Biotechnol. frog. 1994, 10, 451-459
451
TOPICAL PAPER 5-Aminolevulinic Acid: A Potential Herbicide/Insecticide from Microorganisms Ch. Sasikala,' Ch. V. Ramana, and P. Raghuveer Rao Microbial Biotechnology Laboratory, Department of Botany, Osmania University, Hyderabad 500 007 (AP), India
5-Aminolevulinic acid (ALA), a n intermediate of the biological tetrapyrrole synthesis, can be used a s a photodynamic herbicidehnsecticide. Among the various microorganisms capable of its production, anoxygenic phototrophic bacteria produce ALA in considerable amounts, making it worthwhile to work toward commercial exploitation. Knowledge of the biochemical synthesis of ALA and its physiological and genetic regulation in microorganisms can enable the biotechnologist to manipulate them for enhancing ALA production for possible practical applications.
Contents Introduction Production of ALA by Microorganisms Biochemistry and Regulation of ALA Synthesis in Microorganisms Cq Pathway Ca Pathway Molecular Biology of ALA Synthesis Physiology of ALA Production Mechanism of Action of ALA as a Herbicide/Insecticideand Field Trials Concluding Remarks
45 1 451 451 45 1 453 454 455 455 456
Introduction An emerging area of biocontrol of weeds and pests involving the application of microbial cells or products thereof (Heisey et al., 1988) can provide a better alternative to the existing pesticides, most of which are deleterious to humans either during application or at the time of manufacture. 5-Aminolevulinic acid (5-amino-4-oxopentanoic acid) (ALA), an aliphatic precursor for tetrapyrrole synthesis in animals, plants, algae, and bacteria, is one such microbial product, which is the only naturally occurring photodynamic compound (those that convert molecular oxygen into singlet oxygen when excited by the absorption of light) known so far, that can be used as an effective herbicidelinsecticide. In recent years, photodynamic compounds have received attention as potential herbicides (Towers and Amason, 19881, among which the diphenyl ethers have been well studied (Johnson et aZ., 19781, practically applied, and put to commercial use (Duke et al., 1991). However, these compounds are being synthesized chemically and have been found to be skin and eye irritants. In contrast, ALA is harmless for humans and animals (Rebeiz et al., 1984, 1988a,b),in addition to being biodegradable. However, not much attention had been given to the possible application of ALA because of low availability and high cost. Its production by chemical synthesis involves many complex reactions (Tschudy and
Collins, 1959; MacDonald, 1974). Hence, chemists lost interest in this compound and started looking for better alternative photodynamic compounds. In contrast, microbiological production of ALA involves simple reactions and may be expected to become commercially viable.
Production of ALA by Microorganisms Since ALA is a precursor for tetrapyrroles, it is possible that almost all living systems synthesize ALA. Although animals and plants do produce ALA, they do so only in low quantities. Alternatively, microorganisms produce ALA in considerable amounts and are of interest for possible practical applications. Although microbiological formation of ALA by algae and chemotrophic bacteria can be considered as a less expensive method than that of chemical synthesis, the maximum concentration of ALA accumulated by these organisms is still too low for practical exploitation (Table 1). Anoxygenic phototrophic bacteria (APB)are a group of prokaryotic photosynthetic microorganisms that accumulate and excrete high levels of ALA into the medium and, hence, are suitable for commercial exploitation. Quasi-photosynthetic bacteria [a group of bacteria that do not perform true photosynthesis (Gest, 199311 and obligate anaerobic archebacteria have recently been shown to produce high amounts of ALA.
