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Recombinant microorganism producing alkenes from acetyl-COA

A technology for recombining microorganisms and acetylation, applied in the direction of microorganisms, microorganisms, recombinant DNA technology, etc., can solve problems such as inappropriateness and ineffectiveness

Inactive Publication Date: 2017-08-18
GLOBAL BIOENERGIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these disclosed methods have limitations that make them ineffective or unsuitable, especially for large-scale industrial processes

Method used

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  • Recombinant microorganism producing alkenes from acetyl-COA
  • Recombinant microorganism producing alkenes from acetyl-COA
  • Recombinant microorganism producing alkenes from acetyl-COA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0488] Example 1: Production of methane, ethane, ethylene, propane, propylene, n-butane, 1-butene, 2-methylpropene, n-pentane, 1-pentene, n-hexane and 1-hexene

[0489] Construction of expression plasmid SG323 (and all other plasmids described herein) was performed by standard recombinant DNA and molecular cloning techniques using genes from Azotobacter vinelandii CA. Figure 4D Plasmid SG323 (SEQ ID No: 4) is shown, which contains the engineered molybdenum nitrogenase pathway. Electroporate this plasmid into C. ljungdahlii by the following procedure:

[0490] Genetic transformation of Clostridium ljungdahlii cells

[0491] Preparation of electrocompetent (electrocompetent) C. ljungdahlii cells: The method for preparing C. ljungdahlii electrocompetent cells was modified from a previously reported procedure ( , M. et al. Proc Natl Acad Sci U S A. (2010) 107, 29, 13087-13092). All manipulations except centrifugation were performed on ice in an anaerobic chamber. All buffers...

Embodiment approach

[0516] Plasmid SG193 ( Figure 4A and SEQ ID NO:1), plasmid SG211 ( Figure 4B and SEQ ID NO:2) and plasmid SG278 ( Figure 4Cand SEQ ID NO:3) were also used to engineer Clostridium ljungdahlii and Clostridium autoethanogenum cells to produce methane, ethane, ethylene, propane, propylene, n-butane, 1-butene, 2-methylpropene, n-pentane , 1-pentene, n-hexane. The same basic scheme and methods are followed as described in the preferred embodiment. Use the different substrates listed below:

[0517] For all substrates used, and those of the subsequent examples:

[0518] 60% CO, 10% CO 2 , 30%H 2

[0519] 100%CO

[0520] 30%CO 2 and 60%H 2

[0521] 60% CO, 10% CO 2 , 30%H 2 and electronics

[0522] 100% CO and Electronics

[0523] 100%CO 2 and electronics

[0524] 30%CO 2 and 60%H 2 and electronics

Embodiment 2

[0525] Example 2: Chromosomal integration via transposases in Clostridium and positive selection for multi-copy chromosomal integration

[0526] The following examples illustrate methods for integrating heterologous DNA (metabolic gene clusters or any other functional or non-functional nucleic acid sequence) into a given host or chassis microorganism. Positive selection (or direct genetic selection) of mutant bacteria is possible when the survival of recombinant bacteria is dependent on the presence or absence of a specific function encoded by the DNA introduced into the organism. An advantage of selection methods over screening methods is that bacteria harboring a particular mutation desired outgrow bacteria lacking the particular mutation greatly, thereby facilitating the identification of preferred mutants. Since all the functions required for transposition (such as the transposase and the integration cassette flanked by the transposition recognition base sequence) are intr...

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Abstract

Disclosed is a recombinant microorganism, comprising endogenous enzymes that convert CO and / or CO2 to acetyl-CoA. The recombinant microorganism contains a heterologous nucleic acid sequence encoding one or more enzymes that allow the conversion of acetyl-CoA to an alkene with a main chain of 1 to 5 carbon atoms. The heterologous nucleic acid sequence comprises one or more coding sequences encoding one or more enzymes that catalyse the conversion of acetyl-CoA to crotonyl-CoA, and that further catalyse the conversion of crotonyl-CoA to an alkene; or one or more coding sequences encoding one or more enzymes that catalyse the conversion of acetyl-CoA to 3-methylcrotonyl-CoA, and that further catalyse the conversion of 3-methylcrotonyl-CoA to an alkene; or one or more coding sequences encoding one or more enzymes that catalyse the conversion of acetyl-CoA to propionyl-CoA, and that further catalyse the conversion of propionyl-CoA to an alkene. Each coding sequence is operationally linked to a transcriptional promoter.

Description

[0001] Cross References to Related Applications [0002] This application claims the application "Genetically engineered microorganisms and methods for the conversion of gaseous C1-carbon sources and / or gaseous C1-carbon sources and Microorganism and process for converting gaseous C1-carbon sources and / or gaseous C1-carbon sources and electrons into alkenes)” rights and priorities. This application also claims the application filed with the USPTO on September 9, 2014 and officially assigned serial number US 62 / 047,827 "Genetically engineered microorganisms and the conversion of a gaseous C1-carbon source and / or a gaseous C1-carbon source and electrons to 1,3- Butadiene (Genetically engineered microorganism and process for converting gaseous C1-carbon sources and / or gaseous C1-carbon sources and electrons into 1,3-butadiene)" rights and priorities. This application also claims the application filed with the European Patent Office on September 26, 2014 and officially assigned the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P5/02
CPCC12N1/20C12P5/026C12N9/0006C12N9/001C12N9/1025C12N15/00C12N15/52Y02E50/30C12N1/00C12N15/63C12P7/02
Inventor B·盖特纳
Owner GLOBAL BIOENERGIES
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