Growth Parameters for Syringomycin Production in Pseudomonas syringae strain M1 by Nathaly M. Carranza (Faculty mentor: Jon Takemoto) ABSTRACT INTRODUCTION The cyclic lipodepsinonapeptide syringomycin E (SRE) produced by Pseudomonas syringae is being developed as a natural product agrofungicide. The purpose of this research is to identify the most effective P. syringae growth parameters for SRE production. P. syringae strain M1 was grown in 2L capacity fermenters and in capped test tubes both with agitation. A variety of compounds, including amino acids that are components of SRE, were added to the growth medium to study their effects on SRE production. L-histidine was found to be the best amino acid for promoting SRE production. Strain M1 extracts had strong antifungal activities. SRE production was most efficient when the bacteria were grown in capped test tubes than when incubated in aerated fermenters. Cyclic lipodepsinonapeptides (CLPs) are compounds that consist of an unbranched 3-hydroxy fatty acid connected to a lactone ring of nine amino acids (Takemoto et al. 2002). Figure 1 shows the structure of syringomycin E (SRE) which is a well-studied CLP. Strain M1 of the bacterium Pseudomonas syringae has been thoroughly studied for its ability to produce SRE (Takemoto et al. 2005). SRE is a very effective fungicide that acts on a large variety of fungal species including yeasts, and it acts on fungal cell membranes by making 2nm diameter pores. There is interest by the agrobiotechnology industry to develop and then use syringomycin-based materials as broad-spectrum crop fungicides. The purpose of this research is define growth parameters for optimal SRE production that will be useful for application in industry. -hydroxydodecanoic acid - SerSerDabDabArgPheDhbAsp(OH)Thr (Cl) \___________________________/ Figure 1. Structure of syringomycin E. Dab = 2,4-diaminobutanoic acid; Dhb = dehydroamino-butanoic acid. Virtis fermenter METHODS AND RESULTS 1. General Methods: SRE was extracted [2} and activity was determined by performing bioassays with Rhodotorula piliminae [1] and quantitated by HPLC [3}. Strain M1 was grown: 1) with rotary shaking in 20 mL test tubes with tightened screw caps and containing 3 mL of potato dextrose broth (PDB) + casamino acids medium, and 2) in 2 L capacity Virtis fermenters containing 1.2 L PDB medium and stirred with or without aeration. The inoculum volume for the fermenters was 20% of the total volume. 2. Effect of Aeration: Cultures grown in capped test tubes showed high antifungal activities whereas those grown with aeration in fermenters produce no SRE. A possible explanation for this was the difference in oxygen availability in the test tubes (oxygen limited) vs. the aerated fermenter cultures (oxygen rich). When aeration of fermenter cultures was stopped, low and inconsistent production of SRE occurred (Figure 2). 3. Effect of Medium Components: Single amino acids (10 mM to 15mM each), a combination of SRE component amino acids plus L-histidine (AA cocktail), and amino acid precursor, phosphoenolpyruvate (4 mM) were added to PDB medium in the fermenter to test for stimulation of SRE production.. Table 1 shows that L-histidine was best in stimulating the production of SRE in P.syringae strain M1 compared to other amino acids, the AA cocktail, and phosphoenolpyruvate. f tt Figure 2. Antifungal activity bioassay of strain M1 extracts from fermenter (f) and test tube (tt) cultures. Stronger antifungal activity from the test tube culture is observed. “i” is a blank control. Table 1. Effect of growth medium additions on SRE activity of cultures extracts of P. syringae strain M1 grown with aeration in Virtis fermenters 4. Quantifying SRE by HPLC . Extracts from test tube cultures were subjected to HPLC and found to contain around 28 μg of SRE per mL as determined by comparison to a standard curve (Figure 3 and 4). At this level and with large scale (1000 L) fermentative production it is possible to obtain around 3 kg of SRE. This amount would be sufficient for industrial purposes. Component(s) added L-His Arg Glu Phosphoenol-pyruvate AA Cocktail (Arg, Ser, Asp, Thr, Phe, His) Antifungal Activity ++ - REFERENCES 1.Gross, D. C., and J. E. DeVay. 1977. Production and purification of syringomycin, a phytotoxin produced by Pseudomonas syringae. Physiological Plant Pathology 11:15. 2. Takemoto, J. Y., B. J. G., Y. A. Kaulin, V. V. Malev, L. V. Schagina, and K. Blasko. 2002. The syringomycins. In Pore forming peptides and protein toxins, ed. G. Menestrina, M. Dalla Serra, P. Lazarovici, p. 260-271, Taylor and Francis, London 3.Takemoto, J. Y., I. Grgurina, M. Bensaci, G. Pocsfalvi, L. Mannina, O. Cruciani, A. Fiore, V. Fogliano, and K. N. Sorensen. 2005. Novel cyclic lipodepsipeptide from Pseudomonas syringae pv. lachrymans strain 508 and syringopeptin antimicrobial activities. Antimicrobial Agents and Chemotherapy 49:8. Figure 3. HPLC chromatogram of a test tube culture extract. The peak just before minute 15 reveals the presence of SRE. Figure 4. Standard curve for SRE quantitation by HPLC. The R-squared value for the curve is 0.9879 ACKNOWLEDGMENTS CONCLUSIONS The author thanks Dr. Jon Takemoto and Dr. Michelle Grilley for their constant supervision, guidance and teaching, and graduate student Yukie Kawasaki for her invaluable collaboration. SRE production by Pseudomonas syringae strain M1 is promoted by the presence of L-histidine (15mM) in PDB medium. SRE production levels are higher in strain M1 cultures incubated in agitated capped test tubes than in aerated fermenters. Oxygen availability may play a role in the degree of SRE production.