Difference between revisions of "Team:Aachen/Lab/Methanol"

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Most of the annual production of methanol (ca. 70 million tons) is consumed by the chemical industry<ref>http://www.methanol.org/Methanol-Basics.aspx</ref>. Being predominantly obtained from natural gas, it is commonly used as a platform chemical with several applications.  
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Most of the annual production of methanol (70 million tons) is consumed by the chemical industry<ref>http://www.methanol.org/Methanol-Basics.aspx</ref>. Being predominantly obtained from natural gas, it is commonly used as a platform chemical with several applications.  
  
  
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We will approach to unleash this potential by establishing an methanol uptake pathway in ''E.&nbsp;coli'' to form higher metabolites.
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We will approach to unleash this potential by establishing a methanol uptake pathway in ''E.&nbsp;coli'' to form higher metabolites.
  
  
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{{Team:Aachen/Achievements|
 
{{Team:Aachen/Achievements|
 
* Building and testing a synthetic methanol assimilation pathway in ''E.&nbsp;coli''
 
* Building and testing a synthetic methanol assimilation pathway in ''E.&nbsp;coli''
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* Showing our modified ''E.&nbsp;coli'' strain to tolerate higher methanol concentrations  
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* Demonstrating that our modified ''E.&nbsp;coli'' strain can tolerate high methanol concentrations
 
* Characterizing the functional expression of ''Bacillus&nbsp;methanolicus'' methanol dehydrogenase 2 in ''E.&nbsp;coli''
 
* Characterizing the functional expression of ''Bacillus&nbsp;methanolicus'' methanol dehydrogenase 2 in ''E.&nbsp;coli''
 
* Developing an efficient cloning strategy to build a monocistronic diversity library applying the [[Team:Aachen/Notebook/Protocols#RDP_Assembly|RDP standard]]
 
* Developing an efficient cloning strategy to build a monocistronic diversity library applying the [[Team:Aachen/Notebook/Protocols#RDP_Assembly|RDP standard]]
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* 3-hexulose-6-phosphate synthase from ''Bacillus&nbsp;methanolicus'' (Hps)
 
* 3-hexulose-6-phosphate synthase from ''Bacillus&nbsp;methanolicus'' (Hps)
 
* 6-phospho 3-hexuloisomerase from ''Bacillus&nbsp;methanolicus'' (Phi)
 
* 6-phospho 3-hexuloisomerase from ''Bacillus&nbsp;methanolicus'' (Phi)
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* phosphoketolase from ''Bifidobacterium&nbsp;adolescentis'' (Xpk)
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* Xu5P - phosphoketolase from ''Bifidobacterium&nbsp;adolescentis'' (Xpk)
  
 
Using this enzymes, methanol can be converted to acetyl-CoA in an ATP independent way.
 
Using this enzymes, methanol can be converted to acetyl-CoA in an ATP independent way.
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To further optimize the effectivity of the MCC by tuning the expression levels of the four genes to an optimum, we developed an efficient cloning strategy for a [[Team:Aachen/Lab/Methanol/Monocistronic Diversity Library|monocistronic diversity library]] using the RDP standard by synbiota.
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To further optimize the efficiency of the MCC pathway by tuning the expression levels of the four genes to an optimum, we developed an efficient cloning strategy for a [[Team:Aachen/Lab/Methanol/Monocistronic Diversity Library|monocistronic diversity library]] using the RDP standard by synbiota.
  
 
=Results=
 
=Results=

Latest revision as of 03:54, 19 September 2015


Most of the annual production of methanol (70 million tons) is consumed by the chemical industry[1]. Being predominantly obtained from natural gas, it is commonly used as a platform chemical with several applications.


New innovative technologies impressively increased the efficiency of converting CO2 into methanol[2]. Concerning its price, availability and sustainability, it becomes an interesting alternative to conventional carbon sources in bioprocesses.

Therefore, methanol is a chemical with a huge potential not only for the chemical industry, but also for the bioeconomy.


We will approach to unleash this potential by establishing a methanol uptake pathway in E. coli to form higher metabolites.


