JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 68, No. 5, 378-381. 1989

Influence of Iron on the Excretion of 5-Aminolevulinic Acid by a Photosynthetic Bacterium,

Rhodobacter sphaeroides KEN SASAKI, 2 SATOSHI IKEDA, l TOSHIO KONISHI, 1 YOSHINORI NISHIZAWA, l*

ANO MITSUNORI HAYASHP

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Saijo-cho, Higashi-Hiroshima, Hiroshima 724,1 and Hiroshima-Denki Institute of Technology, 20-1

Nakano, Aki-ku, Hiroshima 739-03, 2 Japan

Received 31 March 1989/Accepted 22 August 1989

Fe 2+ and/or Fe 3+ supplemented to the culture of Rhodobacter sphaeroides enhanced intraceilular 5- aminolevulinic acid (ALA) synthetase, but ALA excretion could not be observed, even though the ALA dehydratase inhibitor (levulinic acid) was added. The reason for this was investigated, and it was found that Fe 2+ directly inhibits ALA synthetase activity. The supplemented Fe 2+ was accumulated in the cells.

5-Aminolevulinic acid (ALA) has received attention as a new herbicide effective against weeds but safe for crops, humans or other animals (1). However, its cost is relatively high because chemical synthesis requires many complex steps (2, 3). Although microbiological formation by Chlorella vulgaris (4) or Pseudomonas riboflavina (5) syn- thesizing ALA via the C-5 pathway (5, 6) has been applied as a more inexpensive method, the maximum concentra- tion of ALA accumulated by these organisms was still too low.

We have also attempted the bioproduction of ALA us- ing photosynthetic bacteria, since these have a relatively high ability to synthesize ALA by ALA synthetase (Shemin pathway) (7, 8). In a previous work (7), the addi- tion of levulinic acid (LA), a competitive inhibitor of ALA dehydratase (ALAD), with ALA precursors (glycine and succinate) to the culture of Rhodobacter sphaeroides gave ALA excretion in the absence of cobalt and/or ferric iron (maximum concentration, 2raM with 60h culture). However, ALA excretion could not be found in the presence of iron ions.

Iron is frequently contained in the culture medium when the tap water and natural organic substrates are used for medium preparation. Therefore, it will be very important for industrial ALA production using R. sphaeroides to elucidate the effect of iron on ALA formation.

In this paper, the effect of iron ions (Fe 2+ and Fe 3-) on ALA excretion and the ALA synthetase activity of R. sphaeroides was investigated under conventional culture conditions without addition of ALA precursors. The negative role of iron in ALA excretion is also discussed.

R. sphaeroides IFO 12203 was used throughout since this showed the highest ability to excrete A L A among the photosynthetic bacteria stored in our laboratory (5 genus, 10 strains) (7).

The glutamate-malate medium (9) used was as described previously (7). For preparation of medium, distilled water (iron concentration, less than 0.01 mg./-I) was used. To examine the effects o f iron on ALA formation, Fe 2+ (FeSO4(NHn)2SO4.6H20) or Fe 3~ (ferric citrate) were

* Corresponding author.

378

added in the range of 0.34-13.4mg.l 1. Anaerobic-light cultivation in a 1.5/ Rhoux bottle

(Ogura Glass Co., Ltd., Tokyo, working volume 1/) was carried out as described previously (7) for 3-6 d at 30°C. Levulinic acid (LA) was added at the middle log phase up to 50 mM.

A cell-free extract was prepared by sonication (8) with Tris-HCl buffer (0.02 M, pH 7.4) for ALA synthetase (ALAS) assay and Tris-HC1 buffer (0.02M, pH 8.1) for ALA dehydratase (ALAD) assay, respectively (10, 11). ALAS activity was measured by Burnham's method (10) and ALAD activity was measured by the method of Sato et al. (11). One unit of activity of ALAS and A L A D was defined as the amount of enzyme capable of forming 1 nmol of ALA or of porphobilinogen (PGB) per hour in the assay systems, respectively.

~,a'-Dipyridyl and o-phenanthroline (0.1-10mM) were used as chelating reagents to eliminate iron ions in the culture medium and in the enzyme assay reaction mixture.

