Synthesis Of 5-aminolevulinic Acid Research Articles

Modularized Engineering of Shewanella oneidensis MR-1 for Efficient and Directional Synthesis of 5-Aminolevulinic Acid

Shewanella oneidensis MR-1 has found widespread applications in pollutant transformation and bioenergy production, closely tied to its outstanding heme synthesis capabilities. However, this significant biosynthetic potential is still unexploited so far. Here, we turned this bacterium into a highly-efficient bio-factory for green synthesis of 5-Aminolevulinic Acid (5-ALA), an important chemical for broad applications in agriculture, medicine, and the food industries. The native C5 pathway genes of S. oneidensis was employed, together with the introduction of foreign anti-oxidation module, to establish the 5-ALA production module, resulting 87-fold higher 5-ALA yield and drastically enhanced tolerance than the wild type. Furthermore, the metabolic flux was regulated by using CRISPR interference and base editing techniques to suppress the competitive pathways to further improve the 5-ALA titer. The engineered strain exhibited 123-fold higher 5-ALA production capability than the wild type. This study not only provides an appealing new route for 5-ALA biosynthesis, but also presents a multi-dimensional modularized engineering strategy to broaden the application scope of S. oneidensis.

A novel tetratricopeptide-repeat protein, TTP1, forms complexes with glutamyl-tRNA reductase and protochlorophyllide oxidoreductase during tetrapyrrole biosynthesis.

The biosynthesis of the tetrapyrrole end-products chlorophyll and heme depends on a multifaceted control mechanism that acts primarily at the post-translational level upon the rate-limiting step of 5-aminolevulinic acid synthesis and upon light-dependent protochlorophyllide oxidoreductase (POR). These regulatory processes require auxiliary factors that modulate the activity, stability, complex formation, and subplastidal localization of the relevant proteins. Together, they ensure optimal metabolic flow during the day and at night. As an Arabidopsis homolog of the POR-interacting tetratricopeptide-repeat protein (Pitt) first reported in Synechocystis, we characterize tetrapyrrole biosynthesis-regulating tetratricopeptide-repeat protein1 (TTP1). TTP1 is a plastid-localized, membrane-bound factor that interacts with POR, the Mg protoporphyrin monomethylester cyclase CHL27, glutamyl-tRNA reductase (GluTR), GluTR-binding protein, and FLUORESCENCE IN BLUE LIGHT. Lack of TTP1 leads to accumulation of GluTR, enhanced 5-aminolevulinic acid synthesis and lower levels of POR. Knockout mutants show enhanced sensitivity to reactive oxygen species and a slower greening of etiolated seedlings. Based on our studies, the interaction of TTP1 with GluTR and POR does not directly inhibit their enzymatic activity and contribute to the control of 5-aminolevulinic acid synthesis. Instead, we propose that TTP1 sequesters a fraction of these proteins on the thylakoid membrane, and contributes to their stability.

ZmGluTR1 is involved in chlorophyll biosynthesis and is essential for maize development

Chlorophyll is the most important carrier of photosynthesis in plants and is therefore vital for plant growth and development. Synthesis of 5-aminolevulinic acid (ALA) is initiated and catalyzed by glutamyl-tRNA reductase (GluTR) and is the rate-limiting step in chlorophyll biosynthesis. GluTR is controlled by several regulating factors. Although many studies have investigated the structure and function of GluTR in plants, the maize (Zea mays L.) GluTR has not yet been reported. Here, we isolated and identified the first loss-of-function mutant of GluTR in plants from a maize mutagenic population. The stop-gain mutation in ZmGluTR1 resulted in leaf etiolation throughout the growing season. The level of intermediates of chlorophyll biosynthesis and photosynthetic pigments decreased markedly and abnormal chloroplast structure was also observed in the mutants. Further analysis revealed that the deletion of carboxyl terminal (C-terminal) led to premature transcription termination and this hindered the interaction with FLUORESCENT (FLU), thereby influencing the stability of mutated ZmGluTR1 and leading to abolish interaction with GluTR-binding protein (GluBP). Moreover, mutations in the catalytic domain or nicotinamide adenine dinucleotide phosphate (NADPH) binding domain were lethal under normal growth conditions. These results indicate that ZmGluTR1 plays a fundamental role in chlorophyll biosynthesis and maize development.