Key Achievements

  • Building and testing a synthetic methanol assimilation pathway in E. coli
  • Demonstrating that our modified E. coli strain can tolerate high methanol concentrations
  • Characterizing the functional expression of Bacillus methanolicus methanol dehydrogenase 2 in E. coli
  • Developing an efficient cloning strategy to build a monocistronic diversity library applying the RDP standard


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Design

Few methanol assimilation pathways are known to exist in nature. From literature references, we identified the ribulose monophosphate pathway (RuMP) to be the most efficient naturally occuring one[3][4]. Recently, a modification of the RuMP was developed that theoretically improves the assimilation efficiency[5]. The results of our modeling confirmed the so called "major MCC" (Methanol Condensation Cycle) to be the most promising pathway.

From these literature references, we identified the four enzymes that we had to express heterologously in order to complete the uptake pathway:

Aachen MCC.png
Methanol Condensation Cycle
This figure shows the coherent enzymatic reactions of the MCC, published by Bogorad et al.[5]
  • methanol dehydrogenase 2 from Bacillus methanolicus (Mdh)
  • 3-hexulose-6-phosphate synthase from Bacillus methanolicus (Hps)
  • 6-phospho 3-hexuloisomerase from Bacillus methanolicus (Phi)
  • Xu5P - phosphoketolase from Bifidobacterium adolescentis (Xpk)

Using this enzymes, methanol can be converted to acetyl-CoA in an ATP independent way.


Four new BioBricks were designed, codon optimized for E. coli and synthesized as IDT gBlocks.

In order to express the required assimilation enzymes simultaneously, we designed a polycistronic expression construct and assembled it with Synbiota's[6] RDP Assembly method.


To further optimize the efficiency of the MCC pathway by tuning the expression levels of the four genes to an optimum, we developed an efficient cloning strategy for a monocistronic diversity library using the RDP standard by synbiota.

Results

All four assimilation enzymes were shown to be expressed individually in [http://parts.igem.org/Part:BBa_K1362091 pSB1A30] backbones. These BioBrick-compatible expression backbones made by team Heidelberg 2014 have an IPTG-inducible T7 promoter in front of the BioBrick prefix.

Since the Mdh represents the bottleneck of the MCC, we investigated and proved its functional expression using the Nash Assay.


The polycistronic expression construct was successfully assembled, sequenced and the expression was confirmed.


In shake flask experiments we showed that the strain harbouring the polycistronic expression plasmid is growing with increased maximal growth rate at higher methanol concentrations in the medium (Read more about our characterizations).


By feeding 13C labled methanol and analyzing the central metabolites, we finally investigated, if our strain incorporates carbon atoms from methanol into its metabolism (Read more about our labeling experiment).

References

  1. ↑ http://www.methanol.org/Methanol-Basics.aspx
  2. ↑ Wolfgang Michael Verdegaal, Sebastian Becker und Christian von Olshausen: "Power-to-Liquids: Synthetisches Rohöl aus CO2, Wasser und Sonne"
  3. ↑ Witthoff S, Schmitz K, Niedenführ S, Nöh K, Noack S, Bott M, Marienhagen J. Metabolic engineering of Corynebacterium glutamicum for methanol metabolism. Appl Environ Microbiol. 2015 Mar;81(6):2215-25. doi: 10.1128/AEM.03110-14. Epub 2015 Jan 16. PubMed PMID: 25595770; PubMed Central PMCID: PMC4345391.
  4. ↑ Schrader J, Schilling M, Holtmann D, Sell D, Filho MV, Marx A, Vorholt JA. Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria. Trends Biotechnol. 2009 Feb;27(2):107-15. doi: 10.1016/j.tibtech.2008.10.009. Epub 2008 Dec 26. Review. PubMed PMID: 19111927.
  5. ↑ Bogorad IW, Chen CT, Theisen MK, Wu TY, Schlenz AR, Lam AT, Liao JC. Building carbon-carbon bonds using a biocatalytic methanol condensation cycle. Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15928-33. doi: 10.1073/pnas.1413470111. Epub 2014 Oct 29. PubMed PMID: 25355907; PubMed Central PMCID: PMC4234558.
  6. ↑ https://synbiota.com/beta

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