High level production of 5-aminolevulinic acid by Propionibacterium acidipropionici grown in a low-cost medium

5-Aminolevulinic acid (ALA) is an intermediate in the biosynthesis of tetrapyrroles. Its current production is expensive. We have developed a low-cost medium for Propionibacterium acidipropionici to produce extracellular ALA. When grown at 35?°C on a medium containing 3?% (w/v) food-grade sodium lactate supplemented with 18?g glycine/l, 4.05?g succinate/l, 1.8?g glucose/l, pH 7, it produced ALA up to 7.7?g/l over 6?days. Plant-growth promoting activity assays showed that the ALA was biologically active.     

Production of a herbicide, 5-aminolevulinic acid,by Rhodobacter sphaeroides using the effluent of swine waste from an anaerobic digestor

Summary For the production of a herbicide, 5-amino-levulinic acid (ALA), from anaerobic digestion liquor, the utilization of the photosynthetic bacterium, Rhodobacter sphaeroides was examined. This bacterium could produce ALA extracelularly from this liquor with the addition of levulinic acid (LA), an inhibitor of ALA dehydratase (ALAD), and glycine, a precursor of ALA biosynthesis in the Shemin pathway. Succinate (another precursor) addition was unnecessary for ALA production. When repeated additions of LA were made together with glycine ALA production was significantly enhanced. However, above three additions of LA, ALA production was not further enhanced. The maximum value of ALA production attained was 4.2 mM (0.63 g/ 1), which was over double that of other ALA producers such as Chlorella vulgaris. Propionic acid was predominantly utilized compared with other lower fatty acids, suggesting that this might be converted to ALA via succinyl-coenzyme A (CoA) in the methylmalonyl-CoA pathway.Offprint requests to: Y. Nishizawa     

Chlorophyll formation and glycine metabolism in laevulinic acid treated barley leaves

Laevulinic acid (LA) inhibited chlorophyll formation and δ-aminolaevulinic acid (ALA) accumulation in dark-grown barley leaves. Mole ratios (ALA: chlorophyll × 8) indicate that LA decreased ALA production by about 30%. The turnover of glycine-[¹⁴C] in 7-day-old leaves treated with LA was 70% slower than in control tissue and this resulted in an increase in endogenous glycine. Total amino acid also increased in LA treated leaves. The data indicate that any contribution made by glycine to ALA synthesis in LA-treated barley leaves would be significantly restricted.     

Biosynthesis of intracellular 5-aminolevulinic acid by a newly identified halotolerant <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Of 23 strains of halotolerant (up to 12% w/v NaCl) photosynthetic bacteria isolated from various sources, one isolate, SH5, accumulated intracellular 5-aminolevulinic acid (ALA) at 0.45 μg/g dry cell wt (DCW) growing aerobically in the dark. The strain was identified as Rhodobacter sphaeroides using 16S rDNA sequencing. Biosynthesis of ALA was enhanced to 14 μg/g DCW using modified glutamate/glucose (50 mM) medium with the addition of 10 mM levulinic acid after 24 h cultivation. Addition of 30 μM Fe²⁺ to this medium increased the yield to 226 μg/g DCW.     

