公开(公告)号：US20140030730A1

公开(公告)日：2014-01-30

申请号：US14045683

申请日：2013-10-03

Applicant: DSM IP ASSETS B.V.

Inventor： Ulrike Maria MUELLER ,  Liang WU ,  Lourina Madeleine RAAMSDONK ,  Aaron Adriaan WINKLER

IPC: C12P7/16 ,  C12Q1/32

CPC classification number: C12Q1/32 ,  C07K14/33 ,  C12N9/001 ,  C12P7/16 ,  Y02E50/10

Abstract: The present invention relates to a method of identifying a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in (the cytosol of) a yeast cell comprising: a) providing a mutated yeast cell comprising a deletion of at least one gene of the (PDH) by-pass, selected from the genes encoding the enzymes pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD), and acetyl-CoA synthetase (ACS); b) transforming said mutated yeast cell with an expression vector comprising a heterologous nucleotide sequence encoding a candidate polypeptide having potential enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA; c) testing said recombinant mutated yeast cell for its ability to grow on minimal medium containing glucose as sole carbon source, and d) identifying said candidate polypeptide as a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in (the cytosol of) said yeast cell when growth of said cell is observed. The invention further relates to a method of producing a fermentation production such as butanol.

Abstract translation: 本发明涉及一种鉴定具有用于将丙酮酸，乙醛或乙酸酯转化成酵母细胞的（细胞溶质）中的乙酰辅酶A的酶活性的异源多肽的方法，包括：a）提供包含至少一个缺失的突变的酵母细胞 选自编码丙酮酸脱羧酶（PDC），乙醛脱氢酶（ALD）和乙酰辅酶A合成酶（ACS）的基因的（PDH）旁路的一个基因; b）用包含异源核苷酸序列的表达载体转化所述突变的酵母细胞，所述异源核苷酸序列编码具有将丙酮酸，乙醛或乙酸转化为乙酰辅酶A的潜在酶活性的候选多肽; c）测试所述重组突变酵母细胞在含有葡萄糖作为唯一碳源的基本培养基上生长的能力，以及d）将所述候选多肽鉴定为异源多肽，其具有将丙酮酸，乙醛或乙酸酯转化成乙酰辅酶A 当所述细胞的生长被观察时，所述酵母细胞的胞质溶胶。 本发明还涉及生产发酵生产如丁醇的方法。

公开(公告)号：US20210054433A1

公开(公告)日：2021-02-25

申请号：US17081187

申请日：2020-10-27

Applicant: DSM IP ASSETS B.V.

Inventor： Ulrike Maria MUELLER ,  Liang Wu ,  Lourina Madeleine Raamsdonk ,  Aaron Winkler

IPC: C12Q1/32 ,  C07K14/33 ,  C12N9/02 ,  C12P7/16

Abstract: The present invention relates to a method of identifying a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in (the cytosol of) of a yeast cell comprising: a) providing a mutated yeast cell comprising a deletion of at least one gene of the (PDH) by-pass, selected from the genes encoding the enzymes pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD), and acetyl-CoA synthetase (ACS); b) transforming said mutated yeast cell with an expression vector comprising a heterologous nucleotide sequence encoding a candidate polypeptide having potential enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA; c) testing said recombinant mutated yeast cell for its ability to grown on minimal medium containing glucose as sole carbon source, and d) identifying said candidate polypeptide as a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in (the cytosol of) said yeast cell when growth of said cell is observed. The invention further relates to a method of producing a fermentation production such as butanol.