out using two 100 ml cultures of one of the pMSG-VHb transformants for large-scale plasmid DNA isolation. The DNA was extracted with phenol:chloroform, ethanol precipitated, the pellet resuspended in 1 ml TE (pH 8.0) and stored at -20°C until further use in CHO cell transfection.
  • EXAMPLE 12 TRANSFECTION OF CHO CELL WITH AN EXPRESSION VECTOR
  • a Chinese hamster ovary (CHO) cell line producing tPA was obtained from ATCC (Bethesda, Maryland) . These cells were grown routinely in a non-selection medium containing DMEM (high glucose) (GIBCO, Grand Island, NY) supplemented with IX penicillin-streptomycin-glutamine (Irvine Scientific, Irvine, CA) solution and 5% dialyzed FBS (GIBCO, Grand Island, NY) in a 5% C0 2 humidified incubator at 37°C. Tissue culture dishes (100 & 20mm) were used in all the experiments.
  • DMEM high glucose
  • IX penicillin-streptomycin-glutamine Irvine Scientific, Irvine, CA
  • the selection medium for transfected cells contains 25 mg/1 mycophenolic acid (Grand Island, NY) , 1 X HAT (Gibco, Grand Island, NY) and 250 mg/1 xanthine (Sigma, St. Louis, MO in addition to the non-selections medium components.
  • the selection medium for transfected cells contains 25 mg/1 mycophenolic acid (Grand Island, NY) , 1 X HAT (Gibco, Grand Island, NY) and 250 mg/1 xanthine (Sigma, St. Louis, MO in addition to the non-selections medium components.
  • During regular cultures cells were passaged every 2- 3 days upon semi-confluency. Total cell counts and cell size distribution were monitored by the Coulter counter. Cell viability was determined using the trypan blue exclusion method.
  • the CHO cells producing tPA were transfected with pMSG-VHb using the standard calcium phosphate procedure described in Maniatis (1) . Briefly, one 100 x 200 mm tissue culture dish with 10 ml non- selection medium was inoculated with 1 x 10 6 cells 24 hours prior to transfection. Twenty mg pMSG-VHb DNA was digested overnight with EcoRI at 37°C. This linearized vector DNA was subjected to phenol:chloroform extraction and ethanol precipitation and the resulting pellet was resuspended in an appropriate volume of 0.1 X TE (pH 8.0). This was combined with 20 mg of carrier DNA and the calcium phosphate-DNA precipitate was formed according to the standard protocol.
  • VHb gene probe isolated by miniprep was labeled with biotin using a PolarPlex Kit (Millipore, Bedford, MA) . Hybridization and detection reaction were carried out according to the PolarPlex protocol. A permanent image of the hybridization pattern and the VHb gene was obtained by exposing the membrane to an X-ray film.
  • VHb-CHO clones In a typical autoradiogram obtained as a result of such an experiment, three VHb-CHO clones and the parental CHO-tPA are used. All VHb-CHO clones show two distinct bands hybridized to the VHb probe whereas the parental clone shows one band. The lower band is present in all four clones suggesting that this fragment of DNA exhibits a great deal of homology with the VHb gene and could be the endogenous hemoglobin gene found in Chinese hamster ovary cells. The upper band is present only in VHb- CHO cells indicating that this band corresponds to the VHb gene present in the vector pMSG-VHb that has been integrated in CHO cell chromosome as a result of transfection. Thus, the presence of VHb gene integrated into the chromosome of CHO-VHb cells has been established. EXAMPLE 14 - OXYGEN-BINDING PROTEIN EXPRESSION IN CHO CLONES
  • Cell extracts were prepared for each sample as per the protocol described in Khosla et al. (2) . Briefly, cells were harvested by trypsinization. The cell suspension was centrifuged at 2500 rpm for 10 minutes at 4°C. The resulting cell pellet was resuspended in 40 ml lysis buffer (100 Mm Tris pH 8.0, 10 mM NaCl and 10 mM EDTA pH 8.0). This cell suspension was subjected to freeze-thaw cycles 3 times in a dry ice-ethanol and 37°C waterbath for 5 minutes each. The resulting suspension was centrifuged at 12000 rpm for 2 minutes at 4°C. The supernatant was transferred to an Eppendorf tube and stored at -20°C until SDS-PAGE was performed.
  • 40 lysis buffer 100 Mm Tris pH 8.0, 10 mM NaCl and 10 mM EDTA pH 8.0
  • VHb expression was monitored as a function of dexamethasone concentration (0.01, 0.5, 1.0, and 2.0 ⁇ M) after 50 hours of induction.
  • VHb expression was monitored for a single dexamethasone concentration for induction times of 24, 28, 72, and 96 hours induction period.
  • the clones used for this study were the parental CHO- tPA and the VHb-expressing CHO-tPA clone.
  • dexamethasone concentrations of 0, 0.1 and 0.5 ⁇ M were used. These cells were induced on day 1 of the batch culture. All experiments were carried out using 100 x 20 tissue culture dishes. Cells were inoculated on day 0 at a density of 4 x 10 s cells per dish. On every day of the 5-day batch culture experiment, one dish was removed from the incubator for measurements of cell number, tPA titers and other supernatant metabolite concentrations. Total cell count was monitored using a Coulter Counter.
  • Viability was measured by the trypan blue exclusion method using a hemocytometer. Supernatant was frozen at -20°C for tPA and metabolite analysis. The tPA production was monitored using an ELISA kit (COALIZA, KabiVitrum, Franklin, OH) according to the standard protocol provided by the manufacturer. tPA concentrations in each sample were calculated by using a calibration curve obtained using standard tPA samples provided in the kit.
