Team:Osaka/Project pga

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<h2>Gamma polyglutamic acid</h2>
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<h2>Poly Gamma-Glutamic Acid</h2>
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<h3>Overview</h3>
<p>
<p>
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Gamma polyglutamic acid (γ-PGA) is an interesting biopolymer.
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<div style="float: left;margin: 10px">
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It can be found in "natto", a Japanese traditional food made of soybeans. Bacillus subtilis ferments soybeans and creates sticky-ness product, PGA, to protect itself from other microorganisms.
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<img src="https://static.igem.org/mediawiki/2010/6/6e/Natto2.png" height="180" alt="Fig.1/PGA" >
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It is a promising polymer, because it has many functions.(For instance, highly water-holding, edible, biodegradable...)</p>
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<img src="https://static.igem.org/mediawiki/2010/a/a5/Natto1.png" height="180" alt="Fig.2/PGA" >
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<img src="https://static.igem.org/mediawiki/2010/6/6e/Natto2.png" height="180" width="230" alt="Fig.3" >
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<img src="https://static.igem.org/mediawiki/2010/a/a5/Natto1.png" height="180" width="151" alt="Fig.2" >
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Poly (gamma-glutamic acid), also known as gamma polyglutamic acid (γ-PGA) or gamma polyglutamate, is a very interesting biopolymer that has attracted attention in recent years.
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<br>It is a sticky substance found in "natto", a traditional Japanese fermented food made from soybeans by <i>Bacillus subtilis</i> (formerly <i>Bacillus natto</i>).</p>
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<p>γ-PGA and its derivatives find a wide range of applications as thickeners, humectants, carriers for drug delivery, biodegradable fibers, highly water-absorbent hydrogels, biopolymer flocculants, and heavy metal absorbers<br>
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The high water absorbance of γ-PGA is enhanced by UV crosslinking. Coupled with its non-toxicity, this makes γ-PGA very useful in environmental applications.</p><br><br>
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<h3>The structure of γ-PGA</h3>
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<img src="https://static.igem.org/mediawiki/2010/3/3f/PGAstructure.gif" alt="Fig.3/PGA">
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<p>
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<br>
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Poly glutamate synthetase(PGS) is necessary to produce PGA in Escherichia coli. PGS gene includes three ORF (open reading flame),which are pgsB, pgsC, and pgsA.
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These ORFs code three essential enzymes to create PGA.
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</p>
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<img src="https://static.igem.org/mediawiki/2010/4/45/Structure_pga.png" width="268" height="127" alt="Fig.4">
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<p>γ-PGA is formed by dehydration-condensation reactions between the gamma-position carboxyl and alpha-position amino groups of adjacent glutamate molecules.<br>
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Glutamate molecules are chiral. Although most glutamate in nature exists in L-form, biologically-synthesized PGA also contains a certain proportion of D-glutamate. The ratio of L- to D-glutamate depends on many factors: species, culture condition and so on. These factors also affect the degree of polymerization.</p><br>
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<h3>Polyglutamate synthesis</h3>
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<img src="https://static.igem.org/mediawiki/2010/b/bb/TCA_cycle.png" width="273" height="636" alt="Fig.5">
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<img src="https://static.igem.org/mediawiki/2010/6/65/PGS.png" width="515" height="422" alt="Fig.6">
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<p>Three genes, pgsA, pgsB and pgsC, are required for a poly(gamma-glutamate) synthetic system of <i>B. subtilis</i> IFO 3336 (<i>B. natto</i>), as previously reported. <i>E. coli</i> transformed with these genes produced poly(gamma-glutamate) extracellularly. It was found that the genes encode the essential enzymes necessary to synthesize and secrete PGA. Although the roles of each enzyme are not known in detail, they are all extracellular in localization.<br>
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Another enzyme, glutamate racemase is also important for PGA biosynthesis. Without this enzyme, PGA synthesized by the <i>Bacillus</i> pgs genes is composed of only L-glutamate and in low quantity. Co-expression of pgsBCA genes and racemase increased both the polymer production and D-glutamate content.</p>
<p>
<p>
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PGA contains both D and L glutamate.
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Bacillus subtilis has enzymes to biodegrade PGA; however, E. coli does not. Therefore, we postulate that overproduction of PGA can be done with E. coli. This can hopefully be harnessed to aid the greening of deserts, based on its water-absorbent properties as discussed earlier.
</p>
</p>
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<p>
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<h3>Strategy/Results</h3>
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To increase the amount of PGA production, co-expression of glutamate racemase and PGS is needed.
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<p>We obtained a plasmids containing all the genes required, but these contained several point mutations and illegal restriction sites. Therefore we tried to make these into standard BioBrick form by Polymerase Chain Reaction (PCR). After some difficulties with PCR optimization, we finally succeeded in integrating these genes into BioBricks!!</p>
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</p>
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<h3>References</h3>
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<p>
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<ul>
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PGS is originally localized in cell membrane, and proposed to produce PGA from D,L-Glutamic acid. Bacillus subtilis has enzymes to biodegrade PGA; however, E. coli does not. Therefore, we want E. coli to produce PGA for greening of the desert.
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<li>Makoto Ashiuchi, Kenji Soda, and Haruo Misono Biochemical and Biophysical Research Communications 263, 6–12 (1999)</li>
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</p>
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<li>Ashiuchi M., Misono H. Appl Microbiol Biotechnol 59:9–14 (2002)</li>
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<img src="https://static.igem.org/mediawiki/2010/b/bb/TCA_cycle.png" width="273" height="636" alt="Fig.5">
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<li>Tanaka, T., Fujita, K., Takenishi, S., and Taniguchi, M. J. Ferment. Bioeng. 84, 361–364. (1997)</li>
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<img src="https://static.igem.org/mediawiki/2010/6/65/PGS.png" width="515" height="422" alt="Fig.6">
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<li>Akio Baba and Ikuya Shibata The Chemical Record, Vol. 5, 352–366 (2005)</li>
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<li>Ashiuchi M.; Kamei T.; Misono H. Journal of Molecular Catalysis B: Enzymatic 23 101–106 (2003)</li>
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<li>Ashiuchi M, Tani K, Soda K, Misono H. J. Biochem. 123, 1156-1163 (1998)</li>
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<li>Shih IL, Van YT. Bioresource Technology 79 207-225 (2001)</li>
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<li>生化学第８０巻第４号，pp.３１６―３２３，２００８</li>
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<li>D-グルタミン酸とポリ--グルタミン酸合成システム</li>
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Latest revision as of 03:09, 28 October 2010

