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US 2024/0093255 A1Published application
Biosynthetic production of 2-fucosyllactose
AssigneeConagen Inc.
InventorsGuohong Mao, Meaghan Valliere, Johnson Wu, Oliver Yu, Sean Robert Johnson
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Abstract
The present invention provides a novel biosynthetic production process which converts L-galactose into 2′-fucosyllactose via four enzymatically catalyzed reaction steps. The present process is designed such that co-factors required by the process are regenerated within the four reaction steps, hence making the process cost-effective and efficient. The process can be performed in vitro in a cell-free system. The present invention also provides mutant enzymes that can be used to increase production levels of 2′-fucosyllactose, whether using the novel pathway described herein or the mannose-dependent pathway known in the art.
Claims21
A method for producing 2′-fucosyllactose comprising:
- (i) incubating GDP-L-fucose with an α-1,2-fucosyltransferase in a culture medium comprising lactose for a sufficient time to convert said GDP-L-fucose and lactose into 2′-fucosyllactose and GDP;
- wherein said α-1,2-fucosyltransferase is selected from the group consisting of a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:
- 109, SEQ ID NO:
- 29, SEQ ID NO:
- 107, SEQ ID NO:
- 89, SEQ ID NO:
- 91, SEQ ID NO:
- 93, SEQ ID NO:
- 95, SEQ ID NO:
- 99, SEQ ID NO:
- 101, SEQ ID NO:
- 103, or SEQ ID NO:
- 105;
- or
- (ii) incubating GDP-mannose and/or GDP-L-galactose with a dehydratase and a reductase in the presence of NADPH and/or NADP+ in a culture medium for a sufficient time to convert said GDP-mannose and/or GDP-L-galactose into GDP-L-fucose;
- and incubating said GDP-L-fucose with an α-1,2-fucosyltransferase and lactose for a sufficient time to convert said GDP-L-fucose and lactose into 2′-fucosyllactose and GDP:
- wherein said dehydratase is selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:
- 79, SEQ ID NO:
- 77, SEQ ID NO:
- 75, or SEQ ID NO:
- 5;
- and a polypeptide comprising the amino acid sequence of SEQ ID NO:
- 85, SEQ ID NO:
- 83, SEQ ID NO:
- 81, or SEQ ID NO:
- 9;
- or
- (iii) incubating GDP-L-galactose with a dehydratase and a reductase in the presence of NADPH and/or NADP+ for a sufficient time to convert said GDP-L-galactose into GDP-L-fucose;
- and incubating said GDP-L-fucose with lactose and an α-1,2-fucosyltransferase for a sufficient time to convert said GDP-L-fucose and lactose into 2′-fucosyllactose and GDP;
- or
- (iv) incubating GDP-L-fucose with lactose and an α-1,2-fucosyltransferase for a sufficient time to convert said GDP-L-fucose and lactose into 2′-fucosyllactose, wherein the α-1,2-fucosyltransferase is an enzyme comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:
- 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61;
- or
- (v) providing a reaction mixture comprising a fucokinase/guanylyltransferase, a dehydratase, a reductase, an α-1,2-fucosyltransferase, ATP, GTP, NADP+, and NADPH;
- and adding L-galactose to the reaction mixture;
- and incubating said reaction mixture for a sufficient time to produce 2′-fucosyllactose;
- wherein the reaction mixture further comprises:
- (a) a first regenerating enzyme and a first substrate for said first regenerating enzyme, wherein said first regenerating enzyme is capable of catalyzing a first regeneration reaction involving the first substrate that uses NADP+ as a co-factor, thereby regenerating NADPH:
- (b) a second regenerating enzyme and a second substrate for said second regenerating enzyme, wherein said second regenerating enzyme is capable of catalyzing a second regeneration reaction involving the second substrate that uses ADP as a co-factor, thereby regenerating ATP;
- and (c) a third regenerating enzyme and a third substrate for said third regenerating enzyme, wherein said third regenerating enzyme is capable of catalyzing a third regeneration reaction involving the third substrate that uses GDP as a co-factor, thereby regenerating GTP.
37 more elements
(canceled)
An engineered microorganism for enhanced production of 2′-fucosyllactose, said microorganism comprising at least the following heterologous genes for producing 2′-fucosyllactose:
- (i) a first heterologous gene that encodes a mutant dehydratase for producing GDP-L-fucose, said mutant dehydratase being a polypeptide comprising the amino sequence selected from SEQ ID NO:
- 79, SEQ ID NO:
- 77, SEQ ID NO:
- 75, SEQ ID NO:
- 85, SEQ ID NO:
- 83, and SEQ ID NO:
- 81;
- and
- (ii) a second heterologous gene that encodes a mutant α-1,2-fucosyltransferase for converting GDP-L-fucose to 2′-fucosyllactose, said mutant α-1,2-fucosyltransferase being a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:
- 109.
7 more elements
A polypeptide comprising:
- a. mutant dehydratase for producing GDP-L-fucose, said mutant dehydratase being a polypeptide comprising the amino sequence selected from the group consisting of SEQ ID NO:
- 79, SEQ ID NO:
- 77, SEQ ID NO:
- 75, SEQ ID NO:
- 85, SEQ ID NO:
- 83, and SEQ ID NO:
- 81;
- or
- b. a mutant α-1,2-fucosyltransferase for producing 2′-fucosyllactose, said mutant α-1,2-fucosyltransferase being a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:
- 109, SEQ ID NO:
- 29, SEQ ID NO:
- 107, SEQ ID NO:
- 89, SEQ ID NO:
- 91, SEQ ID NO:
- 93, SEQ ID NO:
- 95, SEQ ID NO:
- 99, SEQ ID NO:
- 101, SEQ ID NO:
- 103, or SEQ ID NO:
- 105.
17 more elements
(canceled)
-58. (canceled)
Dependencies are inferred from the claim text.