Utilization of volatile fatty acids from the anaerobic digestion liquor of sewage sludge for 5-aminolevulinic acid production by photosynthetic bacteria

Using volatile fatty acids (VFA) from the anaerobic digestion liquor of sewage sludge, up to 9.2 mm 5-aminolevulinic acid (ALA) could be produced by Rhodobacter sphaeroides under anaerobic-light (5 kLux) conditions with repeated addition of levulinic acid (LA) and glycine and using a large inoculum (approx. 2 g/l of cells, initially from glutamate/malate medium). As the VFA medium also contained organic nitrogen sources such as glutamic acid, the cells were later grown up to about 2 g/l in the VFA medium instead of the glutamate/malate medium. ALA production was then again promoted by adding LA and glycine. Using this improved method, up to 9.3 mm ALA was produced by feeding propionate and acetate together with LA and glycine, indicating that VFA medium formed from sewage sludge could be useful for ALA production.     

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.

     

5-Aminolevulinic acid production from inexpensive glucose by engineering the C4 pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

5-Aminolevulinic acid (ALA), the first committed intermediate for natural biosynthesis of tetrapyrrole compounds, has recently drawn intensive attention due to its broad potential applications. In this study, we describe the construction of recombinant Escherichia coli strains for ALA production from glucose via the C4 pathway. The hemA gene from Rhodobacter capsulatus was optimally overexpressed using a ribosome binding site engineering strategy, which enhanced ALA production substantially from 20 to 689 mg/L. Following optimization of biosynthesis pathways towards coenzyme A and precursor (glycine and succinyl-CoA), and downregulation of hemB expression, the production of ALA was further increased to 2.81 g/L in batch-fermentation.     

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 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     

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。     

Genetic control of chlorophyll biosynthesis by the plastome in some Oenothera species (subgenus Munzia)

Reciprocal differences in the rates of chlorophyll (Chl) formation during early stages of greening are observed in hybrid seedlings with identical genomes derived from reciprocal crosses between Oenothera berteriana (=villaricae) and Oe. odorata (=picensis), subgenus Munzia. In the presence of levulinic acid (LA), a competitive inhibitor of 5-aminolevulinic acid (ALA) dehydratase, ALA accumulated in the cotyledons and chlorophyll production was reduced in a stoichometric ratio. Accumulation of both Chl in untreated tissue and of ALA in seedlings incubated with LA is much more rapid in cotyledons with berteriana plastids than in those with odorata plastids. No difference was found between the inhibitor constants for LA of ALA dehydratase extracted from seedlings with either berteriana or odorata plastids. ALA formation is not limited by the availability of possible precursors. ALA dehydratase and the porphobilinogenase complex (PBGase) are present in abundance and in equal amounts in cotyledons with either berteriana or odorata plastids. It is concluded that the different capacities of the ALA synthesizing system fully account for the different rates of Chl formation in the seedlings with identical genomes and different plastid types.Abbreviations Chl chlorophyll - ALA 5-aminolevnlinic acid - ALAD 5-aminolevulinic acid dehydratase - LA levulinic acid - PBG porphobilinogen - PBGase porphobilinogenase - Oe Oenothera - bert berteriana - od odorata - Pl plastids     

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Herein, we report the development of a microbial bioprocess for high‐level production of 5‐aminolevulinic acid (5‐ALA), a valuable non‐proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5‐ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5‐ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl‐CoA for enhanced 5‐ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high‐level 5‐ALA biosynthesis from glycerol (~30 g L^?1) under both microaerobic and aerobic conditions, achieving up to 5.95 g L^?1 (36.9% of the theoretical maximum yield) and 6.93 g L^?1 (50.9% of the theoretical maximum yield) 5‐ALA, respectively. This study represents one of the most effective bio‐based production of 5‐ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio‐based production.     

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.     

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).     