  • the total amount of tPA produced each day was calculated by multiplying the concentration of tPA obtained by the volume of supernatant present in each dish. This was done to account for the progressive reduction in volume of the supernatant due to evaporation of water during the course of the batch culture.
  • cell extracts were prepared and stored as described earlier for analysis of VHb expression during the batch culture.
  • the day 1 sample from VHb-expressing cells corresponds to the uninduced level of VHb expression whereas the CHO-tPA sample serves as negative control for VHb expression since these cells lack the VHb gene. This experiment was carried out two times in order to establish reproducibility and consistency of our experiments. The results of such an experiment are discussed below.
  • FIGS 4 A, B, C, and 5 A, B and C show the total cell number, total tPA produced and total tPA produced/10 6 cells as a function of batch culture time for these two independent experiments. From both these experiments, it is clear that the specific growth rates of VHb-CHO cells is about 20-30% lower than that of the parental CHO-tPA clone. However, this effect is not due to VHb expression since the uninduced VHb-CHO cells show almost the same growth characteristics as the induced VHb-CHO cells. This effect is probably due to some unknown host-vector interaction that manifests itself as a result of integration of transfected DNA sequences into the host cell chromosomes.
  • the tPA productivity characteristics are significantly different in the VHb-CHO clone as compared to the parental CHO-tPA clone.
  • the total tPA amounts as well as the amount of tPA produced per cell are about 50-100% higher in VHb-CHO cells compared to these properties in the parental CHO-tPA cell culture.
  • 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, pIJ699
  • 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 screened with polyclonal antiserum generated 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 amine Type I, 1% yeast
  • HEE extract, and 1% v/v trace elements mix (0.1% FeS0 4 -7H 2 0, 0.1% MnS0 4 -7H 2 0, 0.0025% CuCl 2 - 2H 2 0, 0.01% CaCl 2 -2H 2 0, 0.00056% H 3 B0 3 , 0.002% ZnS0 4 ' 7H 2 0, 0.0019% (NH 4 ) 6 Mo 7 0 24 - 4H 2 0) . 5 ug/mL of thiostrepton was added to the TK64:pWLD5 culture.
  • the first condition was a 50 mL culture volume in a 250 mL unbaffled erlenraeyer 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 (0.10-0.11 h_ x ) 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 Springs instruments biological oxygen monitor. The rates were normalized to cell weights and compared throughout the growth curve (Table 1) . Although the OUR's of the two strains were similar throughout the experiment with high aeration (Table 1A) , they were consistently higher in the hemoglobin- expressing strain with lower aeration, especially at the later stages of growth (Table IB) .
  • the OUR for the plasmid-free strain was 0.22 mM 0 2 /h-g whereas the OUR for TK64:pWLD5 was 0.29 mM 0 2 /h-g, a difference of 32%.
  • EXAMPLE 18 GROWTH ENHANCEMENT OF HEMOGLOBIN- EXPRESSING STREPTOMYCES GROWN UNDER TWO ADDITIONAL CONDITIONS OF REDUCED OXYGEN.
  • TK64 no plasmid
  • TK65:pWLD5 The enhanced growth of hemoglobin-expressing Streptomyces was examined under two additional conditions of low aeration in shake flask cultures.
  • Strains TK64 (no plasmid) and TK65:pWLD5 were cultured in 12.5 and 25 mL culture volumes in 250 mL flasks for 72 hours at 150 rpm at 30°C. The medium used was the same as in Example 17. The final cell densities were measured at OD 590 . in the 12.5 mL culture, TK64:pWLD5 reached a final OD 590 of 5.8 while TK64 reached an OD 590 of only 4.0, a difference of 45%.
  • 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.
  • EXAMPLE 19 EXPRESSION OF BACTERIAL HEMOGLOBIN IN STREPTOMYCES COELICOLOR.
  • 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.
  • the plasmid 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:PWLDIO 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, 34% sucrose, 5 mM MgCl 2 * 6H 2 0) at 250 rpm at 30°C.
  • a cell extract was prepared by sonication and the proteins separated by SDS-PAGE and screened with anti-Vitreoscilla hemoglobin antiserum. Western analysis indicated that a significant level of hemoglobin of identical molecular weight as pure
  • Vitreoscilla hemoglobin was present in cell extracts of M145:pWLD10 but not in the plasmid-free strain. This indicates that Vitreoscilla hemoglobin is stably expressed in another species of Streptomyces.
  • 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 468 of the filtrate which is a measure of undecylprodigiosin, was then determined. While the hemoglobin-expressing strain had an OD 68 of 1.4, the non-expressing strain had an OD 68 of only 0.6. This indicated that greater than twice as much antibiotic is produced in a hemoglobin-expressing strain of Streptomyces.
  • EXAMPLE 21 - EXPRESSION OF BACTERIAL HEMOGLOBIN IN CORYNEBACTERIA A plasmid was constructed for the expression of a bacterial hemoglobin in Corynebacteria.
  • This plasmid, pBHb3 contains the Vitreoscilla hemoglobin gene (Khosla and Bailey, Mol. Gen. Genet.. 214:158, 1988) cloned into a common Corynebacterium plasmid pBKlO.
  • pINTl a 5.5 kilobase plasmid pINTl (Khosla and Bailey, J. Mol. Biol.. 210:79, 1989) which consists of the E.
  • coli plasmid pBR322 with a 1.2 kilobase insert consisting of the Vitreoscilla hemoglobin gene and the 122 base pair tac promoter (P.L. Biochemicals) was digested with Sal 1 and EcoRI. The plasmid ends were then made blunt by filling in with DNA polymerase 1 (Klenow fragment) , and the 1.5 kilobase fragment containing the Vitreoscilla hemoglobin gene, tac promoter and flanking pBR322 sequences was isolated. This fragment was ligated to EcoRI linearized plasmid pBKlO (Paradis, et al. , Gene. 61:199, 1987), the ends of which had also been made blunt.
