Title of Technology:  Extracellular production of heme protein in fungi

Abstract: This technology provides a method for the extracellular production of heme proteins in filamentous fungi in yields which far exceed those obtainable for the same protein in yeast. Accordingly, the the method comprises a)  transforming a suitable filamentous fungus with a recombinant DNA vector which comprises a DNA sequence encoding a heterologous heme protein, and a DNA sequence encoding a preregion permitting secretion of the expressed heme protein, and (b) culturing the transformed filamentous fungus in a suitable culture medium under conditions conducive to the production of the heme protein.

Furthermore, this technology can increase the total yield of such heme protein considerably. This is possible by adding hemin or another material containing heme groups to a fermentation medium for growing microorganisms which overproduce the apoprotein of a heme protein, the heme group is bound to the protein whereby the apoprotein is activated and acquires a conformation in which it is more stable against proteolytic degradation. In this way, endogenous heme synthesis in the host organism, which is often a bottle-neck in the expression of heme proteins, may be overcome.

 

Detailed Description:  In this context, the term "heme protein" is intended to include any member of a group of proteins containing heme (e.g. protoporphyrin IX) as a prosthetic group. The term "apoprotein" indicates a form of the heme protein lacking the prosthetic group. The term "extracellular heme protein" is understood to indicate that unlike the heme proteins provided in the prior art by production in bacteria or yeast, the apoprotein form of the heme protein is secr eted from the host cell into the culture medium where it recombines (to the holoprotein) with the prosthetic heme group provided by addition of heme or heme-containing material to the medium.

The cloning and expression of varius heme proteins in bacteria has previously been described. The enzyme is expressed intracellularly as an insoluble aggregate so that it has to be purified from lysed cells. Furthermore, the enzyme is not expressed in active form and must be folded separately in the presence of heme and Ca2+ to become functional.  Expression of human hemoglobins in yeast has also been described. In yeast, hemoglobin is expressed as a fully assembled, heme-containing tetramer. However, the protein is not secreted from the yeast cells, but remains in the cytoplasmic space and must be purified therefrom.

However, the technology presented here, and covered by the patents below, provides a method whereby it is possible not only to produce heme proteins but also of exporting them through the cell membrane in active form, thereby simplifying purification procedures. Further, these basic method allows for the production of heme proteins in filamentous fungi in yields which far exceed those obtainable for the same protein in yeast.

A specific process represented by one  granted US patent (US5958724) consists of the following: 

A process for the extracellular production of a heterologous heme pro-tein in a strain of Aspergillus sp., the process comprising:(a) transforming a suitable  strain of an Aspergillus sp. with a recom-binant DNA vector which comprises a DNA sequence encoding a het-erologous heme protein, and a DNA sequence encoding a preregion permitting secretion of the expressed heme protein, and(b) culturing the transformed  strain of Aspergillus sp. in a suitable culture medium under conditions conducive to the production of the heme protein.

The heme protein produced by the process of the present invention could e.g. be an enzyme, e.g. an oxidoreductase such as a peroxidase,  lignin peroxidase, Mn-peroxidase, or haloperoxidase.

Another important aspect of this technology is obtain significantly increased yields of protein by adding hemin or another material containing heme groups to a fermentation medium for growing microorganisms which overproduce the apoprotein of a heme protein. Hereby the heme group is bound to the protein whereby the apoprotein is activated and acquires a conformation in which it is more stable against proteolytic degradation. The total yield of heme protein is significantly increased. In this way, endogenous heme synthesis in the host organism, which is often a bottle-neck in the expression of heme proteins, may be overcome. Thus method comprises culturing a heme apoprotein producing microorganism in a fermentation medium containing heme or a heme-containing material under conditions permitting the production of active, recombined heme protein, and recovering the resulting heme protein from the medium. The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host organism in question. The heme or heme-containing material added to the medium to obtain recombination of the secreted apoprotein with the heme group may suitably be supplied by the addition of hemin or, preferably, hemoglobin or red blood cells as the heme group remains functional on heating, permitting autoclaving of media containing one of these substances.

Glossary of Terms

Term:	Definition:
Heme protein	"heme protein" is intended to include any member of a group of proteins containing heme (e.g. protoporphyrin IX) as a prosthetic group
Apoprotein	"apoprotein" is intended to indicate a form of the heme protein lacking the prosthetic group.
Extracellular protein	"extracellular heme protein" is understood to indicate that unlike the heme proteins provided in the prior art by production in bacteria or yeast, the apoprotein form of the heme protein is secreted from the host cell into the culture medium where it recombines (to the holoprotein) with the prosthetic heme group provided by addition of heme or heme-containing material to the medium.
heterologous	The term "heterologous" is meant to indicate proteins which are not, in nature, produced by the host organism in question.
filamentous fungus	The term "filamentous fungus" is intended to include fungi belonging to the groups Phycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes or fungi imperfecti, icluding Hyphomycetes such as the genera Aspergillus, Trichoderma, Penicillium, Fusarium or Humicola.

Patent References
Patent Number:   

AT505311, BE505311, CH505311, DE505311, DK505311, ES505311, FR505311, GB505311, GR505311,IT505311,NL505311,SE505311,US5744323, US5958724, AT180837, DE69325169.7, DK631631, ES631631, FR631631, GB631631, IT631631,NL631631, US5681725,