Jan 27, 1992 - the relation between growth temperature and fatty acid composition of ... acids were analyzed (19) as being pentadecanoic, palmitic, isomyristic ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
July 1992, P. 2338-2340
Vol. 58, No. 7
0099-2240/92/072338-03$02.00/0 Copyright © 1992, American Society for Microbiology
Changes in Fatty Acid Branching and Unsaturation of Streptomyces griseus and Brevibacterium fermentans as a Response to Growth Temperature MERJA SUUTARI* AND SIMO LAAKSO
Laboratory of Biochemistry and Microbiology, Department of Chemical Engineering, Helsinki University of Technology, SF-02150 Espoo, Finland Received 27 January 1992/Accepted 22 April 1992
Streptomyces griseus showed three different modes of changing fatty acids in response to temperature change. In Brevibacterium fermentans, two such responses were found. The responses involved changes in fatty acid branching, unsaturation, or chain length, depending on growth temperature range. Changes in unsaturation of branched-chain acids were characteristic at low growth temperatures.
Straight-chain, iso-C2n' iso-C2n+11 and anteiso-C2n1j acids of Streptomyces griseus and Brevibacterium fermentans are supposed to be primed by acetyl acyl carrier protein (ACP) and isobutyryl-, isovaleryl-, and 2-methylbutyrylcoenzyme A (CoA), which might be formed from malonylCoA, valine, leucine, and isoleucine, respectively. Fatty acid synthetase (FAS II) adds repeatedly two carbons from malonyl-CoA to the intermediate (1, 3, 6, 9, 14, 17), and the long-chain fatty acids formed can further be desaturated to monoenes in the presence of oxygen (5, 6, 8, 20). However, unsaturated acids are proposed to have a minor role in microbes containing branched-chain acids (6, 7, 13), although unsaturation is often regarded as one of the most important regulators of membrane flexibility and stability (4, 10, 16). The proportions of branched-chain to saturated straight-chain acids and those of anteiso- to iso-branched acids are supposed to increase with reducing temperature instead of fatty acid unsaturation (6, 7, 16). Nevertheless, the relation between growth temperature and fatty acid composition of S. griseus and B. fermentans appeared not to be studied in spite of their biotechnological interest as producers of antibiotics and amino acids. Therefore, S. griseus ATCC 3326 and B. fermentans NCIB 9943 were cultivated in nutrient broth containing 0.3% (wt/vol) yeast extract and 0.5% (wt/vol) peptone at 10, 15, 20, 26, 30, 35, and 40°C as described previously (18). Fatty acids were analyzed (19) as being pentadecanoic, palmitic, isomyristic (iso-C14:0), isopentadecanoic (iso-C15:o), isOpalmitic (iso-C16:0), anteisopentadecanoic (anteiso-C15:0), and anteisoheptadecanoic (anteiso-C17:0) (Fig. 1). In addition, S. griseus contained palmitoleic (n-C16:1), isoheptadecanoic (iso-C17:0), isopalmitoleic (iso-C16:1), isoheptadecenoic (iso-C17:1), and anteisoheptadecenoic (anteiso-C17:1) acids, and B. fermentans contained anteisopentadecenoic (anteiso-C15:1) acid. The acids accounted for more than 90 and 95% of total fatty acids in S. griseus and B. fermentans, respectively, and represented typical profiles for the microbes (9, 17, 22), with the exception of unsaturated anteisoC15:1 of B. fermentans and iso-C16:1, iso-C17:1, and anteisoC17:1 of S. griseus, which were found for the first time in the microbes probably because of their appearance in significant amounts only at low growth temperatures. Cellular fatty acid *
Corresponding author. 2338
content varied between 3.2 to 4.5% and 4.5 to 5.9% in S.
and B. fermentans, respectively, and showed a slight increasing trend as the temperature was reduced. Temperature effects on the fatty acids of S. griseus and B. fermentans were different (Fig. 1). In addition, in both microbes certain growth temperature ranges could be identified, within which they responded by different fatty acid interchanges at temperature changes. Therefore, when the whole temperature range studied is considered, changes in fatty acid branching, unsaturation, and chain length were all involved. In S. griseus, three such growth temperature ranges were identified. From 35°C to 26°C, lower-meltingpoint anteiso-branched and unsaturated acids increased and saturated iso-branched acids decreased. Within 26 to 20°C, the higher-melting-point even-numbered-carbon isobranched acids and fatty acid unsaturation increased, while anteiso-branched acids and odd-numbered-carbon isobranched acids decreased abruptly. From 20 to 15°C, only anteiso-branched acids increased and even-numbered-carbon iso-branched acids decreased (Fig. 2A). Within the range of 35 to 26°C, the totals of anteiso-branched and unsaturated acids increased because of increases in anteisoC15:0 and n-C16:1 acids, and also slightly because of increases in anteiso-C17:0, anteiso-C17:1, and iso-C16:1, while the totals of iso-branched acids decreased because of decreases in iso-C14:0, iso-C15:0, iso-C16:0, and iso-C17:0. From 20 to 15°C, only anteiso-branched acids anteiso-C15:0 and anteiso-C17:0 increased and even-numbered-carbon iso-branched acids iso-C14:0 and iso-C16:0 decreased. Thus, from 35 to 26°C, the primers of straight-chain and anteiso-branched acids, acetylACP and 2-methylbutyryl-CoA, respectively, increased, and the primers of iso-branched acids, isobutyryl- and isovalerylCoA, decreased. From 20 to 10°C, only 2-methylbutyrylCoA increased and isobutyryl-CoA decreased. In conclusion, the changes in fatty acid branching and unsaturation can be regarded to serve in the maintenance of membrane fluidity properties both above 26°C and below 20°C. Within 26 to 20°C, unsaturated straight-chain and even-numberedcarbon iso-branched acids iso-C14:0, iso-C16:0, iso-C16:1, and n-C16:1 increased abruptly, while anteiso-C15:0 and anteisoC17:0 decreased. Accordingly, isobutyryl-CoA and acetylACP, the primers of even-numbered-carbon iso-branched and straight-chain acids, respectively, were incorporated more efficiently than 2-methylbutyryl-CoA, the primer of anteiso-branched acids. The shift from anteiso-type acids to griseus
VOL. 58, 1992
NOTES
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Temperature (°C)
FIG. 2. Temperature effect on fatty acid branching and unsaturation of S. griseus (A) and B. fermentans (B). Symbols: *, anteisobranched acids; 0, odd-numbered-carbon iso-branched acids; 0, even-numbered-carbon iso-branched acids; O, straight-chain fatty acids; A, unsaturated fatty acids.
