Production of 3-hydroxycarboxylic acid using nitrilase
View Patent ↗This invention relates to nitrilase mutants having improved nitrilase activity for converting 3-hydroxynitriles to 3-hydroxycarboxylic acids. More specifically, the Acidovorax facilis 72W (ATCC 55746) nitrilase gene was mutated using error-prone PCR and site-directed mutagenesis to create nitrilase enzymes having improved nitrilase activity for converting 3-hydroxynitriles (e.g., 3-hydroxybutyronitrile or 3-hydroxyvaleronitrile) to the corresponding 3-hydroxycarboxylic acids. A process using these improved mutants to produce the 3-hydroxycarboxylic acids is also provided.
1. An isolated nucleic acid molecule encoding an enzymatically-active polypeptide having the amino acid sequence of SEQ ID NO:4; said isolated nucleic acid molecule further comprising at least one mutation that results in at least one amino acid substitution of SEQ ID NQ:4, selected from the group consisting of:
a) a substitution at position 210 with alanine, isoleucine, or cysteine;
b) a substitution at position 65 with cysteine;
c) a substitution at position 168 with lysine, valine or leucine; and
d) a substitution at position 174 with isoleucine,
wherein said nucleic acid molecule encodes a polypeptide having nitrilase activity at least 1.8-fold higher relative to the activity of the A. facilis 72W (ATCC 55746) nitrilase when converting 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid under identical reaction conditions.
2. The isolated nucleic acid molecule of claim 1 , having a nucleic acid sequence seiected from the group consisting of SEQ ID NOs:5, 7, 11, 13, 15, and 17.
3. The isolated nucleic acid molecule of claim 1 , encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 12, 14, 16 and 18.
4. A chimeric gene comprising the isolated nucleic acid molecule of claims 1 , 2 , or 3 , operably linked to a suitable regulatory sequence.
5. An expression cassette comprising the chimeric gene of claim 4 .
6. A transformed microorganism comprising the chimeric gene of claim 4 .
7. A transformed microorganism comprising the expression cassette of claim 5 .
8. The transformed microorganism of claim 6 wherein the microorganism is selected from the group consisting of Comamonas sp., Corynebacterium sp., Brevibacterium sp., Rhodococcus sp., Azotobacter sp., Citrobacter sp., Enterobacter sp., Clostridium sp., Klebsiella sp., Salmonella sp., Lactobacillus sp., Aspergillus sp., Saccharomyces sp., Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp., Candida sp., Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp., Torulopsis sp., Methylobacteria sp., Bacillus sp., Escherichia sp., Pseudomonas sp., Rhizobium sp., and Streptomyces sp.
9. The transformed microorganism of claim 8 wherein the transformed microorganism is an Escherichia coli strain selected from the group consisting of MG1655 (ATCC 47076), FM5 (ATCC 53911), W3110 (ATCC 27325), MC4100 (ATCC 35695). and W1485 (ATCC 12435).