Team:Osaka/Project pga

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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.
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.
<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>
<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><br>In this sticky product secreted by <i>B. subtilis</i>, γ-PGA is contained.γ-PGA and its derivatives offer a wide range of unique applications including being used as thickener, humectant, drug carrier, biodegradable fibers, highly water absorbable hydrogels, biopolymer flocculants, and heavy metal absorber. ( Ing-Lung Shih <i>et al,.</i> 2001<a href="http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V24-43F8HMH-1&_user=9152734&_coverDate=09/30/2001&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011799&_version=1&_urlVersion=0&_userid=9152734&md5=ca162614ee76294eb1dbebd32405ac00&searchtype=a#toc17">→</a>)</br>
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<p>γ-PGA and its derivatives offer a wide range of unique applications in thickeners, humectants, drug delivery carriers, biodegradable fibers, highly water-absorbent hydrogels, biopolymer flocculants, and heavy metal absorbers<br>
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Most famous products of PGA is highly water absorbable hydrogels. Its PGA is from <i>B.natto</i>. PGA from<i>B.natto</i> have high ability to absorb and hold water. This character is strengthened by UV-crosslink among PGA chains and very useful to environmental problems.
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The most important property of γ-PGA is its high water absorbance. This property is strengthened by UV-induced crosslinking. Coupled with its non-toxicity, this makes γ-PGAand very useful in environmental applications.</p>
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<br>Further more, PGA is very safe, not having toxicity to animals.</br><p>
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<h4>The structure of γ-PGA</h4>
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<img src="https://static.igem.org/mediawiki/2010/3/3f/PGAstructure.gif" alt="Fig.3/PGA">
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<h3>PGA STRUCTURE</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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<br>( Ing-Lung Shih <i>et al,.</i> 2001<a href="http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V24-43F8HMH-1&_user=9152734&_coverDate=09/30/2001&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011799&_version=1&_urlVersion=0&_userid=9152734&md5=ca162614ee76294eb1dbebd32405ac00&searchtype=a#toc17">→</a>)</br>
 
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<br>
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<p><br>It is Dehydration-condensation reaction between gamma-position carboxyl group and alpha-position amino group of glutamate to compose PGA . </br>
 
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Glutamate have chirality. Although almost glutamate exist in L-form in nature, PGA don't have only L-form Glutamate but also D-form Glutamate.The ratio with L and D depends on some factors, species, culture condition and so on. These factors effect on the degree of polymerization.</p><br>
 
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<h3>POLY GLUTAMATE SYNTHETASE</h3>
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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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<p>
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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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<br>Three genes ware required for a poly-gamma-glutamate synthetic system of <i>B. subtilis</i> IFO 3336 (<i>B. natto</i>), previously reported.The E. coli cloned these genes produced poly-gamma-glutamate extracellularly.( H. Misono <i>et al,.</i> 1999<a href="http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-45V7V30-3&_user=9152734&_coverDate=09/16/1999&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011799&_version=1&_urlVersion=0&_userid=9152734&md5=9cf8843cf0ce14d56845be2a633550c1&searchtype=a">→</a>)</br>
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Three genes, pgsB, pgsC, and pgsA, code three essential enzymes to creat and secrete PGA .Unfortunately, the roles of these enzymes are not confirmed, but the localizations are known, all extracellularly.
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</p>
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<h3>Polyglutamate synthesis</h3>
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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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glutamate racemase is also important enzyme for this synthesis because PGA made of only L-glutamate is synthesized without this enzyme. Therefore, Co-expression of pgsB,C,A genes and racemase increased both the polymer production and D-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.
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content in the polymer.( H. Misono <i>et al,.</i> 1999<a href="http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-45V7V30-3&_user=9152734&_coverDate=09/16/1999&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011799&_version=1&_urlVersion=0&_userid=9152734&md5=9cf8843cf0ce14d56845be2a633550c1&searchtype=a">→</a>)
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</p>
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<p>
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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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</p>
</p>
<img src="https://static.igem.org/mediawiki/2010/b/bb/TCA_cycle.png" width="273" height="636" alt="Fig.5">
<img src="https://static.igem.org/mediawiki/2010/b/bb/TCA_cycle.png" width="273" height="636" alt="Fig.5">
<img src="https://static.igem.org/mediawiki/2010/6/65/PGS.png" width="515" height="422" alt="Fig.6">
<img src="https://static.igem.org/mediawiki/2010/6/65/PGS.png" width="515" height="422" alt="Fig.6">
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<h3>STRATEGY / RESULT</h3>
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<h3>Strategy/Results</h3>
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<p>Our plasmid containing genes for four enzymes had several point mutations and restriction sites. These plasmid were contributed. Therefore We tried to make these sequence optimized for BioBricks with Polymerase Chain Reaction (PCR).However, we faced many difficulties about PCR.Finally, we succeeded to integrate these genes to BioBricks!! </p>
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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>
<h3>References</h3>
<h3>References</h3>
<ul>
<ul>

Revision as of 00:17, 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 offer a wide range of unique applications in thickeners, humectants, drug delivery carriers, biodegradable fibers, highly water-absorbent hydrogels, biopolymer flocculants, and heavy metal absorbers
The most important property of γ-PGA is its high water absorbance. This property is strengthened by UV-induced crosslinking. Coupled with its non-toxicity, this makes γ-PGAand 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

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.

Fig.5 Fig.6

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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