Genencor International, Inc.

Genentech, Inc.

Other

Genencor, Inc.

Virgil B Lawlis

Kirk James Hayenga

Randy M Berka

Gregory Lawrence Gray

Daniel Cullen

Michael C Ward

Henry G Heinsohn

Enrique F Baliu

Bradley Richard Snedecor

William S Hancock

John E Battersby

Todd Becker

Kirk J Kayenga

This invention relates to novel mutant filamentous fungi which are deficient in the gene for the corresponding aspartic proteinase. These organisms are useful production hosts in the production of heterologous polypeptides such as chymosin.

Aspartic proteinase deficient filamentous fungi

Novel vectors are disclosed for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence. Preferably functional polyadenylation sequences are operably linked to the 3&prime; terminus of the DNA sequence encoding the heterologous polypeptides. Each of the above described vectors are used in novel processes to transform a filamentous fungus wherein the DNA sequences encoding the signal sequence and heterologous polypeptide are expressed. The thus synthesized polypeptide is thereafter secreted from the filamentous fungus.

Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same

Heterologous polypeptides expressed in filamentous fungi, process for making same, and vectors for making same

The invention includes novel fusion DNA sequences encoding fusion polypeptides which when expressed in a filamentous fungus result in the expression of fusion polypeptides which when secreted result in increased levels of secretion of the desired polypeptide as compared to the expression and secretion of such polypeptides from filamentous fungi transformed with previously used DNA sequences. The fusion DNA sequences comprise from the 5' terminus four DNA sequences which encode a fusion polypeptide comprising, from the amino to carbonyl-terminus, first, second, third and fourth amino acid sequences. The first DNA sequence encodes a signal peptide functional as a secretory sequence in a first filamentous fungus. The second DNA sequence encodes a secreted polypeptide or portion thereof which is normally secreted from the same filamentous fungus or a second filamentous fungus. The third DNA sequence encodes a cleavable linker polypeptide while the fourth DNA sequence encodes a desired polypeptide. When the fusion DNA sequence is expressed either in the first or second filamentous fungus, increased secretion of the desired polypeptide is obtained as compared to that which is obtained when the desired polypeptide is expressed from DNA sequences encoding a fusion polypeptide which does not contain the second polypeptide normally secreted from either of the filamentous fungi.

DNA sequences, vectors, and fusion polypeptides to increase secretion of

Novel vectors are disclosed for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence. Preferably functional polyadenylation sequences are operably linked to the 3' terminus of the DNA sequence encoding the heterologous polypeptides. Each of the above described vectors are used in novel processes to transform a filamentous fungus wherein the DNA sequences encoding the signal sequence and heterologous polypeptide are expressed. The thus synthesized polypeptide is thereafter secreted from the filamentous fungus.

Heterologous polypeptides expressed in filamentous fungi, processes for

This invention provides a method for killing cells in fermentation mixtures in order to prepare the fermentation mixture for processing to recover or extract a desired product from the fermentation mixture. A preferred method of this invention comprises in either order, adjusting the pH of the fermentation mixture to a value equal to or less than about two pH units below the pK.sub.a of the compatible organic acid using a mineral acid, and adding a sufficient amount of a compatible organic acid and/or organic acid salt to the mixture to effect a substantially complete cell kill. The method of this invention is useful for killing microorganisms such as yeast, bacteria or fungi in any culture or fermentation mixture and is particularly useful in systems where it is desired to kill the cells without lysing them.

Method for killing cells without lysis and enzyme recovery

The invention includes novel fusion DNA sequences encoding fusion polypeptides which when expressed in a filamentous fungus result in increased levels of secretion of the desired polypeptide as compared to the expression and secretion of such polypeptides from filamentous fungi transformed with previously used DNA sequences. The fusion DNA sequences comprise from the 5' terminus four DNA sequences which encode a fusion polypeptide comprising, from the amino to carbonyl-terminus, first, second, third and fourth amino acid sequences. The first DNA sequence encodes a signal peptide functional as a secretory sequence in a first filamentous fungus. The second DNA sequence encodes a secreted polypeptide or portion thereof which is normally secreted from the same filamentous fungus or a second filamentous fungus. The third DNA sequence encodes a cleavable linker polypeptide while the fourth DNA sequence encodes a desired polypeptide. When the fusion DNA sequence is expressed either in the first or second filamentous fungus, increased secretion of the desired polypeptide is obtained as compared to that which is obtained when the desired polypeptide is expressed from DNA sequences encoding a fusion polypeptide which does not contain the second polypeptide normally secreted from either of the filamentous fungi.

DNA sequences, vectors and fusion polypeptides to increase secretion of

This invention provides a method for killing fungal cells without lysing in fermentation processes in order to prepare the fermentation mixture for processing to recover or extract an extracellularly expressed enzyme from the fermentation mixture. A preferred method of this invention comprises adjusting the pH of the fermentation mixture to less than 2.79 using a mineral acid, then adding sufficient acetic acid to the mixture to affect a substantially complete cell kill in mixture. A salt of the acetic acid can be used. The organic acid or salt can be added, then the pH adjusted to the desired level. Other organic acids can be used, in which case the pH of the mixture is adjusted to the pK.sub.a of the selected organic acid before the organic acid is added to the mixture. The method of this invention is useful for stopping the growth and killing the cells in any micro-organism, culture or fermentation such as those containing yeast, bacteria or fungi and is particularly useful in systems where it is desired to kill the cells without lysing them.

Killing cells without lysis in a method for enzyme recovery from a

Heterologous polypeptides expressed in aspergillus

Container systems for stable storage of pharmaceutical compounds are disclosed. The container systems prevent the formation of adducts between pharmaceutical compounds and aldehydes during storage of these compounds in containers sealed with elastomeric stoppers that leach aldehydes. Methods for determining the presence of these adducts are also disclosed.

Process for preparing storage stable pharmaceuticals

Crystalline subtilisin is produced by adding a halide salt, such as sodium chloride or calcium chloride, to a concentrated subtilisin solution (at least about 40 g/1). This process does not produce amorphous subtilisin even at high salt concentrations in the solution. Optionally, subtilisin seed crystals also may be added to the concentrate to speed up the crystallization process.

Subtilisin crystallization process

Cheese is prepared using rennin obtained from insoluble refractile bodies of a recombinant microbial host cell. The rennin is obtained by rupturing the recombinant hose cell, isolating and solubilizing the insoluble refractile bodies, and recovering active rennin. Recombinant techniques involve preparing cDNA corresponding to the coding sequence for calf rennin, introducing into an expression vector and expressing in a host cell. As much as 200 mg rennin per liter of culture may be recovered. Prorennin or preprorennin may be produced and rennin derived therefrom.

Growing community of inventors

San Mateo, CA, United States of America

Virgil B Lawlis