  • the resulting fragment was made circular with T4 DNA ligase.
  • This plasmid, pBHb3 was transformed into E. coli. and was stably maintained by selection with the antibiotic kanamycin.
  • Corynebacterium glutamicum strain ATTC 39022 a variant of the wild type C. glutamicum strain ATTC 13032, was transformed with pBHb3 DNA out of E. coli. A single kanamycin resistant colony was isolated. This clone was designated 39022:pBHb3-7. The wild-type C.
  • glutamicum strain ATTC 13032 was then transformed with pBHb3 DNA isolated out of clone 39022:pBHb3-7 and a kanamycin resistant colony, designated 13032:pBHb3 15 was selected for further experiments.
  • Hemoglobin expression in 13032:pBHb3-15 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 a nitrocellulose membrane and screened with polyclonal antiseru generated against Vitreoscilla hemoglobin purified from E. coli. harboring plasmid pRED2 (Khosla and Bailey, Mol. Gen. Genet. , 1988) .
  • a band of identical molecular weight as pure hemoglobin was detected in the cell extracts.
  • Lysine production in Corynebacterium glutamicum ATCC 13287 and in similar cells transformed with the plasmid pBHb3 was compared in a shake flask culture experiment. Equal amounts of exponential phase cells were inoculated into 75 mL (250 mL flasks) of the following medium: glucose, 175 g/L; yeast extract, 2 g/L; ammonium sulphate, 55 g/L; magnesium sulfate (heptahydrate) , 0.8 g/L; potassium phosphate, 1 g/L; manganese sulfate (tetrahydrate) , 0.01 g/L; ferrous sulfate (heptahydrate), 0.01 g/L; biotin, 100 mg/L; thiamine-HCl, 200 mg/L; L-leucine, L-methionine and L-threonine, 0.001 mM each.
  • the pH of the culture was maintained at neutrality by adding 50 g/L of calcium carbonate to each flask.
  • the cells were grown at 250 rpm shaking speed and 30°C. Samples were taken at different times, and the culture optical density was measured at 600 nm in a spectrophotometer. An OD 600 of 1.0 was determined to correspond to a dry cell weight of 0.35g/Liter. Glucose and lysine concentrations in the sample supernatants were measured by high performance liquid chromatography. Cell extracts were prepared from samples taken from each time point throughout the experiment. Proteins were separated by SDS-PAGE and screened with antiserum against Vitreoscilla hemoglobin. Western analysis confirmed that hemoglobin was being expressed throughout the experiment. This hemoglobin was demonstrated to be functional by a carbon monoxide difference spectrum technique (Webster and Liu, J. Biol. Chem. , 1974) .
  • Table 2-1 shows the results obtained 48 hours after inoculation.
  • the cell yield per glucose consumed is similar in both strains (110 g/kg vs 100 g/kg)
  • the lysine yield per glucose consumed is 43% higher in the hemoglobin containing cells (100 g/kg va 0.07 g/kg) .
  • the lysine produced per cell mass is 25% higher in the hemoglobin containing cells (910 g/kg vs 730 g/kg) .
  • EXAMPLE 37 GROWTH ENHANCEMENT OF E. COLI IS DUE TO THE OXYGEN BINDING PROPERTIES OF VITREOSCILLA HEMOGLOBIN
  • LE392:pINTl Vitreoscilla hemoglobin
  • LE392:pBST truncated version of the E. coli hemoglobin-like protein described in example yy with similar plasmid- containing (LE392:pUC18) and plasmid free (LE392) cells.
  • LE392:pBST cells give a negative CO-binding spectrum indicating that the truncated E. coli hemoglobin protein is not biologically active.
  • Experimental medium and conditions were as described in example 36.
  • Cells containing a biologically active hemoglobin grow to higher cell densities than cell containing a hemoglobin that does not bind oxygen and thus is biologically inactive (LE392:pBST) .
  • Cells expressing inactive hemoglobin reach cell densities similar to control cells containing the parent plasmid pUC18 and to no-plasmid control cells.
  • C. glutamicum ATCC 13287:pBHb3 and C. glutamicum ATCC 13287:no-plasmid cells were grown in 250-mL shake flasks at 30°C and 250 rpm in the following synthetic medium: glucose, 75 g/L; yeast extract, 2 g/L; ammonium sulfate, 55 g/L; magnesium sulfate (heptahydrate), 0.8 g/L; potassium phosphate, 1 g/L; 2manganese sulfate (tetrahydrate) , 0.01 g/L; ferrous sulfate (heptahydrate), 0.01 g/L; biotin, 100 mg/L; thiamine-HCl, 200 mg/L; L-leucine, L-methionine and L-threonine, 200 mg/L each; p 7.0. These cells were used to seed the fermentors.
  • Fermentations were conducted in 3-L B. Braum MD fermentors in the medium described above, at 30°C and under constant air sparging (0.5 L/min). Initially, the impeller agitation rate was constant. During that time, the dissolved oxygen concentration gradually decreased. When the dissolved oxygen concentration reached 5% of air saturation, the agitation rate was adjusted by the fermentor controller in order to maintain the dissolved oxygen concentration at 5% of air saturation until the end of the fermentations. The culture pH was maintained at pH 7.0 by the fermentor pH controller by periodic additions of 4 N sodium hydroxide.