Biochemistry and Regulation of ALA Synthesis in Microorganisms ALA can be synthesized biologically using two distinct metabolic pathways (Figure 1). In the four-carbon (C4) pathway, succinyl-CoA and glycine are condensed by the enzyme 5-aminolevulinic acid synthetase (ALA synthetase). This pathway is observed in animals and fungi and is very common among the purple non-sulfur photosynthetic group of bacteria and a few chemotrophs (Avissar et aZ.,1989). In the five-carbon (C5) pathway, glutamate is converted into ALA with the involvement of tRNA, as observed in higher plants, algae, and a few bacteria. A few microorganisms have both C4 and C g pathways, as is distinct in Euglena gracilis (Weinstein and Beale, 1983). Cr Pathway. The key enzyme involved in the C4 pathway of ALA formation is 5-ALA synthetase (EC
8756-7938/94/3010-0451$04.50/0 0 1994 American Chemical Society and American Institute of Chemical Engineers
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Dr. Ch. Sasikala, born in 1964, graduated in science in 1983 with microbiology as one of her main subjects and in education in 1984 from Osmania University, Hyderabad, India. She obtained her Master's degree in applied microbiology from Bharathiar University, Tamilnadu, India, in 1986 and her doctorate in microbiology from Osmania University, Hyderabad, India, in 1990. Dr. Sasikala is the recipient of a fellowship from the University Grants Commission, Government of India, for work in the field of hydrogen photoproduction using anoxygenic phototrophic bacteria. She is presently working as Research Scientist in the Department of Botany, Osmania University, Hyderabad, India.
L
Dr. Ch. Venkata Ramana;-born in 1962, graduated from Osmania University, Hyderabad, India, in 1983 with microbiology and biochemistry as his main subjects of study. He obtained a Master's degree in botany from M.S. University, Baroda, India, in 1985 and a doctorate in botany from Osmania University, Hyderabad, India, in 1989. Dr. Ramana is the recipient of a fellowship from the Department of Non-ConventionalEnergy Sources, Government of India, for work in the field of oxygenic phototrophic bacterial (cyanobacterial)hydrogen photoproduction. He is currently working as Research Associate in the Department of Botany, Osmania University, Hyderabad, India. The husband and wife pair of Dr. Ch. V. Ramana and Dr. Sasikala are actively working on anoxygenic phototrophic bacteria-their ecology, taxonomy, physiology, and various biotechnological potential applications, specifically hydrogen photoproduction, ammonia photobiosynthesis, anoxygenic phototrophic bacteria as biofertilizers, and ALA synthesis. They are also currently working on the physiology and biochemistry of the photoanaerobic biodegradation of heterocyclic aromatic compounds.
2.3.1.37; succinyl-CoAglycine C-succinyl transferase) catalyzing the condensation of succinyl-CoA and glycine (Figure 1). Two forms of ALA synthetase were first reported by Marriott and his co-workers in Rhodobacter sphaeroides (Marriott et al., 1969),which was confirmed in other photosynthetic and also quasi-photosynthetic bacteria by others (Neuberger et al., 1973a; FranicaGaignier and Clement-Metral, 1973a; Tuboi et al., 1970a; Sato, 1978; Sat0 et al, 1985a,b). Molecular weights of form I and form I1 were about 100 000 and 64 000, with optimal pH values of 8.0 and 6.4 for Protaminobacter ruber and 7.4 and 7.8 for R. sphaeroides respectively, (Sato et al., 1985b). The synthesis of the two forms of ALA synthetase greatly depends upon the age and illumination of the
Professor P. hghuveer fcao, born in 1932,graduated in 1951 and obtained a Master's degree in botany in 1954 and a Ph.D. in botany in 1962. Prof. Rao was a postdoctoral Fulbright fellow, visiting the United States and working with Dr. C. G. Shaw at Washington State University, Pullman, WA, from 1967 to 1969. He is the author of a number of books on botany and a Fellow of the Indian Pathological Society (FIPS). Presently a Professor Emeritus, Dr. Rao is working with various projects on the biotechnological potential of anoxygenic phototrophic bacteria, sponsored by the Government of India. Other areas of his work include mycology, plant pathology, and aerial microbiology of plant parts.
