METHODS AND COMPOSITIONS FOR THE AUGMENTATION OF PYRUVATE AND ACETYL-COA FORMATION
The present disclosure identifies methods and compositions for modifying photoautotrophic organisms as hosts, such that the organisms efficiently convert carbon dioxide and light into pyruvate or acetyl-CoA, and in particular the use of such organisms for the commercial production of molecules derived from these precursors, e.g., ethanol.
1 . An engineered photosynthetic microbe, wherein said engineered photosynthetic microbe comprises a recombinant MdhP enzyme.
2 . The engineered photosynthetic microbe of claim 1 , wherein said recombinant MdhP enzyme is a Pisum sativum MdhP enzyme.
3 . The engineered photosynthetic microbe of claim 1 , wherein said recombinant MdhP enzyme is at least 95% identical to SEQ ID NO: 1.
4 . The engineered photosynthetic microbe of claim 1 , wherein said recombinant MdhP enzyme is at least 95% identical to SEQ ID NO: 2.
5 . The engineered photosynthetic microbe of claim 1 , wherein said engineered photosynthetic microbe comprises an additional mutation which reduces the expression or activity of its endogenous Mdh enzyme.
6 . The engineered photosynthetic microbe of claim 5 , wherein said mutation is a knockout of the gene encoding said endogenous Mdh enzyme.
7 . The engineered photosynthetic microbe of claim 1 , wherein said engineered photosynthetic microbe further comprises a recombinant phosphoenol pyruvate carboxylase.
8 . The engineered photosynthetic microbe of claim 1 , wherein said engineered photosynthetic microbe further comprises a recombinant NADPH-linked malic enzyme.
9 . The engineered photosynthetic microbe of claim 1 , wherein said engineered photosynthetic microbe further comprises a recombinant phosphoenol pyruvate carboxylase and a recombinant NADPH-linked malic enzyme.
10 . The engineered photosynthetic microbe of claim 7 or 9 , wherein said recombinant phosphoenol pyruvate carboxylase is the S8D mutant phosphoenol pyruvate carboxylase.
11 . The engineered photosynthetic microbe of claim 10 , wherein said S8D mutant phosphoenol pyruvate carboxylase is derived from Sorghum bicolor Ppc.
12 . The engineered photosynthetic microbe of claim 10 , wherein said recombinant phosphoenol pyruvate carboxylase is at least 95% identical to SEQ ID NO: 4.
13 . The engineered photosynthetic microbe of claim 8 or 9 , wherein said recombinant NADPH-linked malic enzyme is the Synechococcus elongatus PPC 7002 NADPH-linked malic enzyme.
14 . The engineered photosynthetic microbe of claim 8 or 9 , wherein said recombinant NADPH-linked malic enzyme is at least 95% identical to SEQ ID NO: 5.
15 . An engineered photosynthetic microbe, wherein said engineered photosynthetic microbe comprises a recombinant oxaloacetate decarboxylase.
16 . The engineered photosynthetic microbe of claim 15 , wherein said recombinant oxaloacetate decarboxylase is Corynebacterium glutamicum oxaloacetate decarboxylase.
17 . The engineered photosynthetic microbe of claim 15 , wherein said recombinant oxaloacetate decarboxylase is at least 95% identical to SEQ ID NO: 6.
18 . The engineered photosynthetic microbe of claim 15 , wherein said engineered photosynthetic microbe further comprises a recombinant phosphoenol pyruvate carboxylase.
19 . The engineered photosynthetic microbe of claim 18 , wherein said recombinant phosphoenol pyruvate carboxylase is at least 95% identical to SEQ ID NO: 4.
20 . The engineered photosynthetic microbe of claim 15 , wherein said engineered photosynthetic microbe comprises an endogenous, non-recombinant phosphoenol pyruvate carboxylase.
21 . The engineered photosynthetic microbe of claim 15 , wherein said engineered photosynthetic microbe further comprises a recombinant phosphoenolpyruvate carboxykinase.
22 . The engineered photosynthetic microbe of claim 21 , wherein said recombinant phosphoenolpyruvate carboxykinase is derived from E. Coli.