  • Oxygen uptake rates (OUR) and respiration coefficients were calculated using the off-gas data. Hemoglobin expression was stable throughout the fermentation as demonstrated by Western electroblotting of the samples taken. The hemoglobin produced by 13287:pBHb3 was biologically active as demonstrated by carbon monoxide binding assay.
  • Table 3.1 shows the optical density, glucose and lysine concentration as a function of time for 13287:pBHb3 and 13287:np.
  • the cell yield per glucose consumed is similar in both strains: 196 g cells/kg glucose for 13287:pBHb3 and 201 g cells/kg glucose for 13287:np.
  • the lysine yield per glucose consumed is 360 g/kg glucose for 13287:pBHb3 and 289 g/kg for 13287:np.
  • the hemo ⁇ globin containing cells have a lysine yield 25% higher than the no-plasmid cells.
  • the lysine produced per cell mass is 31% higher in the hemoglobin-producing cells (1,840 g/kg cells for 13287:pBhb3 vs 1,400 g/kg cells for 13287:np). Accordingly, lysine productivity per cell mass is 31% higher in the hemoglobin containing cells (32.9 g/kg cells/h for 13287:pBHb3 and 25.0 g/kg cells/h for 13287:np).
  • Table 3.2 shows the results from the off-gas analysis during the lysine production period for the 13287:pBHb3 and 13287:np fermentations. Table 3.2:
  • the oxygen uptake rate (OUR) of 13287:pBHb3 averages 10.8 mmol/L/h, a 57% higher than the average OUR of 13287:np during the same time period (6.87 mmol/L/h).
  • Plasmid pENT 10 is a vector which contains the VHb gene driven by the strong fungal promoter TR-1 isolated from Trichoderma reesei . Selection in fungi is by the Sh Jble gene product, isolated from Streptoalloteichus hindustanus , which confers phleomycin resistance to the host. This gene is driven by the fungal promoter GPD isolated from Aspergillus nidulans . For bacterial manipulations, selection is tetracycline resistance.
  • Tet r Tet resistance
  • the Tet r vector was constructed by isolating the Tet r gene from a pBR322 based vector and cloning it into pUC 18 (Yanisch-Perron et al.. Gene 33:103 (1985)) so as to disrupt the Amp r gene.
  • the tet r gene carried on pVU-2 on an Aval fragment was inserted via blunt ends into Seal-Avail sites of pUC 18, which inactivtes the amp r gene.
  • the resulting vector was named pTAS 18R.
  • the cloning of the VHb gene was accomplished by isolating the VHb gene from plasmid p TacHb (Example 5) via a Xba I-Sph I fragment.
  • the TR-1 promoter fragment was isolated from plasmid pUT 737 (purchased from CAYLA, France) on a Nde I-Spe I fragment. These DNA fragments were ligated to vector pTAS 18R cut with Nde I-Sph I. The resulting intermediate vector was named pENT IB.
  • the Sh ble gene, GPD promoter and Trp C terminator region were isolated from vector pUT 720 (CAYLA, France) by a Eco Rl-Xba I digest, Klenowed, and inserted into the Bam HI site of pENT IB which had also been treated with Klenow to remove protruding DNA ends and create a blunt end cloning site.
  • the resulting vector was named pENT 10.
  • Transformation into fungi is accomplished by breaking down cell walls via enzymatic digestion to form protoplasts which are then selectively permeable to uptake of exogenous DNA.
  • DNA which is transformed into fungi is not maintained as plasmid DNA as in E. coli but integrated into the host genome.
  • Buffers used in procedure KMC 700mM KC1 50mMC CaC12 10 mM MES (buffering agent)
  • - mycelia are recovered by filtration through a 30 ⁇ m filter and washed with a 0.9% solution.
  • penicillin production was studied in hemoglobin-expressing P. chrysogenum and in non-expressing control cells in batch fermentations.
  • the plasmid pENTIO described in Example 24 was used for integration of the Vitreoscilla hemoglobin gene into the genome of Penicillium chrysogenum ATCC 48271.
  • the seed medium was as follows (per liter) : 30 g glucose, 10 g lactose monohydrate, 30 ml corn steep liquor, 2 g ammonium sulfate, 5 g calcium carbonate, 0.5 g potassium dihydrogen phosphate, 10 g Pharmamedia, 10 g yeast extract. Two hundred ml of the seed medium was inoculated with spores to a final concentration of 1 x 10 8 spores/ml. The seed cultures were grown at 30°C for 48 hours at 220 rpm and were used to inoculate 2-liter fermentors.
  • the fermentation medium consisted of the following (per liter) : 120 g lactose monohydrate, 27.5 g Pharmamedia, 10 g ammonium sulfate, 10 g calcium carbonate, 10 g lard oil, 0.5 g fermentation cultures were grown at 30°C for 24 hours in B. Braun MD fermentors. The air flow rate was maintained at 1 l/min and the dissolved oxygen was controlled at 30% of air saturation throughout the fermentation. At 24 hours, the temperature was reduced to 25°C, keeping all other conditions the same, and a phenylacetic acid (potassium salt) feed was initiated at a rate of 0.07 g/h. Throughout the duration of the fermentation, the off-gas was analyzed by mass spectrometry (model 1200, Perkin-Elmer, USA) .
  • HPLC High pressure liquid chromatography
  • Buffer B was a 0.015 M solution of ammonium acetate in 30% (v/v) methanol.
  • the peaks were eluted over a binary gradient and were detected at 220 nm in a UV spectrophotometer (model SPD-6AV, Shimadzu, Japan) . Peak areas were calculated by an integrator (model CR501, Shimadzu, Japan) .
  • the levels of penicillin concentrations obtained in shake flask cultures were elevated by greater than 20% in strains transformed with the VHb gene relative to control strains.
