Method for identifying and using biosynthetic genes for enhanced production of secondary metabolites
AssigneeGist-Brocades N.V.
InventorsMartien A. M. Groenen, Annemarie E. Veenstra, Pieter van Solingen, Bertus P. Koekman
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Abstract
Novel methods and compositions are provided for the enhanced production of secondary metabolites. Methods are provided for identifying sequences which when transformed into secondary metabolite producing hosts, enhance the production of secondary metabolite. The process is exemplified for penicillin. In addition, the P. chrysogenum acyltransferase gene is isolated and sequenced.
Claims22
A method for enhancing the production of a secondary metabolite in a bacterium or fungus host which produces said secondary metabolite, said method comprising:
- screening a DNA library prepared from a first host producing said secondary metabolite with probes obtained from mRNA or DNA derived therefrom from a second host of the same species lacking the production of said secondary metabolite;
- screening a genomic library of said first host with sequences which do not hybridize to said probes to identify fragments comprising genes transcribed in said first host which are not transcribed in said second host;
- preparing DNA constructs comprising said transcribed fragments and a marker for selection;
- transforming a candidate host capable of production of said secondary metabolite with said constructs and cloning the resulting transformants;
- and
- identifying clones producing said secondary metabolite at a higher level than said candidate host.
A vector comprising (a) a gene encoding a protein involved in production of a .beta.-lactam antibiotic selected from the group consisting of an acyltransferase gene, cryptic gene Y and a cryptic gene present on any of clones L12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16, and B23, (b) a marker for selection in a host producing said .beta.-lactam antibiotic and optionally (c) a sequence for enhancing transformation efficiency of said vector in said host.
A transformed host capable of increased expression of a .beta.-lactam antibiotic selected from the group consisting of Penicillium, Aspergillus, Acremonium, and Actinomycetes, comprising as a result of transformation an extra copy of a sequence comprising a gene selected from the group consisting of an acyltransferase gene, cryptic gene Y, and a cryptic gene present on any of clones L12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16, and B23, and encoding a protein involved in production of a .beta.-lactam antibiotic.
A transformed Penicillium host capable of increased expression of penicillin comprising as a result of transformation an extra copy of a sequence comprising a gene selected from the group consisting of an acyltransferase gene, cryptic gene Y, and a cryptic gene present in any of clones K12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16, and B23, and encoding a protein involved in production of a penicillin.
A method for providing improved yields of a .beta.-lactam antibiotic comprising:
- growing a transformed host comprising an extra copy of a sequence comprising a gene selected from the group consisting of an acyltransferase gene, cryptic gene Y, and a cryptic gene present on any of clones L12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16, and B23, encoding a protein involved in production of said .beta.-lactam antibiotic resulting in enhanced production of said .beta.-lactam antibiotic.
A gene encoding a protein involved in production of a secondary metabolite, said gene prepared according to a method comprising (1) screening a DNA library prepared from a first host producing said secondary metabolite with probes obtained from mRNA or DNA derived therefrom from a second host of the same species lacking the production of said secondary metabolite;
- and (2) screening a genomic library of said first host with sequences which do not hybridize to said probes to identify fragments comprising genes transcribed in said first host which are not transcribed in said second host.
A DNA construct comprising (a) a gene encoding a protein involved in the production of a secondary metabolite and (b) a marker for selection in a host producing said secondary metabolite, said gene prepared according to a method comprising (1) screening a DNA library prepared from a fist host producing said secondary metabolite with probes obtained from mRNA or DNA therefrom from a second host of the same species lacking production of said secondary metabolite;
- and (2) screening a genomic library of said first host with sequences which do not hybridize to said probes to identify fragments comprising genes transcribed in said first host which are not transcribed in said second host.
Dependencies are inferred from the claim text.