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WO1992003546A1 - Augmentation de la production de produits naturels dans corynebacterium par expression d'hemoglobines bacteriennes clonees - Google Patents

Augmentation de la production de produits naturels dans corynebacterium par expression d'hemoglobines bacteriennes clonees Download PDF

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Publication number
WO1992003546A1
WO1992003546A1 PCT/US1991/005825 US9105825W WO9203546A1 WO 1992003546 A1 WO1992003546 A1 WO 1992003546A1 US 9105825 W US9105825 W US 9105825W WO 9203546 A1 WO9203546 A1 WO 9203546A1
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host
coryneform
protein
expression
vector
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PCT/US1991/005825
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English (en)
Inventor
Fred C. Sander
Deborah L. Leenutaphong
Jorge L. Galazzo
Dallas E. Hughes
James E. Bailey
Chaitan S. Khosla
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Exogene Corporation
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Publication of WO1992003546A1 publication Critical patent/WO1992003546A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

Definitions

  • This invention relates to the expression of Vitreoscilla hemoglobin Coryneform bacteria to enhance amino acid production and growth characteristics.
  • This invention relates to all members of the Coryneform group of bacteria including the genera Corvnebacterium, revibacterium, Arthrobacter, Microbacterium and Cellulomonas.
  • the globins represent a family of heme-containing proteins that reversibly bind oxygen and are thus able to enhance the oxygen transfer rate to cells in multicellular organisms.
  • the expression of bacterial hemoglobin has been shown to enhance the growth properties of the bacterium Escherichia coli. especially under conditions of reduced oxygen (Khosla and Bailey, Nature, 331:633, 1986). Such enhancement may result in part from an altered intracellular redox state.
  • the bacteria of the Coryneform group are the single most important bacterial group used for the commercial production of amino acids (Atkinson and Mauituma, Biochemical Engineering and Biotechnology Handbook. MacMillan, England, 1987).
  • Examples of widelyused amino acid products produced in Corvnebacterium fermentations include taste enhancers in human food, especially mono-sodium glutamate, and animal feed supplements, notably L-lysine.
  • production by Corvnebacterium of aromatic amino acids for example, L-phenylalanine, L-tyrosine, and L-tryptophan, is of increasing industrial interest (Ito, et al. , Agricultural and Biological Chemistry 54 : 70 , 1990) .
  • Phenylalanine is an important component of the new sweetener known as "Aspartame", and tryptophan, is used as an ingredient in animal feeds and in medicines.
  • Corvnebacterium are also used in the production of nucleotides and related compounds (Shiio and Nakamoir, Fermentation Process - Development of Industrial Organisms, J. O. Newary, ed., Marcel Dekker, N.Y., 1989).
  • Extensive "classical” mutagenesis of Corynebacterium has been used to develop strains which overproduce a variety of nucleotides, nucleosides, and amino acids.
  • further increase in production by such traditional techniques is limited since the cumulative effect of many mutations results in almost complete physiological deregulation (Martin, et al., Bio ⁇ Technology, 5:137, 1987).
  • Corvnebacterium are facultative aerobes in which the accumulation of amino acids depends upon a metabolic balance that is greatly affected by environmental factors. Among these factors, the most important are the concentrations of oxygen, ammonium ion, phosphate, biotin, and the culture pH.
  • a typical Corynebacterium qlutamicum fermentation begins with the growth of cells to high densities (growth phase). There is little glutamic acid production during growth phase. The final cell densities achieved are usually limited by the supply of the growth factor biotin, or by the addition of penicillin to the fermentation media late in the growth phase.
  • the mechanism by which glutamic acid production in Corynebacterium is regulated is most influenced by two major factors: oxygen demand and biotin concentration (Hirose, "The Microbial Production of Amino Acids", Halsted Press, 1970) .
  • Oxygen demand is closely correlated with cell growth, glutamic acid formation, and utilization of substrate.
  • the biotin concentration in the medium controls the rate of substrate utilization and hence the cell oxygen demand. Higher concentrations of biotin result in increased rate of substrate utilization and oxygen demand.
  • the hemoglobin is a soluble, dimeric protein that combines with oxygen and displays a spectral response to carbon monoxide binding characteristic of eukaryotic hemoglobins (Wakabayashi, et al., Nature. 332:481, 1986). It was conjectured by
  • the gene for the Vitreoscilla hemoglobin has been isolated along with its native transcriptional regulatory sequences (Khosla and Bailey, Mol. Gen. Genet..214:158, 1988). Interestingly, this gene was expressed from its native promoter when introduced into E. coli. Of particular interest was the observation that expression of hemoglobin was regulated by the culture oxygen content such that maximal induction occurred under microaerobic conditions. Under fed-batch fermentation conditions, E. coli cells expressing hemoglobin displayed significantly higher specific growth rates and achieved final cell densities 2-3-fold those attained by non- expression strains (Khosla and Bailey, Nature. 331:633, 1988).
  • the present invention relates to oxygen-binding proteins, particularly hemoglobins, a recombinant-DNA method of producing same, and to portable DNA sequences capable of directing intracellular production of these oxygenbinding proteins in Coryneform.