Glycine metabolism and chlorophyll synthesis in barley leaves

α-Hydroxypyridine methane sulphonic acid (HPMS), isonicotinyl hydrazide (INH) and nialamide inhibit chlorophyll synthesis in etiolated barley leaves exposed to light. HPMS lowered the rate of protochlorophyllide regeneration but had little effect on the synthesis of protochlorophyll (P630) from exogenous $\text{δ}$-aminolaevulinic acid (ALA). The addition of glycine to HPMS treated leaves partially overcame the inhibition of chlorophyll synthesis. Glycine-[¹⁴C] was readily incorporated into ALA in dark-grown leaves. HPMS treatment increased the sp. act. of ALA in leaves fed glycine-[¹⁴C]. Glycollate oxidation was lower in extracts from HPMS treated leaves. Plants may therefore have two pathways for ALA production with the glutamate pathway becoming more important in conditions where photorespiration is high.     

琥珀酸脱氢酶或琥珀酰辅酶A合成酶缺失促进大肠杆菌积累5-氨基乙酰丙酸

5-氨基乙酰丙酸 (ALA) 是生物体内四吡咯类化合物的合成前体,在农业及医药领域应用广泛,是极具开发价值的高附加值生物基化学品。目前利用外源C4途径的重组大肠杆菌发酵生产ALA的研究主要利用LB培养基并添加葡萄糖和琥珀酸、甘氨酸等合成前体,成本较高。琥珀酸在C4途径中以琥珀酰辅酶A的形式直接参与ALA的合成。文中在以葡萄糖为主要碳源的无机盐培养基中研究了琥珀酰辅酶A下游代谢途径琥珀酸脱氢酶编码基因sdhAB和琥珀酰辅酶A合成酶编码基因sucCD缺失对ALA积累的影响。与仅表达异源ALA合成酶的对照菌株相比,sdhAB和sucCD缺失菌株ALA的产量分别提高了25.59%和12.40%,且ALA的积累不依赖于琥珀酸的添加和LB培养基的使用,从而大幅降低了生产成本,显示出良好的工业应用前景。     

Comparative effects of well-balanced diets enriched in α-linolenic or linoleic acids on LC-PUFA metabolism in rat tissues

The intake of the essential fatty acid precursor α-linolenic acid (ALA) contributes to ensure adequate n-3 long-chain polyunsaturated fatty acid (LC-PUFA) bioavailability. Conversely, linoleic acid (LA) intake may compromise tissue n-3 PUFA status as its conversion to n-6 LC-PUFA shares a common enzymatic pathway with the n-3 family. This study aimed to measure dietary ALA and LA contribution to LC-PUFA biosynthesis and tissue composition. Rats were fed with control or experimental diets moderately enriched in ALA or LA for 8 weeks. Liver Δ6- and Δ5-desaturases were analyzed and FA composition was determined in tissues (red blood cells, liver, brain and heart). Hepatic Δ6-desaturase activity was activated with both diets, and Δ5-desaturase activity only with the ALA diet. The ALA diet led to higher n-3 LC-PUFA composition, including DHA in brain and heart. The LA diet reduced n-3 content in blood, liver and heart, without impacting n-6 LC-PUFA composition. At levels relevant with human nutrition, increasing dietary ALA and reducing LA intake were both beneficial in increasing n-3 LC-PUFA bioavailability in tissues.     

Isolation and Characterisation of a High-Efficiency Desaturase and Elongases from Microalgae for Transgenic LC-PUFA Production

The production of long-chain polyunsaturated fatty acids from precursor molecules linoleic acid (LA; 18:2ω6) and α-linolenic acid (ALA; 18:3ω3) is catalysed by sequential desaturase and elongase reactions. We report the isolation of a front-end Δ6-desaturase gene from the microalgae Ostreococcus lucimarinus and two elongase genes, a Δ6-elongase and a Δ5-elongase, from the microalga Pyramimonas cordata. These enzymes efficiently convert their respective substrates when transformed in yeast (39–75% conversion for ω3 substrate fatty acids), and the Δ5-elongase in particular displays higher elongation efficiency (75% for conversion of eicosapentaenoic acid (20:5ω3) to docosapentaenoic acid (22:5ω3)) than previously reported genes. In addition, the Δ6-desaturase is homologous with acyl-CoA desaturases and shows a strong preference for the ω3 substrate ALA.     

