Dsm IP Assets B.v.

Viktor Marius Boer

Marco Alexander Van Den Berg

Johannes Andries Roubos

Cornelis Maria Jacobus Sagt

Priscilla Zwartjens

Johannes Gustaaf Ernst Van Leeuwen

Liang Wu

René Marcel De Jong

Jos Van Vugt

Erwin Suir

Bernard Meijrink

Igor Galaev

Robertus Antonius Mijndert Van Der Hoeven

Eric Van Den Berg

Catharina Petronella Antonia Maria Kolen

Nicolette Jasmijn Broers

Nathalie Wiessenhaan

Yvonne Johannes Odilia Arendsen

Adam G Lawrence

Johannes Gustaaf Emst Van Leeuwen

Martin Lehmann

Van Der Robertus Antonius Mijndert Hoeven

Manoj Kumar

Joshua Truehart

A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least about 50% sequence identity thereto. A recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least about 50% sequence identity thereto.

Steviol glycoside transport

The disclosure provides a recombinant cell capable of producing a steviol glycoside, wherein the cell comprises a nucleic acid coding for a variant of a parent polypeptide, wherein the variant has steviol glycoside transport mediating activity, wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence of the parent polypeptide, comprises at least one modification of the amino acid residue corresponding to any of the amino acids in the amino acid sequence of the parent polypeptide, wherein the variant has an improved ability to produce rebaudioside M and optionally other steviol glycosides extracellularly if compared with the parent polypeptide when measured under the same conditions.

The present invention relates a polypeptide having kaurenoic acid 13-hydroxylase activity, which polypeptide comprises an amino acid sequence which, when aligned with a kaurenoic acid 13-hydroxylase comprising the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 3, comprises at least one substitution of an amino acid corresponding to any of amino acids at positions 136, 248, 336 or 403, said positions being defined with reference to SEQ ID NO: 1 or SEQ ID NO: 3 and wherein the polypeptide has one or more modified properties as compared with a reference polypeptide having kaurenoic acid 13-hydroxylase activity. A polypeptide of the invention may be used in a recombinant host for the production of steviol or a steviol glycoside.

Kaurenoic acid hydroxylases

The present invention relates a variant polypeptide having geranylgeranyl pyrophosphate synthase activity, which variant polypeptide comprises an amino acid sequence which, when aligned with a geranylgeranyl pyrophosphate synthase comprising the sequence set out in SEQ ID NO: 1, comprises at least one substitution of an amino acid residue corresponding to any of amino acids at positions 92, 100 or 235 said positions being defined with reference to SEQ ID NO: 1 and wherein the variant has one or more modified properties as compared with a reference polypeptide having geranylgeranyl pyrophosphate synthase activity. A variant polypeptide of the invention may be used in a recombinant host for the production of steviol or a steviol glycoside.

Geranylgeranyl pyrophosphate synthases

The present invention relates to a recombinant microorganism comprising one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity, whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol.

Microorganisms for diterpene production

The present disclosure relates a polypeptide having UGT activity, which polypeptide comprises an amino acid sequence which, when aligned with a polypeptide having UGT activity comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid corresponding to any of amino acids at positions

UDP-glycosyltransferases

The present invention relates to a recombinant host comprising a recombinant nucleic acid sequence encoding a polypeptide having at least about: a. 85% identity to the amino acid sequence set forth in SEQ ID NO: 1; b. 85% identity to the amino acid sequence set forth in SEQ ID NO: 3; c. 85% identity to the amino acid sequence set forth in SEQ ID NO: 6; d. 85% identity to the amino acid sequence set forth in SEQ ID NO: 9; e. 85% identity to the amino acid sequence set forth in SEQ ID NO: 11; f. 85% identity to the amino acid sequence set forth in SEQ ID NO: 14; g. 85% identity to the amino acid sequence set forth in SEQ ID NO: 17; h. 85% identity to the amino acid sequence set forth in SEQ ID NO: 20; i. 85% identity to the amino acid sequence set forth in SEQ ID NO: 22; or j. 85% identity to the amino acid sequence set forth in SEQ ID NO: 25.

A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto. A recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

The present invention relates a variant polypeptide having kaurenoic acid 13-hydroxylase activity, which variant polypeptide comprises an amino acid sequence which, when aligned with a kaurenoic acid 13-hydroxylase comprising the sequence set out in SEQ ID NO: 1, comprises at least one substitution of an amino acid residue corresponding to any of amino acids 72, 85, 108, 127, 129, 141, 172, 195, 196, 197, 199, 226, 236, 291, 302, 361 or 464, said positions being defined with reference to SEQ ID NO: 1 and wherein the variant has one or more modified properties as compared with a reference polypeptide having kaurenoic acid 13-hydroxylase activity. A variant polypeptide of the invention may be used in a recombinant host for the production of steviol or a steviol glycoside.

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Echt, Netherlands

Viktor Marius Boer