Cell-free and minimized metabolic reaction cascades for the production of chemicals

A chemical, cell-free enzyme system technology, applied in the field of preparation of target chemicals, can solve the problems of expensive methods, unstable technology, low efficiency, etc., and achieve the effect of high cost efficiency and complexity elimination.

Inactive Publication Date: 2015-06-24
CLARIANT PROD DEUT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All in all, the documented method would be expensive, inefficient and technically unstable

Method used

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  • Cell-free and minimized metabolic reaction cascades for the production of chemicals
  • Cell-free and minimized metabolic reaction cascades for the production of chemicals
  • Cell-free and minimized metabolic reaction cascades for the production of chemicals

Examples

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preparation example Construction

[0140] According to a preferred aspect, the preparation method of the present invention includes the following 4 steps:

[0141] Step I: Use microbial cells to prepare enzymes ("target enzymes") for the conversion of carbon sources into chemicals (also referred to herein as "target chemicals" or "target organic compounds");

[0142] Step II: Release the target enzyme from the microbial cell used in Step I, preferably combined with the release of cofactors and the inactivation of other non-target enzyme activities; or purify the target enzyme from the non-target enzyme activity, preferably with the cofactor Release combined

[0143] Step III: Contact the target enzyme of Step II with the carbon source under conditions suitable for the conversion of the carbon source to the target chemical;

[0144] Step IV: Separate the target chemical from the reaction mixture.

[0145] Enzyme selection and preparation:

[0146] In step I, the target enzyme is prepared using microbial cells. In one emb...

Embodiment 1

[0308] Example 1 (ethanol synthesis)

[0309] A general example of the flexibility of the cell-free synthesis toolbox is the use of an enzyme cascade that converts glucose or galactose to pyruvate with four enzymes to convert glucose or galactose to pyruvate. The four enzymes include glucose dehydrogenation. Enzymes (GDH), gluconic acid / glycerate / dihydroxy acid dehydratase (DHAD), 2-keto-3-deoxygluconic acid aldolase (KDGA) and glyceraldehyde dehydrogenase (ALDH). The ALDH used in this example is defined by SEQ ID NO 10 constructed in Example 4.

[0310] In the next two-step reaction, pyruvate passes through pyruvate decarboxylase (PDC) (J. Mol. Catal. B-Enzym. 2009, 61, 30-35) and alcohol dehydrogenase (ADH) (Protein Eng. 1998, 11, 925-930) and converted into acetaldehyde and then into ethanol. Due to its relatively high thermal tolerance and activity, PDC derived from Zymomonas mobilis was chosen. Although it is a mesophilic source, Zymomonas mobilis PDC is heat stable up to ...

Embodiment 2

[0315] Example 2 (Isobutanol synthesis)

[0316] This example shows the successful conversion of pyruvate to isobutanol using only four additional enzymes (see Figure 2 and Table 2) in a completely cell-free environment. Initially, two pyruvate molecules are combined by acetolactate synthase (ALS) (FEMS Microbiol. Lett. 2007, 272, 30-34) to obtain acetolactate, which is further passed by ketol acid reductoisomerase (KARI) ( Accounts Chem. Res. 2001, 34, 399-408) to obtain natural DHAD substrate dihydroxyisovaleric acid. After DHAD, dihydroxyisovaleric acid is converted to 2-ketoisovaleric acid.

[0317] Table 2:

[0318]

[0319] Table 2: Enzymes used in the cell-free synthesis of isobutanol. a: For the activity of natural substrates, DHAD (respectively) for gluconic acid, glyceric acid and dihydroxyisovaleric acid as substrates, and ADH for acetaldehyde and isobutyraldehyde as substrates; b: higher than solubility, c: Enzyme is engineered; E50: ethanol concentration that causes...

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Abstract

Provided are enzymatic processes for the production of chemicals from carbon sources. In particular, a process for the production of a target chemical is disclosed using a cell-free enzyme system that converts carbohydrate sources to the intermediate pyruvate and subsequently the intermediate pyruvate to the target chemical.

Description

Technical field [0001] The present invention relates to an enzymatic method for preparing chemicals from carbon sources. Specifically, a method for preparing a target chemical is disclosed, which uses a cell-free enzyme system that converts a sugar source into an intermediate product pyruvate and then converts the intermediate product pyruvate into a target chemical. Background technique [0002] The development of sustainable and biomass-based production strategies requires effective depolymerization into intermediate carbohydrates, and requires flexible and effective techniques for converting these intermediate carbohydrates into chemical products. Currently, biotechnological approaches for converting biomass into chemicals focus on well-established microbial fermentation processes. [0003] However, these fermentation pathways are still limited to the physiological limits of the cell production system. The main obstacles to cost-effective fermentation processes are their low t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/06C12P7/16C12N15/10C12N9/00
CPCC12P7/06C12P7/16C12N9/0008C12N15/52Y02E50/10C12N9/0006C12N9/1022C12N9/88C12Y101/01001C12Y101/01047C12Y101/01086C12Y102/01003C12Y202/01006C12Y401/02014C12Y402/01009C12P7/14
Inventor M·克劳斯A·科尔特曼U·克特林D·加尔贝T·布吕克J-K·古特尔V·西贝尔
Owner CLARIANT PROD DEUT GMBH
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