MBTH-LIKE PROTEINS IN EUKARYOTIC NRPS-CATALYZED PROCESSES
The present invention relates to a method to improve the production of a secondary metabolite catalyzed by a non-ribosomal peptide synthetase comprising contacting in a eukaryotic host a eukaryotic non-ribosomal peptide synthetase with an MbtH-like protein. The present invention further relates to a composition comprising a eukaryotic non-ribosomal peptide synthetase that is not a hybrid and a prokaryotic MbtH and to a eukaryotic host cell comprising a non-ribosomal peptide synthetase and a polynucleotide allowing the expression of an MbtH-like protein.
1 . A method to improve the production of a secondary metabolite or a precursor occurring in the pathway leading to said secondary metabolite catalyzed by a non-ribosomal peptide synthetase comprising contacting in a eukaryotic host said non-ribosomal peptide synthetase with an MbtH-like protein, characterized in that said non-ribosomal peptide synthetase is from eukaryotic origin and is not a hybrid.
2 . The method according to claim 1 wherein said eukaryotic host is a fungus.
3 . The method according to claim 1 wherein said secondary metabolite is a β-lactam, a pigment or a mycotoxin.
4 . The method according to claim 3 wherein said β-lactam is 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid, adipyl 7-aminodesacetoxycephalosporanic acid, cephalosporin C, penicillin G or penicillin V, wherein said pigment is a chrysogenin or wherein said mycotoxin is a roquefortin.
5 . The method according to claim 2 wherein said fungus is Penicillium chrysogenum.
6 . The method according to claim 5 wherein said eukaryotic host is a multi copy strain.
7 . The method according to claim 1 wherein said MbtH-like protein has SEQ ID NO: 1-7 or a sequence that is at least 70% homologous to SEQ ID NO: 1-7.
8 . The method according to claim 1 wherein said MbtH-like protein comprises the amino acid code of SEQ ID NO: 17.
9 . The method according to claim 8 wherein said MbtH-like protein comprises the amino acid code of any one of SEQ ID NO: 18 to SEQ ID NO: 53 or SEQ ID NO: 57 to SEQ ID NO: 92.
10 . A composition comprising a eukaryotic non-ribosomal peptide synthetase that is not a hybrid and a prokaryotic MbtH.
11 . The composition according to claim 10 which is
SEQ ID NO: 54 with SEQ ID NO: 1; or
SEQ ID NO: 54 with SEQ ID NO: 3; or
SEQ ID NO: 54 with SEQ ID NO: 4; or
SEQ ID NO: 54 with SEQ ID NO: 6; or
SEQ ID NO: 54 with SEQ ID NO: 7; or
SEQ ID NO: 55 with SEQ ID NO: 1; or
SEQ ID NO: 55 with SEQ ID NO: 3; or
SEQ ID NO: 55 with SEQ ID NO: 4; or
SEQ ID NO: 55 with SEQ ID NO: 6; or
SEQ ID NO: 55 with SEQ ID NO: 7; or
SEQ ID NO: 56 with SEQ ID NO: 1; or
SEQ ID NO: 56 with SEQ ID NO: 3; or
SEQ ID NO: 56 with SEQ ID NO: 4; or
SEQ ID NO: 56 with SEQ ID NO: 6; or
SEQ ID NO: 56 with SEQ ID NO: 7; or
sequences that are at last 90% homologous thereto.
12 . A eukaryotic host cell comprising a non-ribosomal peptide synthetase that is not a hybrid and a polynucleotide allowing the expression of an MbtH-like protein, characterized in that said non-ribosomal peptide synthetase is from eukaryotic origin.
13 . The eukaryotic host cell according to claim 12 wherein said MbtH-like protein has SEQ ID NO: 1-7 or a sequence that is at least 70% homologous to SEQ ID NO: 1-7.
14 . The eukaryotic host cell according to claim 12 wherein said MbtH-like protein comprises the amino acid code of SEQ ID NO: 17.
15 . The eukaryotic host cell according to claim 12 which is Penicillium chrysogenum.