NTRC and TRX-f Coordinately Affect the Levels of Enzymes of Chlorophyll Biosynthesis in a Light-Dependent Manner.

Redox regulation of plastid gene expression and different metabolic pathways promotes many activities of redox-sensitive proteins. We address the question of how the plastid redox state and the contributing reducing enzymes control the enzymes of tetrapyrrole biosynthesis (TBS). In higher plants, this metabolic pathway serves to produce chlorophyll and heme, among other essential end products. Because of the strictly light-dependent synthesis of chlorophyll, tight control of TBS requires a diurnal balanced supply of the precursor 5-aminolevulinic acid (ALA) to prevent the accumulation of photoreactive metabolic intermediates in darkness. We report on some TBS enzymes that accumulate in a light intensity-dependent manner, and their contents decrease under oxidizing conditions of darkness, low light conditions, or in the absence of NADPH-dependent thioredoxin reductase (NTRC) and thioredoxin f1 (TRX-f1). Analysis of single and double trxf1 and ntrc mutants revealed a decreased content of the early TBS enzymes glutamyl-tRNA reductase (GluTR) and 5-aminolevulinic acid dehydratase (ALAD) instead of an exclusive decrease in enzyme activity. This effect was dependent on light conditions and strongly attenuated after transfer to high light intensities. Thus, it is suggested that a deficiency of plastid-localized thiol-redox transmitters leads to enhanced degradation of TBS enzymes rather than being directly caused by lower catalytic activity. The effects of the proteolytic activity of the Clp protease on TBS enzymes were studied by using Clp subunit-deficient mutants. The simultaneous lack of TRX and Clp activities in double mutants confirms the Clp-induced degradation of some TBS proteins in the absence of reductive activity of TRXs. In addition, we verified previous observations that decreased chlorophyll and heme levels in ntrc could be reverted to WT levels in the ntrc/Δ2cp triple mutant. The decreased synthesis of 5-aminolevulinic acid and porphobilinogen in ntrc was completely restored in ntrc/Δ2cp and correlated with WT-like levels of GluTR, ALAD, and other TBS proteins.

Enhanced recombinant carbonic anhydrase in T7RNAP-equipped Escherichia coli W3110 for carbon capture storage and utilization (CCSU)

Sulfurihydrogenibium yellowstonense carbonic anhydrase (SyCA) is a well-known thermophilic CA for carbon mineralization. To broaden the applications of SyCA, the activity of SyCA was improved through stepwise engineering and in different cultural conditions, as well as extended to co-expression with other enzymes. The engineered W3110 strains with 4 different T7 RNA polymerase levels were employed for SyCA production. As a result, the best strain WT7L cultured in modified M9 medium with temperature shifted from 37 to 30 °C after induction increased SyCA activity to 9122 U/mL. The SyCA whole-cell biocatalyst was successfully applied for carbon capture and storage (CCS) of CaCO3. Furthermore, SyCA was applied for low-carbon footprint synthesis of 5-aminolevulinic acid (5-ALA) and cadaverine (DAP) by coupling with ALA synthetase (ALAS) and lysine decarboxylase (CadA), suppressing CO2 release to −6.1 g-CO2/g-ALA and −2.53 g-CO2/g-DAP, respectively. Harnessing a highly active SyCA offers a complete strategy for CCSU in a green process.

Glutamate 1-semialdehyde aminotransferase is connected to GluTR by GluTR-binding protein and contributes to the rate-limiting step of 5-aminolevulinic acid synthesis