  • a plasmid was constructed for the purpose of enhancing the expression of the hmp gene
  • hmp amino acid sequence bears great similarity to that of the
  • VHb Vitreoscilla hemoglobin
  • a 25-oligomer encodes the first 18 nucleotides of the hmp gene starting from the first ATG, preceded by a synthetic restriction enzyme site (Xbal) and consists of the sequence 5' CTC-TAG-AAT- GCT-TGA-CGC-TCA-AAC-C 3' .
  • the second 25-obigomer encodes the complement to the end of the hmp gene starting with the first stop codon, preceded by a synthetic restriction enzyme site (Kpn 1) and consists of the sequence 5' AGG-TAC-CTT-ACA-GCA-CCT- TAT-GCG-A 3' .
  • Plasmid pTac-Bst was constructed by digesting pTac-HMP with Xba 1 and Mlu 1. An approx. 400bp fragment was isolated which consists of the first portion of the hmp gene from the first ATG to a unique Mlu 1 site, and encodes the first 118 amino acids of the hmp protein.
  • Plasmid pTac-VHb which had been cut with Xba 1 and Mlul in-order to remove the first 120 amino acids of the VHb gene.
  • Plasmid pTac-Bst thus contains a DNA sequence which encodes the first 118 amino acids of the hmp gene followed by the last 26 amino acids of the VHb gene. These two segments are joined at a unique Mlu 1 site which both genes have in-common. Plasmid pTac-Bst was transformed into E ⁇ . coli cells and expression was confirmed by Western analysis. A crude cell extract was generated by sonication and the proteins separated by SDS-polyacrylamide gel electrophoresis.
  • the proteins were transferred to a nitrocellulose membrane and screened with polyclonal antiserum generated against Vitreoscilla hemoglobin. A band of identical molecular weight as pure hemoglobin was detected in the cell extract. A carbon monoxide difference spectrum was performed, however no functional protein could be detected by this method.
  • the third plasmid was constructed by using PCR to generate a truncated version of the hmp gene and then ligating the product into pTacVHb from which the VHb sequences had been removed.
  • a 23- oligomer was designed which encodes the complement to 17 nucleotides of the hmp DNA sequence from a region aprox. 7 amino acids past the end of the portion of the hmp gene which is similar to the VHb gene. These 17 nucleotides are preceded by an additional 6 nucleotides which encode the restriction enzyme site Bsu36I.
  • the Bs36 I site was designed to encode a stop codon.
  • This 23-oligomer consists of the sequence 5' ACC-TTA-GGC-TTT-GCT-GGC-GTT-TT 3' .
  • a PCR reaction was carried out using this 23-oligomer and the 25-oligomer which codes for the first portion of the hmp gene, and using plasmid pTac-HMP DNA for template.
  • a PCR product of aprox. 440bp was generated which restriction analysis showed to be a portion of the hmp gene. The 3' end of this product was digested with the restriction enzyme Bsu36 I and made blunt by filling in with DNA polymerase I (Klenow fragment) .
  • This DNA fragment was then cut with the restriction enzyme Xba 1 and ligated into plasmid pTacVHb which had been cut with EcoRI, been made blunt-ended by filling-in with Klenow, and then digested with Xba 1 to remove the VHb sequence.
  • the ligation mix was transformed into competent ⁇ ___ coli cells and a transformant which contained a correct plasmid construct as judged by restriction analysis was isolated. This plasmid was designated pTac-K12Hb.
  • the ________ coli clone transformed with pTac-K12Hb was used for two experiments.
  • Plasmid pTac- Bst was constructed by digesting pTac-HMP with Xba 1 and Mlu 1. An aprox. 400bp fragment was isolated which consists of the first portion of the hmp gene from the first ATG to a unique Mlu 1 site, and encodes the first 118 amino acids of the hmp protein.
  • Plasmid pTac-Bst thus contains a DNA sequence which encodes the first 118 amino acids of the hmp gene followed by the last 26 amino acids of the VHb gene. These two segments are joined at a unique Mlu 1 site which both genes have in-common. Plasmid pTac-Bst was transformed into E. coli cells and expression was confirmed by Western analysis. A crude cell extract was generated by sonication and the proteins separated by SDS-polyacrylamide gel electrophoresis.
  • the proteins were transferred to a nitrocellulose membrane and screened with polyclonal antiserum generated against Vitreoscilla hemoglobin. A band of identical molecular weight as pure hemoglobin was detected in the cell extract. A carbon monoxide difference spectrum was performed, however no functional protein could be detected by this method.
  • the third plasmid was constructed by using PCR to generate a truncated version of the hmp gene and then ligating the product into pTacVHb from which the VHb sequences had been removed.
  • a 23- oligomer was designed which encodes the complement to 17 nucleotides of the hmp DNA sequence from a region aprox. 7 amino acids past the end of the portion of the hmp gene which is similar to the VHb gene.
  • the 17 nucleotides are preceded by an additional 6 nucleotides which encode the restriction enzyme site
  • Bsu36I The Bsu36 I site was designed to encode a stop codon.
  • This 23-oligomer consists of the sequence 5• ACC-TTA-GGC-TTT-GCT-GGC-GTT-TT 3' .
  • a PCR reaction was carried out using this 23-oligomer and the 25-oligomer which codes for the first portion of the hmp gene, and using plasmid pTac-HMP DNA for template.
