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United States Patent (19) 11 Patent Number: 5,824,511 Matt00n Et Al

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United States Patent (19) 11 Patent Number: 5,824,511 Matt00n Et Al USOO582451 1A United States Patent (19) 11 Patent Number: 5,824,511 Matt00n et al. (45) Date of Patent: Oct. 20, 1998 54 METHOD FOR ENHANCING THE De Llano et al., “Recombinant Human Sickle Hemoglobin PRODUCTION OF HEMOPROTEINS Expressed in Yeast', Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 918-922, Feb., 1993. 75 Inventors: James R. Mattoon; George Bajszár, Grandchamp et al., “Enzymes of the Name Biosynthesis both of Colorado Springs, Colo. Pathway: Recent Addresses in Molecular Genetics”, Semi nars in Hematology, vol. 25, No. 4, pp. 303-311, Oct., 1988. 73 Assignee: University Technology Corporation, Mattoon, et al., “Coordinate Regulation of Mitochondrial Boulder, Colo. Cytochrome Formation in Saccharomyces cerevisiae' Genetics of Respriratory Enzymes in Yeasts (Materials of the 21 Appl. No.: 509,951 International Conference Karpacz in 1990), pp. 15-22. 22 Filed: Aug. 1, 1995 Mattoon, “Molecular Genetics of Heme and Cytochrome Biosynthesis”, Anal. Acad. Nac. Cs. Ex. Fis. Nat., Buenos (51) Int. Cl. ................................................ C12P 21/06 Aires, tomo. 40, 1988, pp. 87-94. 52 U.S. Cl. ........................ 435/69.6; 435/70.1; 435/69.1 Meussdorfer et al., Effect of Mitochondrial Cytochromes 58 Field of Search .................................. 435/69.1, 69.6, and Home Content on Cytochrome P450 in Saccharomyces 435/70.1, 71.1, 71.2 cerevisiae, Journal of General Microbiology vol. 132, pp. 2187-2193, 1986. 56) References Cited Myers et al., “Characterization of the Yeast HEM2 Gene and Transcriptional Regulation of COX5 and COR1 by Heme”, U.S. PATENT DOCUMENTS The Journal of Biological Chemistry, vol. 262, No. 35, pp. 4,849,348 7/1989 Marcker et al. ....................... 435/69.1 16822-16828, December Issue, 1987. 4,902,620 2/1990 Bard et al. ... ... 435/172.3 Oechsner et al., Expression of Yeast Cytochrome c' is 5,049,493 9/1991 Khosla et al. ... 435/69.1 Controlled at the Transcriptional Level by Glucose, Oxygen 5,242,892 9/1993 Rebeiz .......... ... 504/129 and Haem, Mol. Gen. Genet., 192, pp. 447-459. 5,635,375 6/1997 Kraus et al. ........................... 435/71.1 Ogden et al., Expression and ASSembly of Functional FOREIGN PATENT DOCUMENTS Human Hemoglobin in S. Cerevisiae, Biomat., Art, Cells & WO 90/13645 11/1990 WIPO. Immob. Biotech, 20 (2-4) pp. 473–475 (1992). WO 92/16634 10/1992 WIPO. Schauer et al., Heterologous Expression of Human 5-Ami nolevulinate Dehydratase in Saccharomyces cerevisiae, OTHER PUBLICATIONS Current Genetics, vol. 17, pp. 106, 190. Adachi et al., Oxygen Binding and Other Physical Properties Wagenbach et al., Synthesis of Wild Type and Mutant Hman of Human Hemoglobin Made in Yeast, Protein Engineering, Hemoglobins in Saccharomyces Cerevisiae, Biotechnology, vol. 5, No. 8, pp. 807-810, 1992. vol. 9, pp. 57-61, Jan., 1991. Borralho et al., In Situ Assay for 5-Aminolevulinate Dehy dratase and Application to the Study of a Carbolite Repres Primary Examiner Terry A. McKelvey Sion-Resistant Sacchromyces cerevisiae Mutant Journal of Attorney, Agent, or Firm-Sheridan Ross P.C. Bacteriology, Oct., 1983, pp. 141-147, vol. 156, No. 1. 