cultures (Sato et al., 1985b). While form I was constitutive, form I1 was inducible in light and was responsible for the biosynthesis of bacteriochlorophyll. The constitutive enzyme participates in the syntheses of vitamin B12 and cytochromes. Dark semiaerobic conditions increased ALA synthetase activity, which was due to an increase in form I but not form 11, while in light semianaerobic conditions both forms I and I1 markedly increased (Tuboi et al., 1970b), suggesting that the syntheses of forms I and I1 are controlled independently. Conditions such as aerobiddark, anaerobidight, or microaerobiddark greatly influenced the synthesis of ALA synthetase (Tanaka et al., 1991). Changing the culture conditions from aerobic to microaerobic increased the activity of ALA synthetase by 2-4-fold. Enzyme synthesis in light was repressed by oxygen, and the effect could be overcome upon the restoration of anaerobic conditions (Viale et al., 1983). Light (484-654 nm), on the other hand, has no significant effect on the cellular level of ALA synthetase in intact cells of R. sphaeroides (Amheim and Oelze, 1983);however, in cell-free extracts (Oelze, 1986), ALA synthetase was inhibited upon irradiation (422, 522, and 522-556 nm). The ALA synthetase activity was highest from cells harvested at the logarithmic phase of growth (Sato et al., 1985a), and pyridoxal phosphate was the cofactor required (Gibson et al., 1961). The ALA synthetase activity of R. sphaeroides was regulated in vivo by low molecular weight sulfur compounds of trisulfides of cystine or glutathione (Neuberger et al., 1973b,c). These activators were identified as cystine trisulfide (CySSSCy), glutathione trisulfide (GSSSG), glutathione and cystine trisulfide (GSSSCy), and trisulfanedisulfonate (&Os2-) (Sandy et al., 1975). Among poly(su1fane)disulfonates (-03S-Sn-SO3-, n > l), activator activity was exhibited only by compounds with n > 3. In general, an activator of ALA synthetase was found to have an R-S,-R' (where R and R' are organic or inorganic groups) structure with n > 3. Oxygenation is one of the important factors affecting ALA synthetase activity (Sandy et al., 1975). With the APB in particular, oxygenation results in the loss of pigmentation due to the decrease in the ALA synthetase activators, cystine trisulfide and glutathione trisulfides.
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Table 1. Production of ALA by Different Groups of Microorganisma inhibitor C and N source Phototrophs algae Agmenellum quadruplicatum Cyanidium cala'urium bacteria, oxygenic phototrophic Anacystis nidulans Anabaena variabilis bacteria, anoxygenic phototrophic R . palustris R. sphaeroides -do-doChlorobium limicola Chloroflexusaurantiacus
+ + + + + 4+ + + +
L-alanine succinate and glycine
+ +
glucose and m y s t h e methanoV2-oxoglutarate Hz + COz
+ +
glutamate glutamate glutamate glutamate succinate and glycine succinate and glycine succinate and glycine swine waste' glutamate glutamate Chemotrophic Bacteria
aerobes Pseudomonas riboflavim Propionibacterium shermanii anaerobes Clostridium thermoaceticum Methanosarcinu barkeri Methanobacterium thermoautotrophicum
(LA)
-
ALA
01M)
reference
0.225 0.483
Kipe-Nolt and Stevens, 1980 Jugenson et al, 1976
0.380 0.019
Anderson et al., 1983 Avisser, 1980
0.750 2-4 160.0 4200.0 3.950 0.580
Andersen et al., 1983 Sasaki et al., 1991 Ishii et al., 1990 Sasaki et al., 1990 Anderson et al., 1983 Anderson et al., 1983
0.200 0.040
Rhee et al., 1987 Menon and Shemin, 1967
155.0 0.400 0.200
Shoji et al., 1989 Lin et al, 1989 Lin et al, 1989
a Swine waste (feces:urine:tap water = 1:2:2 (w/w) contained an abundant amount of lower fatty acids such as acetic, propionic, and butyric acids.
y 2 FH2 COOH
D
Transaminase
Glutamate
0
FmH FH2 FH2 F=O
Cog
CoASH
\
f,
0: ketoglutarate
COOH oc-ketoglutarate
dehydrogenase Mq" TPP, LIP
FWH
yH2 FH2
Co-SCoA
-
V ALA
Porphobilinogen
COOH Glutamyl-tRNA
W! Hydroxy amno tetrahydro pyranone
Figure 1. Biosynthesis of 5-aminolevulinic acid by microorganisms from glutamate in the C4 and c5 pathways; genes involved in its synthesis and the sites of inhibition of various inhibitors are indicated (Beale and Castelfranco, 1974; Hansson et al., 1991; Grimm et al., 1991; Ilag et al., 1991; Ikemi et al., 1992; Smith and Grimm, 1992; Ellen and Kaplan, 1993a,b). PALP, pyridoxyl phosphate; WP, thiamine pyrophosphate; ALA,5-aminolevulinic acid; ALAD,5-aminolevulinic acid dehydratase.
