CONTROL OF GROWTH-INDUCTION-PRODUCTION PHASES
The present invention provides various combinations of genetic modifications to a transformed host cell that provide increase conversion of carbon to a chemical product. The present invention also provides methods of fermentation and methods of making various chemical products.
1 . A method of producing a chemical product from a renewable carbon source through a bioproduction process that comprises a controlled multi-phase production process.
2 . The method of claim 1 wherein the initiation or completion of one or more phases of the production process is controlled by genetic modifications to the organism producing the chemical product.
3 . The method of claim 1 wherein the initiation or completion of one or more phases of the production process is controlled by changes made to the cell environment.
4 . A bioproduction method of producing a chemical product from a renewable carbon source comprising utilizing an genetically modified organism to convert the carbon source to the chemical product, wherein said conversion is a controlled multi-phase production process wherein the initiation and/or completion of one or more phases of the production process is controlled by genetic modifications to the organism and/or is controlled by changes made to the organism's environment.
5 . The method of claim 1 or 4 wherein the bioproduction process includes two or more of the following phases: (1) growth phase; (2) induction phase; and (3) production phase.
6 . The method of claim 5 wherein the growth phase and induction phase are conducted at a temperature of about 25 to about 35, about 28 to about 32, or about 30° C.
7 . The method of claim 5 wherein the production phase is conducted at a temperature of about 35 to about 45, about 35 to about 40, or about 36 to about 38° C.
8 . The method of claim 5 wherein the production phase temperature is higher than the induction phase temperature
9 . The method of claim 5 wherein the increase in temperature between the production phase occurs over a period of about 1 to about 5 hours, about 1 to about 3 hours, about 2 hours, or about 1 hour.
10 . The method of claim 5 wherein during the growth phase, the organism replicates itself and biocatalyst needed to produce the chemical product is built up.
11 . The method of claim 5 wherein during the induction phase, expression of key enzymes critical to the production of the chemical are induced and the enzymes accumulate to carry out reactions required to produce the chemical product.
12 . The method of claim 5 wherein during the production phase the organism produces the chemical product.
13 . The method of claim 5 wherein the growth phase is dependent on the presence of a critical external reactant that will initiate growth, and the initiation and completion of the growth phase is controlled by the addition and amount of the initiating reactant added to a composition comprising the carbon source and the organism.
14 . The method of claim 13 wherein the chemical product is 3-HP, the organism is E. coli , and the critical external reactant is phosphate.
15 . The method of claim 14 wherein the growth phase is initiated by the addition of phosphate to the composition comprising the carbon source and the organism, and the duration of the growth phase is controlled by the amount of phosphate added.
16 . The method of claim 5 , wherein the induction phase is controlled by a genetic modification to the organism.
17 . The method of claim 16 wherein the genetic modification comprises encoding expression of a gene encoding an enzyme in a biosynthetic pathway for converting the carbon source to the chemical product, wherein the gene is encoded into the organism using promoters that are activated by phosphate depletion.
18 . The method of claim 17 wherein the chemical product is 3-HP and the organism is E. coli , and the gene is selected from the group consisting of mcr, mmsB, ydfG, rutE, nemA, NDSD, genes that encode individual or fused subunits of ACCase.
19 . The method of claim 17 wherein the chemical product is 3-HP and the organism is E. coli , and the promoter is selected from the group consisting of a promoter that directs expression of an E. coli gene selected from the group consisting of amn, tktB, xasA, yibD, ytfK, pstS, phoH, phnC, and other phosphate-regulated genes.
20 . The method of claim 17 wherein the chemical product is 3-HP and the organism is E. coli , and the gene is selected from the group consisting of mcr, mmsB, ydfG, rutE, nemA, NDSD, genes that encode individual or fused subunits of ACCase; and the promoter is selected from the group consisting of a promoter that directs expression of an E. coli gene selected from the group consisting of amn, tktB, xasA, yibD, ytfK, pstS, phoH, phnC, and other phosphate-regulated genes.
21 . The method of claim 5 , wherein the production phase is also controlled by genetic modifications to the organism.
22 . The method of claim 21 , where the genetic modification comprises encoding expression of a gene that is mutated to become activated or deactivated at a given temperature range.
23 . The method of claim 21 , where the genetic modification comprises encoding expression of a gene that is mutated such that as a result of a change in the organisms temperature the mutated gene: (1) becomes active and serves a key function in the conversion of the carbon source to the chemical product; or (2) becoming inactive and turns off a branch pathway or other competitive pathway that prevents or limits the pathway leading to the conversion of the carbon source to the chemical product.
24 . The method of claim 22 or 23 , wherein the chemical product is 3-HP, the organism is E. coli , and the mutated gene is selected from the group consisting of fabl, fabB, and fabD.
25 . A method of producing a chemical product from a renewable carbon source through a bioproduction process comprising:
(a) constructing a genetically modified organism capable of converting said renewable carbon source to said chemical product, wherein said genetically modified organism requires inorganic phosphate for growth and comprises: (a) at least one heterologous gene whose expression is regulated by a promoter sensitive to inorganic phosphate levels within a culture system, wherein said gene provides a critical function in converting said carbon source to said chemical product and is not required for the genetically modified organism to replicate; and (b) a gene encoding a temperature-sensitive enzyme;
(b) forming a culture system comprising said carbon source in an aqueous medium and said genetically modified microorganism;
(c) maintaining the culture system under conditions that allow the genetically modified microorganism to replicate comprising maintaining a sufficient level of inorganic phosphate within said culture system;
(d) allowing the inorganic phosphate to deplete thereby triggering the expression of the gene regulated by a promoter sensitive to inorganic phosphate levels; and
(e) changing the temperature of the culture system thereby activating or deactivating said temperature-sensitive enzyme and initiating the production of said chemical product.