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FIG. 1. Effect of temperature on the fatty acid profiles of S. griseus (A) and B. fennentans (B). Symbols: 0, palmitic acid; O, palmitoleic acid; A, isomyristic acid; El, isopentadecanoic acid; *, isopalmitic acid; A, isoheptadecanoic acid; 0, isopalmitoleic acid; M, anteisopentadecanoic acid; *, anteisoheptadecanoic acid; +, anteisopentadecenoic acid; x, anteisoheptadecenoic acid.
even-numbered-carbon iso-branched and straight-chain acids was found in all four growth phases studied, and as such is opposite to changes supposed to be ideal for membrane functions upon temperature change. The same kind of change has been observed in the fatty acid profile of the antibiotic-producing genus Streptomyces. This is of interest since the fatty acids and the antibiotics have the same precursors (2, 11, 12, 15). Therefore, the changes in fatty acids within 26 to 20°C might indicate a peak in antibiotic production. Moreover, the growth rate decreased stepwise to one-third within 26 to 20°C, as is reported to occur during production of secondary metabolites (21). In B. fermentans, characteristic fatty acid interchanges were observed within two growth temperature ranges. Elongation of anteiso-branched acids and the ratio of oddnumbered- to even-numbered-carbon iso-branched acids decreased down to 30°C, and below that fatty acid unsaturation and branching increased as the temperature was reduced. Down to 30°C, lower-melting-point anteiso-C,5:0 increased nearly as much as anteiso-C17:0, which is two carbons longer, decreased, indicating reducing fatty acid elongation. Concomitantly iso-branched acids showed unexpected behavior, since the higher-melting-point iso-C16:0 increased and iso-C150 decreased as temperature reduced, which indicate increasing use of isobutyryl-CoA instead of isovalerylCoA as a primer of fatty acid biosynthesis. From 30 to 10°C, anteiso-C15:1 increased while anteiso-C15:0 and palmitic acids decreased, and hence slightly more 2-methylbutyryl-CoA than acetyl-ACP was probably incorporated. Changes both
in fatty acid composition and growth rate were minor within 300C. In conclusion, S. griseus showed two modes of changing fatty acid branching and unsaturation when the temperature was reduced from 35 to 20°C, and below that only fatty acid branching was changed. In B. fermentans, two growth temperature ranges in which the fatty acid changes were different were found; above 300C, the chain length of anteiso-branched acids changed, and below 30°C, fatty acid unsaturation was varied. Fatty acid branching varied over the whole growth temperature range. Unsaturated fatty acids with branched and straight chains appeared to be more important in adaptation of the actinomycetes to low growth temperatures. Saturated branched-chain acids dominated at high temperatures and, interestingly, they are often the principal fatty acids of thermophiles (16). The desaturase(s) of S. griseus seemed to use mainly 16-carbon (iSO-C16:0 and n-C16:0) and also 17-carbon (iSO-C17:0 and anteiso-C17:0) acids as substrates, while the main unsaturated acid of B. fermentans was anteiso-C15:1l REFERENCES 1. Ariga, N., K. Maruyama, and A. Kawaguchi. 1984. Comparative studies of fatty acid synthases of corynebacteria. J. Gen. Appl. Microbiol. 30:87-95. 2. Arima, K., H. Okazaki, H. Ono, K. Yamada, and T. Beppu. 1973. Effect of exogenous fatty acids on the cellular fatty acid composition and neomycin formation in a mutant strain of Streptomyces fradiae. Agric. Biol. Chem. 37:2313-2317. 3. Butterworth, P. H. W., and K. Bloch. 1970. Comparative aspects of fatty acid synthesis in Bacillus subtilis and Escherichia coli. Eur. J. Biochem. 12:496-501. 4. Cronan, J. E., Jr., and E. P. Gelmann. 1975. Physical properties of membrane lipids: biological relevance and regulation. Bacteriol. Rev. 39:232-256. 5. Fulco, A. J. 1967. The effect of temperature on the formation of A5-unsaturated fatty acids by bacilli. Biochim. Biophys. Acta 144:701-703. 6. Kaneda, T. 1977. Fatty acids of the genus Bacillus: an example of branched-chain preference. Bacteriol. Rev. 41:391-418. 7. Kaneda, T., and E. J. Smith. 1980. Relationship of primer specificity of fatty acid de novo synthetase to fatty acid composition in 10 species of bacteria and yeasts. Can. J. Microbiol. 26:893-898. 8. Kikuchi, M., T. Kanamaru, and Y. Nakao. 1973. Relation between the extracellular accumulation of L-glutamic acid and
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