  • a PCR product of aprox. 440bp was generated which restriction analysis showed to be a portion of the hmp gene. The 3' end of this product was digested with the restriction enzyme Bsu36 I and made blunt by filling in with DNA polymerase I
  • Plasmid pGYM was digested with restriction enzymes Xbal and SSP1. The larger of two fragments was gel isolated. This fragment contained all of the myoglobin gene and some pUC sequence but no promoter. This fragment was ligated to a fragment of plasmid pTac-Bst which had been first digested with Mlul, made blunt with DNA polymerase 1 (Klenow fragment) , then digested with Xbal, the smaller of two fragments having been gel isolated. This smaller fragment consists of the Tac promoter and some pUC sequence. The two fragments were ligated together and transformed into competent E. coli cells.
  • Transformants were screened for plasmid DNA consisting of the pUC vector having a Tac-Myoglobin insert.
  • One such transformant was found and its plasmid DNA, pUC-TacMyo, was isolated for further manipulations.
  • Plasmid pUC-TacMyo was digested with restriction enzyme Hind III. This liberated an 800bp fragment consisting of the myoglobin gene preceded by the Tac promoter. This fragment was gel isolated, made blunt using Klenow, and ligated with a deleted version of plasmid pFSl (pFSl del) .
  • Plasmid pFSl del had been prepared by first digesting Nhel to remove the Tac-VHb sequence, then filling in the ends with Klenow.
  • the pFSl del - TacMyo. ligation mix was transformed into competent Coryneform strain ATTC 13287.
  • One Coryneform transformant showed a reddish pigment and plasmid isolated from this transformant was determined to be a correct construction by restriction analysis and in particular by the presence of a unique EcoRI site which is present in the myoglobin sequence but is not present in the VHb sequence.
  • This Coryneform transformant was designated 13287:pFS-TacMyo.
  • the expression of functional myoglobin in 13287:pFS- TacMyo was demonstrated by a carbon monoxide difference spectrum.
  • a 5.2Kb plasmid was constructed for the expression of a soybean leghemoglobin protein (LHbcl) in E___ coli.
  • This plasmid, pKK-LHbcl contains the cDNA to soybean leghemoglobin cl cloned into the expression vector pKK 233-2 (Pharmacia).
  • pKK 233-2 Pharmacia
  • a 3.5Kb plasmid pCDl which consists of the E_j_ coli plasmid pBluescript SK (Stratagene) with the 600bp insert consisting of a cDNA sequence to the soybean leghemoglobin cl gene, was used as template in a polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • the oligomer which is complementary to the 3' end of the - strand of leghemoglobin cl cDNA starts with a Hind III site and consists of the sequence 5'-caa-gct-ttt-ttt-ttt-ttt-ttt-ttt-t-3* .
  • a polymerase chain reaction amplified the 600bp LHb cl sequence resulting in a PCR product having a Ncol site at the 5' end and a Hind III site at the 3' end. This PCR product was ligated with the cloning plasmid pCR 1000 (Invitrogen) .
  • Plasmid DNA was isolated from several transformants and screened for the presence of full-length LHb cl cDNA sequence by restriction analysis and gel electrophoresis. One clone was found to be correct and was designated plasmid pTA- LHbcl. Plasmid pTA-LHbcl was linearized with Spel which cuts 40bp past the 3' terminus of the LHbcl insert. The plasmid ends were then made blunt by filling in with DNA polymerase 1 (Klenow fragment) .
  • the LHbcl sequence was removed from the pCR 1000 vector by digestion with Ncol, and this 600bp fragment was purified by agarose gel electrophoresis.
  • An expression vector, pKK 233-2 (Pharmacia) was prepared by first linearizing with Hind III and filling in the ends with DNA polymerase 1 (Klenow fragment) , then digesting with Ncol. The purified 600bp LHbcl fragment and the linearized pKK 233-2 were ligated together and transformed into competent _______ coli cells. Transformants were selected by resistance to the antibiotic ampicillin. Plasmid DNA was isolated from several transformants and screened for the presence of full-length LHbcl sequence by restriction analysis and gel electrophoresis.
  • clone pKK-LHbcl Plasmid pKK-LHbcl was transformed into E. coli strains JMlOl and LE 392. A single ampicillin resistant transformant of each strain was isolated and designated JMlOl: pKK-LHbcl, and LE 392: pKK- LHbcl. Expression of functional leghemoglobin was confirmed in both transformants by carbon monoxide binding assay (Webster, D.A. , 1974, J. Biol. Chem. 249:4257-4260). A crude cell extract was prepared from each transformant as well as control cells by sonication.
  • the medium was further supplemented with 1.0ml of the following vitamin solution: in 500mls 0.21g riboflavin, 2.7g pantothenic acid, 3.0g niacin/nicotanic acid, 0.7g pyridoxine, 0.03g biotin, and 0.02g folic acid.
  • the pH of the medium was adjusted to pH7.3 and maintained with 12.1g/L Trisma base.
  • the cells were grown at 350rpm shaking speed and 37 deg. C.
  • Cell density was monitored by measuring absorbance at 600nm in a spectrophotometer (OD600) . Cell density was monitored at hourly intervals from 19 to 23 hours after inocultion. Maximum cell density was determined to be when OD600 did not increase within one hour.
  • the maximum cell density obtained for the parent _____ coli strain LE 392 and four transformants is shown in Table C-l.
  • Table C-l Effect of the expression of four oxygen- binding proteins on maximum cell density attained b E coli strain
  • a plasmid used for the expression of myoglobin in Escherichia coli was constructed in the following manner.
  • the structural gene encoding horse heart myoglobin was synthesized based on its amino acid sequence [Eur. J. Biochem. 11, 267-277, 1969].
  • the purified gene was then inserted into the E.coli pEMBL18+ [Methods Enzymol. 155. 111-119, 1987].
  • the myoglobin gene is under the control of the tac promoter.
