Difference between revisions of "Part:BBa K2963021"

Latest revision as of 02:27, 22 October 2021

pgsBCA- encoding a poly-γ-glutamic acid synthetase

PgsBCA complex, consisting of three subunits, is composed of PgsB、PgsC and PgsA. PgsB catalyzes poly-γ-glutamic acid synthesis．And PgsC links PgsB and PgsA in the membrane. While PgsA transports poly-γ-glutamic acid outside the cell. In our project, we use the PgsBCA complex to biosynthesize poly-γ-glutamic acid.

Usage and Biology

The BCA genes from Bacillus sp. encode a γ-PGA synthetase located on the cell membrane which is capable of polymerizing glutamic acid to form poly-γ-glutamic acid. In Bacillus subtilis, BCA are called pgsBCA. This part is used producing L-glutamate-rich γ-PGA.

Characterization

We used NMR to detect γ-PGA and HPLC to analyze L- glutamate ratio of γ-PGA. The results show we have successfully produced L-glutamate-rich γ-PGA.

By the NMR detection of the fermentation product, the specific hydrogen peaks a, b, and c on the γ-amide bond on the γ-polyglutamic acid could be detected at the corresponding time points. It is indicated that the synthetase pgsBCA genes of Bacillus subtilis heterologously expressed in Corynebacterium glutamicum, and the target product γ-polyglutamic acid is successfully produced.

We used HPLC to detect the L-glutamate monomer ratio of the γ-PGA we have produced. The results show that the L-glutamic acid monomer ratio reaches more than 90%. This part is working and we have produced L-glutamate-rich γ-PGA.

References

1. Xu P, Vansiri A, Bhan N, et al. ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli[J]. ACS Synthetic Biology, 2012, 1(7): 256-266.

2. Peng Yingyun. Study on the production, synthesis mechanism and antifreeze of γ-polyglutamic acid. Diss. Jiangnan University, 2015.

3. Sung M H, Park C, Kim C J, et al. Natural and edible biopolymer poly-gamma-glutamic acid: synthesis, production, and applications [J]. Chemical Record, 2005, 5(6): 352-366.

Contribution From CAU_China 2021

Group: CAU_China,2021 https://2021.igem.org/Team:CAU_China

Author: Geng Wanying & Xiong Liuchang

Summary:Literature information about other functions and the product of this part

Literature Information

We noticed that there was a lack of information on the Bacterial capsule and stress resistance in the registry. Despite some producting functions about pgsB(capB)、pgsC(capC) and pgsA(capA) , this lack of information will make the future team ignore the application potential of these genes. To facilitate the research of future iGEM teams, we added information about other functions of this part. We also supplemented information about its product.

γ-PGA is the mian product this part synthesize.It was first isolated from the capsule of Bacillus anthracis in 1937. It was found in Bacillus subtilis in 1942 and can be secreted into the culture medium as fermentation product. In both B. subtilis and B. anthracis, the membrane γ-PGA synthetic proteins encoded by the capB,capC,capA</i> operon catalyse synthesis of the capsule polypeptide.

B.subtilis can utilize both D- and L-glutamate as nitrogen sources. D-Glutamate catabolism by this bacterium proceeds after conversion to the L-form by glutamate racemases (the racE and yrpC products). Mutants of racE or yrpC accumulate D-glutamate in latestationary-phase cultures, indicating that B. subtilis cells degrade capsule γ-PGA into its constituent glutamates outside the cells, and utilize them as nitrogen sources during late stationary phase.

Besides, γ-Glutamyltransferase (EC 2.3.2.2; GGT) is widely distributed in nature, from bacteria to animals. For example, independent of the growth phase, Escherichia coli produces GGT in the periplasmic space to utilize γ-glutamylpeptides as amino acid sources.

There are reasons why organisms synthesize a specific secondary metabolite. Why do organisms synthesize γ-PGA?