ALA synthetase is inhibited by aminomalonate (Gibson et al., 19611, iron protoporphyrin (haemin) and other iron porphyrins ( B u " and Lascelles, 1963),helium (ClarkWalker et al., 1967), adenosine triphosphate (ATP), guanosine triphosphate (GTP), and pyrophosphates (Fanica-Gaignier and Clement-Metral, 1973b1, metal ions, particularly Fez+, Cu2+ (Sasaki et al., 1989), and CaZ+, and nitrate (Tait, 1973). Helium had no effect on the ALA synthetase of P. ruber, and ALA synthetase was not inhibited by its end product vitamin BIZ (Sat0 et al., 1985a). Ca Pathway. There are groups of prokaryotes in which the a-ketoglutarate-oxidizing enzyme system is lacking in the citric acid cycle (Smith and Hoare, 1977), and in these bacteria ALA is formed from glutamate or a-ketoglutarate via a path that does not involve the ALA synthetase reaction. There is increasing evidence to
show that the C g pathway of ALA synthesis is operative in plants (Beale and Castelfranco, 19741,algae (Anderson et al., 1983; Jahn, 19921, anaerobic archaebacteria including the green sulfur bacteria (Oh-Hamaet al., 1986a, 1988; Zeikus, 1983; Avissar et al., 1989; Friedmann and Thauer, 1986; Smith and Huster, 1987), and aerobic bacteria like Pseudomonas riboflavim (Wee et al., 19871, Escherichia coli (Grimm et al., 1991; Ikemi et al., 1992), Salmonella typhimurium (Elliott and Roth, 1989),Bacillus subtilis (Hansson et al., 19911, and Propionibacterium freudenreichii (Katsuji et al., 1993). Bacteria in which the Cg pathway is operative can be divided into two groups on the basis of the pathway used for glutamate synthesis. Glutamate synthesis via the citrate pathway (oxaloacetate acetyl-coA citrate isocitrate oxoglutarate glutamate) occurs in Clostridium kluyueri (Stern and Bambers, 19661, C. thermoace-
-
-
+
-
-
Biotechnol. Prog., 1994,Vol. 10, No. 5
454
a
CHNH2 I
(H N HE-CHNH2-CHO ~
c= 0 I
$CHo H2 $ H2 COOH
COOH
GSA
4,5-Diamino valerate
"O°CJ
\
"O°C\
,OO°C\
*Oooc\
\ coon
'COOH
COOH
A LA
Amino-hemi Schiff Amino-hemi ALA ALA acetal base acetal Figure 2. Two catalytic mechanisms proposed for the conversion of glutamate l-semialdehyde to ALA (Grimm et al.,1991)(a, top, and b, bottom). GSA GSA
tium (Oh-Hama et al., 1988), Chromatium uinosum (OhHama et al., 1986a1, and Anacystis nidulans (Laycock and Wright, 1981). The second pathway (oxoglutarate malate fumarate -. succinate COz oxoglutarate glutarate) is operative in all green sulfur bacteria (OhHama et al., 1986b) and Methanobacterium thermoautotrophicum (Zeikus, 1983). Recent purification of the C5 pathway enzymes (TienEn et al., 1990; Hansson et al., 1991; Grimm et al., 1991; Ilag et al., 1991) indicates that glutamate is reduced to ALA in three steps (Figure 1). These include (1)ligation of tRNA to glutamate catalyzed by glutamyl-tRNA synthetase, (2) reduction of glutamyl tRNA to generate glutamate l-semialdehyde (GSA) catalyzed by glutamyltRNA reductase, and (3) transamination of GSA to generate ALA catalyzed by GSA aminotransferase (GSAAT). The details of the reaction mechanism of GSA-AT (EC 5.4.3.8, glutamate-l-semialdehyde2,l-aminomutase or (S)-4-amino-5-oxopentanoate 4,5-aminomutase) are still a subject of intensive research. Two catalytic mechanisms (Figure 2a,b) have been proposed for the conversion of GSA to ALA (Grimm et al., 1991). An amino group is donated from the enzyme or cofactor (vitamin Bg) to GSA and 4,5-diaminovalerate (DAVA) is formed; subsequently the amino group at position 4 is transferred to the enzyme or cofactor and ALA is formed (Figure 2a). Evidence for the occurrence of this mechanism comes from recent studies with the anomalous enantiomeric reactions of the enzyme GSA-AT using enantiomers of GSA and DAVA (Smith et al., 1992). Alternatively, two molecules of GSA oriented head-to-tail form an amino hemiacetal dimer, which is converted into a double Schiff base and rearranged into the amino hemiacetal, exchange amino groups, and subsequently dissociate into two molecules of ALA (Figure 2b). The primary structure of GSA-AT appears to be a dimer of identical subunits with a molecular mass of 46 kDa (Grimm, 1990). The enzyme has an exceptionally high affinity for its substrate and is inhibited by gabaculine, i.e., 3-amino-2,3-dihydrobenzoic acid (Grimm et al., 1991). Not much is known about the regulation of the enzymes of the C5 pathway.