  • the resulting plasmid, called pGYM was transformed into E. coli strain LE392 (supE44 supF58 hsdR514 galK2 galT22 metBl trpR55 lacYl) .
  • the experimental cultures contained 12.5 mL of glucose semi-defined medium (see EXAMPLE 32) in 250 mL erlenmeyer flasks shaken at 250 rpm in a New Brunswick G24 incubator at 37°C. The flasks were seeded with a 0.5% (v/v) inoculum of late expoinential-phase cells. The culture optical densities (ODs) were measured between 22-24 hrs, when the cultures had reached their maximum ODs. The maximum cell ODs are shown below:
  • E__ coli strain MG1655 expressing myoglobin demonstrates a 27% improvement in growth over the control, indicating that the beneficial effect of myoglobin is not strain specific.
  • the growth properties are compared of the recombinant strains expressing Vitreoscilla hemoglobin (LE392:pINTl) , horse heart myoglobin (LE392:pGYM) , soybean leg hemoglobin (LE392:pKK-LHbcl) , and E. coli hemoprotein (LE392:pTac-HMP) with similr plasmid- containing (LE392:pUC18) and plasmid free (LE392) cells under typical batch fermentation conditions.
  • DO dissolved oxygen
  • glutamicum ATCC 13287:pFSl _____ glutamicum ATCC 13287:pMYO, and C ⁇ .
  • glutamicum ATCC 13287:no-plasmid cells were grown in 250-mL shake flasks at 30°C and 250 rpm in the following synthetic medium: glucose, 60 g/L; yeast extract, 2 g/L; ammonium sulfate, 55 g/L; magnesium sulfate (heptahydrate), 0.8 g/L; potassium phosphate, 1 g/L; manganese sulfate (tetrahydrate) , 0.01 g/L; ferrous sulfate (heptahydrate), 0.01 g/L; biotin, 100 ⁇ g/L; thiamine- HCl, 200 ⁇ g/L; L-leucine, L-methionine and L- threonine, 200 mg/L each; pH 7.0. These cells were used to seed the fermentors.
  • Fermentations were conducted in 3-L B. Braun MD fermentors in the medium described above, at 30°C and udner constant air sparging (1.0 L/min). Initially, the impeller agitation rate was constant. During that time, the dissolved oxygen concentration gradually decreased. When the dissolved oxygen concentration reached 5% of air saturation, the agitation rate was adjusted by the fermentor controller in order to maintain the dissolved oxygen concentration at 5% of air saturation until the end of the fermentations. The culture pH was maintained at pH 7.0 by the ferrrmentor pH controller by periodic additions of 4 N sodium hydroxide.
  • Table 2.1 shows the optical density, glucose and lysine concentration as a function of time for 13287:pFSl, 13287:pMY0, and 13287:np.
  • the cell yield per glucose consumed is similar in all the strains: 305 gcells/kgglucose for 13287:pFSl, 302 gcells/kgglucose for 13287:pMY0, and 310 gcells/kgglucose for 13287:np.
  • the lysine yield per glucose consumed is 200 g/kkglucose for 13287:pFSl, 188 g/kgglucose for 13287:pMY0, and 155 g/kgglucose for 13287:np.
  • Vitreoscilla hemoglobin-containing cells have a lysine yield 29% higher than the no-plasmid cells, and myoglobin-containing cells have a lysine yield 21% higher than the no-plasmid cells.
  • Lysine productivity per cell mass is 31% higher in the Vitreoscilla hemoglobin containing cells (23.4 g/kgcells/h for 13287:pFSl and 17.9 g/kgcells/h for 13287:np).
  • lysine productivity per cell mass is 25% higher in the myoglobin containing cells (22.3 g/kgcells/h for 13287:pMYO and 17.9 g/kgcells/h for 13287:np) .
  • MOLECULE TYPE cDNA to genomic RNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GCC AAA CAC CCT GAA GTA CGT CCT TTG TTT GAT ATG GGT CGC CAA GAA TCT TTG GAG CAG 300
  • ORGANISM Vitreoscilla sp.

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  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Méthodes d'utilisation d'ADN codant des protéines fixatrices d'oxygène et de plasmides apparentés renfermant de telles protéines. Ces méthodes se prêtent à toute une série d'applications: apport d'oxygène aux cellules; stimulation de la croissance; expression de divers produits génétiques; soutien des processus nécessitant de l'oxygène; liaison et séparation de l'oxygène des liquides et des gaz; ainsi qu'à toute une série de réactions oxydatives.