Concentrations of cell mass, extracellular ALA and LA, and soluble protein in the cell-free extract were measured using the same methods as described previously (7).

Fe 2+ and Fe 3~ concentrations in the medium were measured by the colorimetric method using o-phenan- throline (JIS K 0102), with reducing reagent (NH2OH. HC1) for Fe 2+ and Fe 3-, or without for Fe 2+, because the reaction of Fe 3 ~ for o-phenanthroline (forming a red pigment) is negligible within ca. 30 rain under the measure- ment conditions. The total iron in the cells was measured by an atomic absorption analyzer (Shimadzu, AA640, Shimadzu, Co., Ltd., Kyoto) after decomposing cells harvested at various growth phases with a HC104-HNO3 mixture according to the Japanese Industrial Standards (JIS K 0102).

Figure 1 shows the effects of the iron (Fe z + ) concentra- tion on cell growth and ALA excretion with the intracellular enzyme activities of ALA synthetase (ALAS) and ALA dehydratase (ALAD). As reported previously (7), the addi- tion of LA under iron-free conditions retarded cellular growth and enhanced ALAS activity (see Fig. lb). When Fe 2~ was present in the medium (Fig. lc-Fig, lg), the growth retardation caused by LA addition was recovered

VoL 68, 1989 NOTES 379

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FIG. 1. Effects of Fe ~+ concentration on growth, ALA formation, ALA synthetase (ALAS) and ALA dehydratase (ALAD) activities in anaerobic-light culture of R. sphaeroides (30°C, 5 klx). Levulinic acid (LA 15 mM, arrow) was added at the middle log phase, a: Control; b: Fe 2+ = 0 plus LA; c: Fe 2. - 0 . 3 4 mg .1 ~ plus LA; d: Fe 2+ - 1.7 mg .l J plus LA; e: Fe 2÷ =3.4 mg. l ~ plus LA; f: Fe 2~ =6.8 mg .1 ~ plus LA; g: Fe 2+ = 13.6 mg. l ~ plus LA. Symbols: G, cell mass; O, extracellular ALA; ±, ALAS activity; n , ALAD activity.

and intracellular A L A S activity was significantly enhanced except for the case of the highest Fe 2+ concentrat ion (Fig. lg, 13rag Fe2+ . l - l ) . However, A L A excretion did not follow the enhanced level of A L A S activity as it did in the absence of Fe 2÷ (Fig. lb) . A L A D activity seemed to be in- hibited by the addit ion of LA, decreasing rapidly and return- ing to its former level in ca. 60 h (see Fig. la , lb) . This pat tern of A L A D activity was also found in the presence of Fe z* (Fig. le). In addi t ion, A L A excretion could not be observed, even if the LA concentra t ion added was increased up to 5 0 m M in the Fe2~-containing medium (3 .4mg FeZ+.1-1, da ta not shown) in the expectat ion of more effective inhibi t ion of A L A D activity in the cells.

In experiments with Fe 3+ (0-13.4mg.1-1) in place of Fe 2+, almost the same phenomena with respect to growth, A L A S activity and A L A excretion were observed (data not shown). These results suggest that Fe 2÷ and Fe 3+ have a similar effect on the growth and A L A excretion of R. sphaeroides with added LA.

It is well known that Fe 2+ inhibits A L A S activity in the par t ia l ly purified enzyme react ion of R. sphaeroides (8, 9). Therefore, to further elucidate why A L A excretion did not appear in spite of a higher level of A L A S activity, the effects of Fe 2+ and Fe 3+ on the A L A react ion using the cell-free extract obta ined f rom an iron-free medium (Fig. lb) were determined with or without a chelating reagent. The results are summarized in Table 1.

While Fe 3+ up to 300mg Fe 3~.1 ~ did not influence A L A S activity at all, strong inhibi t ion was caused by Fe 2+ as the concentra t ion in the reaction mixture increased. On the other hand, the addi t ion of ct,ct'-dipyridyl was not only able to cause a recovery of enzyme activity in the presence of Fe 2÷, but could also enhance it. However , in the case of o-phenanthrol ine , excess chelating reagent residue had a negative effect on the enzyme reaction.