- -
+
-
-
Molecular Biology of ALA Synthesis The regulation of ALA synthesis at a molecular level and the development of genetically engineered strains may result in an improvement in production, as was observed in several genetically altered strains that overproduced ALA (Tanaka et al., 1991; Gloria and Dailey, 1993; Ikemi et al., 1993). A large number of mutants auxotrophic for ALA isolated from S. typhimurium (Elliott and Roth, 19891, E. coli (Ikemi et al., 19921, R. sphaeroides (Lascelles and Altshuler, 1969; Ellen and Kaplan, 1993a1, and R. capsulatus (Wright et al., 1987; Hornberger et al., 1990)have enabled the study of genes coding for ALA synthesis. ALA synthetase genes have been cloned and sequenced from Rhizobium meliloti (Leong et al., 1985),Bradyrhizobium japonicum (Robertson McClung et al., 19871, and R. capsulatus (Hornbergeret al., 1990). ALA synthetases are evolutionarily related to two enzymes that catalyze ligase similar reactions: 2-amino-3-ketobutyrate-CoA and 7-keto-8-aminopelargonic acid synthetase (Ellen and Kaplan, 1993b). Two different ALA synthetase genes hemA and hemT encoded the two ALA synthetase isoenzymes (form I and form 11)in R. sphaeroides (Tai et al., 1988),and their molecular sequences (Ellen and Kaplan, 1993b) and regulation (Ellen and Kaplan, 1993a) are well established. Contrary to the R. sphaeroides case, there is a probability of only one gene coding for ALA synthetase in R. capsulatus (Hornberger et al., 1990). The genes encoding ALA synthesis through the Cg pathway have been cloned and sequenced from E. coli (Ilag et al., 1991; Grimm et al., 19911,B. subtilis (Hansson et al., 1991), S. typhimurium (Elliott et al., 19901, P. freudenreichii (Katsuji et al., 1993), and Synechococcus (Grimm et al., 1991). These organisms required two genes (hemA and hemL) for ALA synthesis. hemA coded a structural component of glutamyl-tRNA dehydrogenase, while the hemL gene encoded GSA-AT. However, recent findings (Ikemi et al., 1992) with Ala- mutants of E. coli suggest a new gene (hemM) that is essential for the synthesis of ALA. The hemM gene, which was adjacent to hemA, encodes glutamyl-tRNA dehydrogenase or its subunits, thus raising questions about the role of hemA in the synthesis of ALA. The product of hemA appears to act in an alternative, minor pathway that is
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Table 2. Inhibition of 5-Aminolevulinate Dehydratase by Substrate and Product Analogues and Their Effective Concentrations (&) (Luond et al., 1992) compound Substrate (ALA) Analogues 4-amino-3-oxobutanesulfonicacid 3-oxobutanephosphonic acid 4-nitro-2-butanone rac-2-hydroxy-4-oxopentanoicacid rac-3-hydroxy-4-oxo-pentanoic acid acetylglycine lewlinic acid 5-hydroxy-4-oxo-pentanoic acid 5-nitrilo-4-oxo-pentanoic acid N-(2-carboxybenzoyl)-5-amino-4oxopentanoic acid 2-oxoglutaric acid Product Analogues chloropyrroles 3-(2-carboxyethyl)-4-methylmaleimide 3-(2-mercaptoimidazol-4-yl)propanoicacid 3-(2-~arboxymethy1)-4-(carboxymethyl)-5methylpyrazole
Kl
no inhibition 25f3mM 18 f 3pM 0.43 f 0.13 mM 1.2 f 0.4 mM 28f3mM 1 f 0.15 mM 0.25 f 0.05 mM 60 f 15 pM 27f4mM
Gabaculine Lwulinate
distinct from the C5 pathway. Extensive work in progress on the molecular genetics of ALA synthesis is expected to provide possible solutions for the existing questions.
