Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 9, Num. 1/02, 1998

FATTY ACID PROFILE OF A PHOSPHATE-DEREGULATED AND ANTIBIOTIC HYPERPRODUCER MUTANT OF STREPTOMYCES HYGROSCOPICUS

V. GESHEVA and R. RACHEV

Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Code Number:AC98001
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ABSTRACT.

Fatty acid profiles of a phosphate-deregulated and antibiotic hyperproducer mutant of Streptomyces hygroscopicus showed notable differences in respect to the parent strain. It is suggested that the higher content of saturated branched fatty acids in the mutant strain may be associated with membrane permeability and altered antibiotic synthesis.

Considerable attention has been devoted to the occurrence and synthesis of fatty acids in antibiotic producing streptomycetes. The ratio of branched iso and anteiso fatty acids is thought to affect membrane function (Vancura et al., 1987; David et al., 1992, Rezanka et al., 1992; Adamidis and Sherman, 1995). Streptomyces hygroscopicus, strain 111-81, produces many antibiotics (Gesheva et al., 1985) among which the nonpolyenic macrolide complex AK-111- 81 with antibacterial and antifungal activity. It has been shown that some phosphate-deregulated mutants overproduce the AK-111-81 complex (Gesheva, 1994) and an investigation has been carried out to determine the fatty acid profile.

MATERIALS and METHODS

Organisms and cultivation. Strain 111-81 of S. hygroscopicus and its phosphate deregulated mutant, strain 84, were grown in 500 ml Erlenmeyer flasks on a rotary shaker (220 rpm) for 40 hrs at 28 C and then the cultures were used to inoculate other flasks and harvested at the mid- exponential phase. Medium composition was as follows (g/l): glucose, 30; KNO3, 1; K2HPO4, 0.5; MgSO4, 1; CaCO3, 1.

Antibiosis. Antibiotic production was analysed by thin-layer chromatography according to Gesheva (1994), using AK-111-81 as standard. Activity was measured by the agar diffusion method against Candida utilis.

Fatty acid analysis. The mycelium was harvested by centrifugation and washed twice with distilled water. Lipids were extracted three times with chloroform:methanol (2:1, v/v). The extracts were mixed with a 1% solution of NaCl and the lower layer separated and dried under reduced pressure. Lipids were transesterified with 14% BF^3 in methanol. Gas chromatography was carried out in a C. Erba Science 4300 chromatograph under the following operating conditions: WCOT column DB5 (Varian); 25 m x 0.22 mm x 0.2 um; initial temperature 90 C (5 min); final temperature 220 C (5 min); injection and detection temperatures 240 C; program rate 5 C; detector FID. Fatty acids were identified by comparison of retention time of standards (Polyscience, Co., USA).

RESULTS and DISCUSSION

Fatty acid composition of strains 111-81 and 84 is shown in Table 1.

Table 1. Percentage of fatty acids in strains 111-81 and 84 of S. hygroscopicus. (ai: anteiso acid; i: iso acid; sc: saturated straight- chain acid; u: unsaturated acid; x: unidentified)

----------------------------------------
Fatty acid    Strain 111-81    Strain 84
----------------------------------------
11:0             -              0.20
12:1            2.66            0.71
12:0            0.62            0.54           
i-13:0           -              0.21           
ai-13:0          -              0.54           
13:1            0.66            0.83           
13:0            0.59            1.26           
i-14:0          1.02            0.68           
14:1            1.11            1.20           
14:0            0.72            1.28           
i-15:0          1.83            7.95         
ai-15:0         8.31            6.82         
15:1            1.20            0.79         
 x              1.78            1.44         
i-16:0          4.64           10.16         
16:1            4.78            2.64         
10:0           13.23           16.32         
i-17:0          0.78            2.59         
ai-17:0         2.73            1.59         
17:1            1.50            1.29         
17.0             -              2.97         
i-18:0          0.69             -         
18:1           33.18           35.24         
18:8            4.92            3.05         
19:1             5.11            -         
20:0             0.13            -         
21:0             0.92            -         
i-21:0           3.00            -         
ai-21:0          3.89            -                     
sc              20.51          25.62         
ai              14.93           8.95         
i               1196           21.59         
u               50.20          42.40                
----------------------------------------- 

Strain 84 contains markedly higher levels than the parent strain of some unsaturated (C18:1), saturated straight-chain (C13:0, C14:0, C16:0) and branched (i-C15:0, i-C16:0) fatty acids. Some of the fatty acids (C11:0, i- C13:0, ai-C13:0, C17:0) are present only in the mutant strain, while others are absent. In addition the percentage of saturated straight-chain fatty acid is different. According to Reynolds (1995) crotonyl-CoA reductase is a key enzyme in straight-chain fatty acid formation in S. cinnamonensis and S. hygroscopicus. Possibly the altered amount of straight-chain fatty acids in the mutant strain is due to a higher activity of the enzymes involved in the synthesis. In the same strain there is an almost two-fold increase of the iso branched fatty acids.

Differences in fatty acid composition influence membrane functions (Arima et al., 1973; Vancura et al., 1987; Rezanka et al., 1992) and in particularly membrane permeability and consequently could favour antibiotic biosynthesis.

REFERENCES

Adamidis, T. & D. H. Sherman (1995). Factors affecting fatty acid composition in Streptomyces coelicolor. Abstr. SIM Ann. Meeting, San JosŠ, CA, p. 91

Arima, K., H. Okazaki, H. Hono, K. Yamada & T. Beppu (1973). Effect of exogenous fatty acids on the cellular fatty acid composition and neomycin formation in a mutant strain of Streptomyces fradiae. Agr. Biol. Chem., 37: 2313-2317

David, L., H. Loutheiller, D. Bauchart, S. Ausboiron & J. Asselineau (1992). Effects of exogenous methyloleate on the biosynthesis of nigericin, a polyether carboxylic antibiotic by Streptomyces hygroscopicus NRRL B-1865. Biosci. Biotechnol. Biochem., 56: 330

Gesheva, V. (1994). Isolation of spontaneous Streptomyces hygroscopicus 111-81 phosphate-deregulated mutants hyperproducing its antibiotic complex. Biotechnol. Lett., 16: 443-448

Gesheva, R. L., V. J. Gesheva, M. Z. Darakchieva, V. B. Ivanova, P. R. Panajotov, R. Schlegel & H. Thrum (1985). Taxonomy of Streptomyces hygroscopicus 111-81 producing nonpolyenic macrolide antibiotics. Compt. Rend. Acad. Bulg. Sci., 38: 619-622

Reynolds, K. A. (1995). Butyrate metabolism in Streptomyces. Abstr. SIM Ann. Meeting, San JosŠ, CA, p. 67

Rezanka, T., I. Vancurova, V. Kristufex, T. Koza, J. Zaslavska, V. Prykrylova & N. Blumauerova (1992). Taxonomic studies of Streptomyces virginiae mutant hyperproducing virginiamycin M. Folia Microbiol., 37: 105-110

Vancura, A., T. Rezanka, V. Marsalek, V. Kristan & G. Barasova (1987). Fatty acids and production of tylosin compounds in Streptomyces fradiae. J. Bas. Microbiol., 27: 167-171.

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