MODIFIED PHOTOSYNTHETIC MICROORGANISMS FOR CONTINUOUS PRODUCTION OF CARBON-CONTAINING COMPOUNDS
The present invention relates to a continuous production system for producing carbon-containing compounds, comprising a genetically modified photosynthetic microorganism, such as a Cyanobacterium , that contains one or more mutations or deletions in a glycogen biosynthesis or storage pathway. The system and methods provided herein facilitate the production of carbon-containing compounds under reduced growth conditions, including biofuels, lipids, and other specialty chemicals, such as fatty acids, alkanes, alkenes, wax esters, and triglycerides.
1 .- 63 . (canceled)
64 . A system for producing a carbon-containing compound, comprising:
a modified Cyanobacterium that accumulates a reduced amount of glycogen as compared to a corresponding wild-type Cyanobacterium ; and
a culture system for culturing the modified Cyanobacterium under a stress condition,
wherein the modified Cyanobacterium maintains photosynthetic activity and accumulates reduced biomass when grown under the stress condition as compared to when grown under a non-stress condition.
65 . The system of claim 64 , wherein the modified Cyanobacterium secretes and/or intracellularly accumulates an increased amount of a carbon-containing compound when grown under the stress condition as compared to when grown under non-stress conditions, or as compared to the corresponding wild-type Cyanobacterium grown under the stress condition.
66 . The system of claim 64 , wherein the modified Cyanobacterium has reduced expression of one or more genes of a glycogen biosynthesis or storage pathway as compared to the corresponding wild-type photosynthetic microorganism, and wherein the one or more genes are selected from the group consisting of: a glucose-1-phosphate adenyltransferase (glgC) gene, a phosphoglucomutase (pgm) gene, and a glycogen synthase (glgA) gene.
67 . The system of claim 64 , wherein the stress condition is a reduced level of an essential nutrient, and wherein the essential 5 nutrient is selected from at least one of nitrogen, sulfur, and phosphorous.
68 . The system of claim 64 , wherein the modified Cyanobacterium further: comprises one or more introduced polynucleotides encoding a protein that increases glycogen breakdown; and/or comprises one or more introduced polynucleotides encoding a protein that increases secretion of a glycogen precursor.
69 . The system of claim 64 , wherein the Cyanobacterium is a Synechococcus elongatus.
70 . The system of claim 64 , wherein the carbon-containing compound is a lipid.
71 . The system of claim 64 , wherein the carbon-containing compound is one or more of 2-oxoglutarate, pyruvate, malate, fumarate, succinate, 4-hydroxybutyrate, 1,4 butanediol, glutaconic acid, 3-methyl-2-oxobutyrate, 3methyl-2-oxovalerate, 4-methyl-2-oxopentanoate, isobutaraldehyde, isobutanol, 2methyl-1-butanol, 3-methyl-2-butanol, isopentanol, glucose, glutathione, 3phosphoglycerate, cis-aconitate, agmatine, putrescine, or glycyerin.
72 . The system of claim 64 , wherein photosynthetic activity of the modified Cyanobacterium under the stress condition is substantially greater than photosynthetic activity of the corresponding wild-type Cyanobacterium under the stress condition.
73 . The system of claim 64 , wherein photosynthetic activity of the modified Cyanobacterium under the stress condition:
is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20-fold greater than photosynthetic activity of the corresponding wild-type Cyanobacterium under the stress condition; or
is measured at about day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 post-initiation of the stress condition.
74 . A method for producing a carbon-containing compound other than glycogen, comprising culturing in a culture media under a stress condition a modified Cyanobacterium that accumulates a reduced amount of glycogen as compared to a corresponding wild-type Cyanobacterium , wherein the modified Cyanobacterium maintains photosynthetic activity and accumulates reduced biomass when grown under the stress condition as compared to when grown under non-stress conditions.
75 . The method of claim 74 , wherein the modified Cyanobacterium secretes and/or intracellularly accumulates an increased amount of a carbon-containing compound when grown under the stress condition as compared to when grown under non-stress conditions, or as compared to a corresponding wild-type Cyanobacterium grown under the stress condition.
76 . The method of claim 74 , wherein the modified Cyanobacterium has reduced expression of one or more genes of a glycogen biosynthesis or storage pathway as compared to the corresponding wild-type Cyanobacterium , and wherein the one or more genes are selected from the group consisting of: a glucose-1-phosphate adenyltransferase (glgC) gene, a phosphoglucomutase (pgm) gene, and a glycogen synthase (glgA) gene.
77 . The system of claim 74 , wherein the stress condition is a reduced level of an essential nutrient, and wherein the essential 5 nutrient is selected from at least one of nitrogen, sulfur, and phosphorous.
78 . The method of claim 74 , wherein the modified Cyanobacterium further: comprises one or more introduced polynucleotides encoding a protein that increases glycogen breakdown; and/or comprises one or more introduced polynucleotides encoding a protein that increases secretion of a glycogen precursor.
79 . The method of claim 74 , wherein Cyanobacterium is a Synechococcus elongatus.
80 . The method of claim 74 , wherein the carbon-containing compound is a lipid.
81 . The method of claim 74 , wherein the carbon-containing compound is one or more of 2-oxoglutarate, pyruvate, malate, fumarate, succinate, 4-hydroxybutyrate, 1,4 butanediol, glutaconic acid, 3-methyl-2-oxobutyrate, 3-methyl-2-oxovalerate, 4-methyl-2-oxopentanoate, isobutaraldehyde, isobutanol, 2-methyl-1-butanol, 3-methyl-2-butanol, isopentanol, glucose, glutathione, 3-phosphoglycerate, cis-aconitate, agmatine, putrescine, or glycyerin.
82 . The method of claim 74 , wherein photosynthetic activity of the modified Cyanobacterium under the stress condition is substantially greater than photosynthetic activity of the corresponding wild-type Cyanobacterium under the stress condition.
83 . The method of claim 74 , wherein the maintained photosynthetic activity comprises maintenance of chlorophyll A levels, wherein the chlorophyll A levels:
are at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20-fold greater than chlorophyll A levels of the 10 corresponding wild-type Cyanobacterium under the stress condition.
are measured at about day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 post-initiation of the stress condition.