Physiology of ALA Production Although the carbon source of choice employed for ALA production by many microorganisms is glutamate, ALA can also be produced with other carbon sources (Table 1). A variety of amino acids can be used for ALA production (Rhee et al., 1987; Shoji et al., 1989). For the production of ALA by APB, succinate and glycine were used as precursors in the culture medium (Sasaki et al., 1987). Organic wastes can also be used for the production of ALA (Sasaki and Tanaka, 1991). Various environmental parameters were optimized for ALA production in M e r e n t organisms, which include pH, temperature [Rhee et al. (1987) for P . riboflauinal, and light intensity [Sasaki et al. (1987) for R. sphaeroidesl. The concentration of the precursors (glycine and succinate) regulates ALA production in R. sphaeroides (Sasaki et al., 1987). The intermittent or continuous supplement of a fixed amount of precursors could enhance the ALA accumulation to twice as much as that when the same amount of precursors was supplied all a t once, because this reduces the inhibitory effects of glycine on growth (Sasaki et al., 1991). Pyruvate inhibited the C5 pathway enzyme, resulting in low yields of ALA in P. riboflavina. To eliminate the inhibition of pyruvate, a coupled reaction with lactate dehydrogenase was tried, which improved ALA production (Rhee et al., 1987). Metal ions, particularly iron, played an important role in the excrection of ALA (Sasaki et al., 1989), and nitrite in the growth medium inhibited ALA production (Tait, 1973). Needless to say, all of the factors afTecting either the synthesis or activity of the enzymes involved in ALA synthesis discussed earlier also affect the production and accumulation of ALA in the medium. The ALA formed is immediately metabolized by the bacterial cells into porphobilinogen (PBG) catalyzed by the enzyme 5-aminolevulinic acid dehydratase (ALAD, porphobilinogen synthetase, EC 4.2.1.24; Shemin, 1970). ALAD requires the presence of an exogenous thio, such as 2-mercaptoethanol or dithioerythritol, and a metal ion (K+, Mg2+,Zn2+)to maintain catalytic activity (Nandi et al., 1968; Tsukamoto et al., 1979). For high yields of ALA, the inhibition of ALAD is essential. ALAD can be inhibited very effectively using
+
DEATH OF PLANT
5
-3
A LA
.
c
Cytochromes
:-:A
Singlet 02
Protopoiphyrin IX‘ Protoheme Jl Hehe
I
t
Protopo r phyr inogen
19f2mM 13f2mM 12f2mM 20 f 5 mM 32 f 4 mM
-
c
uo++
J.
2.2’-di~g&!
-+
Mg Protoporphyrin I X 5 MgProto&phyrtn
I X ME
Protochlorophyllide
J.
Chlorophyllide
c
Chlorophyll a
Figure 3. Porphyrin synthesis pathway, sites of inhibition of various inhibitors and modulators, and mechanism of action of photodynamic herbicides. Inhibitors are underlined and sites of inhibiton are indicated [modified from Duke et al. (1991)l.
either substrate or product analogues (Luond et al., 1992). Among those listed in Table 2, substrate analogues proved to be more effective inhibitors of ALAD than product analogues. Utilizing the most widely used substrate analogue, levulinic acid (LA), high amounts of ALA could be produced with various microorganisms (Table l), and its addition at the middle log phase of the culture yielded high ALA (Sasaki et al., 1987). Recently (Fujii et al., 1993),it was shown in a few nonsulfur APB that LA could serve as the sole source of carbode- donor for growth, simultaneously producing ALA and hydrogen (“fuel of the future”). Initially, LA incorporated was metabolized to acetate, propionate, and other unknown substrates under anaerobic illumination conditions (Sasaki et al., 1990). While most of the isolates of various bacteria reported so far required the presence of LA for greater yields, ALA could be produced in its absence by a strain of C. thermoucetium, yielding about 155pM (Shoji et al., 1989). An alternative approach for enhancing ALA production is to isolate mutants that excrete ALA without adding LA. This is the case with a mutant of R. sphaeroides isolated recently (Tanaka et al., 19911, which excreted ALA (64.5 pM)in the absence of inhibitors, making the process more economical for industrial operations.