  • the present invention also relate vectors containing these portable DNA sequences.
  • One object of the present invention is to provide a recombinant-DNA method for the production of these oxygen-binding proteins.
  • To facilitate the recombinantDNA synthesis of these oxygen-binding proteins it is a further object of the present invention to provide portable DNA sequences capable of directing intracellular production of oxygen-binding proteins in Coryneform. It is also an object of the present invention to provide cloning vectors containing these portable sequences.
  • These vectors are capable of being used in recombinant Coryneform to enhance the growth characteristics of organisms, and to produce useful quantities of oxygenbinding proteins. In cells augmented by intracellular synthesis of oxygen-binding proteins, the production of amino acids, nucleotides and other products can also be enhanced.
  • the present invention also provides novel methods and materials for expression of cloned genes in Coryneform.
  • a preferred expression vector, a recombinant-DNA method of producing same, and a preferred portable DNA sequence capable of directing the translation and transcription initiation and control of the expression of desired gene products are disclosed.
  • a preferred recombinant-DNA method which results in manufacture by cells of the genus Coryneform of the oxygen-binding proteins using the preferred portable DNA sequences.
  • Figure 1 is a partial restriction map of plasmid pBHb3.
  • oxygen-binding protein is intended to mean a protein having oxygen-binding activity, with a primary structure as defined by the codons present in the deoxyribonucleic acid sequence which directs intracellular translation and which may or may not include post-translational modifications. Such post-translational modifications may include, for example, association with a heme prosthetic group. It is further intended that the term “oxygen-binding protein” refers to its intracellular forms which are excretable or non-excretable.
  • portable DNA sequences are provided which are capable of directing intracellular production of a hemoglobin biologically equivalent to that 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) isolated from Vitreoscilla.
  • 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 translation to 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 its intracellular forms which are excretable or non-excretable. It is intended that promoter/regulator sequences of the present invention may control and initiate transcription and translation of many other endogenous and/or exogenous foreign proteins, such as, proteins involved in amino acid synthesis.
  • a functional promoter/regulator in Coryneform bacteria is operatively fused with at least a major portion of the following nucleotide sequence which reads 5 to 3 and includes the translation initiation sequence ATG (double underlined) and the nucleotide sequence of the Vitreoscilla structural gene (also underlined): AAGCTTAACG GACCCTGGGG TTAAAAGTAT TTGAGTTTTG
  • the nucleotide coding sequence is operatively fused to the promoter/regulator sequences in a cell when RNA polymerase transcribes the nucleotide coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • the promoter ⁇ regulator thus comprise operational elements for expression.
  • Vitreoscilla hemoglobin should contain at least a major portion of the following amino acid sequence of Vitreoscilla hemoglobin, which portion is capable of binding oxygen.
  • the portable DNA sequences of the present invention may be synthetically created, by hand or with automated apparatus.
  • the methods used 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 if directed to Maniatis, et al. , Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory (1984), and Horvath, et aJL. , "An Automated DNA Synthesizer Employing Deoxynucleoside 3 1 Phosphoramidites, Methods in Enzymology 154:313-326, 1987, hereby incorporated by reference.
  • a portable DNA sequence may be a fragment of a natural sequence, i. e., a fragment of a polynucleotide which occurred in nature.
  • a portable DNA sequence may be a restriction fragment isolated from a genomic library.
  • the genomic library is created from the bacterium Vitreoscilla.
  • a portable DNA sequence may be isolated from other genomic and cDNA libraries.
  • the present invention also provides a series of vectors, each containing at least one portable DNA sequence as described herein. It is contemplated that additional copies of a 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 portable DNA sequences and, optionally, other desirable DNA sequences. Other desirable sequences may encode metabolites normally made by Coryneform.
  • cloning vectors within the scope of the present invention may contain supplemental nucleotide sequences preceding or subsequent to the portable promoter/regulator and/or DNA sequence. These supplemental sequences are those 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, post-translational 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, pBHb3, contains a nucleotide sequence which codes for an oxygen-binding protein derived from Vitreoscilla hemoglobin gene.
  • Plasmid pBHb3 may also contain supplemental nucleotide sequences such as terminators, enhancers, attenuators and the like.
  • terminators 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 in Coryneform contain various operational elements in addition to or instead of the promotor/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 include modifications employing other vectors modified to contain one or more of the preferred portable DNA sequences described herein.
  • such vectors have some or all of the following characteristics: (1) possess a minimal number of host- organisms 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 readily performed by those of ordinary skill in the art in light of the available literature and the teachings herein.