57 ABSTRACT Borralho et al., “Parallel Changes in Catabolite Repression of Haem Biosynthesis and Cytochromes in Repression-re A method for increasing the production of desired hemo sistant Mutants of Saccharomyces cerevisiae', Journal of proteins is described in which the rate-limiting Step in a Gen. Microbiology, vol. 13, pp, 1217–1227, 1989. heme biosynthetic pathway is deregulated to increase the Borralho et al. “Purification of 8-Aminolevulinate Dehy quantity of the rate-limiting enzyme. In yeast, transforming dratase from Genetically Engineered Yeast', Yeast, vol. 6: cells with the HEM2 gene increases the quantity of the 319-330 (1990). rate-limiting enzyme ALA dehydratase. Free heme concen Carvajal, “Isolation and Characterization of a New Mutant trations are reduced in cells by providing heme binding of Saccharomyces cerevisiae with Altered Synthesis of agents that complexes with Overproduced heme, thereby 5-Aminolevulinic Acid”, Journal of Bacteriology, Jun., reducing or eliminating heme feedback inhibition in the cell. 1990, pp. 2855-2861, vol. 172, No. 6. The present invention permits investigation of other factors Coghlin et al. “Structural and Functional Characterisation of effecting the heme biosynthetic pathway by reducing inter Recombinant Human Haemoglobin AExpressed in Saccha ference from heme feedback inhibition within that pathway. romyces cerevisiac,' European Journal of Biochemistry, vol. 207, pp. 931–993, 1992. 16 Claims, 7 Drawing Sheets U.S. Patent Oct. 20, 1998 Sheet 1 of 7 5,824,511 Glycine synthaseA. NUCLEUSHAP1.2, ...3,...4, 5-ALA lycine synthase 5-ALA + -> 5-ALA ALA dehydrates succinvgly ((HEM1 ) (HEM2) Porphobilinogen PBG deaminase (HEM3) MITOCHODRON Uroporphyrinogen HEMEA synthetase APOPROTEINS -1/ Uroporphyrinogen(HEM4) Ill l Protoheme Uroporphyrinogen HEMPROTEINS: FerroSage decarboxylase (Catalase T, ( ) (HEM6) Catalase A, Protoporphyrin IX Coproporphyrinogen Peroxidase, O2 Oxidase Cytochr. p450, Protoporphyrinogen IX (HEM13) Cytochromes, Oxidase Happroteins) (HEM14) Protoporphyrinogen IX Fig. 1 U.S. Patent Oct. 20, 1998 Sheet 2 of 7 5,824,511 Catalase T COR Sac Scal PSr.Nhel del fKpn s ac mo uy Heme CTT1 Mitochodr. binding targeting ligand sequence ActiveSite Fig. 2 U.S. Patent Oct. 20, 1998 Sheet 3 of 7 5,824,511 I6L I69T T69T T6TT– I60T T68– I6L– T69-- I6G– I68- TSN IP2X HILIP8 TáIVIH U.S. Patent Oct. 20, 1998 Sheet 4 of 7 5,824,511 EcoRI, 1 Bc,304 11176,Aatl S SS 10736, Scal Xbal.703 SnaBI, 1042 Nsil, 1396 Nrul,2090 Stu2160 Spe228 | Clal,2266EcoR2241 EcoRV,2685 8558, BSaBl pHEM2 A A NCO,2704 Scal,2809 11250 bps URA3 NApal,2873 Stu,2933 Tc(s) TC'(S Nsil,3322 7862, Nirul ( (S), Smal,3378 7541Sall/1 EcoRV,3594 7452, Sphl promoter HEM2 Nhel,3638 7265, BamHI C/al,4832 5927, Paci Xbal,4901 5856. Af/ BamHI,5082 Kpnl,5090 Nsil,5375 Nhel,5506 Fig. 4 U.S. Patent Oct. 20, 1998 Sheet 5 of 7 5,824,511 HEME ASA COORDINATE REULATOR PRECURSORS AMNO ACDS ( HEME APOCYTOCHROMES CYTOCHROMES Fig. 5 U.S. Patent Oct. 20, 1998 Sheet 6 of 7 5,824,511 Fig. 6A ALADEHYDRATESE EXPRESSION 16 14 12 10 :8 No plasmid Vector only pCTT1 pHEM2 & pCtt1 / -5-ALA N +5-ALA CATALASET EXPRESSION 2 5 O 2 O O l 5 O O No plasmid Vector only pCTT1 pHEM2 & pCtt1 /1 -5-ALA N +5-ALA 2%. Raffinose medium, reg1 -strain Fig. 6B U.S. Patent Oct. 20, 1998 Sheet 7 of 7 5,824,511 Z‘61-I | u - - S. s YZZZZZZZZZ \/TV/VNUG’O+---- LOdR]SWEHd+||L. €LWEHd+||1Od7) G?WEHd+||LOdN| | ///////// Vzd?A+L_1Lod[] ZWEHd+||LOd |09 OZ (ueOudelo fuued Sun) eSeeleC) 5,824,511 1 2 METHOD FOR ENHANCING THE to achieve desired production levels of hemoproteins. PRODUCTION OF HEMOPROTEINS Moreover, it would be desirable to have a method in which the heme biosynthetic pathway can be regulated in Such a FIELD OF THE INVENTION manner that other critical factors in Such pathway can be The present invention is directed to a method to improve further Studied and regulated in order to achieve greater the production of cytoplasmic hemoproteins and is more production of hemoproteins. particularly directed to a method to increase the production SUMMARY OF THE INVENTION of homologous and heterologous hemoproteins in yeast cells One embodiment of the present invention is directed to a by increasing the production of the HEM2 gene product in method to produce hemoproteins by transforming a host cell the heme biosynthetic pathway. with a nucleic acid Sequence encoding a rate-limiting BACKGROUND OF THE INVENTION enzyme in a heme biosynthetic pathway. The host cell is cultured under conditions effective to provide expression of An important class of homologous and heterologous the Sequence to produce desired concentrations of heme. A proteins produced in genetically engineered cells is repre 15 Sufficient amount of a heme binding agent capable of com Sented by proteins carrying a heme prosthetic group. A plexing free heme produced by the cell is Supplied to reduce number of heme proteins are of great importance from a the concentration of free heme within the cell. The coordi pharmaceutical point of view. Many commercial Ventures nation of gene expression of a rate-limiting enzyme, coupled are working to enhance the biosynthesis of the heme protein with the expression of a gene encoding a heme-binding family and efforts are continuing in the development of new agent, prevents undesired feedback inhibition that would Strains of improved biochemical and cell-physiological char otherwise be caused by exceSS concentrations of free heme acteristics in yeast, as well as in bacteria and fungi. in the cell. AS described in greater detail herein, Simulta There is considerable current interest in the potential of neous production of heme and heme binding agents, (e.g., cell-free hemoglobin as a Substitute for red cells in oxygen apoproteins) allows one to study various aspects of heme delivery, Such interest increased due to concern over viral 25 regulation, which can then be manipulated to produce fur contamination of donated blood. Recombinant DNA tech ther improvements in hemoprotein production Systems. niques now make it possible to produce recombinant hemo In yeast, the rate-limiting enzyme is encoded in the HEM2 globin in genetically engineered microorganisms, thus insur gene and thus, one embodiment of the present invention ing a Source of hemoglobin which is free of human blood involves the transformation of a yeast cell with the HEM2 derived infectious agents, such as hepatitis B and HIV. gene to overproduce ALA dehydratase. Preferred yeast Methods using E. coli to produce human hemoglobin have strains include Pichia, Hansenula and strains of S. cerevi been developed, however, production of hemoglobin in this Siae. Other host cells, Such as Filamentous fungus, Specifi way has proved to be inefficient.
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