Tetrapyrroles play fundamental roles in crucial processes including photosynthesis, respiration, and catalysis. In plants, 5-aminolevulinic acid (ALA) is the common precursor of tetrapyrroles. ALA is synthesized from activated glutamate by the enzymes glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase (GSAAT). ALA synthesis is recognized as the rate-limiting step in this pathway. We aimed to explore the contribution of GSAAT to the control of ALA synthesis and the formation of a protein complex with GluTR. In Arabidopsis thaliana, two genes encode GSAAT isoforms: GSA1 and GSA2. A comparison of two GSA knockout mutants with the wild-type revealed the correlation of reduced GSAAT activity and ALA-synthesizing capacity in leaves with lower chlorophyll content. Growth and green pigmentation were more severely impaired in gsa2 than in gsa1, indicating the predominant role of GSAAT2 in ALA synthesis. Interestingly, GluTR accumulated to higher levels in gsa2 than in the wild-type and was mainly associated with the plastid membrane. We propose that the GSAAT content modulates the amount of soluble GluTR available for ALA synthesis. Several different biochemical approaches revealed the GSAAT-GluTR interaction through the assistance of GluTR-binding protein (GBP). A modeled structure of the tripartite protein complex indicated that GBP mediates the stable association of GluTR and GSAAT for adequate ALA synthesis.

Synergistic Improvement of 5-Aminolevulinic Acid Production with Synthetic Scaffolds and System Pathway Engineering.

Engineered synthetic scaffolds to organize metabolic pathway enzymes and system pathway engineering to fine-tune metabolic fluxes play essential roles in microbial production. Here, we first obtained the most favorable combination of key enzymes for 5-aminolevulinic acid (5-ALA) synthesis through the C5 pathway by screening enzymes from different sources and optimizing their combination in different pathways. Second, we successfully constructed a multienzyme complex assembly system with PduA*, which spatially recruits the above three key enzymes for 5-ALA synthesis in a designable manner. By further optimizing the ratio of these key enzymes in synthetic scaffolds, the efficiency of 5-ALA synthesis through the C5 pathway was significantly improved. Then, the competitive metabolism pathway was fine-tuned by rationally designing different antisense RNAs, further significantly increasing 5-ALA titers. Furthermore, for efficient 5-ALA synthesis, obstacles of NADH and NADPH imbalances and feedback inhibition of the synthesis pathway were also overcome through engineering the NADPH regeneration pathway and transport pathway, respectively. Finally, combining these strategies with further fermentation optimization, we achieved a final 5-ALA titer of 11.4 g/L. These results highlight the importance of synthetic scaffolds and system pathway engineering to improve the microbial cell factory production performance.

An alanine to valine mutation of glutamyl-tRNA reductase enhances 5-aminolevulinic acid synthesis in rice.

An alanine to valine mutation of glutamyl-tRNA reductase's 510th amino acid improves 5-aminolevulinic acid synthesis in rice. 5-aminolevulinic acid (ALA) is the common precursor of all tetrapyrroles and plays animportant role in plant growth regulation. ALA is synthesized from glutamate, catalyzed by glutamyl-tRNA synthetase (GluRS), glutamyl-tRNA reductase (GluTR), and glutamate-1-semialdehyde aminotransferase (GSAT). In Arabidopsis, ALA synthesis is the rate-limiting step in tetrapyrrole production via GluTR post-translational regulations. In rice, mutations of GluTR and GSAT homologs are known to confer chlorophyll deficiency phenotypes; however, the enzymatic activity of rice GluRS, GluTR, and GSAT and the post-translational regulation of rice GluTR have not been investigated experimentally. We have demonstrated that a suppressor mutation in rice partially reverts the xantha trait. In the present study, we first determine that the suppressor mutation results from a G → A nucleotide substitution of OsGluTR (and an A → V change of its 510th amino acid). Protein homology modeling and molecular docking show that the OsGluTRA510V mutation increases its substrate binding. We then demonstrate that the OsGluTRA510V mutation increases ALA synthesis in Escherichia coli without affecting its interaction with OsFLU. We further explore homologous genes encoding GluTR across 193 plant species and find that the amino acid (A) is 100% conserved at the position, suggesting its critical role in GluTR. Thus, we demonstrate that the gain-of-function OsGluTRA510V mutation underlies suppression of the xantha trait, experimentally proves the enzymatic activity of rice GluRS, GluTR, and GSAT in ALA synthesis, and uncovers conservation of the alanine corresponding to the 510th amino acid of OsGluTR across plant species.

Post-translational regulation of metabolic checkpoints in plant tetrapyrrole biosynthesis.