23 . The engineered photosynthetic microbe of claim 21 , wherein said recombinant phosphoenolpyruvate carboxykinase is at least 95% identical to SEQ ID NO: 7.
24 . The engineered photosynthetic microbe of claim 15 , wherein said engineered photosynthetic microbe lacks an endogenous or recombinant malate dehydrogenase activity, or wherein said engineered photosynthetic microbe comprises a mutation which attenuates or knocks out endogenous malate dehydrogenase activity in said engineered photosynthetic microbe.
25 . The engineered photosynthetic microbe of any of claims 1 - 24 , wherein said engineered photosynthetic microbe further comprises a mutation which attenuates or knocks out endogenous pyruvate dehydrogenase activity in said photosynthetic microbe.
26 . An engineered photosynthetic microbe, wherein said engineered photosynthetic microbe comprises a recombinant NADPH-producing transhydrogenase system.
27 . The engineered photosynthetic microbe of claim 26 , further comprising a recombinant MdhP enzyme.
28 . The engineered photosynthetic microbe of claim 26 , wherein said recombinant NADPH-producing transhydrogenase system comprises PntA transhydrogenase, PntB transhydrogenase, or PntAB transhydrogenase.
29 . The engineered photosynthetic microbe of claim 28 , wherein said PntA transhydrogenase comprises a sequence at least 95% identical to SEQ ID NO: 8.
30 . The engineered photosynthetic microbe of claim 28 , wherein said PntB transhydrogenase comprises a sequence at least 95% identical to SEQ ID NO: 9.
31 . The engineered photosynthetic microbe of claim 27 , wherein said PntAB transhydrogenase comprises a sequence at least 95% identical to SEQ ID NO: 8 and further comprises a sequence at least 95% identical to SEQ ID NO: 9.
32 . An engineered photosynthetic microbe, wherein said engineered photosynthetic microbe comprises a recombinant NADPH-generating pyruvate dehydrogenase.
33 . The engineered photosynthetic microbe of claim 32 , further comprising a recombinant MdhP enzyme.
34 . The engineered photosynthetic microbe of claim 32 , wherein said recombinant NADPH-generating pyruvate dehydrogenase is Euglena gracilis Pno or Cryptosporidium parvum Pno.
35 . The engineered photosynthetic microbe of claim 34 , wherein said recombinant NADPH-generating pyruvate dehydrogenase is at least 95% identical to SEQ ID NO: 10.
36 . The engineered photosynthetic microbe of claim 34 , wherein said recombinant NADPH-generating pyruvate dehydrogenase is at least 95% identical to SEQ ID NO: 11.
37 . The engineered photosynthetic microbe of claim 32 , wherein said engineered photosynthetic microbe naturally lacks an endogenous pyruvate dehydrogenase activity or comprises a mutation which attenuates or knocks out endogenous pyruvate dehydrogenase activity.
38 . An engineered photosynthetic microbe, wherein said engineered photosynthetic microbe comprises a recombinant pyruvate:ferredoxin oxidoreductase, wherein expression of said recombinant pyruvate:ferredoxin oxidoreductase is expressed by a gene, wherein said gene is controlled by a promoter which leads to increased expression of said pyruvate:ferredoxin oxidoreductase relative to that obtained with the endogenous gene under the control of its native promoter, or wherein said gene is present in a copy number which leads to increased expression of said pyruvate:ferredoxin oxidoreductase relative to that obtained with an otherwise identical photosynthetic microbe with a lower copy number.
39 . The engineered photosynthetic microbe of claim 38 , further comprising a recombinant MdhP enzyme.
40 . The engineered photosynthetic microbe of claim 38 , wherein said recombinant pyruvate:ferredoxin oxidoreductase is at least 95% identical to SEQ ID NO: 12.
41 . An engineered photosynthetic microbe, wherein said engineered photosynthetic microbe comprises a recombinant NADPH-generating pyruvate dehydrogenase system, wherein said recombinant NADPH-generating pyruvate dehydrogenase system comprises a pyruvate decarboxylase, an NADP-dependent acetaldehyde dehydrogenase, and an acetyl-CoA synthetase.
