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AssigneeGenomatica, Inc.
InventorsStefan Andrae, Michael Patrick Kuchinskas, Jingyi Li, Harish Nagarajan, Priti Pharkya
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Described herein are non-natural NAD+-dependent alcohol dehydrogenases (ADHs) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its unmodified counterpart. Nucleic acids encoding the non-natural alcohol dehydrogenases, as well as expression constructs including the nucleic acids, and engineered cells comprising the nucleic acids or expression constructs are described. Also described are engineered cells expressing a non-natural NAD+-dependent alcohol dehydrogenase, optionally include one or more additional metabolic pathway transgene(s), methanol metabolic pathway genes, target product pathway genes, cell culture compositions including the cells, methods for promoting production of the target product or intermediate thereof from the cells, compositions including the target product or intermediate, and products made from the target product or intermediate.
An engineered 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 at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to an engineered 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 at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to an engineered 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.
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A method for increasing the conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, comprising a step of (a) culturing an engineered cell expressing a NAD+-dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase in a culture medium comprising methanol or ethanol, where in said culturing the cell provides at least two fold greater conversion of the methanol or ethanol to formaldehyde or acetaldehyde respectively, as compared to an engineered cell expressing the corresponding alcohol dehydrogenase without amino acid substitution or comprising a step of (b) providing a reaction composition having a pH in the range of 6-9.5, or 7-9, or 8.5, the composition comprising a NAD+-dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase and methanol or ethanol, where in the composition said NAD+-dependent non-natural alcohol dehydrogenase provides at least two fold greater conversion of methanol or ethanol to a formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution.
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A NAD+-dependent non-natural alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase capable, when expressed in a cell, of at least two fold greater conversion of a methanol or ethanol to formaldehyde or acetaldehyde respectively, as compared to the corresponding alcohol dehydrogenase without amino acid substitution.
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Dependencies are inferred from the claim text.