Mechanism of Action of ALA as a Herbicide/ Insecticide and Field Trials The mechanism of action of this compound as a herbicidehnsecticide depends on its conversion to tetrapyrroles within plants/insects (Figure 3) after application in the dark (Rebeiz et al., 198813; Rebeiz and Hopen, 1984). The six tetrapyrroles that are important for this effect are magnesium mono and divinylprotoporphyrin M, their monoesters, and mono- and divinylprotochlorophyllide. Uncontrolled protoporphyrin biosynthesis caused the death of the treated plants and insects in light. According to Rebeiz and Hopen (1984), a t sunrise the excess concentrations of tetrapyrroles act on photosen-
Biotechnol. frog., 1994,Vol. 10, No. 5
456 Table 3. Patents That Have Appeared So Far on ALA Production and Applications product assigneeb) 5-aminolevulinic acid Haruhiko (Cosmo Oil Co. Ltd.) 1. microbial production Sasaki and Tanaka Ikemi and co-workers Rebeiz 2. use as a herbicide Tanaka and co-workers 3. plant growth stimulator 4. bacteriochlorophyll Uragami and Yoshida 5. tetrapyrroles Tanaka and co-workers
sitizers, converting triplet oxygen to the potently oxidizing singlet form. Singlet oxygen oxidizes the phospholipids of membranes within plants in free-radical chain reactions. Damaged membranes become leaky, the plant loses sap, and death occurs in 1-4 h. Susceptibility to tetrapyrrole-photosensitizing reactions is greater for dicotyledons than for monocotyledons. The herbicidal effect of ALA is short-lived in the light compared to that of protoporphyrinogen M oxidase (Protox) (the enzyme that converts protoporphyrinogen M to protoporphyrin E), inhibiting herbicides (Figure 3). "his is probably due to the fact that protochlorophyllide levels are rapidly reduced by conversion to chlorophyll or by photodestruction in ALA-treated plant tissues (Duke et al., 1991). However, the presence of ALA with several commercial and experimental diphenyl ether herbicides (those that inhibit Protox) had an additive effect on the phytotoxicity. These include ALA with low concentrations of diphenyl ether herbicides like acifluod e n [5-[2-chloro-4-(trifluoromethyl)phenoxy]S-nitrobenzoic acid1 (Lydon and Duke, 1988)and TNPP ether [ethyl 24 1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxylpropionatel (Yanase and Andoh, 1989). It is possible to chemically modulate the activity of ALA with tetrapyrrole dependent photodynamic herbicides that act in concert with ALA. ALA serves as a building block of tetrapyrrole accumulation, while the modulator(s) quantitatively and qualitatively alters the pattern of tetrapyrrole accumulation (Rebeiz et al., 1984, 198813). Modulators have been classified into four distinct groups (Nandihalli and Rebeiz, 1991): (a) enhancers of ALA conversion to divinylprotochlorophyllide and (b) monovinylprotochlorophyllide,(c) inducers of tetrapyrrole accumulation, which induces the plant tissue to form and accumulate large amounts of tetrapyrroles in the absence of added ALA, and (d) inhibitors of monovinylprotochlorophyllide accumulation, which appear to block the detoxification of divinyltetrapyrroles by inhibiting their conversion to monovinyltetrapyrroles. A number of novel tetrapyrrole dependent photodynamic herbicidal modulators have already been screened (Rebeiz et al., 1990). o-Phenanthroline (1,lO-phenanthroline) and its derivatives are one such group of potent photodynamic herbicide modulators (Nandihalli and Rebeiz, 1991) effective against a large number of weed species (Mayasich et al., 1990). Another very effective modulator is ethyl nicotinate (a simple vitamin derivative used for the defoliation of field-grown apple trees). The differential molecular basis for tetrapyrrole dependent photodynamic herbicide selectivity and susceptibility by various plant species has been discussed (Rebeiz et al., 1990). Extensive laboratory experiments concluded that ALA, along with a modulator, is very effective against many weed species. The target species and the ALA modulators are well worked out (Rebeiz et al., 1990; Mayasich et al., 1990). Apart from the laboratory studies, several field studies carried out with ALA along with a modulator proved that the combination is effective in controlling
patent no. J P 04 356 193 (1992) J P 03 172 191 (1991) JP 05 227 976 (1993) WO 9 1 016 820 (1991) EP 514 776 (1992) J P 6 1 192 296 (1986) J P 05 244 937 (1993)
