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Recombinant microorganisms exhibiting increased flux through a fermentation pathway

A technology for recombining microorganisms and microorganisms, applied in the field of identifying and solving reaction bottlenecks in fermentation pathways, and can solve problems such as inability to identify rate-limiting reactions

Inactive Publication Date: 2016-01-27
LANZATECH NEW ZEALAND LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a limiting factor of this approach is that it cannot identify rate-limiting reactions (bottlenecks)

Method used

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  • Recombinant microorganisms exhibiting increased flux through a fermentation pathway
  • Recombinant microorganisms exhibiting increased flux through a fermentation pathway
  • Recombinant microorganisms exhibiting increased flux through a fermentation pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] Example 1 - Identification of Bottlenecks

[0193] The fermentation pathways of carboxydotrophic bacteria, such as Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium rasseri, for the production of ethanol and 2,3-butanediol were analyzed for bottlenecks using an enzyme assay. For this, oxidoreductase reactions are particularly suitable because they involve one or more cofactors whose reduction or oxidation can be measured. Synthetic redox dyes such as methyl viologen or benzyl viologen can also be used for this purpose.

[0194] The oxidoreductase steps of the Wood-Yendal pathway and the fermentative pathways producing ethanol and 2,3-butanediol were assayed to determine their activity. Enzymes in these pathways are involved in autotrophic growth (including CO, CO 2 and H 2 gas absorption and utilization) and product formation.

[0195] The enzymes assayed and their activities are detailed in figure 1 middle. All assays performed were tested usi...

Embodiment 2

[0214] Example 2: Bottleneck of ethanol production

[0215] as in figure 1 As seen in the ethanol fermentation pathway depicted in , the bottleneck for ethanol production is the alcohol dehydrogenase reaction. While all other measured reactions showed at least 1.1 U / mg activity, the alcohol dehydrogenase reaction step only had a total activity of 0.35 U / mg (or 31%), i.e. 0.2 U / mg (18%) with NADH and 0.15U / mg (13%) with NADPH. This is 69% smaller than all other reactions in the pathway. In a similar manner, the aldehyde dehydrogenase reaction had only 0.16 U / mg (14%) of total activity, ie 0.08 U / mg (7%) with NADH and 0.08 U / mg (7%) with NADPH. This is 86% smaller than all other reactions in the pathway. However, this reaction can be bypassed via acetate and aldehyde:ferredoxin oxidoreductase (AOR), which has an activity of 1.9 U / mg and has Phosphorylation of the substrate level in the reaction produces ATP, which provides the cell with the advantage of more energy. To he...

Embodiment 3

[0216] Example 3: Improving flux through ethanol production pathways by removing bottlenecks

[0217] The reactions catalyzing the conversion of acetyl-CoA to acetaldehyde and the conversion of acetaldehyde to ethanol have been identified as the rate-limiting steps in the formation of ethanol in Clostridium autoethanogenum, Clostridium ljungdahlii or Clostridium rasseri. This can be overcome by:

[0218] i. overexpressing a native bifunctional alcohol / aldehyde dehydrogenase,

[0219] ii. expressing a heterologous bifunctional alcohol / aldehyde dehydrogenase, or

[0220] iii. Expressing a heterologous aldehyde dehydrogenase and alcohol dehydrogenase.

[0221] These results can be achieved by using the methods described below.

[0222] Overexpression of a native bifunctional alcohol / aldehyde dehydrogenase in Clostridium autoethanogenum

[0223] Selective overexpression of the native bifunctional alcohol / aldehyde dehydrogenase gene (SEQ ID NO: 1) of Clostridium autoethanologen...

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Abstract

The invention provides methods of increasing the production of fermentation products by increasing flux through a fermentation pathway by optimising enzymatic reactions. In particular, the invention relates to identifying enzymes and / or co-factors involved in metabolic bottlenecks in fermentation pathways, and fermenting a CO-comprising substrate with a recombinant carboxydotrophic Clostridia microorganism adapted to exhibit increased activity of the one or more of said enzymes, or increased availability of the one or more of said co-factors, when compared to a parental microorganism.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to Provisional Application No. 61 / 831,591, filed June 5, 2013, the contents of which are hereby incorporated by reference. technical field [0003] The present invention relates to methods of increasing flux through fermentation pathways by optimizing enzymatic reactions. More specifically, the invention relates to identifying and resolving reaction bottlenecks in fermentation pathways. Background technique [0004] Acetogenic microorganisms are known to be useful in the production of fuels and other chemicals such as ethanol, butanol or butanediol by fermenting substrates including, for example, carbon monoxide, carbon dioxide, and hydrogen. [0005] Efforts to improve product concentration and substrate utilization to date have focused on strain selection, optimization of fermentation conditions and parameters, and optimization of media formulation or process conditions (Abubackar e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/04C12P7/06C12P7/16
CPCC12P7/065C12N15/52C12N9/0006C12N9/0008C12N9/1007C12P7/18C12Y101/01001C12Y102/0101C12Y102/07001C12Y102/07005Y02E50/10C12P7/06C12N15/74C12Y201/01196
Inventor M·考普克A·P·穆勒
Owner LANZATECH NEW ZEALAND LTD
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