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Characterization of Violacein Producing Host Pseudoduganella sp. NI28 and Production of Deoxyviolacein with Recombinant Escherichia coli Strain

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Title
Characterization of Violacein Producing Host Pseudoduganella sp. NI28 and Production of Deoxyviolacein with Recombinant Escherichia coli Strain
Author
Choi, Seong Yeol
Advisor
Mitchell, Robert J.
Keywords
Violacein; Deoxyviolacein
Issue Date
2015-08
Publisher
Graduate School of UNIST
Abstract
Violacein is bacteria pigment made several strain such as J.lividum, C.violaceum, Duganella sp., Pseudoduganella sp., Collimonas sp. which found various environment from glacial to subtropical climate. The usage of violacein in the bio-application in the field is diverse to block growth of several type of living organism: gram positive bacteria, fungus, virus, protozoa, nematodes, parasites and some cancer cell line like Hep2, KM12, MOLT-4. Because of this versatile application potent, needs of amount of violacein and its derivation will be increase near future. Nevertheless this positive application prediction, the production of violacein and its derivation is just beginning stage. The crude violacein contained violacein and its derivation specific productivity is reported 12.4mg/L?h and its final concentration is 0.44g/L using C.violaceum natural violacein producing host. In recombinant strain violacein producing field, recent reported recombinant E. coli produce pure violacein 0.710g/L, however, specific productivity is 3.1mg/ L?h. On the other hands violacein measurement method is not standardized, spectrophotometry is generally used for measurement violacein but extinction coefficient doesn’t have unity every study. Moreover purity of crude violacein is also questionable without HPLC analysis. Thus violacein production field need more study for enhance productivity and standardizing measurement of crude violacein. Characterization of violacein producing strain Pseudoduganella sp. NI28 found forest soil sample. P. sp. NI28 is similar to Psedoduganella violanceinigra YIM 31327 98.8% for 16s rRNA, 95.9% for gyrB, 88.8% for vioA. Even this similarity NI28 shown better crude violacein production tendency in NB media two Pseudoduganella strains shown significantly different production of crude violacein, NI28 produced 14mg/L but type strain produced under 0.1mg at 24 hours culture. Following HPLC analysis, NI28 crude violacein contain more than 95% violacein in the extracted violacein almost similar purity of commercial violacein (Sigma Aldrich. USA). Antibacterial effect of extracted violacein from NI28 shown 78%, 96% and 99.5% S. aureus population decrease in violacein concentration 15μM, 30μM and 60μM. Co-culture of violacein producing strain with S. aureus was tested with NI28 and type strain in the NB media using violacein productivity difference, violacein producing is more helpful to compete co-culture condition to eliminate viability of S. aureus in the media. For enhance of production of violacein, E. coli GPT vio and E. coli GPT pCOM10vio transformed tryptophan over-producing E. coli was produced crude violacein in 37°C fermentation. GPT vio produce more 95% concentration of deoxyviolacein form HPLC analysis. The final concentration of deoxyviolacein form GPT vio is 584.8mg/L, specific productivity of 24 hours fermentation is 20.91mg/L?h which was 1.65 fold high specific productivity compare than recent reported strain of deoxyviolacein productivity.
Description
Department of Biological Sciences
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