several broad leaf weeds in lawns and the defoliation of field-grown apple trees (Rebeiz et al., 1990). A spray of 75 g of N a c r e with activators (modulator) like a,a'dipyridyl gave good results for these authors. A novel porphyric insecticide developed by Rebeiz and co-workers (Rebeiz et al., 1988a) consists of modulators of the porphyrin-hem biosynthetic pathway (30 mM ALA 30 mM 2,2'-dipyridyl at pH 3.51, which when sprayed on the larvae of Trichoplusia ni Hubner insects induced the massive accumulation of protoporphyrin M, causing death in darkness and in light. In light, death appeared to be photodynamic in nature. Further, it was also shown that photodynamic damage could be induced by treating the insects with exogenous photoporphyrin or Mg-protoporphyrin. In insects, 59% (on a unit protein basis) of protoporphyrin M accumulated in the hemolymph, 35% in the gut, and 6% in the integument. Contary to what was observed in plants, the dark accumulation of protoporphyrin M in T. ni is accompained by larval death in darkness. The molecular basis for this phenomenon is not yet understood. In addition to being nontoxic to nontarget organisms, i.e., other crops, animals, and humans, ALA has another important advantage in that it may be more difficult for insects to develop resistance aganist ALA, since chemical modulation of the porphyrin pathway appears to involve more than one metabolic step. Even if some insects were to succeed in developing resistance by destroying the accumulated tetrapyrroles, it is unlikely to protect the mutated insect against photodynamic damage in light (Rebeiz et al., 1988a). In addition to the herbicidal property of ALA, it is a very good growth stimulator when used at low concentrations (Tanaka et al., 1992). ALA acts by enhancing photosynthesis, suppressing respiration, and stimulating COz uptake. Soaking rice seedlings in 1-3 ppm ALA for 1-48 h increased plant height, plant weight, and the number of roots.
+
Concluding Remarks The yields of ALA from chemical synthesis of 4,5dioxovalerate and its nonenzymatic transamination to ALA are very low (933.3nM with glycine as amino donor) (Beale et al., 1979) compared to microbial production, particularly using APB, which produce ALA in considerable amounts. A n added advantage with APB is their ability to produce ALA from wastes (Sasaki et al., 1990). ALA thus produced was at a concentration level (ca. 4.2 mM) sufficient for practical use as a herbicide (3-5 mM; Rebeiz et al., 1984). Spraying of the culture broth directly was found to be effective aganist a number of common dicotyledonous weeds in the field (Sasaki et al., 1990). Additionally, the culture broth has bacterial cells that are good nitrogen fixers with proven potential as biofertilizers in rice fields (Kobayashi and Haque, 1971), a cereal crop field where ALA can be used effectively as a selective herbicide (Rebeiz et al., 1984). Large scale cultivation of APB for the production of ALA and the process for product recovery has been
Biotechnol. Prog,, 1994,Vol. 10, No. 5 patented recently (T. Haruhiko, Cosmo Oil Co., Ltd., J a p a n , 1992). A number of other patents have also appeared involving ALA production and usage (Table 3). Apart from the usage of ALA as an effective herbicide, its use in photodynamic therapy for the cure of superficial epithelial skin tumors (Peter et al., 1993) is also significant. Thus, ALA finds importance in the present-day world, and its production by microorganisms (more specifically, APB) has the potential to become an industrial process, provided appropriate technologies are develoDed for making industrial production technically feasible.
Notation
APB
anoxygenic phototrophic bacteria ALA 5-aminolevulinic acid ALAD 5-aminolevulinic acid dehydratase DAVA 4,5-diaminovaleric acid GSA glutamate 1-semialdehyde GSA-AT glutamate 1-semialdehyde aminotransferase LA levulinic acid PBG porphobilinogen Protox protoporphyrinogen M oxidase tRNA transfer ribonuclic acid
Acknowledgment Ch.S. and P.R.R. thank the U.G.C. Government of India for t h e awards of Research Scientistship and Professor Emeritus, respectively. Ch.V.R. thanks the CSIR (New Delhi) for the award of Research Associateship. Financial support received from ICAR and CSIR (New Delhi) is gratefully acknowledged.
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* Abstract published in Advance ACS Abstracts, September 1, 1994.