  • Coryneform Any strain of Coryneform which admits stable insertion of cloned DNA may serve as a host for the practice of this invention.
  • Examples of Coryneform strains which can be transformed or transduced are: Corynebacterium glutamicum - Thierbach, et al., Applied Microbiology and Biotechnology. 29:356, 1988.
  • Coryneform plasmids are useful vectors:
  • Phages used as Coryneform vectors include derivatives of f1A (Miwa, et al., Gene. 39:281, 1985). Synthesis and/or isolation of necessary and desired component parts of such cloning vectors, and their assembly is believed to be within the capability of those of ordinary skill in the art and, such , are capable of being performed without undue experimentation.
  • the Coryneform host may produce greater amounts per vector of the cloned protein. The number of multiple copies of the DNA sequence which may be inserted into the vector is limited by the ability of the resultant vector, due to its size, to be transferred into and replicated and expressed in the Coryneform host.
  • a 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 kanamycin resistance is preferred and is thus included in the preferred vector pBHb3.
  • 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. A biologically pure culture of the transformed Coryneform host would be obtained by culturing under conditions which require the induced phenotype for survival.
  • a portable DNA sequence of the present invention may be used as a selectable marker, in that it provides enhanced growth characteristics in low oxygen circumstances.
  • promoter/regulators useful in the practice of this invention are not restricted to the tac promoter, but may also include other promoter/regulations used in Coryneform vectors, for example: trp - Morinaga, et al., J. of Biotechnology.
  • the present invention also provides a recombinant-DNA method for the production of oxygen-binding proteins comprising the steps of:
  • Maturation of the protein to its oxygen-binding active structure occurs by folding into an active configuration and incorporation of heme.
  • 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.
  • the portable DNA sequences may be inserted directly into the Coryneform host chromosome, or alternatively may utilize a plasmid cloning system.
  • the cloning vector pBHb3 is used in the disclosed method.
  • a vector thus obtained may be transferred into the appropriate Coryneform species. It is believed that any Coryneform species having the ability to take up exogenous DNA and express genes and attendant operational elements thereof may be used.
  • 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.
  • the Coryneform host cells 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.
  • This invention also provides a recombinant-DNA method for the use in Coryneform of the Viteoscilla hemoglobin promoter/regulator.
  • a selected DNA sequence for example, a structural gene or a sequence in which a foreign protein is encoded
  • external control in Coryneform under given environmental conditions which comprises the steps of:
  • a SNA expression vector comprising at least one selected isolated structural gene or foreign DNA that is transcriptionally and/or translationally responsive to a Vitreoscilla hemoglobin promoter/regulator DNA sequence in Coryneform under the given environmental conditions, which is operatively fused with that promoter/regulator DNA sequence;
  • the products and processes of the present invention find usefulness in the production of amino acids and other metabolites using Coryneform in labortory and industrial applications.
  • the invention provides metabolically engineered cells with enhanced growth characteristics for increasing production of proteins, amino acids or other metabolites in Coryneform.
  • a plasmid was constructed for the expression of a bacterial hemoglobin in Corynebacterium.
  • This plasmid, pBHb3 contains the Vitreoscilla hemoglobin gene (Khosla and Bailey, Mol. Gen. Genet..214:158, 1988) cloned into a common Corvnebacterium plasmid pBK10.
  • pINTl 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 EcoR1. 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 EcoR1 linearized plasmid pBK10 (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 ATTC13032 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 antiserum generated against Vitreoscilla hemoglobin purified from E. coli. harboring plasmid pRed 2 (Khosla and Bailey, Mol. Gen. Genet., 1988). A band of identical molecular weight as pure hemoglobin was detected in the cell extracts.
  • 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 anti-Vitreoscilla hemoglobin antisera. 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.
  • Table 2-1 Effect of hemoglobin expression on the production of L-lysine in C. glutamicum ATCC 13287
  • 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). Also, the lysine produced per cell mass is 25% higher in the hemoglobin containing cells (910 g/kg vs 730 g/kg).
  • 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; 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, 200 mg/L each; p 7.0.
  • Table 3.1 shows the optical density, glucose and lysine concentration as a function of time for 13287 :pBHb3 and 13287:np. Table 3.1
  • 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
  • the hemoglobin containing cells have a lysine yield 25% higher than the no-plasmid cells. Also, the lysine produced per cell mass is 31% higher in the hemoglobin-producing cells (1,840 g/kg cells for
  • 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
  • Table 3.2 shows the results from the off-gas analysis during the lysine production period for the 13287 :pBHb3 and 13287 :np fermentations.
  • 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).