Even if Fe 2+ was not added, A L A S activity was enhanced by the chelating reagent, suggesting that a trace amount of i ron contained in the enzyme solution was removed by the chelating reaction, as well as that the chelating reagent could convert A L A S protein to the act ive-form of A L A S , as observed by Tuboi et al. (12) with an ion-exchange resin (Dowex I) t reatment which could convert A L A S from an inactive to an active form.

Since Fe 2÷ in the medium suppresses A L A excretion and inhibits A L A S activity in the cell-free extract, it can be con- sidered that intracellular A L A S activity may be inhibited by Fe 2+ if it is accumulated in the ceils at a high concentra- tion.

To observe the profile of Fe 2~ uti l ization, the time course of Fe 2 ~ during culture with added L A was examined under the same condit ions as in Fig. le. The results are shown in Fig. 2. Fe 2+ in the medium was quite stable without being oxidized to Fe 3 + for at least ca. 100 h culture under i l luminated condit ions. The iron content in the cells (initial content was 0.03-0.06 mg. g - t ) tended to decrease gradual ly after it increased due to the rapid incorpora t ion of iron at the beginning of the growth phase. An increase of iron content was observed again when the growth, re tarded once by the addi t ion of LA, was recovered with the decrease of LA at 50-80 h of the culture (cf. Fig. le).

The iron content is expressed based on the cell volume (per 1 cm3). The culculat ion was as follows: i ron content in the c e l l s - 6.11 - 0.99. The value 6.11 indicates the propor - t ion of wet cells to dry bases, while 0.99 indicates the cell specific gravity (weight /volume) measured directly after centr ifugat ion of the culture bro th (30,000 x g, 20 min). As shown in Fig. 2, the iron content during culture was in the range 0.17-0.30 on the basis of mg iron per cm 3 wet cells. These values correspond to 170-300 mg Fe 2÷ per liter i.e., quite a high concentrat ion of Fe 2~, giving the strong en-

380 SASAKI ET AL. J. FERMENT. BIOENG.,

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FIG. 2. Time courses of Fe 2+ and growth on Fe 2- sufficient medium (3.4 mg.l t) under anaerobic-light conditions (30°C, 5 klx). Levulinic acid (LA 15 raM, arrow) was added at the middle log phase. Symbols: ©, cell mass; zx, iron content in the cells (dry base per g); A, iron content in the cells (wet base, per cm3); [], residual iron concentration in the broth; *, residual Fe 2+ concentration in the broth.

zyme reaction inhibit ion shown in Table 1, while the Fe 2+ concentrat ion was 1.7 mg. / -1 (Fig. ld), the iron content was almost the same or slightly lower (0. t3-0.25 mg per cm 3 cells, data not shown), suggesting that this organism tends to accumulated iron in the cells. In addition, Fe 2+ might become more concentrated on the membrane frac- tion of the cells, because it will be an essential element to synthesize cytochrome (heme compound) required for growth.

From these results, the reason why extracellular A L A was not observed in the presence of iron might be con- sidered to be as follows: First, A L A S activity might be in- hibited even though a high level of A L A S was synthesized. Second, a small amount of A L A formed in the cells by the residual activity of ALAS from Fe z+ inhibition might be utilized for tetrapyrrol synthesis to maintain cellular growth.

As the addition of a chelating reagent was able to recover the A L A S activity in the cell-free system (Table 1), the effects o f cr,a'-dipyridyl addition on A L A excretion in the culture were examined. As shown in Fig. 3b, the addi-

t ion of chelating reagent and LA at the same time did not enhance A L A excretion. This may cause strong suppres- sion of growth. However, 24-h-delayed addition of a , c { -

dipyridyl after LA addition resulted in the obvious enhancement of A L A excretion up to 63 pM (Fig. 3c). In- crease of the a,a '-dipyridyl concentrat ion (5 mM, data not shown) suppressed the growth almost completely. This means that excess supplementat ion of chelating reagent will chelate other elements essential for growth, it addition to removal of iron. It was, however, clear that added c~,a'- dipyridyl could recover A L A excretion by removing iron from the cells. Indeed, the iron content after a,a '-dipyridyl addition (Fig, 3c) was kept in the range of 0.06- 0.08 m g / c m 3, indicating the relief of intracellular inhibi- t ion of Fe z+ for ALAS by supplementing the chelating reagent into the culture medium. However, it is difficult to remove iron completely to compare with the iron level of cells grown in an iron-free medium.

