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                    <p>Your place:&nbsp;<a href="https://2015.igem.org/Team:Nankai">Home</a>&nbsp;&gt;&nbsp;<a href="https://2015.igem.org/Team:Nankai/Description">Project</a></p>
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<h2 class="page-title">Project Description</h2>
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<h4>What is γ-PGA?</h4>
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<p>Poly-γ-glutamic acid (γ-PGA) is an important, naturally occurring polyamide consisting of D/L-glutamate monomers. Unlike typical peptide linkages, the amide linkages in γ-PGA are formed between the α-amino group and the γ-carboxyl group. γ-PGA exhibits many favorable features such as biodegradable, water soluble, edible and non-toxic to humans and the environment. Therefore, it has been widely used in fields of foods, medicines, cosmetics and agriculture and many unique applications, such as a sustained release material and drug carrier, curable biological adhesive, biodegradable fibres, and highly water absorbable hydrogels.</p>
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<h4>How can we produce it?</h4>
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<p>Strains capable for producing γ-PGA are divided into two categories based on their requirement for glutamate acid: glutamate-dependent strains and glutamate-independent strains. Glutamate-independent strains are preferable for industrial production because of their low cost and simplified fermentation process. However, compared with glutamate-dependent strains, their lower γ-PGA productivity limits their industrial application.Therefore, the construction of a glutamate-independent strain with high γ-PGA yield is important for industrial applications.</p>
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<h4>Who can produce it?</h4>
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<p>Bacillusamyloliquefaciens LL3, isolated from fermented food, is a glutamate-independent strain, which can produce 3-4 g/L γ-PGA with sucrose as its carbon source and ammonium sulfate as its nitrogen source. The B. amyloliquefaciens LL3 strain was deposited in the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 208109 and its whole genome has been sequenced in 2011. In this study, we aimed to improve the γ-PGA production based on the B. amyloliquefaciens NK-1 strain (a derivative of LL3 strain with its endogenous plasmid and upp gene deleted).</p>
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<h4>What did we do?</h4>
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<p>In order to improve γ-PGA production, we employed two strategies to fine-tune the synthetic pathways and balance the metabolism in the glutamate-independent B. amyloliquefaciens NK-1 strain. Firstly, we constructed a metabolic toggle switch in the NK-1 strain to inhibit the expression of ODHC (2-oxoglutarate dehydrogenase complex) by adding IPTG in the stationary stage and distribute the metabolic flux more frequently to be used for γ-PGA precursor-glutamate synthesis. As scientists had found that the activity of ODHC was rather low when glutamate was highly produced in a Corynebacterium glutamicum strain. Second, to balance the increase of endogenous glutamate production, we optimized the expression level of pgsBCA genes (responsible for γ-PGA synthesis) by replacing its native promoter to seven different strength of promoters. Through these two strategies, we aimed to obtain a γ-PGA production improved mutant strain.<a href="https://2015.igem.org/Team:Nankai/Experiments">Click for more detail.</a></p>
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<h6><a href="https://2015.igem.org/Team:Nankai/Description">Description</a></h6>
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<h6><a href="https://2015.igem.org/Team:Nankai/Experiments">Experiments & Protocols</a></h6>
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<h6><a href="https://2015.igem.org/Team:Nankai/Results">Results</a></h6>
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<h6><a href="https://2015.igem.org/Team:Nankai/Design">Design</a></h6>
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                                                <p>Preparing for LB medium.</p>
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                                                <p>Cultured LL3.</p>
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                                                <p>In the progress of fermentation.</p>
  
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<h6>References</h6>
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<p>1. Ashiuchi, M., Misono, H., 2002. Biochemistry and molecular genetics of poly-γ-glutamate synthesis. Appl. Biochem. Biotechnol. 59, 9–14.</br>
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2. Kunioka, M., 1997. Biosynthesis and chemical reactions of poly(amino acid)s from
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microorganisms. Appl. Microbiol. Biotechnol. 47, 469–475.</br>
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3. Shih, I.L., Van, Y.T., 2001. The production of poly(γ-glutamic acid) from microorganism and its various applications. Bioresour. Technol. 79, 207–225.</br>
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4. Li, C., 2002. Poly(L-glutamic acid)--anticancer drug conjugates. Adv. Drug Deliver. Rev. 54, 695–713.</br>
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5. Liang, H.F., Chen, C.T., Chen, S.C., Kulkarni, A.R., Chiu, Y.L., Chen, M.C., Sung, H.W., 2006. Paclitaxel-loaded poly(γ-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system for the treatment of liver cancer. Biomaterials. 27, 2051–2059.</br>
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6. Richard, A., Margaritis, A., 2001. Poly (glutamic acid) for biomedical applications. Crit. Rev. Biotechnol. 21, 219–232.</br>
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7. Park, Y.J., Liang, J., Yang, Z., Yang, V.C., 2001. Controlled release of clot-dissolving tissue-type plasmmogen activator from a poly(L-glutamic acid) semi-interpenetrating polymer network hydrogel. J. Control. Release. 74, 243–247.</br>
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8. Cao, M.F., Geng, W.T., Liu, L., Song, C.J., Xie, H., Guo, W.B., Jin, Y.H., Wang, S.F., 2011. Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes. Bioresour. Technol. 102, 4251–4257.</br>
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9. Geng, W.T., Cao, M.F., Song, C.J., Xie, H., Liu, L., Yang, C., Feng, J., Zhang, W., Jin, Y.H., Du, Y., Wang, S.F., 2011. Complete genome sequence of Bacillus amyloliquefaciens LL3, which exhibits glutamic acid-independent production of poly-γ-glutamic acid. J. Bacteriol. 193, 3393–3394.</br>
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10. Feng, J., Gao, W.X., Gu, Y.Y., Zhang, W., Cao, M.F., Song, C.J., Zhang, P., Sun, M., Yang, C.,  Wang, S.F., 2014a. Functions of poly-gamma-glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance. Appl. Microbiol. Biotechnol. 98, 6397–6407.</br>
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11. Uy, D., Delaunay S., Germain, P., Engasser, J.M., Goergen, J.L. 2003. Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process. J. Biotech. 104, 173-184.</p>
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Revision as of 11:52, 5 September 2015

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Project Description

What is γ-PGA?