Biosynthesis of δ-aminolevulinic acid from glutamate by Sulfolobus solfataricus

The extremely thermophilic, obligately aerobic bacterium Sulfolobus solfataricus forms the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate by the tRNA-dependent five-carbon pathway. This pathway has been previously shown to occur in plants, algae, and most prokaryotes with the exception of the -group of proteobacteria (purple bacteria). An alternative mode of ALA formation by condensation of glycine and succinyl-CoA occurs in animals, yeasts, fungi, and the -proteobacteria. Sulfolobus and several other thermophilic, sulfur-dependent bacteria, have been variously placed within a subgroup of archaea (archaebacteria) named crenarchaeotes, or have been proposed to comprise a distinct prokaryotic group designated eocytes. On the basis of ribosomal structure and certain other criteria, eocytes have been proposed as predecessors of the nuclear-cytoplasmic descent line of eukaryotes. Because aplastidic eukaryotes differ from most prokaryotes in their mode of ALA formation, and in view of the proposed affiliation of eocytes to eukaryotes, it was of interest to determine how eocytes form ALA. Sulfolobus extracts were able to incorporate label from [1-¹⁴C]glutamate, but not from [2-¹⁴C]glycine, into ALA. Glutamate incorporation was abolished by preincubation of the extract with RNase. Sulfolobus extracts contained glutamate-1-semialdehyde aminotransferase activity, which is indicative of the five-carbon pathway. Growth of Sulfolobus was inhibited by gabaculine, a mechanism-based inhibitor of glutamate-1-semialdehyde aminotransferase, an enzyme of the five-carbon ALA biosynthetic pathway. These results indicate that Sulfolobus uses the five-carbon pathway for ALA formation.Abbreviations AHA 4-amino-5-hexynoic acid - ALA -aminolevulinic acid, Gabaculine, 3-amino-2,3-dihydrobenzoic acid - GSA glutamate 1-semialdehyde     

Two Biosynthetic Pathways to delta-Aminolevulinic Acid in a Pigment Mutant of the Green Alga, Scenedesmus obliquus

Pigment mutant C-2A′ of the unicellular green alga Scenedesmus obliquus develops only traces of chlorophyll and has no detectable amount of δ-aminolevulinic acid (ALA) when grown in the dark. In light it develops ALA and in the presence of levulinic acid (LA), a competitive inhibitor of ALA dehydratase, it accumulates 0.18 mmoles of ALA per 10 microliters of packed cell volume per 12 hours. This amount could be increased up to 15 times by feeding precursors and cofactors.

Incubation with [U-¹⁴C]glutamate, [1-¹⁴C]glutamate, and [2-¹⁴C]glycine yielded significantly labeled ALA, whereas [1-¹⁴C]glycine did not label the ALA specifically. Thus, two pathways using either glycine/succinyl-coenzyme A or incorporating the whole C-5-skeleton of glutamate into ALA are present in this alga. The efficiency of the glycine/succinyl-coenzyme A pathway seems to be three times higher than that of the glutamate pathway. Incubation with [5-¹⁴C]2-ketoglutarate, which can serve both pathways as a precursor, resulted in radioactivity of ALA as high as the sum of both labeling with [1-¹⁴C]glutamate and [2-¹⁴C]glycine.

Since the newly synthesized chlorophyll was radioactive regardless of labeled substrate employed, both pathways culminate in chlorophyll formation.

     

Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells

Summary When MDCK cells are cultured in MEM, they maintain a high concentration of three amino acids: glutamate (25mm), taurine (19 mm) and glycine (9 mm). With incubation of the cells in hypotonic media, the contents of these amino acids measured by HPLC are reduced in different time courses: taurine decreases most rapidly, followed by glutamate and glycine. All these losses are Na⁺ independent. To determine the transport mechanism activated by the hypotonic media, increasing external concentrations reaching 60 mm for nine different amino acids in Na⁺-free media were tested separately. For the five neutral (zwitterionic) amino acids, taurine, glycine, alanine, phenylalanine and tryptophan, cell contents increased linearly with external concentrations in hypotonic media, whereas in isotonic media only a slight rise was observed. The two anionic amino acids, glutamate and aspartate, were also increased linearly with their external concentrations in hypotonic media, but the changes were lower than those found for neutral amino acids. The presence of a negative membrane potential was responsible for this behavior since, using a K⁺ hypotonic medium which clamps the potential to zero, the glutamate content was found to increase linearly with an amplitude similar to the one observed for neutral amino acid. When external concentrations of two cationic amino acids, arginine and lysine, were increased in hypotonic media, only a small change, similar to that in isotonic media, was observed. These results indicate that a diffusion process for neutral and anionic amino acids is activated by a volume increase and it is suggested that an anion channel is involved.     

