MODIFIED ALCOHOL DEHYDROGENASES FOR THE PRODUCTION OF FUELS AND CHEMICALS
The present invention relates to recombinant microorganisms comprising biosynthetic pathways and methods of using said recombinant microorganisms to produce various beneficial metabolites. In various aspects of the invention, the recombinant microorganisms may further comprise one or more modifications resulting in the reduction or elimination of 3 keto-acid (e.g., acetolactate and 2-aceto-2-hydroxybutyrate) and/or aldehyde-derived by-products. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.
1 . A modified alcohol dehydrogenase (ADH) comprising one or more modifications at positions corresponding to amino acids selected from the group consisting of: (a) tyrosine 50 of the wild-type L. lactis AdhA (SEQ ID NO: 2); (b) glutamine 77 of the wild-type L. lactis AdhA; (c) valine 108 of the wild-type L. lactis AdhA; (d) asparagine 110 of the wild-type L. lactis AdhA; (e) tyrosine 113 of the wild-type L. lactis AdhA; (f) isoleucine 212 of the wild-type L. lactis AdhA; and (g) leucine 264 of the wild-type L. lactis AdhA.
2 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein said tyrosine 50 residue is replaced with a phenylalanine or tryptophan residue.
3 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein said glutamine 77 residue is replaced with an arginine or serine residue.
4 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein said valine 108 residue is replaced with a serine or alanine residue.
5 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein said asparagine 110 residue is replaced with a serine or cysteine residue.
6 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein said tyrosine 113 residue is replaced with a phenylalanine or glycine residue.
7 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein said isoleucine 212 residue is replaced with a threonine, alanine, or valine residue.
8 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein said leucine 264 residue is replaced with a valine residue.
9 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein the catalytic efficiency for the conversion of isobutyraldehyde to isobutanol of the modified ADH is enhanced by at least about 50% as compared to the wild-type ADH.
10 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein the catalytic efficiency for the conversion of isobutyraldehyde to isobutanol of the modified ADH is enhanced by at least about 100% as compared to the wild-type ADH.
11 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein the catalytic efficiency for the conversion of isobutyraldehyde to isobutanol of the modified ADH is enhanced by at least about 200% as compared to the wild-type ADH.
12 . The modified alcohol dehydrogenase (ADH) of claim 1 , wherein the ADH is derived from a genus selected from Lactococcus, Lactobacillus, Pediococcus, Bacillus, Leptotrichia, Actinobacillus, Streptococcus, Streptobacillus, Staphylococcus, Eikenella, Weissella, Kingella, Rothia , and Exiguobacterium.
13 . The modified alcohol dehydrogenase (ADH) of claim 1 wherein the ADH is codon optimized for expression in a host cell.
14 . The modified alcohol dehydrogenase (ADH) of claim 13 , wherein said host cell is yeast.
15 . The modified alcohol dehydrogenase (ADH) of claim 13 , wherein said host cell is E. coli.
16 . A recombinant microorganism transformed with a nucleic acid construct encoding a modified alcohol dehydrogenase (ADH) according to claim 1 .
17 . The recombinant microorganism of claim 16 , wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway comprising one or more isobutanol metabolic pathway enzymes selected from acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxy acid dehydratase (DHAD), and 2-keto-acid decarboxylase (KIVD).
18 . The recombinant microorganism of claim 16 , wherein said recombinant microorganism is a yeast microorganism.
19 . A method of producing isobutanol, comprising:
(a) providing a recombinant microorganism according to claim 17 ; and
(b) cultivating said recombinant microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of the isobutanol is produced.
20 . A method of producing isobutanol, comprising:
(a) providing a recombinant microorganism according to claim 18 ; and
(b) cultivating said recombinant microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of the isobutanol is produced.