Poly-γ-glutamic acid (γ-PGA) is an important, naturally occurring polyamide consisting of D/L-glutamate monomers. Unlike typical peptide linkages, the amide linkages in γ-PGA are formed between the α-amino group and the γ-carboxyl group. γ-PGA exhibits many favorable features such as biodegradable, water soluble, edible and non-toxic to humans and the environment. Therefore, it has been widely used in fields of foods, medicines, cosmetics and agriculture and many unique applications, such as a sustained release material and drug carrier, curable biological adhesive, biodegradable fibres, and highly water absorbable hydrogels.

How can we produce it?

Strains capable for producing γ-PGA are divided into two categories based on their requirement for glutamate acid: glutamate-dependent strains and glutamate-independent strains. Glutamate-independent strains are preferable for industrial production because of their low cost and simplified fermentation process. However, compared with glutamate-dependent strains, their lower γ-PGA productivity limits their industrial application.Therefore, the construction of a glutamate-independent strain with high γ-PGA yield is important for industrial applications.

Who can produce it?

Bacillusamyloliquefaciens LL3, isolated from fermented food, is a glutamate-independent strain, which can produce 3-4 g/L γ-PGA with sucrose as its carbon source and ammonium sulfate as its nitrogen source. The B. amyloliquefaciens LL3 strain was deposited in the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 208109 and its whole genome has been sequenced in 2011. In this study, we aimed to improve the γ-PGA production based on the B. amyloliquefaciens NK-1 strain (a derivative of LL3 strain with its endogenous plasmid and upp gene deleted).

What did we do?

In order to improve γ-PGA production, we employed two strategies to fine-tune the synthetic pathways and balance the metabolism in the glutamate-independent B. amyloliquefaciens NK-1 strain. Firstly, we constructed a metabolic toggle switch in the NK-1 strain to inhibit the expression of ODHC (2-oxoglutarate dehydrogenase complex) by adding IPTG in the stationary stage and distribute the metabolic flux more frequently to be used for γ-PGA precursor-glutamate synthesis. As scientists had found that the activity of ODHC was rather low when glutamate was highly produced in a Corynebacterium glutamicum strain. Second, to balance the increase of endogenous glutamate production, we optimized the expression level of pgsBCA genes (responsible for γ-PGA synthesis) by replacing its native promoter to seven different strength of promoters. Through these two strategies, we aimed to obtain a γ-PGA production improved mutant strain.Click for more detail.

Description
Experiments & Protocols
Results
Design

    Preparing for LB medium.

    Cultured LL3.

    In the progress of fermentation.

    References

    1. Ashiuchi, M., Misono, H., 2002. Biochemistry and molecular genetics of poly-γ-glutamate synthesis. Appl. Biochem. Biotechnol. 59, 9–14.
    2. Kunioka, M., 1997. Biosynthesis and chemical reactions of poly(amino acid)s from microorganisms. Appl. Microbiol. Biotechnol. 47, 469–475.
    3. Shih, I.L., Van, Y.T., 2001. The production of poly(γ-glutamic acid) from microorganism and its various applications. Bioresour. Technol. 79, 207–225.
    4. Li, C., 2002. Poly(L-glutamic acid)--anticancer drug conjugates. Adv. Drug Deliver. Rev. 54, 695–713.
    5. Liang, H.F., Chen, C.T., Chen, S.C., Kulkarni, A.R., Chiu, Y.L., Chen, M.C., Sung, H.W., 2006. Paclitaxel-loaded poly(γ-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system for the treatment of liver cancer. Biomaterials. 27, 2051–2059.
    6. Richard, A., Margaritis, A., 2001. Poly (glutamic acid) for biomedical applications. Crit. Rev. Biotechnol. 21, 219–232.
    7. Park, Y.J., Liang, J., Yang, Z., Yang, V.C., 2001. Controlled release of clot-dissolving tissue-type plasmmogen activator from a poly(L-glutamic acid) semi-interpenetrating polymer network hydrogel. J. Control. Release. 74, 243–247.
    8. Cao, M.F., Geng, W.T., Liu, L., Song, C.J., Xie, H., Guo, W.B., Jin, Y.H., Wang, S.F., 2011. Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes. Bioresour. Technol. 102, 4251–4257.
    9. Geng, W.T., Cao, M.F., Song, C.J., Xie, H., Liu, L., Yang, C., Feng, J., Zhang, W., Jin, Y.H., Du, Y., Wang, S.F., 2011. Complete genome sequence of Bacillus amyloliquefaciens LL3, which exhibits glutamic acid-independent production of poly-γ-glutamic acid. J. Bacteriol. 193, 3393–3394.
    10. Feng, J., Gao, W.X., Gu, Y.Y., Zhang, W., Cao, M.F., Song, C.J., Zhang, P., Sun, M., Yang, C., Wang, S.F., 2014a. Functions of poly-gamma-glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance. Appl. Microbiol. Biotechnol. 98, 6397–6407.
    11. Uy, D., Delaunay S., Germain, P., Engasser, J.M., Goergen, J.L. 2003. Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process. J. Biotech. 104, 173-184.

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