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Abstract

L'invention concerne un procédé d'expression de l'hémoglobine bactérienne dans Coryneforme et, par ce moyen, d'augmentation de la production d'amino acides ou autres métabolites par Coryneforme. L'invention concerne également l'expression de vecteurs pour l'expression de gènes naturels et hétérologues dans Coryneforme.
PCT/US1991/005825 1990-08-23 1991-08-22 Augmentation de la production de produits naturels dans corynebacterium par expression d'hemoglobines bacteriennes clonees WO1992003546A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025697A1 (fr) * 1992-06-15 1993-12-23 California Institute Of Technology Renforcement de la croissance des cellules par expression de proteines clonees fixatrices d'oxygene
EP0629699A3 (fr) * 1993-06-15 1996-04-24 Mitsubishi Petrochemical Co Fragment de DNA ayant une activité de promoteur et provenant de bacteries corynéformes.
WO2001094569A3 (fr) * 2000-06-02 2002-03-21 Degussa Nouvelles sequences de nucleotides codant le gene glbo
EP0841395B1 (fr) * 1995-06-07 2011-11-02 Ajinomoto Co., Inc. Procede de production de l-lysine
EP0811682B2 (fr) † 1996-06-05 2017-04-19 Ajinomoto Co., Inc. Procédé pour préparer L-lysine
KR20190025615A (ko) * 2016-06-30 2019-03-11 지머젠 인코포레이티드 박테리아 헤모글로빈 라이브러리를 생성하는 방법 및 이의 용도

Citations (3)

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Publication number Priority date Publication date Assignee Title
US4087329A (en) * 1975-08-28 1978-05-02 Kyowa Hakko Kogyo Co., Ltd. Creatinine desimidase and its method of production
US4710471A (en) * 1981-12-17 1987-12-01 Kyowa Hakko Kogyo Co., Ltd. Novel vector plasmids
WO1989003883A1 (fr) * 1987-10-23 1989-05-05 California Institute Of Technology Amelioration de la croissance de cellules par expression d'un gene d'hemoglobine clone

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4087329A (en) * 1975-08-28 1978-05-02 Kyowa Hakko Kogyo Co., Ltd. Creatinine desimidase and its method of production
US4710471A (en) * 1981-12-17 1987-12-01 Kyowa Hakko Kogyo Co., Ltd. Novel vector plasmids
WO1989003883A1 (fr) * 1987-10-23 1989-05-05 California Institute Of Technology Amelioration de la croissance de cellules par expression d'un gene d'hemoglobine clone

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025697A1 (fr) * 1992-06-15 1993-12-23 California Institute Of Technology Renforcement de la croissance des cellules par expression de proteines clonees fixatrices d'oxygene
EP0629699A3 (fr) * 1993-06-15 1996-04-24 Mitsubishi Petrochemical Co Fragment de DNA ayant une activité de promoteur et provenant de bacteries corynéformes.
EP0841395B1 (fr) * 1995-06-07 2011-11-02 Ajinomoto Co., Inc. Procede de production de l-lysine
EP0811682B2 (fr) † 1996-06-05 2017-04-19 Ajinomoto Co., Inc. Procédé pour préparer L-lysine
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
KR20190025615A (ko) * 2016-06-30 2019-03-11 지머젠 인코포레이티드 박테리아 헤모글로빈 라이브러리를 생성하는 방법 및 이의 용도
JP2019519242A (ja) * 2016-06-30 2019-07-11 ザイマージェン インコーポレイテッド 細菌ヘモグロビンライブラリーを生成するための方法およびその使用
EP3478833A4 (fr) * 2016-06-30 2019-10-02 Zymergen, Inc. Procédés de génération d'une bibliothèque d'hémoglobines bactériennes et utilisations associées
US10544390B2 (en) 2016-06-30 2020-01-28 Zymergen Inc. Methods for generating a bacterial hemoglobin library and uses thereof
KR102345899B1 (ko) * 2016-06-30 2021-12-31 지머젠 인코포레이티드 박테리아 헤모글로빈 라이브러리를 생성하는 방법 및 이의 용도

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