These results suggest that materials used for medium preparation, such as the natural carbon and nitrogen sources and tap water, should be selected or pre-treated

TABLE 1. Effects of iron and chelating reagents on ALA synthetase activity in the cell-free extract

Relative specific activity (%) Iron Fe 2 ~

concn. (mg.l 1) (raM) Fe3- c~,¢{-Dipyridyl (mM) o-Phenanthroline (mM)

0.1 1 10 0.1 1 10

0 0 100 100 189 244 204 271 205 53 1.7 0.03 100 99 173 218 191 236 202 79 3.4 0.06 100 96 182 222 191 220 171 70 8.5 0.15 100 85 130 183 206 182 180 69

34.0 0.61 100 72 102 119 199 158 140 110 136 2.43 100 33 62 68 207 73 89 212 200 3.57 100 4 12 3 212 0 4 215 300 5.36 1130 4 4 4 182 4 5 207

The cell-free extract used was prepared from cells grown on Fe2+-deficient medium (Fe 2÷ not added); it contained a trace amount of iron (0.11-0.168mg iron./ ~).

VOL. 68, 1989 NOTES 381

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3.4 mg. l ~). Levulinic acid (LA 15 mM, 0) was added at the middle log phase, a,a'-Dipyridyl (1 mM, .,) was added at 0 or 24 h after LA addi- tion. a: Control; b: LA plus a,a'-dipyridyl 1 mM (0 h); c: LA plus a,a'-dipyridyl 1 mM (24 h). Symbols: G, cell mass; e , extracellular ALA.

w i th a view to r e d u c i n g the i r i r on c o n t e n t fo r the i n d u s t r i a l p r o d u c t i o n o f A L A .

REFERENCES

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2. MacDonald, S.F.: Methyl 5-bromolevulinate. Can. J. Chem., 52, 3257-3258 (1974).

3. Beale, S. I., Gold, M. H., and Granic, S.: Chemical synthesis of 4,5-dioxovaleric acid and its nonenzymatic transamination to 5- aminolevulinic acid. Phytochemistry, 18, 441-444 (1979).

4. Beale, S.I. : The biosynthesis of 5-aminolevulinic acid in Chlorella. Plant Physiol., 45, 504-506 (1970).

5. Rhee, H., Murata, K., and Kimura, A.: Formation of the her- bicide, 5-aminolevulinate, from L-alanine and 4,5-dioxovalerate by Pseudomonas riboflavina. Agric. Biol. Chem., 51, 1701-1702 (1987).

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moautotrophicum. FEMS Microb. Lett., 12, 167-170 (1981). 7. Sasaki, K., Ikeda, S., Nishizawa, Y., and Hayashi, M.: Produc-

tion of 5-aminolevulinic acid by photosynthetic bacteria. J. Fer- ment. Technol., 65, 511-515 (1987).

8. Burnham, B. F. and Laseelles, J.: Control of porphyrin biosyn- thesis through a negative-feedback mechanism. Biochem. J., 87, 462-472 (1963).

9. Lascelles, J.: The biosynthesis of porphyrins and bact- eriochlorophyll by cell suspensions of Rhodopseudomonas spheroides. Biochem. J., 62, 78-93 (1956).

10. Burnham, B.F.: 5-Aminolevulinic acid synthase (Rhodo- pseudomonas spheroides), p. 195-200. In Tubor, H. and Tubor, C. W. (ed.), Methods in enzymology, vol. 17. Academic Press, New York (1970).

11. Sato, K., Ishida, K., Kuno, T., Mizuno, A., and Shimizu, S.: Regulation of vitamin Bj2 and bacteriochlorophyll biosynthesis in a facultative methylotroph, Protoaminobacter ruber. J. Nutr. Sci. Vitaminol., 27, 439-447 (1981).

12. Tuboi, S., Kim, K. J., and Kikuchi, G.: Occurrence of a specific and reversible inhibitor of 5-aminolevulinate synthetase in extracts of Rhodobacter sphaeroides. Arch. Biochem. Biophys., 130, 92-100 (1967).