42 . The engineered photosynthetic microbe of claim 41 , further comprising a recombinant MdhP enzyme
43 . The engineered photosynthetic microbe of claim 41 , wherein said pyruvate decarboxylase is Zymomonas mobilis pyruvate decarboxylase.
44 . The engineered photosynthetic microbe of claim 41 , wherein said pyruvate decarboxylase is at least 95% identical to SEQ ID NO: 13.
45 . The engineered photosynthetic microbe of claim 41 , wherein said NADP-dependent acetaldehyde dehydrogenase is E. coli AldB.
46 . The engineered photosynthetic microbe of claim 41 , wherein said NADP-dependent acetaldehyde dehydrogenase is at least 95% identical to SEQ ID NO: 14.
47 . The engineered photosynthetic microbe of claim 41 , wherein said acetyl-CoA synthetase is E. coli Acs.
48 . The engineered photosynthetic microbe of claim 41 , wherein said acetyl-CoA synthetase is at least 95% identical to SEQ ID NO: 15.
49 . The engineered photosynthetic microbe of any of claims 1 - 48 , wherein said engineered photosynthetic microbe further comprises at least one recombinant gene selected from the group consisting of pyruvate decarboxylase and alcohol dehydrogenase.
50 . A method for improving production of a carbon-based compound of interest by a photosynthetic microbe, wherein said carbon-based compound of interest is synthesized by said photosynthetic microbe using pyruvate, at least in part, as a source of carbon, comprising: (a) culturing said photosynthetic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an MdhP enzyme in said photosynthetic microbe.
51 . The method of claim 50 , wherein said recombinant expression of said MdhP enzyme in said photosynthetic microbe results in increased carbon flux to pyruvate in said photosynthetic microbe.
52 . The method of claim 50 , wherein said MdhP enzyme is a Pisum sativum MdhP enzyme.
53 . The method of claim 50 , wherein said MdhP enzyme is at least 95% identical to SEQ ID NO: 1.
54 . The method of claim 50 , wherein said MdhP enzyme is at least 95% identical to SEQ ID NO: 2.
55 . The method of claim 50 , wherein said photosynthetic microbe comprises an additional mutation which reduces the expression or activity of its endogenous Mdh enzyme.
56 . The method of claim 54 , wherein said mutation is a knockout of the gene encoding said endogenous Mdh enzyme.
57 . The method of claim 50 , wherein said method further comprises recombinantly expressing a phosphoenolpyruvate carboxylase enzyme.
58 . The method of claim 50 , wherein said method further comprises recombinantly expressing a recombinant NADPH-linked malic enzyme.
59 . The method of claim 50 , wherein said method further comprises recombinantly expressing a phosphoenolpyruvate carboxylase enzyme and an NADPH-linked malic enzyme.
60 . The method of any of claims 50 - 59 , wherein said recombinant expression results in increased carbon flux to pyruvate in said photosynthetic microbe.
61 . The method of claim 57 or 59 , wherein said recombinant phosphoenol pyruvate carboxylase is the S8D mutant phosphoenol pyruvate carboxylase.
62 . The method of claim 61 , wherein said S8D mutant phosphoenol pyruvate carboxylase is derived from Sorghum ppc.
63 . The method of claim 61 , wherein said S8D mutant phosphoenol pyruvate carboxylase is at least 95% identical to SEQ ID NO: 4.
64 . The method of claim 58 or 59 , wherein said recombinant NADPH-linked malic enzyme is the Synechococcus elongatus PPC 7002 NADPH-linked malic enzyme.
65 . The method of claim 58 or 59 , wherein said recombinant NADPH-linked malic enzyme is at least 95% identical to SEQ ID NO: 5.
66 . A method for improving production of a carbon-based compound of interest by a photosynthetic microbe, wherein said carbon-based compound of interest is synthesized by said photosynthetic microbe using pyruvate, at least in part, as a source of carbon, comprising: (a) culturing said photosynthetic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an oxaloacetate decarboxylase enzyme in said photosynthetic microbe.
