Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
2000-07-18
2001-07-17
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S254300, C435S183000, C530S385000
Reexamination Certificate
active
06261827
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods of producing hemoproteins in filamentous fungi and to filamentous fungal cells capable of producing hemoproteins.
2. Description of the Related Art
Heme, a chelate complex of protoporphyrin IX and iron, serves as a prosthetic group of hemoproteins. Protoporphyrin IX consists of a porphyrin ring, substituted with four methyl groups, two vinyl groups, and two propionic acid groups, which acquires an iron atom to form heme. The biosynthesis of heme from glycine and succinyl-CoA involves eight enzymatic steps which are catalyzed by 5-aminolevulinic acid synthase (EC 2.3.1.37), porphobilinogen synthase (EC 4.2.1.24), porphobilinogen deaminase (EC 4.3.1.8), uroporphyrinogen III synthase (EC 4.2.11.75), uroporphyrinogen III decarboxylase (EC 4.1.1.37), coproporphyrinogen III oxidase (EC 1.3.3.3), protoporphyrinogen IX oxidase (EC 1.3.3.4), and ferrochelatase (EC 4.99.1.1). 5-Aminolevulinic acid synthase catalyzes the condensation of glycine and succinyl-CoA to form 5-aminolevulinic acid. Porphobilinogen synthase (also called 5-aminolevulinic acid dehydratase or 5-aminolevulinic acid dehydrase) catalyzes the condensation of two molecules of 5-aminolevulinic acid to form porphobilinogen. Porphobilinogen deaminase (also called hydroxymethylbilane synthase or uro I synthase) catalyzes the tetrapolymerization of pyrole porphobilinogen into preuroporphyrinogen. Uroporphyrinogen III synthase (also called uro III synthase or uro III cosynthase) catalyzes a rearrangement of the fourth ring of preuroporphyrinogen followed by cyclization to produce uroporphyrinogen III. Uroporphyrinogen III decarboxylase (also called uro D or uroporphyrinogen decarboxylase) catalyzes the decarboxylation of all four acetic acid side chains of uroporphyrinogen III to methyl groups to yield coproporphyrinogen III. Coproporphyrinogen III oxidase (also called coproporphyrinogenase) catalyzes the oxidative decarboxylation of two propionate groups at positions 2 and 4 on the A and B rings of coproporphyrinogen III to vinyl groups yielding protoporphyrinogen IX. Protoporphyrinogen IX oxidase catalyzes a six electron oxidation of protoporphyrinogen IX to yield protoporphyrin IX. Ferrochelatase (also called ferrolyase, heme synthase, or protoheme ferrolyase) catalyzes the insertion of iron into the protoporphyrin to yield heme.
The conversion of an apoprotein into a hemoprotein depends on the availability of heme provided by the heme biosynthetic pathway. The apoprotein form of the hemoprotein combines with heme to produce the active hemoprotein which acquires a conformation which makes the hemoprotein more stable against proteolytic attack than the apoprotein. If the amount of heme produced by a microorganism is less relative to the amount of the apoprotein produced, the apoprotein will accumulate and undergo proteolytic degradation lowering the yield of the active hemoprotein.
In order to overcome this problem, Jensen showed that the addition of heme or a heme-containing material to a fermentation medium led to a significant increase in the yield of a peroxidase produced by
Aspergillus oryzae
(WO 93/19195). While heme supplementation of a fermentation medium results in a significant improvement in the yield of a hemoprotein, it is non-kosher, costly, and difficult to implement on a large scale.
Wu et al. (1991,
Journal of Bacteriology
173:325-333) disclose a method for overexpression of an
E. coli
NADPH-sulfite reductase, a sirohemoprotein, comprising introducing a
Salmonella typhimurium
cysG gene, which encodes a uroporphyrinogen III methyltransferase required for the synthesis of siroheme, in a plasmid.
It is an object of the present invention to provide improved methods for increasing production of hemoproteins in filamentous fungal strains to yield commercially significant quantities.
SUMMARY OF THE INVENTION
The present invention relates to methods of producing a hemoprotein, comprising:
(a) introducing into a filamentous fungal cell,
(i) one or more first control sequences capable of directing the expression of a heme biosynthetic enzyme encoded by a first nucleic acid sequence endogenous to the filamentous fungal cell, wherein the one or more of the first control sequences are operably linked to the first nucleic acid sequence; and/or
(ii) one or more copies of one or more second nucleic acid sequences encoding a heme biosynthetic enzyme;
(b) cultivating the filamentous fungal cell in a nutrient medium suitable for production of the hemoprotein and the heme biosynthetic enzymes; and
(c) recovering the hemoprotein from the nutrient medium of the filamentous fungal cell.
The present invention also relates to recombinant filamentous fungal cells comprising one or more first control sequences capable of directing the expression of a heme biosynthetic enzyme encoded by a first nucleic acid sequence endogenous to the filamentous fungal cell and/or one or more copies of one or more second nucleic acid sequences encoding heme biosynthetic enzymes.
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patent: WO 89/03883 (1989-05-01), None
patent: WO 93/25697 (1993-12-01), None
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Cherry Joel R.
Elrod Susan L.
Jones Aubrey
Carlson Karen Cochrane
Novozymes Biotech Inc.
Starnes Robert L.
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