PCT/US1993/005527 1992-06-15 1993-06-15 Renforcement de la croissance des cellules par expression de proteines clonees fixatrices d'oxygene WO1993025697A1 (fr)

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WO1996010583A1 (fr) * 1994-10-04 1996-04-11 Mallinckrodt Veterinary, Inc. Vaccin contenant une hemoproteine
FR2736930A1 (fr) * 1995-07-17 1997-01-24 Biocem Procede de production, par des cellules vegetales, de proteines heminiques, proteines ainsi obtenues et produits contenant ces proteines
WO1997047746A1 (fr) * 1996-06-10 1997-12-18 Novo Nordisk Biotech, Inc. Procede permettant d'augmenter la production d'hemoproteines dans des champignons filamenteux
WO1998053084A1 (fr) * 1997-05-22 1998-11-26 Solidago Ag Augmentation de la production d'erythromycine par l'expression d'une proteine clonee fixant l'oxygene
WO1999002687A1 (fr) * 1997-07-08 1999-01-21 Commonwealth Scientific And Industrial Research Organisation Procede pour augmenter la teneur en fer des cellules des plantes
WO2001094569A3 (fr) * 2000-06-02 2002-03-21 Degussa Nouvelles sequences de nucleotides codant le gene glbo
US8293696B2 (en) 2009-02-06 2012-10-23 Ecolab, Inc. Alkaline composition comprising a chelant mixture, including HEIDA, and method of producing same
US9011949B2 (en) 2011-07-12 2015-04-21 Impossible Foods Inc. Methods and compositions for consumables
US9700067B2 (en) 2011-07-12 2017-07-11 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US9808029B2 (en) 2011-07-12 2017-11-07 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US9826772B2 (en) 2013-01-11 2017-11-28 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US10039306B2 (en) 2012-03-16 2018-08-07 Impossible Foods Inc. Methods and compositions for consumables
US10172380B2 (en) 2014-03-31 2019-01-08 Impossible Foods Inc. Ground meat replicas
US10986848B2 (en) 2013-01-11 2021-04-27 Impossible Foods Inc. Methods and compositions for consumables
CN116179586A (zh) * 2021-11-26 2023-05-30 上海凯赛生物技术股份有限公司 用于发酵生产l-赖氨酸的表达盒、菌株及其应用
WO2024167695A1 (fr) * 2023-02-08 2024-08-15 Danisco Us Inc. Compositions et procédés de production de globines hétérologues dans des cellules fongiques filamenteuses

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Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010583A1 (fr) * 1994-10-04 1996-04-11 Mallinckrodt Veterinary, Inc. Vaccin contenant une hemoproteine
FR2736930A1 (fr) * 1995-07-17 1997-01-24 Biocem Procede de production, par des cellules vegetales, de proteines heminiques, proteines ainsi obtenues et produits contenant ces proteines
WO1997004115A3 (fr) * 1995-07-17 1997-02-27 Biocem Procede de production, par des cellules vegetales, de proteines heminiques, proteines ainsi obtenues et produits contenant ces proteines
US6916787B2 (en) 1995-07-17 2005-07-12 Institut National De La Sante Et De Recherche Medicale Method for producing hemin proteins using plant cells, resulting proteins and products containing same
US6344600B1 (en) 1995-07-17 2002-02-05 Meristem Therapeutics Method for producing human hemoglobin proteins using plant cells
US6100057A (en) * 1996-06-10 2000-08-08 Novo Nordisk Biotech, Inc. Method for increasing hemoprotein production in filamentous fungi
US6261827B1 (en) 1996-06-10 2001-07-17 Novozymes Biotech, Inc. Method for increasing hemoprotein production in filamentous fungi
WO1997047746A1 (fr) * 1996-06-10 1997-12-18 Novo Nordisk Biotech, Inc. Procede permettant d'augmenter la production d'hemoproteines dans des champignons filamenteux
WO1998053084A1 (fr) * 1997-05-22 1998-11-26 Solidago Ag Augmentation de la production d'erythromycine par l'expression d'une proteine clonee fixant l'oxygene
WO1999002687A1 (fr) * 1997-07-08 1999-01-21 Commonwealth Scientific And Industrial Research Organisation Procede pour augmenter la teneur en fer des cellules des plantes
WO2001094569A3 (fr) * 2000-06-02 2002-03-21 Degussa Nouvelles sequences de nucleotides codant le gene glbo
US6759218B2 (en) 2000-06-02 2004-07-06 Degussa-Ag Nucleotide sequences coding for the glbO gene
US8293696B2 (en) 2009-02-06 2012-10-23 Ecolab, Inc. Alkaline composition comprising a chelant mixture, including HEIDA, and method of producing same
US10863761B2 (en) 2011-07-12 2020-12-15 Impossible Foods Inc. Methods and compositions for consumables
US10327464B2 (en) 2011-07-12 2019-06-25 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US9808029B2 (en) 2011-07-12 2017-11-07 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US9700067B2 (en) 2011-07-12 2017-07-11 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US9943096B2 (en) 2011-07-12 2018-04-17 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US9011949B2 (en) 2011-07-12 2015-04-21 Impossible Foods Inc. Methods and compositions for consumables
US10039306B2 (en) 2012-03-16 2018-08-07 Impossible Foods Inc. Methods and compositions for consumables
US11224241B2 (en) 2013-01-11 2022-01-18 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US11219232B2 (en) 2013-01-11 2022-01-11 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US10172381B2 (en) 2013-01-11 2019-01-08 Impossible Foods Inc. Methods and compositions for consumables
US9826772B2 (en) 2013-01-11 2017-11-28 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US10314325B2 (en) 2013-01-11 2019-06-11 Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables
US10986848B2 (en) 2013-01-11 2021-04-27 Impossible Foods Inc. Methods and compositions for consumables
US10993462B2 (en) 2013-01-11 2021-05-04 Impossible Foods Inc. Methods and compositions for consumables
US11013250B2 (en) 2013-01-11 2021-05-25 Impossible Foods Inc. Methods and compositions for consumables
US10172380B2 (en) 2014-03-31 2019-01-08 Impossible Foods Inc. Ground meat replicas
US10798958B2 (en) 2014-03-31 2020-10-13 Impossible Foods Inc. Ground meat replicas
US11439166B2 (en) 2014-03-31 2022-09-13 Impossible Foods Inc. Ground meat replicas
US11819041B2 (en) 2014-03-31 2023-11-21 Impossible Foods Inc. Ground meat replicas
CN116179586A (zh) * 2021-11-26 2023-05-30 上海凯赛生物技术股份有限公司 用于发酵生产l-赖氨酸的表达盒、菌株及其应用
WO2024167695A1 (fr) * 2023-02-08 2024-08-15 Danisco Us Inc. Compositions et procédés de production de globines hétérologues dans des cellules fongiques filamenteuses

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