Yeast microorganisms with reduced by-product accumulation for improved production of fuels, chemicals, and amino acids
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 .- 221 . (canceled)
222 . A recombinant microorganism comprising a biosynthetic pathway which uses a 3-keto acid as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of said 3-keto acid to a 3-hydroxyacid by-product.
223 . The recombinant microorganism of claim 222 , wherein said 3-keto acid intermediate is acetolactate and said 3-hydroxyacid by-product is 2,3-dihydroxy-2-methylbutanoic acid (DH2MB).
224 . The recombinant microorganism of claim 223 , wherein said recombinant microorganism produces an acetolactate-derived product.
225 . The recombinant microorganism of claim 224 , wherein said acetolactate-derived product is selected from isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A.
226 . The recombinant microorganism of claim 222 , wherein said 3-keto acid intermediate is 2-aceto-2-hydroxybutyrate and said 3-hydroxyacid by-product is 2-ethyl-2,3-dihydroxybutanoate.
227 . The recombinant microorganism of claim 226 , wherein said recombinant microorganism produces a 2-aceto-2-hydroxybutyrate-derived product.
228 . The recombinant microorganism of claim 227 , wherein said 2-aceto-2-hydroxybutyrate-derived product is selected from 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
229 . The recombinant microorganism of claim 222 , wherein said enzyme catalyzing the conversion of a 3-keto acid to a 3-hydroxyacid by-product is a 3-keto acid reductase.
230 . The recombinant microorganism of claim 229 , wherein said 3-keto acid reductase is the S. cerevisiae YMR226 (SEQ ID NO: 1) or a homolog or variant thereof.
231 . The recombinant microorganism of claim 229 , wherein said 3-keto acid reductase is selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, or homologs or variants thereof.
232 . A recombinant microorganism comprising a biosynthetic pathway which uses an aldehyde as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of said aldehyde to an acid by-product.
233 . The recombinant microorganism of claim 232 , wherein said biosynthetic pathway which uses an aldehyde as intermediate is selected from a pathway for the biosynthesis of isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
234 . The recombinant microorganism of claim 232 , wherein said enzyme catalyzing the conversion of an aldehyde to an acid by-product is an aldehyde dehydrogenase.
235 . The recombinant microorganism of claim 234 , wherein said aldehyde dehydrogenase is the S. cerevisiae ALD6 (SEQ ID NO: 25) or a homolog or variant thereof.
236 . The recombinant microorganism of claim 234 , wherein said aldehyde dehydrogenase is selected from SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, or homologs or variants thereof.
237 . A recombinant microorganism comprising a biosynthetic pathway which uses a 3-keto acid and an aldehyde as an intermediate, wherein said recombinant microorganism is:
(i) engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of said 3-keto acid to a 3-hydroxyacid by-product; and
(ii) engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of said aldehyde to an acid by-product.
238 . The recombinant microorganism of claim 237 , wherein said enzyme catalyzing the conversion of a 3-keto acid to a 3-hydroxyacid by-product is a 3-keto acid reductase.
239 . The recombinant microorganism of claim 238 , wherein said 3-keto acid reductase is the S. cerevisiae YMR226 (SEQ ID NO: 1) or a homolog or variant thereof.
240 . The recombinant microorganism of claim 238 , wherein said 3-keto acid reductase is selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, or homologs or variants thereof.
241 . The recombinant microorganism of claim 237 , wherein said enzyme catalyzing the conversion of an aldehyde to an acid by-product is an aldehyde dehydrogenase.