Production of extracellular 5-aminolevulinic acid byClostridium thermoaceticum grown in minimal medium

Summary A growth associated formation of extracellular 5-aminolevulinic acid (ALA) was found in the homoacetogenesis of glucose byClostridium thermoaceticum grown in minimal defined medium. The growth and ALA production was enhanced by L-cysteine HCl both in complex medium (UM) and minimal defined medium (MDM). The amount of ALA produced extracellularly in MDM wasca. 15 mg/L after 90-h anaerobic cultivation (cell-mass: 1.5 g/l; glucose consumed: 20 g/l).     

光合细菌嗜酸柏拉红菌5-氨基乙酰丙酸合成酶基因的克隆与原核表达

5-氨基乙酰丙酸(ALA)可作为除草剂、杀虫剂和植物生长调节剂在农业上应用,但由于其成本较高而限制了它的大面积使用。利用常规基因工程操作方法结合载体介导PCR法(Vecterette PCR)克隆了嗜酸柏拉红菌(Rhodoblastus acidophilus)的5-氨基乙酰丙酸合成酶(ALAS)基因。并将编码ALAS的基因插入到原核表达载体pQE30中,在大肠杆菌不同菌株(E.coli JM109、M15及BL21(DE3))中进行诱导表达。对产物进行SDS-PAGE分析表明,ALAS基因已在细菌中成功表达。使用Ni-NTA亲和层析法对表达的ALAS进行分离、纯化,得到大小约为44kD的ALAS蛋白。通过优化工程菌株的培养条件,建立了发酵生产ALA的方法,其胞外分泌ALA产量达5.379g/L,ALAS酶活力高达333U/min.mg。这是目前国内外利用生物法生产ALA产量最高的报道,为ALA的产业化应用打下了良好的基础。     

Extracellular 5-aminolevulinic acid production by Escherichia coli containing the Rhodopseudomonas palustris KUGB306 hemA gene

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'- phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).     

The role of malate in ammonia assimilation in cotyledons of radish (Raphanus sativus L.)

The relationships between the metabolism of malate, nitrogen assimilation and biosynthesis of amino acids in response to different nitrogen sources (nitrate and ammonium) have been examined in cotyledons of radish (Raphanus sativus L.). Measurements of the activities of some key enzymes and pulse-chase experiments with [¹⁴C]malate indicate the operation of an anaplerotic pathway for malate, which is involved in the synthesis of glutamine during increased ammonia assimilation. It is most likely that the tricarboxylicacid cycle is supplied with carbon through entry of malate, formed via the phosphoenolpyruvate (PEP)-carboxylation pathway, when 2-oxoglutarate leaves the cycle to serve as precursor for an increased synthesis of glutamine via glutamate. This might occur predominantly in the cytosol via the activity of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle, the NADH-dependent GOGAT being the rate-limiting activity.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - GDH glutamate dehydrogenase - GOGAT glutamate synthase (glutamine: 2-oxoglutarate aminotransferase) - GOT aspartate aminotransferase (glutamate: oxaloacetate transaminase) - GS glutamine synthetase - HPLC high-performance liquid chromatography - MCF extraction medium of methanol: chloroform: 7M formic acid, 12:5:3, by vol. - MDH malate dehydrogenase - MSO L-methionine, sulfoximine - PEPCase phosphoenolpyruvate carboxylase - TLC thin-layer chromatography     

Coproporphyrin production by Rhodobacter sphaeroides under aerobic in the dark and dissolved-oxygen-controlled conditions

Porphyrin production under aerobic in the dark condition was carried out using the photosynthetic bacterium, Rhodobacter sphaeroides IFO12203 and its mutant, CR 386 which can produce 5-aminolevulinic acid (ALA) under aerobic in the dark conditions. IFO12203 produced about 1.0 mg/l of porphyrin even if 2.0 mg of ALA/l was added to the glucose–glutamate–yeast extract (GGY2) medium. However, CR 386 produced 15.0 mg/l of porphyrin after 55 h culture with the addition of 2.0 g of ALA/l and sufficient oxygen supply (dissolved oxygen, DO > 7.0 mg/l). The porphyrin produced by CR 386 consisted only of coproporphyrin III. Under conditions of strict DO control (DO = 2.0 ± 0.2 mg/l), the maximum porphyrin production attained 56.3 mg/l. Low DO (1.0 ± 0.2 mg/l) and high DO control (3.0 ± 0.2 mg/l) did not enhance porphyrin production. It is suggested that oxygen supply seems to control the step(s) of porphyrin biosynthesis of CR 386 in the stages after ALA synthase in the Shemin pathway.     