67 . The method of claim 66 , wherein said recombinant expression of said oxaloacetate decarboxylase enzyme in said photosynthetic microbe results in increased carbon flux to pyruvate in said photosynthetic microbe.
68 . The method of claim 66 , wherein said oxaloacetate decarboxylase is Corynebacterium glutamicum oxaloacetate decarboxylase.
69 . The method of claim 66 , wherein said oxaloacetate decarboxylase is at least 95% identical to SEQ ID NO: 6.
70 . The method of claim 66 , wherein said photosynthetic microbe further comprises a recombinant phosphoenol pyruvate carboxylase.
71 . The method of claim 70 , wherein said recombinant phosphoenol pyruvate carboxylase is at least 95% identical to SEQ ID NO: 4.
72 . The method of claim 66 , wherein said photosynthetic microbe comprises an endogenous, non-recombinant phosphoenol pyruvate carboxylase.
73 . The method of claim 66 , wherein said method further comprises recombinantly expressing a phosphoenolpyruvate carboxykinase in said photosynthetic microbe.
74 . The method of claim 73 , wherein said phosphoenolpyruvate carboxykinase is derived from E. Coli.
75 . The method of claim 73 , wherein said phosphoenolpyruvate carboxykinase is at least 95% identical to SEQ ID NO: 7.
76 . The method of claim 66 , wherein said photosynthetic microbe lacks an endogenous or recombinant malate dehydrogenase activity, or wherein said engineered photosynthetic microbe comprises a mutation which attenuates or knocks out endogenous malate dehydrogenase activity in said engineered photosynthetic microbe.
77 . The method of any of claims 50 - 76 , wherein said engineered photosynthetic microbe further comprises a mutation which attenuates or knocks out endogenous pyruvate dehydrogenase activity in said photosynthetic microbe.
78 . A method for improving production of a carbon-based compound of interest by a photosynthetic microbe, wherein said carbon-based compound of interest is synthesized by said photosynthetic microbe using acetyl-CoA, at least in part, as a source of carbon, comprising: (a) culturing said photosynthetic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an NADPH-producing transhydrogenase system in said photosynthetic microbe.
79 . The method of claim 78 , wherein said method further comprises recombinantly expressing an MdhP enzyme in said photosynthetic microbe.
80 . The method of claim 78 , wherein said recombinant expression of said NADPH-producing transhydrogenase in said photosynthetic microbe results in increased carbon flux to acetyl-CoA in said photosynthetic microbe.
81 . The method of claim 78 , wherein said NADPH-producing transhydrogenase system comprises PntA transhydrogenase, PntB transhydrogenase, or PntAB transhydrogenase.
82 . The method of claim 81 , wherein said PntA transhydrogenase is at least 95% identical to SEQ ID NO: 8.
83 . The method of claim 81 , wherein said PntB transhydrogenase is at least 95% identical to SEQ ID NO: 9.
84 . The method of claim 81 , wherein said PntAB transhydrogenase is at least 95% identical to SEQ ID NO: 8 and further comprises a sequence at least 95% identical to SEQ ID NO: 9.
85 . A method for improving production of a carbon-based compound of interest by a photosynthetic microbe, wherein said carbon-based compound of interest is synthesized by said photosynthetic microbe using acetyl-CoA, at least in part, as a source of carbon, comprising: (a) culturing said photosynthetic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an NADPH-generating pyruvate dehydrogenase in said photosynthetic microbe.
86 . The method of claim 85 , wherein said method further comprises recombinantly expressing an MdhP enzyme in said photosynthetic microbe.
87 . The method of claim 85 , wherein said recombinant expression of said NADPH-generating pyruvate dehydrogenase in said photosynthetic microbe results in increased carbon flux to acetyl-CoA in said photosynthetic microbe.
88 . The method of claim 85 , wherein said NADPH-generating pyruvate dehydrogenase is Euglena gracilis Pno or Cryptosporidium parvum Pno.
89 . The method of claim 85 , wherein said NADPH-generating pyruvate dehydrogenase is at least 95% identical to SEQ ID NO: 10.
90 . The method of claim 85 , wherein said NADPH-generating pyruvate dehydrogenase is at least 95% identical to SEQ ID NO: 11.
