Due to this versatile application power, the quantity needs of violacein and its derivative will increase in the near future. However, this positive prediction of the application, the production of violacein and its derivative is only in the initial phase. The crude violacein containing violacein and its derivative specific productivity is reported 12.4 mg/L∙h and its final concentration is 0.44 g/L using the natural violacein-producing host C.violaceum.
Therefore, more studies are needed in the field of violacein production to increase productivity and standardize the measurement of crude violacein.
Violacein: Properties and Production of a Versatile Bacterial Pigment
- Summary
- Natural Violacein-Producing Strains and Their Locales are Quite Diverse
- Violacein as an Indicator of Quorum Sensing
- Violacein’s Function in Nature - Predator Defense?
- Production of Violacein by Natural Host Strains
- Production of Violacein within E. coli and Other Heterologous Hosts
- Deoxyviolacein and Oxyviolacein
- Conclusions
In most violacein-producing bacterial strains isolated from nature, this bisindole is a secondary metabolite associated with biofilm production5. B2 was able to produce 1.6 g/l of crude violacein, usually referring to the naturally produced mixture of violacein and deoxyviolacein. However, the impact of violacein was dose-dependent as larger additions of this bisindole led to greater concomitant losses in HepG2 viability.
As presented in this report, the production and characterization of violacein is not without its own obstacles and struggles, and much work remains to be done.
Isolation, Identification and Characterization of Violacein-Producing Pseudoduganella sp
Summary
Introduction
Materials and Methods
- Isolation of Pseudoduganella sp. NI28
- Bacterial Strains and Growth
- Identification of Pseudoduganella sp. NI28 by 16S using phylogenetic markers
- Fatty Acid Methyl Ester Preparation and Analysis
- Phenotypic Assessment of P. sp. NI28
- Extraction and HPLC Analysis of the Violacein Produced by P. sp. NI28
- Standard curve of Commercial Violacein with HPLC and Spectrophotometry Analysis
- Violacein Killing of Staphylococcus aureus
- Co-culture of violacein producing strain with Staphylococcus aureus
- Violacein Gene Cloning
- Data Analysis
NI28 Violacein producer This study Pseudoduganella violaceinigra Violacein producer YIM 31327 Janthinobacterium lividum Violacein producer ATCC 12473 Chromobacterium pseudoviolaceum Violacein producer ATCC 7461. To prepare the samples, several colon samples were transferred with #1 mL reagents of NaOH (1 mL) (ACS Grade, Sigma-Aldrich Aldrich, USA), 150 ml HPLC grade methanol (Sigma-Aldrich, USA) and 150 ml deionized distilled water) were added. To initiate methylation of the fatty acids, 2 ml of Reagent #2 (325 ml of 6 N HCl (Sigma-Aldrich, USA) mixed with 275 ml of HPLC Grade methanol (Sigma-Aldrich, USA)) was added to each tube which was vortexed and then incubated at 80ºC for 10 minutes.
In each tube, 1.25 mL of Reagent #3 (prepared using an equal volume of HPLC grade hexane (Sigma-Aldrich, USA) and HPLC grade methyl tert-butyl ether (Sigma-Aldrich, USA)) was added and the contents stirred gently. end-over-end for 10 minutes using a rolling pin. NI28 and YIM 31327 were performed using API 20NE and API ZYM test kits (bioMerieux, France) according to the protocol suggested by the manufacturer. After growth for 0, 24, and 48 h, 1 mL of the culture was pelleted by centrifugation for one minute at 16,200 xg and room temperature.
This value was used to calculate the concentration of violacein using an extinction coefficient of 73.4 L mg-1 cm-1 43. The mobile phase was 50% denatured ethanol (HPLC Grade, Sigma-Aldrich, USA) and detection was performed at 575 nm using an Agilent 1260 Infinity ELSD. The concentration of violacein within the ethanol extracts was determined as described above using violacein purchased from Sigma-Aldrich (USA, Cat.
The extracted violacein was concentrated by drying several preparations under nitrogen gas and resuspending the violacein in DMSO. The violacein gene was cloned by primers VPA3 and VPA4, what was used for vioA sequencing, vioB, vioC and vioD were cloned by primers VioASeqF (5'-CCCAAGCGGCA TCATCGCCTGC-3') and VioBSeqR (5'-CGCCGTACCACCATCATC VioBSeqF1 (5'- TTCGGCCTGGACACG GAATTTGC- 3') and VioDSeqR(5' –CACAGSGTSACGCCGGTGCTCAT- 3') for vioC, vioDNF (5'-AACATATGAAAATCCTCGTCATCGG-3') and vioDXGATCGAGGCGTGG-CCCGATCGAGGTAGG-CCCGATCGAGGTAGG cloning which primer was designed by J.
Results
- Isolation and Identification of Pseudoduganella sp. NI28
- P. sp. NI28 is Phenotypically Variant from P. violaceinigra YIM 31327
- Pseudoduganella sp. NI28 is a Prolific Violacein Producer
- Standard Curve of Commercial Violacein with Spectrophotometry and HPLC Analysis
- Pseudoduganella sp. NI28 Violacein is Bacteriocidal Towards Staphylococcus aureus
- Pseudoduganella sp. NI28 violacein gene cluster sequencing
NI28 cultures showing the predominant presence of violacin (7.9 min) and a small amount of deoxyviolazine (13.5 min). However, of all the strains tested, the new isolate was the best violacein producer, especially when grown for 24 hours (Figure 2.5B). This rapid and high level production by P. NI28 in NB clearly distinguishes this race from the other strains, especially its closest relative, P. violaceinigra YIM 31327, which was the weakest violacein producer tested in this study.