26 . A method of producing a chemical product from a renewable carbon source through a bioproduction process comprising:
(a) constructing a genetically modified organism capable of converting said renewable carbon source to 3-HP, wherein said genetically modified organism requires inorganic phosphate for growth and comprises: (a) at least one heterologous gene whose expression is regulated by a promoter sensitive to inorganic phosphate levels within a culture system, wherein said gene is selected from the group consisting of mcr, mmsB, ydfG, rutE, nemA, NDSD, accA, accB, accC, accD, accDA fusion, and accCB fusion; and (b) a gene encoding a temperature-sensitive enzyme selected from the group consisting of fabl, fabB and fabD;
(b) forming a culture system comprising said carbon source in an aqueous medium, phosphate and said genetically modified microorganism, and thereby initiating a growth phase during which the genetically modified microorganism replicates;
(c) maintaining a sufficient level of inorganic phosphate within said culture system until the desired level of cell growth is achieved;
(d) allowing the inorganic phosphate to deplete thereby initiating an induction phase which begins the expression of said gene regulated by a promoter sensitive to inorganic phosphate levels; and
(e) changing the temperature of the culture system thereby activating or deactivating said temperature-sensitive enzyme and initiating a growth phase during which said genetically modified microorganism produces 3-HP.
27 . A genetically modified organisms capable of producing a chemical product of interest from a carbon source, wherein the genetic modification includes introduction of nucleic acid sequences coding for polynucleotides encoding expression of a gene encoding an enzyme in a biosynthetic pathway for converting the carbon source to the chemical product, wherein the gene is encoded into the organism using promoters that are activated by phosphate depletion.
28 . A genetically modified organism of claim 27 , wherein the chemical product is 3-HP, the organism is E. coli , and the gene is selected from the group consisting of mcr, mmsB, ydfG, rutE, nemA, NDSD, genes that encode individual or fused subunits of ACCase.
29 . A genetically modified organism of claim 27 wherein the chemical product is 3-HP, the organism is E. coli , and the promoter is selected from the group consisting of a promoter that directs expression of an E. coli gene selected from the group consisting of amn, tktB, xasA, yibD, ytfK, pstS, phoH, phnC, and other phosphate-regulated genes.
30 . A genetically modified organism of claim 27 wherein the chemical product is 3-HP, the organism is E. coli , the gene is selected from the group consisting of mcr, mmsB, ydfG, rutE, nemA, NDSD, genes that encode individual or fused subunits of ACCase, and the promoter is selected from the group consisting of a promoter that directs expression of an E. coli gene selected from the group consisting of amn, tktB, xasA, yibD, ytfK, pstS, phoH, phnC, and other phosphate-regulated genes.
31 . A genetically modified organisms capable of producing a chemical product of interest from a carbon source, wherein the genetic modification includes introduction of nucleic acid sequences coding for polynucleotides encoding expression of a gene that is mutated to become activated or deactivated at a given temperature range.
32 . A genetically modified organism of claim 31 wherein the temperature range is 35 to 39° C.
33 . A genetically modified organisms capable of producing a chemical product of interest from a carbon source, wherein the genetic modification includes introduction of nucleic acid sequences coding for polynucleotides encoding expression of a gene that is mutated such that as a result of a change in the organisms temperature the mutated gene: (1) becomes active and serves a key function in the conversion of the carbon source to the chemical product; or (2) becoming inactive and turns off a branch pathway or other competitive pathway that prevents or limits the pathway leading to the conversion of the carbon source to the chemical product.
34 . A genetically modified organism of claim 31 or 33 wherein the chemical product is 3-HP, the organism is E. coli , and the mutated gene is selected from the group consisting of fabl, fabB, and fabD.
36 . A method of producing a chemical product using the genetically a modified organisms of any of the above claims.
37 . Products made from the genetically modified organisms of any of the above claims.
38 . Products made from the methods of any of the above claims.
39 . The products of claims 37 and 38 , wherein said product is selected from the group consisting of acetyl-CoA, malonyl-CoA, malonate semialdehyde, 3-hydroxypropionic acid (3-HP), acrylic acid, 1,3 propanediol, malonic acid, ethyl 3-HP, propiolactone, acrylonitrile, acrylamide, methyl acrylate, a polymer, a superabsorbent polymer, polyacrylic acid, and a consumer product.
40 . The method of any one of the proceeding claims, wherein said chemical is 3-hydroxypropionic acid or a derivative of 3-HP, 1,4-butanediol, butanol, isobutanol, polyketide chemical product, or C4-C18 fatty acid chain.
41 . The method of any one of the proceeding claims, wherein said chemical is converted to acrylic acid, acrylates, 1,3-propanediol, malonic acid, ethyl-3-hydroxypropionate, ethyl ethoxy propionate, propiolactone, acrylamide, or acrylonitrile to make consumer products.
42 . The method of any one of the proceeding claims, wherein said chemical is oligomerized or polymerized to form polyacrylic acid, methyl acrylate, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxyethyl acrylate, isobutyl acrylate, 2-ethylhexyl acrylate, and acrylic acid ester to which an alkyl or aryl addition may be made, and/or to which halogens, aromatic amines or amides, and aromatic hydrocarbons may be added to make consumer products.