Alcohol dehydrogenase variants

An engineered microbial cell either (a) expressing a non-natural NAD+-dependent alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase and capable of greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to an engineered microbial cell expressing the corresponding alcohol dehydrogenase without amino acid substitution or (b) expressing a first sequence that is a non-natural NAD+-dependent alcohol dehydrogenase comprising at least one amino acid substitution capable of greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to an engineered microbial cell expressing a second sequence that is an NAD+-dependent alcohol dehydrogenase, wherein the first and second sequences differ with regards to the at least one amino acid substitution,

• wherein the non-natural alcohol dehydrogenase has a sequence identity of 75% or greater to an NAD+-dependent alcohol dehydrogenase template selected from the group consisting of SEQ ID NO:
• 1 (MDH MGA3_17392), SEQ ID NO:
• 3, SEQ ID NO:
• 5, SEQ ID NO:
• 7, SEQ ID NO:
• 9, SEQ ID NO:
• 11, SEQ ID NO:
• 13, SEQ ID NO:
• 15, SEQ ID NO:
• 17, SEQ ID NO:
• 19, SEQ ID NO:
• 21, SEQ ID NO:
• 23, SEQ ID NO:
• 25, SEQ ID NO:
• 27, SEQ ID NO:
• 29, SEQ ID NO:
• 31, SEQ ID NO:
• 33, SEQ ID NO:
• 35, SEQ ID NO:
• 37, SEQ ID NO:
• 39, SEQ ID NO:
• 41, SEQ ID NO:
• 43, SEQ ID NO:
• 45, SEQ ID NO:
• 47, SEQ ID NO:
• 49, SEQ ID NO:
• 51, SEQ ID NO:
• 53, SEQ ID NO:
• 55, SEQ ID NO:
• 57, SEQ ID NO:
• 59, SEQ ID NO:
• 61, SEQ ID NO:
• 63, SEQ ID NO:
• 65, SEQ ID NO:
• 67, SEQ ID NO:
• 69, SEQ ID NO:
• 71, SEQ ID NO:
• 73, SEQ ID NO:
• 75, and SEQ ID NO:
• 77, or a fragment of said template having said dehydrogenase activity with an amino-terminal deletion, carboxy-terminal deletion, or both, the fragment having a sequence identity of 75% or greater, to the template, and
• wherein the non-natural alcohol dehydrogenase comprises one or more amino acid substitutions based on formula:
• R1XR2, wherein R1 is an original amino acid at position X of the template, and R2 is the variant amino acid that replaces R1 at a position on the template corresponding to X, wherein XR2 is selected from the group consisting of (a) 11T, 38N, 42Q, 48D, 53I, 56K, 60E, 61A, 63F, 65Q, 70N, 71I, 71T, 71V, 74S, 81G, 84R, 86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R, 115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 130F, 130I, 130Y, 134T, 143T, 145M, 146N, 147R, 148A, 148F, 148G, 148I, 148T, 148V, 148W, 149L, 149M, 149T, 149V, 150A, 150I, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G, 161Q, 161S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T, 186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y, 296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A, 344G, 345E, 350K, 354M, 355D, 355I, 355K, 358G, 360A, 360G, 360K, 360R, 360S, 361N, 361R, 363K, and 379M.