Chlorophyll biosynthesis from glutamate or 5-aminolevulinate in intact Euglena chloroplasts

Chloroplasts observed, by electron microscopy, to be intact and uncontaminated, with high rates of light-dependent protein synthesis and CO₂ fixation were isolated from cells grown on low-vitamin-B₁₂ medium in the light or from cells grown in the same medium in the dark and then exposed to light for 36 h. Both types of chloroplasts were active but less variability was encountered with developing chloroplasts from 36-h cells. The 36-h chloroplasts showed good light-dependent incorporation of 5-amino-levulinic acid (ALA) or l-glutamic acid into chlorophyll (Chl) a which was linear for approx. 1 h. The specific activity of the Chl a remained the same after conversion to pheophytin a, methylpheophorbide a or pyromethylpheophorbide a and rechromatography, indicating that the label was in the tetrapyrrole. Incorporation of ALA was inhibited by levulinic acid, and by chloramphenicol and other inhibitors of translation of 70S-type chloroplast ribosomes at concentrations which did not appreciably inhibit photosynthesis but which blocked plastid protein synthesis nearly completely. Cycloheximide, an inhibitor of translation on 87S cytoplasmic ribosomes of Euglena, was without effect. The 70S inhibitors did not block uptake of labeled ALA. Although labeled glycine was taken up by the plastids, no incorporation into Chl a was observed. Thus the developing chloroplasts appear to contain all of the enzymatic machinery necessary to convert glutamic acid to Chl via the C5 pathway of ALA formation but the Shemin pathway from succinyl coenzyme A and glycine to ALA appears to be absent. The requirement for plastid protein synthesis concomitant with Chl synthesis indicates a regulatory interaction and also indicates that at least one protein influencing Chl synthesis is synthesized on 70S-type plastid ribosomes and is subject to metabolic turnover.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll     

Quantitative relationships between ingested and intestinal flows of linoleic and alpha-linolenic acids,body weight and milk performance in mid-lactation dairy cows

Linoleic acid (LA) and alpha-linolenic acid (ALA) are essential fatty acids found in variable quantities in ruminant feedstuffs. Revision of French feed unit systems in 2018 has proposed the reassessment of energy requirements through a between-experiment approach expressing metabolisable energy supply as a function of the energy expenditures for maintenance and production, with these expenditures that reflect homeorhetic regulations. Based on the same approach, LA and ALA intake can be related to animal characteristics (i.e., BW) reflecting maintenance expenditures and secretion characteristics (i.e., milk yield, milk fat content and contents of LA and ALA in milk fat). Therefore, the objective of this work was to analyse the between-experiment relationships between ingested, duodenal, or absorbed flows of LA and ALA, BW and milk LA and ALA secretion by meta-analysis in mid-lactation dairy cows. These relationships were analysed using LA and ALA subsets of 96 and 99 experiments, respectively. Between-experiment regressions of daily flows of ingested, duodenal or absorbed LA and ALA on BW and milk LA and ALA flows were studied, with statistical unit defined as the mean of within-experiment treatments. For LA, the BW-associated coefficient was 0.019 (±0.0034) g absorbed LA/d per kg BW and milk LA secretion-associated coefficient was 0.70 (±0.081) g absorbed LA/g of LA secreted into milk. For ALA, the BW-associated coefficient was 0.0058 (±0.00093) g absorbed ALA/d per kg BW and milk ALA secretion-associated coefficient was 0.57 (±0.097) g absorbed ALA/g of ALA secreted into milk. When coding the diets as either control or milk fat depression diets, the BW-associated coefficient for LA was 0.017 (±0.0032) g absorbed LA/d per kg BW for both diets. For milk fat depression diets, milk LA secretion-associated coefficient was 1.02 (±0.119) g absorbed LA/g of LA secreted into milk, whereas it was 0.70 (±0.075) g absorbed LA/g of LA secreted into milk for control diets. Significant BW and milk performance coefficients were obtained in all LA and ALA equations, allowing the calculation of ingested and intestinal flows of LA and ALA based on measured BW, milk fat yield and milk fat content of LA and ALA. The relationships between ingested and intestinal flows of LA and ALA, BW and milk performance obtained in the present work could be integrated into renewed feed unit systems for energy and protein in dairy cows.     