When the amount of violacein produced by all six strains was normalized using the OD, the newly isolated P. This was especially true for the 24 h sample, where the yield was 6.1 mg violacein/OD, a value that was more than that double that obtained for the next closest strain, C. NI28's high production of violacein implies that this strain contains more. Note the faster colony development and violacein production by P. B) Growth of both strains in NB media confirming that P. The data for each strain were obtained from three independent cultures.
Concentration of violacein in each culture at the same time points, demonstrating the rapid and high formation of violacein by P. NI28 compared to the other strains. The relative concentration of violacein, based on culture density, illustrates that P. NI28 produced the largest yields. violacein per cell than the other strains, a finding that may help facilitate the downstream purification of violacein. The standard curve of spectrophotometry analysis is shown in Figure 2.6A. The measured extinction coefficient of commercial violacein is 68.4 L mg-1 cm-1.
HPLC analysis for the quantification of pure violacein in crude violacein is performed with commercial violacein containing a small amount of deoxyviolacein (Figure 2.6B). Thus, for the HPLC standard curve, only the peak integration value of violacein is calculated. Several recent papers highlighted the activity of violacein against the human pathogen Staphylococcus aureus and showed that a concentration of 17 µM was able to inhibit the growth of this strain32,51. The optical density of co-culture is increased for both Pseudoduganella species in Figure 2.8A, rather than for J.
-culture of the violacein-producing strain with S. A) Single culture and co-culture S. Single culture was measured to compare the co-culture.
Discussion
NI28, when used at a slightly lower concentration, not only inhibited but actually caused a significant loss of S. Compared to initial CFU numbers, a 24-hour treatment with 5 µg/mL, or about 15 mM, resulted in a 69% reduction in viability in the ATCC strain and a 78% reduction in in a clinical isolate. Although it is not clear why the preparation in this study appears to be more potent, the data clearly indicate that violacein purified from cultures of P.
It shows that violacein production affects the growth of S.aureus not only violacein in the media but also in the cell. Therefore, there is a possibility that improved violacein production by NI28 than type strains aids the competition between Gram-positive strains and violacein-producing strains. In conclusion, a new violacein-producing strain was isolated from a forest soil sample in Ulsan, South Korea.
Although the concentrations produced in this study are low, a group recently reported achieving as much as 0.82 g/L with C. NI28 was generally superior to the other strains tested in this study, and expanding its growth and violacin production through similar techniques is likely to increase yields. Moreover, other recent studies have shown the successful expression of the vioABCDE operon in other bacterial hosts to produce even higher levels of violacin.
NI28 is an attractive strain for violacein production and for further studies, especially for cloning, sequencing and heterologous expression of its vioABCDE genes. Fermentative production of deoxyviolacein and construction of its producer strain from the improved Escherichia L-Tryptophan synthesis pathway.
Fermentative Production of Deoxyviolacein and Construction of Its Producing Strain from
- Summary
- Introduction
- Material and Methods
- Transformation of violacein production strain
- Microbial production extract and purification of crude violacein
- Violacein quantification and purity measurement with HPLC
- Recombinant plasmid for converting violacein production
- Violacein derivation fermentation
- Results and discussions
- Recombinant strain violacein production strain
- Fermentation of crude violacein production with GPT vio and GPT pCOM10vio
- HPLC purity analysis of crude violacein from GPT vio and pCOM10 vio
- vioD gene cloning and its crude violacein HPLC analysis
- Conclusion
Recent study of the production of violacein is optimization of natural violacein producing stem culture, recombinant violacein production is not activated. Even the early stage of violacein production, this confusing standard is delayed violacein production study and its application. In this study, we improved specific deoxyviolasin production for pre-stage of violasin production using tryptophan overproducing E.coli strains and recombinant plasmid for conversion of deoxyviolasin to violasin production.
A 1% starter inoculum is used for the second culture to produce violacein GPT via violacein. Violacein GPT pCOM10vio requires 25 °C for violacein production, so the second culture is performed at this temperature. Crude violacein extracted from GPT vio contained deoxyviolacein, an additional plasmid is required to convert deoxyviolacein into violacein production.
The transformed violacein-producing strain GPT vio prepared with the plasmid BBa J72214 BBa J72090 showed that the colony was black colored due to the production of crude violacein. Violacein production is also delayed due to low temperature conditions for violacein production pCOM10 vio needs 25 °C. When violacein production is rapid, the pH of the violacein-producing strain contained in the media shows an increasing tendency.
Nevertheless, this possibility of violacein production with high purity inside recombinant strain is shown GPT pCOM10 vio, crude violacein production by GPT vio partial deoxyviolacein. Nevertheless, fastidious mode reenact of pCOM10vio violacein production to avoid vioD denaturation in different temperatures. For this reason, violacein production will be supported for mass production conversion by its use.
One of attractive point in violacein production is easy to establish, because of the production only need five continuous genes and its expression with tryptophan.
Concluding Remarks
Summary and Conclusions
Further study
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Yanischperron, C.; Vieira, J.; Messing, J., Improved M13 phage cloning vectors and host strains - Nucleotide sequences of the M13mp18 and Puc19 vectors. M; Wittmann, C., Systems Metabolic Engineering of Escherichia Coli for the gram-scale production of the antitumor drug Deoxyviolacein from glycerol.
