Biotica Technology Limited

Other

Pfizer Corporation

James Staunton

Peter Francis Leadlay

Jesus Cortes

Hamish Alastair Irvine McArthur

Michael Stephen Pacey

Lake Ee Khaw

Kim Jonelle Stutzman-Engwall

Johannes Laurenz Kellenberger

Sabine Gaisser

Hamish Alastair Irvine Mcarthur

A hybrid type I polyketide synthase gene typically containing a starter module and a plurality of heterologous extender modules is used to synthesise novel polyketides. It is preferably under the control of type II polypolyketide synthase promoter e.g. act I or

Method for producing hybrid polyketide synthases

The present invention relates to hybrid glycosylated products, and in particular, to natural products such as polyketides and glycopeptides, and to processes for their preparation. The invention is particularly concerned with recombinant cells in which a cloned microbial glycosyltransferase can be conveniently screened for its ability to generate specific glycosylated derivatives when supplied with polyketide, peptide, or polyketide-peptides as substrates. The invention demonstrates that cloned glycosyltransferases when rapidly screened for their ability to attach a range of activated sugars to a range of exogenously supplied or endogenously generated aglycone templates, show a surprising flexibility towards both aglycone and sugar substrates, and that this process allows the production of glycosylated polyketides in good yield. This overcomes the problem not only of supplying novel sugar attachments to individual polyketides, including polyketides altered by genetic engineering, but also of increasing the diversity of polyketide libraries by combinatorial attachment of sugars.

Hybrid glycosylated products and their production and use

The rapamycin gene cluster is an example of a gene cluster which includes a gene (rapL) leading to the formation of a precursor compound (pipecolic acid, in this case) which is required for inclusion in the larger product (rapamycin) produced by the enzymes encoded by the cluster. We have produced a mutant strain containing a rapamycin gene cluster in which the rapL gene is disabled. The strain does not naturally produce rapamycin but does so if fed with pipecolic acid. By feeding with alternative carboxylic acids we have produced variants of rapamycins. Tests have shown biological activity.

Polyketides and their synthesis and use

A polyketide synthase ('PKS') of Type I is a complex multienzyme including a loading domain linked to a multiplicity of extension domains. The first extension module receives an acyl starter unit from the loading domain and each extension module adds a further ketide unit which may undergo processing (e.g. reduction). We have found that the Ksq domain possessed by some PKS's has decarboxylating activity, e.g. generating (substituted) acyl from (substituted) malonyl. The CLF domain of type II PKS's has similar activity. By inserting loading modules including such domains into PKS's not normally possessing them it is possible to control the starter units used.

Polyketides and their synthesis

Hybrid polyketide synthases and novel loading domains

Nucleic acid molecules encoding at least part of a Type I polyketide synthase, and having a polylinker with multiple restriction enzyme sites in place of one or more PKS genes encoding enzymes associated with reduction, optionally further including nucleic acid incorporated into the polylinker, the further nucleic acid encoding one or more reductive enzymes; plasmids incorporating such nucleic acids; host cells transfected with such plasmids; methods relating thereto.

Nucleic acid molecules encoding modified polyketide synthases containing polylinkers and uses thereof

A hybrid type I polyketide synthase gene typically containing a starter module and a plurality of heterologous extender modules is used to synthesize novel polyketides. It is preferably under the control of a type II polypolyketide synthase promoter e.g. act I of

Hybrid polyketide synthases combining heterologous loading and extender modules

Erythromycins, particularly ones with novel C-13 substituents R1 (e.g. C -C cycloalkyl or cycloalkenyl groups) are prepared by fermenting suitable organisms in the presence of R CO H. A preferred organism is preferably containing an integrated plasmid capable of directing synthesis of desired compounds.

Erythromycins and process for their preparation

Erythromycins, particularly with C-13 substituents R1 (e.g. C,-C,cycloalkyl or cycloalkenyl groups) are prepared by fermenting suitable organisms in the presence of R,CO,H. A preferred organism is,preferably containing an integrated plasmid capable of directing synthesis of desired compounds.

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Cambridge, United Kingdom

James Staunton