Variation in the Fatty‐Acid Content in Seeds of Various Black,Red, and White Currant Varieties

Currant seeds, a by‐product of juice production, are recognized as a valuable source of oil rich in polyunsaturated fatty acids. We have evaluated 28 currant varieties for their oil content and fatty‐acid composition. The oil content in the seeds ranged from 18.2–27.7%, and no statistical difference between varieties of different fruit color were recorded. Furthermore, the estimated oil yields in the field production ranged from 26.4–212.4 kg/ha. The GC and GC/MS chemical profiles of the seed oils extracted from all examined varieties were common for currants. Linoleic acid (LA) was the major component, with contents ranging from 32.7–46.9% of total fatty acids, followed by α‐linolenic acid (ALA; 2.9–32.0 %), oleic acid (OA; 9.8–19.9%), γ‐linolenic acid (GLA; 3.3–18.5%), palmitic acid (PA; 4.4–8.1%), stearidonic acid (SDA; 2.2–4.7%), and stearic acid (SA; 1.2–2.4%). Quantitative differences in the fatty‐acid profiles between varieties of different fruit color were observed. Blackcurrant varieties showed significantly higher contents of LA, GLA, and PA than red and white currant varieties, whereas significantly higher amounts of ALA and OL were detected in the red and white varieties. Cluster analysis based on the chemical oil profiles joined the blackcurrants in one group, while most of the red and white cultivars joined in a second group at the same linkage distance.     

Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

Investigating the bifunctionality of cyclizing and “classical” 5‐aminolevulinate synthases

The precursor to all tetrapyrroles is 5‐aminolevulinic acid, which is made either via the condensation of glycine and succinyl‐CoA catalyzed by an ALA synthase (the C4 or Shemin pathway) or by a pathway that uses glutamyl‐tRNA as a precursor and involves other enzymes (the C5 pathway). Certain ALA synthases also catalyze the cyclization of ALA‐CoA to form 2‐amino‐3‐hydroxycyclopent‐2‐en‐1‐one. Organisms with synthases that possess this second activity nevertheless rely upon the C5 pathway to supply ALA for tetrapyrrole biosynthesis. The C₅N units are components of a variety of secondary metabolites. Here, we show that an ALA synthase used exclusively for tetrapyrrole biosynthesis is also capable of catalyzing the cyclization reaction, albeit at much lower efficiency than the dedicated cyclases. Two absolutely conserved serines present in all known ALA‐CoA cyclases are threonines in all known ALA synthases, suggesting they could be important in distinguishing the functions of these enzymes. We found that purified mutant proteins having single and double substitutions of the conserved residues are not improved in their respective alternate activities; rather, they are worse. Protein structural modeling and amino acid sequence alignments were explored within the context of what is known about the reaction mechanisms of these two different types of enzymes to consider what other features are important for the two activities.     

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.     

Occurrence of two 5-aminolevulinate biosynthetic pathways in Streptomyces nodosus subsp. asukaensis is linked with the production of asukamycin

We report the results of cloning genes for two key biosynthetic enzymes of different 5-aminolevulinic acid (ALA) biosynthetic routes from Streptomyces. The genes encode the glutamyl-tRNAGlu reductase (GluTR) of the C5 pathway and the ALA synthase (ALAS) of the Shemin pathway. While Streptomyces coelicolor A3(2) synthesizes ALA via the C5 route, both pathways are operational in Streptomyces nodosus subsp. asukaensis, a producer of asukamycin. In this strain, the C5 route produces ALA for tetrapyrrole biosynthesis; the ALA formed by the Shemin pathway serves as a precursor of the 2-amino-3-hydroxycyclopent-2-enone moiety (C5N unit), an antibiotic component. The growth of S. nodosus and S. coelicolor strains deficient in the GluTR genes (gtr) is strictly dependent on ALA or heme supplementation, whereas the defect in the ALAS-encoding gene (hemA-asuA) abolishes the asukamycin production in S. nodosus. The recombinant hemA-asuA gene was expressed in Escherichia coli and in Streptomyces, and the encoded enzyme activity was demonstrated both in vivo and in vitro. The hemA-asuA gene is situated within a putative cluster of asukamycin biosynthetic genes. This is the first report about the cloning of genes for two different ALA biosynthetic routes from a single bacterium.