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Microorganisms for producing 1,4-butanediol and methods related thereto

Inactive Publication Date: 2013-02-07
GENOMATICA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about creating microbial organisms that can produce certain chemicals, such as 1,4-butanediol (BDO), 4-hydroxybutanal (4-HBal), 4-hydroxybutyryl-CoA (4-HBCoA) and putrescine. These organisms have been altered with genetic material to increase their ability to produce these chemicals. The invention also describes methods for using these altered organisms to produce BDO, 4-HBeck's patent is about creating bacteria that have the ability to produce certain chemicals, such as BDO, 4-HBeck's "technical effect" is the creation of microbial organisms that can produce specific chemicals that are not normally found in nature. These organisms have been modified to increase their ability to produce these chemicals.

Problems solved by technology

However, succinic acid is costly to isolate and purify and requires high temperatures and pressures for catalytic reduction to butanediol.

Method used

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  • Microorganisms for producing 1,4-butanediol and methods related thereto
  • Microorganisms for producing 1,4-butanediol and methods related thereto
  • Microorganisms for producing 1,4-butanediol and methods related thereto

Examples

Experimental program
Comparison scheme
Effect test

example i

Biosynthesis of 4-Hydroxybutanoic Acid

[0362]This example describes exemplary biochemical pathways for 4-HB production.

[0363]Previous reports of 4-HB synthesis in microbes have focused on this compound as an intermediate in production of the biodegradable plastic poly-hydroxyalkanoate (PHA) (U.S. Pat. No. 6,117,658). The use of 4-HB / 3-HB copolymers over poly-3-hydroxybutyrate polymer (PHB) can result in plastic that is less brittle (Saito and Doi, Intl. J. Biol. Macromol. 16:99-104 (1994)). The production of monomeric 4-HB described herein is a fundamentally distinct process for several reasons: (1) the product is secreted, as opposed to PHA which is produced intracellularly and remains in the cell; (2) for organisms that produce hydroxybutanoate polymers, free 4-HB is not produced, but rather the Coenzyme A derivative is used by the polyhydroxyalkanoate synthase; (3) in the case of the polymer, formation of the granular product changes thermodynamics; and (4) extracellular pH is not...

example ii

Biosynthesis of 1,4-Butanediol from Succinate and Alpha-Ketoglutarate

[0369]This example illustrates the construction and biosynthetic production of 4-HB and BDO from microbial organisms. Pathways for 4-HB and BDO are disclosed herein.

[0370]There are several alternative enzymes that can be utilized in the pathway described above. The native or endogenous enzyme for conversion of succinate to succinyl-CoA (Step 1 in FIG. 1) can be replaced by a CoA transferase such as that encoded by the cat1 gene C. kluyveri (Sohling and Gottschalk, Eur. J Biochem. 212:121-127 (1993)), which functions in a similar manner to Step 9. However, the production of acetate by this enzyme may not be optimal, as it might be secreted rather than being converted back to acetyl-CoA. In this respect, it also can be beneficial to eliminate acetate formation in Step 9. As one alternative to this CoA transferase, a mechanism can be employed in which the 4-HB is first phosphorylated by ATP and then converted to the C...

example iii

Biosynthesis of 4-Hydroxybutanoic Acid, γ-Butyrolactone and 1,4-Butanediol

[0402]This Example describes the biosynthetic production of 4-hydroxybutanoic acid, γ-butyrolactone and 1,4-butanediol using fermentation and other bioprocesses.

[0403]Methods for the integration of the 4-HB fermentation step into a complete process for the production of purified GBL, 1,4-butanediol (BDO) and tetrahydrofuran (THF) are described below. Since 4-HB and GBL are in equilibrium, the fermentation broth will contain both compounds. At low pH this equilibrium is shifted to favor GBL. Therefore, the fermentation can operate at pH 7.5 or less, generally pH 5.5 or less. After removal of biomass, the product stream enters into a separation step in which GBL is removed and the remaining stream enriched in 4-HB is recycled. Finally, GBL is distilled to remove any impurities. The process operates in one of three ways: 1) fed-batch fermentation and batch separation; 2) fed-batch fermentation and continuous sepa...

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Abstract

The invention provides non-naturally occurring microbial organisms comprising a 1,4-butanediol (BDO), 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine pathway comprising at least one exogenous nucleic acid encoding a BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine pathway enzyme expressed in a sufficient amount to produce BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine and further optimized for expression of BDO. The invention additionally provides methods of using such microbial organisms to produce BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine.

Description

[0001]This application claims the benefit of priority of U.S. Provisional application Ser. No. 61 / 500,120, filed Jun. 22, 2011, and U.S. Provisional application Ser. No. 61 / 502,837, filed Jun. 29, 2011, each of which the entire contents are incorporated herein by reference.[0002]Incorporated herein by reference is the Sequence Listing being concurrently submitted via EFS-Web as an ASCII text file named 12956-144-999 SEQLIST.TXT, created Jun. 21, 2012, and being 227,850 bytes in size.[0003]This invention relates generally to in silico design of organisms and engineering of organisms, more particularly to organisms having 1,4-butanediol, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine biosynthesis capability.BACKGROUND OF THE INVENTION[0004]The compound 4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB) is a 4-carbon carboxylic acid that has industrial potential as a building block for various commodity and specialty chemicals. In particular, 4-HB has the potent...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/02C12P17/04C12N1/19C12P7/18C12P7/42
CPCC12N15/52Y02E50/10C12P7/18C12P7/16C12N9/0008C12Y102/99006C12P7/42
Inventor BURGARD, ANTHONY P.OSTERHOUT, ROBIN E.SUN, JUNPHARKYA, PRITI
Owner GENOMATICA INC
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