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Fermentative production of acetone from renewable resources by means of novel metabolic pathway

A technology of acetone and coenzyme, applied in the field of enzymes and nucleic acids, to achieve the effects of increasing space-time yield, simplifying fermentation process, and simplifying separation and processing

Active Publication Date: 2009-05-06
EVONIK OPERATIONS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no examples to date that the above enzymes can hydrolyze acetoacetyl-CoA to form acetoacetate but not CoA in vitro or in vivo

Method used

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  • Fermentative production of acetone from renewable resources by means of novel metabolic pathway
  • Fermentative production of acetone from renewable resources by means of novel metabolic pathway
  • Fermentative production of acetone from renewable resources by means of novel metabolic pathway

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preparation example Construction

[0032] The method for preparing acetone of the present invention and the use of the polypeptides and nucleic acids in implementing the method of the present invention are described by the following exemplary methods, and the present invention is not limited to the following exemplary embodiments. It is intended that the documents cited in the description of the present invention incorporate all the contents disclosed therein in the present invention. Polypeptide refers to any chain length. Thus, it also includes any protein, enzyme, in particular acetoacetate-CoA hydrolase, acyl-CoA thioesterase, acyl-CoA synthetase or acyl-CoA thiokinase. "Spontaneous conversion" refers to a chemical reaction that produces energy. "Under stringent conditions" hybridization refers to experimental conditions, such as the application of a washing solution at 50-70° C., preferably 60-65° C. in Northern blotting techniques, such as the application of 0.1x SSC buffer containing 0.1% SDS (2Ox SSC ...

Embodiment 1

[0109] Example 1: Thioesterase II (TEII) from Bacillus subtilis srf )

[0110] This enzyme is related to the non-ribosomal peptide synthase used in the formation of surfactant peptides (peptide antibiotics) in Bacillus subtilis ATCC 21332. Schwarzer et al. were able to clone the corresponding gene into the plasmid pQE60 that mediates the fusion of the target protein with the C-terminal His tag (pTEIIsrf) and purify the protein (28kDa) (Schwarzer D., H.D.Mootz, U.Linne, M.A.Marahiel .2002.Regeneration of misprimed nonribosomal peptidesythetases by type II thioesterases.PNAS 99:14083-14088). In the following study, the hydrolytic activity of the protein with acetyl-CoA and propionyl-CoA as substrates was demonstrated.

[0111] Plasmid pTEIIsrf with SEQ ID NO.13 was prepared as described by Schwarzer D, Mootz HD, Linne U, Marahiel MA (Regeneration of misprimed nonribosomal peptide sythetases by type II thioesterases. Proc Natl AcadSci US A. 2002 Oct 29; 99 (22 ): 14083-8).

...

Embodiment 2

[0118] Example 2: Acetoacetyl-CoA Synthetase (AACS) from S. meliloti

[0119] With the amino acid sequence shown in SEQ ID No.3 and the corresponding cDNA sequence shown in SEQ ID No.4, the acetoacetyl-CoA synthetase (AACS) from Sinorhizobium meliloti catalyzes the consumption of ATP by acetoacetate and coenzyme A is converted to acetoacetyl-CoA and AMP. Within the framework of the present invention, however, the reverse reaction is of interest.

[0120] Such as Aneja, P., R.Dziak, G.-Q.Cai, T.C.Charles.2002. Identification of an Acetoacetyl Coenzyme A synthetase-Dependent Pathway for Utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium meliloti.J.Bacteriol. The corresponding gene acsA2 was cloned into the expression vector pET30Xa / LIC as described in 184:1571-1577. The plasmid "pRD112" obtained in this way mediates the fusion of the N-terminus of the target protein with the His tag, thereby facilitating the 2+ -Purification by affinity chromatography on NTA agarose.

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Abstract

The invention describes a process for the preparation of acetone starting from acetyl-coenzyme A comprising the process steps A. enzymatic conversion of acetyl-CoA to give acetoacetyl-CoA B. enzymatic conversion of acetoacetyl-CoA into acetoacetate and CoA, and C. decarboxylation of acetoacetate to give acetone and CO2, which process is characterized in that, in process step B, the coenzyme A is not transferred to an acceptor molecule. Moreover, surprisingly, process step B is catalyzed by enzymes from the classes acyl-CoA thioesterase, acyl-CoA synthetase or acyl-CoA thiokinase. This is an entirely novel metabolic pathway since the enzymatic hydrolysis of acetoacetyl-CoA without the simultaneous transfer of CoA to a receptor molecule has never been described before for any microbial enzyme.

Description

technical field [0001] The present invention relates to a new enzymatic biosynthetic pathway for the production of acetone, which differs from traditional fermentation methods in that it is not coupled to the formation of ethanol and butanol, and to enzymes and nucleic acids for this purpose. Background technique [0002] ABE method in Clostridium [0003] The traditional ABE fermentation method, which uses microorganisms to produce acetone, butanol and ethanol, has long been the second largest biotechnology method worldwide, second only to the method of using yeast fermentation to produce ethanol. Commercial ABE fermentation started in England in 1916, especially when Chaim Weizmann discovered the ability of Clostridium acetobutylicum to form the solvents acetone, butanol and ethanol. This method was used in the Western world until the end of the 1850s, and in South America even until 1981. [0004] There are two basic reasons for abandoning this approach: first, the incr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/28C12N9/16C12N9/00C12N15/55C12N15/52
CPCC12Y301/02C12N9/93C12P7/28C12Y301/02018C12N9/16
Inventor S·弗塞克S·沙弗W·弗赖塔格F·G·施米特M·奥尔谢尔G·格伦德W·施米特H·J·巴尔R·-J·费希尔A·梅
Owner EVONIK OPERATIONS GMBH
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