Tetrapyrrole biosynthesis produces metabolites that are essential for critical reactions in photosynthetic organisms, including chlorophylls, heme, siroheme, phytochromobilins, and their derivatives. Due to the paramount importance of tetrapyrroles, a better understanding of the complex regulation of tetrapyrrole biosynthesis promises to improve plant productivity in the context of global climate change. Tetrapyrrole biosynthesis is known to be controlled at multiple levels-transcriptional, translational and post-translational. This review addresses recent advances in our knowledge of the post-translational regulation of tetrapyrrole biosynthesis and summarizes the regulatory functions of the various auxiliary factors involved. Intriguingly, the post-translational network features three prominent metabolic checkpoints, located at the steps of (i) 5-aminolevulinic acid synthesis (the rate-limiting step in the pathway), (ii) the branchpoint between chlorophyll and heme synthesis, and (iii) the light-dependent enzyme protochlorophyllide oxidoreductase. The regulation of protein stability, enzymatic activity, and the spatial organization of the committed enzymes in these three steps ensures the appropriate flow of metabolites through the tetrapyrrole biosynthesis pathway during photoperiodic growth. In addition, we offer perspectives on currently open questions for future research on tetrapyrrole biosynthesis.

Inhibiting Glutamine Metabolism with CB-839 Reduces Erythrocytosis in MPN Mice

Metabolic reprogramming is one of the hallmarks of cancer, as these rapidly dividing cells need to adapt their metabolism to cope with an increased energy demand. We have previously showed that in mouse models of myeloproliferative neoplasms (MPN), JAK2-mutant cells display metabolic alterations, including increased oxidative phosphorylation and glycolysis. The glutamine-glutamate-alphaKetoglutarate (aKG) axis, besides fueling the Krebs cycle and anabolic processes, contributes to the synthesis of the heme precursor 5-aminolevulinic acid (5-ALA), thus making glutaminolysis a potential target for MPN therapy.Here, we examined the effects of pharmacological inhibition of the conversion of glutamine to glutamate by glutaminase 1 by Telaglenastat (CB-839) in a mouse model of JAK2-V617F driven MPN. We performed competitive transplantations of bone marrow co-expressing JAK2-V617F with a GFP-reporter and wildtype mice in a 1:10 mixture into lethally irradiated mice. At 8 weeks post-transplantation, the mice developed MPN phenotype and were randomized into 4 treatment arms. They were then treated for 6 weeks with vehicle, CB-839, 3PO (6-phosphofructo-2-kinase inhibitor) and the combination of CB-839 + 3PO. We found that hemoglobin levels were significantly reduced in CB-839 treated mice and were fully normalised in mice that received a combination of CB-839 + 3PO (Fig. 1A). Other peripheral blood cell counts did not change (not shown). Hypoglycemia was noted in the vehicle group, as previously described, but an increase in blood glucose levels was seen already after one week of treatment with CB-839. 3PO alone had less impact on hypoglycemia and mice receiving CB-839 + 3PO had similar values to CB-839 alone (Fig. 1A). In line with these findings, splenomegaly was also reduced (Fig. 1A).To characterise the changes in metabolic activity, we determined the metabolic profiles of unfractionated bone marrow (BM) cells using seahorse assays. As expected, basal and maximal glycolysis and oxidative phosphorylation were higher in vehicle treated MPN cells than cells from wildtype controls. However, when BM cells from CB-839 or CB-839 + 3PO groups were examined, they displayed a strong downmodulation of both glycolysis and oxidative phosphorylation, indicating that chronic exposure to CB-839 reprograms the metabolic machinery by reducing not only the basal glycolytic and respiratory levels, but also the maximal capacity (Fig. 1B).Glutamine is the primary source of aKG needed to produce the heme precursor 5-ALA, and hemoglobin synthesis consumes the majority of ATP in erythroid progenitors. We therefore hypothesize that 5-ALA shortage caused by CB-839 limits hemoglobin production, leading to reduced catabolic activity and drop in ATP demand. To evaluate this model, we examined the stages of erythroid differentiation from vehicle or treated mice. Early and intermediate erythroid cells were enriched at the expense of later stages in CB-839 and CB-839+3PO treatment arms (Fig. 1C). The accumulation of erythroid precursors at the stage before hemoglobin is assembled supports the hypothesis that targeting glutamine usage impairs excessive erythropoiesis by limiting hemoglobinization rate.Our data show that inhibiting glutaminolysis ameliorated MPN phenotype and that the combination with the glycolytic inhibitor 3PO was able to induce complete hematological response. Since CB-839 is already in clinical trials for other cancers, our data suggest that it could also be tested for treatment of patients with polycythemia vera. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In vivo functional analysis of the structural domains of FLUORESCENT (FLU).