Effect of culture pH on the extracellular production of 5-aminolevulinic acid by Rhodobacter sphaeroides from volatile fatty acids

Summary 5-Aminolevulinic acid(ALA) production by Rhodobacter sphaeroides was investigated at various pH with levulinic acid addition using a volatile fatty acids medium prepared from the mandarin orange peel supplemented with glycine. At neutral pH (6.8 and 7.0), extracellular ALA production was up to 16 mM, while low production of ALA(less than 3.5 mM) was observed at acidic pH (lower than 6.5) and less than 3.9 mM of ALA produced at alkaline pH (higher than 7.5). The higher ALA synthase activity observed at neutral pH might enhance the ALA production compared with that observed in acidic and alkaliphilic cultures.     

Biosynthesis of poly (γ-glutamic acid) from l-glutamine,citric acid and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. PGA was only slightly produced from medium (100 ml) containing 2 g citric acid and 0.5 g ammonium sulfate in B. subtilis IFO3335. When 0.01 g/100 ml l-glutamine was added to this medium, a large amount of PGA (0.45 g/100 ml), without any by-products such as polysaccharides, was produced. The changes in cell growth, and PGA, glutamic acid, citric acid and ammonium sulfate concentrations in this medium during cultivation were investigated. It was found that PGA was effectively produced for the short time of 20 h after an induction period and that glutamic acid was scarcely excreted during PGA production. PGA could be effectively produced using this medium containing l-glutamine, citric acid and ammonium sulfate. It is suggested that a small amount of l-glutamine added to the medium activated enzymes in the pathway of PGA synthesis in B. subtilis IFO3335. It can be presumed that the enzyme catalyzing the reaction from 2-oxoglutaric acid to l-glutamic acid was glutamate synthase in this bacterium.     

Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)-hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6g/l via fed-batch fermentation.     

Design of acidogenic reactors for the anaerobic treatment of the organic fraction of solid food waste

Volatile Fatty Acids (VFA) production by anaerobic fermentation of organic solid wastes was studied at laboratory scale. The influence of initial substrate concentration was evaluated on VFA production. Completely mixed reactors (0.9?l) were used at mesophilic temperature (35?°C). Food wastes had 43.8% Total Solids content. Three dilutions of substrate (1/25, 1/10 and 1/5) corresponding to 1.75%, 4.38% and 8.76% of Total Solids and five values of Organic Loading Rates: 2, 5, 10, 12.5 and 25?kg COD/m³?d were studied. It was found that substrate 1/10 led to 14?g VFA/l at a loading rate of 12.5?kg COD/m³?d and an hydraulic retention time of 3.7 d. The main VFA produced were especially acetate and butyrate. Substrate diluted 1/5 led to 26.1?g VFA/l at a loading of 5?kg COD/m³?d and an hydraulic retention time of 15.1 d, but biomass production was not optimal. In a second study, a cascade of three reactors was used. An effluent with 42?g VFA/l was obtained at steady-state conditions at a loading of 12.5?kg of COD/m³?d and an hydraulic retention time of 12.5?d. The distribution of VFA was the following: 36% of propionate, 34% of acetate and 22.5% of butyrate.     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.