91 . The method of claim 85 , wherein said photosynthetic microbe naturally lacks an endogenous pyruvate dehydrogenase activity or comprises a mutation which attenuates or knocks out endogenous pyruvate dehydrogenase activity.
92 . A method for improving production of a carbon-based compound of interest by a photosynthetic microbe, wherein said carbon-based compound of interest is synthesized by said photosynthetic microbe using acetyl-CoA, at least in part, as a source of carbon, comprising: (a) culturing said photosynthetic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing a pyruvate:ferredoxin oxidoreductase in said photosynthetic microbe, wherein expression of said recombinant pyruvate:ferredoxin oxidoreductase is expressed by a gene, wherein said gene is controlled by a promoter which leads to increased expression of said pyruvate:ferredoxin oxidoreductase relative to that obtained with the endogenous gene under the control of its native promoter, or wherein said gene is present in a copy number which leads to increased expression of said pyruvate:ferredoxin oxidoreductase relative to that obtained with an otherwise identical photosynthetic microbe with a lower copy number.
93 . The method of claim 92 , wherein said method further comprises recombinantly expressing an MdhP enzyme in said photosynthetic microbe.
94 . The method of claim 92 , wherein said recombinant expression of said pyruvate:ferredoxin oxidoreductase in said photosynthetic microbe results in increased carbon flux to acetyl-CoA in said photosynthetic microbe
95 . The method of claim 92 , wherein said pyruvate:ferredoxin oxidoreductase is at least 95% identical to SEQ ID NO: 12.
96 . A method for improving production of a carbon-based compound of interest by a photosynthetic microbe, wherein said carbon-based compound of interest is synthesized by said photosynthetic microbe using acetyl-CoA, at least in part, as a source of carbon, comprising: (a) culturing said photosynthetic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an NADPH-generating pyruvate dehydrogenase system in said photosynthetic microbe, wherein said NADPH-generating pyruvate dehydrogenase system comprises a pyruvate decarboxylase, an NADP-dependent acetaldehyde dehydrogenase, and an acetyl-CoA synthetases.
97 . The method of claim 96 , wherein said method further comprises recombinantly expressing an MdhP enzyme in said photosynthetic microbe.
98 . The method of claim 96 , wherein said recombinant expression of said NADPH-generating pyruvate dehydrogenase system in said photosynthetic microbe results in increased carbon flux to acetyl-CoA in said photosynthetic microbe.
99 . The method of claim 96 , wherein said pyruvate decarboxylase is Zymomonas mobilis pyruvate decarboxylase.
100 . The method of claim 96 , wherein said pyruvate decarboxylase is at least 95% identical to SEQ ID NO: 13.
101 . The method of claim 96 , wherein said NADP-dependent acetaldehyde dehydrogenase is E. coli AldB.
102 . The method of claim 96 , wherein said NADP-dependent acetaldehyde dehydrogenase is at least 95% identical to SEQ ID NO: 14.
103 . The method of claim 96 , wherein said acetyl-CoA synthetase is E. coli Acs.
104 . The method of claim 96 , wherein said acetyl-CoA synthetase is at least 95% identical to SEQ ID NO: 15.
105 . The method of any of claims 50 - 104 , wherein said carbon-based compound of interest is produced at a greater rate or in greater yields in said engineered photosynthetic microbe relative to an otherwise identical photosynthetic microbe lacking the recited recombinant enzymes or mutations.
106 . The method of claim 105 , wherein said engineered photosynthetic microbe further comprises at least one recombinant gene selected from the group consisting of pyruvate decarboxylase and alcohol dehydrogenase.
107 . The method of claim 106 , wherein said carbon-based compound of interest is ethanol.
108 . The method of any of claims 50 - 104 , wherein said carbon-based compound of interest is selected from the group consisting of: alcohols, alkenes, and alkanes.
109 . An engineered heterotrophic microbe, wherein said engineered heterotrophic microbe comprises a recombinant MdhP enzyme.
110 . An engineered heterotrophic microbe, wherein said engineered heterotrophic microbe comprises a recombinant oxaloacetate decarboxylase.