Poly Gamma-Glutamic Acid

Overview

Fig.1/PGA Fig.2/PGA
Poly (gamma-glutamic acid), also known as gamma polyglutamic acid (γ-PGA) or gamma polyglutamate, is a very interesting biopolymer that has attracted attention in recent years.
It is a sticky substance found in "natto", a traditional Japanese fermented food made from soybeans by Bacillus subtilis (formerly Bacillus natto).

γ-PGA and its derivatives find a wide range of applications as thickeners, humectants, carriers for drug delivery, biodegradable fibers, highly water-absorbent hydrogels, biopolymer flocculants, and heavy metal absorbers
The high water absorbance of γ-PGA is enhanced by UV crosslinking. Coupled with its non-toxicity, this makes γ-PGA very useful in environmental applications.



The structure of γ-PGA

Fig.3/PGA

γ-PGA is formed by dehydration-condensation reactions between the gamma-position carboxyl and alpha-position amino groups of adjacent glutamate molecules.
Glutamate molecules are chiral. Although most glutamate in nature exists in L-form, biologically-synthesized PGA also contains a certain proportion of D-glutamate. The ratio of L- to D-glutamate depends on many factors: species, culture condition and so on. These factors also affect the degree of polymerization.


Polyglutamate synthesis

Fig.5 Fig.6

Three genes, pgsA, pgsB and pgsC, are required for a poly(gamma-glutamate) synthetic system of B. subtilis IFO 3336 (B. natto), as previously reported. E. coli transformed with these genes produced poly(gamma-glutamate) extracellularly. It was found that the genes encode the essential enzymes necessary to synthesize and secrete PGA. Although the roles of each enzyme are not known in detail, they are all extracellular in localization.
Another enzyme, glutamate racemase is also important for PGA biosynthesis. Without this enzyme, PGA synthesized by the Bacillus pgs genes is composed of only L-glutamate and in low quantity. Co-expression of pgsBCA genes and racemase increased both the polymer production and D-glutamate content.

Bacillus subtilis has enzymes to biodegrade PGA; however, E. coli does not. Therefore, we postulate that overproduction of PGA can be done with E. coli. This can hopefully be harnessed to aid the greening of deserts, based on its water-absorbent properties as discussed earlier.

Strategy/Results

We obtained a plasmids containing all the genes required, but these contained several point mutations and illegal restriction sites. Therefore we tried to make these into standard BioBrick form by Polymerase Chain Reaction (PCR). After some difficulties with PCR optimization, we finally succeeded in integrating these genes into BioBricks!!

References

  • Makoto Ashiuchi, Kenji Soda, and Haruo Misono Biochemical and Biophysical Research Communications 263, 6–12 (1999)
  • Ashiuchi M., Misono H. Appl Microbiol Biotechnol 59:9–14 (2002)
  • Tanaka, T., Fujita, K., Takenishi, S., and Taniguchi, M. J. Ferment. Bioeng. 84, 361–364. (1997)
  • Akio Baba and Ikuya Shibata The Chemical Record, Vol. 5, 352–366 (2005)
  • Ashiuchi M.; Kamei T.; Misono H. Journal of Molecular Catalysis B: Enzymatic 23 101–106 (2003)
  • Ashiuchi M, Tani K, Soda K, Misono H. J. Biochem. 123, 1156-1163 (1998)
  • Shih IL, Van YT. Bioresource Technology 79 207-225 (2001)
  • 生化学第８０巻第４号，pp.３１６―３２３，２００８
  • D-グルタミン酸とポリ--グルタミン酸合成システム
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