The main functions of γ-PGA are as follows:

① Protective effect: the cell capsule of B. anthracis contains γ-PGA, which can help bacteria escape phagocytosis, enhance toxicity, make bacteria have non immune characteristics and prevent bacteria from being attacked by cellular antibodies.[9][10] In addition, it can also protect bacteria from phage infection and resist antimicrobial peptides;

② Tolerance to diverse environment: most bacteria synthesize PGA and secrete it into the environment, which can isolate toxic metal ions and reduce the high salt environment, so as to make bacteria tolerate the adverse environment.[11-13]

③ Energy reserve: it can be degraded into glutamic acid for cell growth when there is a lack of energy in the environment.[14]

④ Promote biofilm formation and related to motility.[15]

γ-PGA plays an important role in microbial stress resistance, which has been confirmed by many studies. For example, an research has demonstrated that S. epidermidis secretes poly-γ- DL -glutamic acid (PGA) to facilitate growth and survival in the human host. Importantly, PGA efficiently sheltered S. epidermidis from key components of innate host defense, namely ntimicrobial peptides and neutrophil phagocytosis, and was indispensable for persistence during device-related infection.[10]

These features of γ-PGA provide us with new usage of this part.

References For Contribution From CAU_China

1.Ivanovics G, Bruckner V.1937.Chemical and immunologic studies on themechanism of anthrax infection and immunity. The chemical structure of capsulesubstance of anthrax bacilli and its identity with that of the B. mesentericus[J].Z.Immunitaetsforsch, 90:304-318.
2.Ivanovics G,Bruckner V. 1937.The chemical nature of the immuno-specificcapsule substance of an thrax bacillus[J. Naturwissenschaften,25:250.
3. Bovarnick M. The formation of extracellular D(-)-glutamic acid polypeptideby Bacillus subtillis[J].Journal of Biological Chemistry,145:415-424.
4. Kimura, K. & Itoh, Y. (2003). Characterization of poly-c-glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly-c-glutamate. Appl Environ Microbiol 69, 2491–2497.
5. Kimura, K., Tran, L.-S. P. & Itoh, Y. (2004). Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis. Microbiology 150, 2911–2920.
6.Suzuki, H., Kumagai, H. & Tochikura, T. (1986). c-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties. J Bacteriol 168, 1325–1331.
7.Suzuki, H., Hashimoto, W. & Kumagai, H. (1993). Escherichia coli K-12 can utilize an exogenous c-glutamyl peptide as an amino acid source, for which c-glutamyltranspeptidase is essential. J Bacteriol 175, 6038–6040.
8. Kimura K, Tran L S P, Uchida l, et al. 2004. Characterization of Bacillussubtilis v-glut amyltransferase and its involvement in the degradation of capsulepoly--glutamate[J]. Microbiology, 150:4115-4123.
9. Mesnage s,Tosi-Couture E，Gounon P, et al. 1998. The capsule andS-Layer: two independent and yet compatible macromolecular structures in Bacillusan thracis[J].Journal of Bacteriology,180( 1):52-58.
10. Kocianov s, Vuong C, Yao Y, et al. 2005. Key role of poly-γ-DL-glutamicacid in immune evasion and virulence of Staphylococcus epidermidis. The Journalof Clinical Investigation,115:688-694.
11. Mclean R JC，Beauchemin D,Clapham L, et al. 1990.Metal-bindingcharacteristics of the gamma-Glutamyl capsular polymer of Bacillus licheniformis ATCC 9945[J].Applied and Environmental Microbiology,56(12):3671-3677.
12. Kandler o, Konig H, Wiegel J, et al. 1982. Occurrence of Poly-γ-D-Glutamic Acid and Poly-α-L-Glutamine in the Genera Xanthobacter, Flexithrix Sporosarcina and Planococcus[J].Systematic and Applied Microbiology,4:34—-41.
13. Minami H, Suzuki H, Kumagai H, 2004. γ-Glutamyltranspeptidase, but notYwrD,is important in utilization of extracellular glutathione as a sulfur source in Bacillus subtilis[J]. Journal of Bacteriology, 186:1213-1214.
14. Kimura K, Tran L S P, Uchida l, et al. 2004. Characterization of Bacillussubtilis v-glut amyltransferase and its involvement in the degradation of capsulepoly--glutamate[J]. Microbiology, 150:4115-4123.
15. Liu J, He D, Li XZ, et al. 2010.γ-Polyglutamic acid (γ-PGA) produced byBacillus amyloliquefaciens C06 promoting its colonization on fruit surface.International Journal of Food Microbiology,142:190-197.

Sequence and Features