111 . An engineered heterotrophic microbe, wherein said engineered heterotrophic microbe comprises a recombinant NADPH-producing transhydrogenase system.
112 . An engineered heterotrophic microbe, wherein said engineered heterotrophic microbe comprises a recombinant NADPH-generating pyruvate dehydrogenase.
113 . An engineered heterotrophic microbe, wherein said engineered heterotrophic microbe comprises a recombinant pyruvate:ferredoxin oxidoreductase, wherein expression of said recombinant pyruvate:ferredoxin oxidoreductase is expressed by a gene, wherein said gene is controlled by a promoter which leads to increased expression of said pyruvate:ferredoxin oxidoreductase relative to that obtained with the endogenous gene under the control of its native promoter, or wherein said gene is present in a copy number which leads to increased expression of said pyruvate:ferredoxin oxidoreductase relative to that obtained with an otherwise identical heterotrophic microbe with a lower copy number.
114 . An engineered heterotrophic microbe, wherein said engineered heterotrophic microbe comprises a recombinant NADPH-generating pyruvate dehydrogenase system, wherein said recombinant NADPH-generating pyruvate dehydrogenase system comprises a pyruvate decarboxylase, an NADP-dependent acetaldehyde dehydrogenase, and an acetyl-CoA synthetase.
115 . A method for improving production of a carbon-based compound of interest by a heterotrophic microbe, wherein said carbon-based compound of interest is synthesized by said heterotrophic microbe using pyruvate, at least in part, as a source of carbon, comprising: (a) culturing said heterotrophic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an MdhP enzyme in said heterotrophic microbe.
116 . A method for improving production of a carbon-based compound of interest by a heterotrophic microbe, wherein said carbon-based compound of interest is synthesized by said heterotrophic microbe using pyruvate, at least in part, as a source of carbon, comprising: (a) culturing said heterotrophic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an oxaloacetate decarboxylase enzyme in said heterotrophic microbe.
117 . A method for improving production of a carbon-based compound of interest by a heterotrophic microbe, wherein said carbon-based compound of interest is synthesized by said heterotrophic microbe using acetyl-CoA, at least in part, as a source of carbon, comprising: (a) culturing said heterotrophic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an NADPH-producing transhydrogenase system in said heterotrophic microbe.
118 . A method for improving production of a carbon-based compound of interest by a heterotrophic microbe, wherein said carbon-based compound of interest is synthesized by said heterotrophic microbe using acetyl-CoA, at least in part, as a source of carbon, comprising: (a) culturing said heterotrophic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an NADPH-generating pyruvate dehydrogenase in said heterotrophic microbe.
119 . A method for improving production of a carbon-based compound of interest by a heterotrophic microbe, wherein said carbon-based compound of interest is synthesized by said heterotrophic microbe using acetyl-CoA, at least in part, as a source of carbon, comprising: (a) culturing said heterotrophic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing a pyruvate:ferredoxin oxidoreductase in said heterotrophic microbe, wherein expression of said recombinant pyruvate:ferredoxin oxidoreductase is expressed by a gene, wherein said gene is controlled by a promoter which leads to increased expression of said pyruvate:ferredoxin oxidoreductase relative to that obtained with the endogenous gene under the control of its native promoter, or wherein said gene is present in a copy number which leads to increased expression of said pyruvate:ferredoxin oxidoreductase relative to that obtained with an otherwise identical heterotrophic microbe with a lower copy number.
120 . A method for improving production of a carbon-based compound of interest by a heterotrophic microbe, wherein said carbon-based compound of interest is synthesized by said heterotrophic microbe using acetyl-CoA, at least in part, as a source of carbon, comprising: (a) culturing said heterotrophic microbe in the presence of light and an inorganic carbon source, and (b) recombinantly expressing an NADPH-generating pyruvate dehydrogenase system in said heterotrophic microbe, wherein said NADPH-generating pyruvate dehydrogenase system comprises a pyruvate decarboxylase, an NADP-dependent acetaldehyde dehydrogenase, and an acetyl-CoA synthetases.