Method of selecting a polypeptide of interest
AssigneeImperial College Innovations Limited
InventorsLara Vidal, John Heap
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Abstract
The invention relates to methods for identifying polypeptides and polynucleotides of interest, be they novel or variant polypeptides and polynucleotides, by expressing a plurality of polypeptides in an obligate or facultative anaerobe that is incapable of, or displays a reduction in, the oxidation of NADH and/or NADPH under anaerobic fermentation conditions and selecting an obligate or facultative anaerobe that grows or displays a growth advantage under said conditions. The invention is also concerned with novel enzymes per se, and their use in enzymatic production processes.
Claims21
A method of identifying a variant polypeptide of interest, or its encoding polynucleotide, the method comprising:
- (i) generating a plurality of variant polypeptides;
- (ii) expressing the plurality of variant polypeptides in an obligate or facultative anaerobe that is incapable of, or displays a reduction in, the oxidation of NADH and/or NADPH under anaerobic fermentation conditions;
- (iii) culturing, in growth media, the obligate or facultative anaerobe under anaerobic fermentation conditions in the presence of a substrate, wherein the polypeptide of interest enables the obligate or facultative anaerobe to oxidise, or to increase oxidation of, NADH and/or NADPH in the presence of the substrate;
- (iv) selecting an obligate or facultative anaerobe that grows or displays a growth advantage in the growth media;
- and
- (v) identifying the variant polypeptide of interest expressed, or its encoding polynucleotide, in the obligate or facultative anaerobe of step (iv).
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A kit for identifying a variant polypeptide of interest, or its encoding polynucleotide, the kit comprising:
- i. an obligate or facultative anaerobe that is rendered incapable of, or displays a reduction in, the oxidation of NADH and/or NADPH;
- and
- ii. growth media comprising a substrate;
- wherein a variant polypeptide of interest will enable the obligate or facultative anaerobe to oxidise, or to increase oxidation of, NADH and/or NADPH in the presence of the substrate when grown under anaerobic fermentation conditions, the kit optionally further comprising providing a library of variant polypeptides.
A variant of:
- Clostridium beijerinckii alcohol dehydrogenase, which comprises a modification of one or more amino acids relative to the wild-type sequence of SEQ ID NO:
- 1, wherein the variant has altered cofactor specificity compared to its corresponding wild-type, such that it utilises NADH instead of NADPH;
- (ii) Myxococcus stipitatus imine reductase, which comprises a modification of one or more amino acids relative to the wild-type sequence of SEQ ID NO:
- 34, wherein the variant has altered cofactor specificity compared to its corresponding wild-type, such that it utilises NADH instead of NADPH, optionally wherein the variant of Myxococcus stipitatus imine reductase comprises an amino acid sequence substantially as set out in SEQ ID NO:
- 35, or a fragment or variant thereof or
- (iii) Enterobacter cloacae nitroreductase, which comprises a modification of one or more amino acids relative to the wild-type sequence of SEQ ID No:
- 37, wherein the variant has altered substrate specificity, such that it is able to catalyse the reduction of 2-nitrobenzoic acid (2-NBA) and/or 4-nitrobenzyl alcohol for efficiently that the wild-type nitroreductase, optionally wherein the variant of Enterobacter cloacae nitroreductase comprises an amino acid sequence substantially as set out in SEQ ID No 38 or 40, or a fragment or variant thereof.
21-31. (canceled)
Dependencies are inferred from the claim text.