Unilever Patent Holdings B.v.

Internationale Octrooi Maatschappij 'Octropa' Bv

Christiaan Visser

Cornelis T Verrips

Adrianus M Ledeboer

Jan Maat

Cornelis P Hollenberg

Leon Gerardus Frenken

Zbigniew A Janowicz

Jannetje Wilhelmina Bos

The production of an active Pseudomonas glumae lipase isolated from P. glumae PG1 (CBS 322.89) and other lipases suitable for application in detergent systems is described in homologous, but particularly in heterologous hosts, e.g. Bacillus subtilis. For the latter a 'helper function' or 'lipase-specific stabilization/translocation protein' is needed, for which a gene is provided which, when expressed in concert with the lipase gene, can improve the stabilization of the intermediates involved in the production and translocation/secretion of the lipase. The hosts are transformed by recombinant DNA methods and modified lipases can be made by site-directed mutation or classical mutation techniques. The lipase gene and the gene encoding the helper function can be part of one operon that can be transcribed to form a polycistronic messenger or be present as separate genes yielding two mRNA's on transcription. The production level can be further improved by optimising the regulation sequences. It can be advantageous to use at least part of the signal sequence of the lipase gene in addition to a signal sequence homologous to the host in which the lipase is produced.

Production of active Pseudomonas glumae lipase in homologous or

The structural genes and their regulatory DNA sequences of an alcohol oxidase (MOX) and a dihydroxyacetone synthase (DHAS) of Hansenula polymorpha have been isolated and the nucleotide sequences determined. The invention relates to the use of the MOX gene, as well as the use of the regulatory DNA sequences of MOX and/or DAS in combination with the MOX gene, optionally after modification thereof, or other oxidase genes, or other genes, to produce engineered microorganisms, 0in particular yeasts. Said engineered microorganisms can produce oxidases or other enzymes in yields that allow industrial application on a large scale. Moreover, said engineered microorganisms can produce oxidases having improved properties with respect to their application in oxidation reactions and/or in bleaching and detergent products.

Expression and production of polypeptides using the promoters of the

The production of an active Pseudomonas glumae lipase isolated from P. glumae PG1 (CBS 322.89) and other lipases suitable for application in detergent systems is described in homologous, but particularly in heterologous hosts, e.g. Bacillus subtilis. For the latter a 'helper function' or 'lipase-specific stabilization/translocation protein' is needed, for which a gene is provided which, when expressed in concert with the lipase gene, can improve the stabilization of the intermediates involved in the production and translocation/secretion of the lipase. The hosts are transformed by recombinant DNA methods and modified lipases can be made by site-directed mutation or classical mutation techniques. The lipase gene and the gene encoding the helper function can be part of one operon that can be transcribed to form a polycistronic messenger or be present as separate genes yielding two mRNA's on transcription. The production level can be further improved by optimizing the regulation sequences. It can be advantageous to use at least part of the signal sequence of the lipase gene in addition to a signal sequence homologous to the host in which the lipase is produced.

The structural genes and their regulatory DNA sequences of an alcohol oxidase (MOX) and a dihydroxyacetone synthase (DHAS) of Hansenula polymorpha have been isolated and the nucleotide sequences determined. The invention relates to the use of the MOX gene, as well as the use of the regulatory DNA sequences of MOX and/or DAS in combination with the MOX gene, optionally after modification thereof, or other oxidase genes, or other genes, to produce engineered microorganisms, in particular yeasts. Said engineered microorganisms can produce oxidases or other enzymes in yields that allow industrial application on a large scale. Moreover, said engineered microorganisms can produce oxidases having improved properties with respect to their application in oxidation reactions and/or in bleaching and detergent products.

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Capelle a/d IJssel, Netherlands

Christiaan Visser