Microorganisms for the production of 1,4-butanediol and related methods
View Patent ↗The invention provides non-naturally occurring microbial organisms comprising a 1,4-butanediol (BDO) pathway comprising at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO and further optimized for expression of BDO. The invention additionally provides methods of using such microbial organisms to produce BDO.
1. A non-naturally occurring Escherichia coli , comprising a 1,4-butanediol (BDO) pathway comprising one or more heterologous polynucleotides encoding BDO pathway enzymes expressed in a sufficient amount to produce BDO, wherein said E. coli:
(A) comprises a BDO pathway comprising:
(a) alpha-ketoglutarate decarboxylase; or alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase; or glutamate:succinate semialdehyde transaminase and glutamate decarboxylase;
(b) 4-hydroxybutyrate dehydrogenase;
(c) 4-hydroxybutyryl-CoA transferase; or 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and
(d) 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase; or aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol; and
(B) comprises disruption of a gene encoding a protein in an aerobic respiratory control regulatory system; or expresses an exogenous NADH insensitive citrate synthase.
2. The non-naturally occurring E. coli of claim 1 , wherein said E. coli comprises disruption of a gene encoding a protein in an aerobic respiratory control regulatory system.
3. The non-naturally occurring E. coli of claim 2 , wherein said gene encoding the protein in the aerobic respiratory control regulatory system is an arcA gene.
4. The non-naturally occurring E. coli of claim 1 , wherein said E. coli expresses an exogenous NADH insensitive citrate synthase.
5. The non-naturally occurring E. coli of claim 4 , wherein said NADH insensitive citrate synthase is encoded by a gltA gene or a mutant gltA gene encoding an R163L mutant NADH insensitive citrate synthase.
6. The non-naturally occurring E. coli of claim 1 , wherein said E. coli further expresses an exogenous phosphoenolpyruvate carboxykinase.
7. The non-naturally occurring E. coli of claim 1 , further comprising disruption of a gene encoding malate dehydrogenase.
8. The non-naturally occurring E. coli of claim 1 , wherein one or more of said one or more heterologous nucleotides encoding BDO pathway enzymes are integrated into the fimD locus of the E. coli.
9. The non-naturally occurring E. coli of claim 1 , wherein said E. coli further expresses an exogenous non-phosphotransferase sucrose uptake system.
10. The non-naturally occurring E. coli of claim 1 , wherein said E. coli further comprises disruption of endogenous lactate dehydrogenase, endogenous alcohol dehydrogenase, and endogenous pyruvate formate lyase.
11. The non-naturally occurring E. coli of claim 1 , wherein said E. coli further expresses an exogenous pyruvate dehydrogenase.
12. The non-naturally occurring E. coli of claim 11 , wherein one or more genes encoding pyruvate dehydrogenase subunits is under the control of a pyruvate formate lyase promoter.
13. The non-naturally occurring E. coli of claim 11 , wherein said exogenous pyruvate dehydrogenase is NADH insensitive.
14. The non-naturally occurring E. coli of claim 11 , wherein said exogenous pyruvate dehydrogenase is encoded by the Klebsiella pneumonia lpdA gene.
15. The non-naturally occurring E. coli of claim 1 , wherein said succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase and 4-hydroxybutyryl-CoA/acetyl-CoA transferase are encoded by Porphyromonas gingivalis W83 genes.
16. The non-naturally occurring E. coli of claim 1 , wherein said 4-hydroxybutyryl-CoA reductase is encoded by the Clostridium beijerinckii ald gene.
17. The non-naturally occurring E. coli of claim 1 , wherein said E. coli further expresses an exogenous succinyl-CoA synthetase.
18. The non-naturally occurring E. coli of claim 17 , wherein said succinyl-CoA synthetase is encoded by the Escherichia coli sucCD genes.
19. The non-naturally occurring E. coli of claim 1 , wherein said alpha-ketoglutarate decarboxylase is encoded by the Mycobacterium bovis sucA gene.
20. The non-naturally occurring E. coli of claim 1 , wherein said 4-hydroxybutyrate kinase and said phosphotrans-4-hydroxybutyrylase are encoded by the Clostridium acetobutylicum buk1 and ptb genes.
21. The non-naturally occurring E. coli of claim 1 , wherein said 4-hydroxybutyryl-CoA reductase is encoded by the Clostridium beijerinckii ald gene.
22. The non-naturally occurring E. coli of claim 1 , wherein said 4-hydroxybutyraldehyde reductase is encoded by the Geobacillus thermoglucosidasius adh1 gene.
23. The non-naturally occurring E. coli of claim 6 , wherein said phosphoenolpyruvate carboxykinase is encoded by the Haemophilus influenza phosphoenolpyruvate carboxykinase gene.