The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme protochlorophyllide oxidoreductase (POR). The interruption of Chl synthesis during darkness requires suppression of the synthesis of 5-aminolevulinic acid (ALA), the first precursor molecule specific for Chl synthesis. The inactivation of glutamyl-tRNA reductase (GluTR), the first enzyme in tetrapyrrole biosynthesis, accomplished the decreased ALA synthesis by the membrane-bound protein FLUORESCENT (FLU) and prevents overaccumulation of protochlorophyllide (Pchlide) in the dark. We set out to elucidate the molecular mechanism of FLU-mediated inhibition of ALA synthesis, and explored the role of each of the three structural domains of mature FLU, the transmembrane, coiled-coil and tetratricopeptide repeat (TPR) domains, in this process. Efforts to rescue the FLU knock-out mutant with truncated FLU peptides revealed that, on its own, the TPR domain is insufficient to inactivate GluTR, although tight binding of the TPR domain to GluTR was detected. A truncated FLU peptide consisting of transmembrane and TPR domains also failed to inactivate GluTR in the dark. Similarly, suppression of ALA synthesis could not be achieved by combining the coiled-coil and TPR domains. Interaction studies revealed that binding of GluTR and POR to FLU is essential for inhibiting ALA synthesis. These results imply that all three FLU domains are required for the repression of ALA synthesis, in order to avoid the overaccumulation of Pchlide in the dark. Only complete FLU ensures the formation of a membrane-bound ternary complex consisting at least of FLU, GluTR and POR to repress ALA synthesis.

Downregulating of hemB via synthetic antisense RNAs for improving 5-aminolevulinic acid production in Escherichia coli.

The online version contains supplementary material available at 10.1007/s13205-021-02733-8.

Cold acclimation can specifically inhibit chlorophyll biosynthesis in young leaves of Pakchoi

BackgroundLeaf color is an important trait in breeding of leafy vegetables. Y-05, a pakchoi (Brassica rapa ssp. chinensis) cultivar, displays yellow inner (YIN) and green outer leaves (GOU) after cold acclimation. However, the mechanism of this special phenotype remains elusive.ResultsWe assumed that the yellow leaf phenotype of Y-05 maybe caused by low chlorophyll content. Pigments measurements and transmission electron microscopy (TEM) analysis showed that the yellow phenotype is closely related with decreased chlorophyll content and undeveloped thylakoids in chloroplast. Transcriptomes and metabolomes sequencing were next performed on YIN and GOU. The transcriptomes data showed that 4887 differentially expressed genes (DEGs) between the YIN and GOU leaves were mostly enriched in the chloroplast- and chlorophyll-related categories, indicating that the chlorophyll biosynthesis is mainly affected during cold acclimation. Together with metabolomes data, the inhibition of chlorophyll biosynthesis is contributed by blocked 5-aminolevulinic acid (ALA) synthesis in yellow inner leaves, which is further verified by complementary and inhibitory experiments of ALA. Furthermore, we found that the blocked ALA is closely associated with increased BrFLU expression, which is indirectly altered by cold acclimation. In BrFLU-silenced pakchoi Y-05, cold-acclimated leaves still showed green phenotype and higher chlorophyll content compared with control, meaning silencing of BrFLU can rescue the leaf yellowing induced by cold acclimation.ConclusionsOur findings suggested that cold acclimation can indirectly promote the expression of BrFLU in inner leaves of Y-05 to block ALA synthesis, resulting in decreased chlorophyll content and leaf yellowing. This study revealed the underlying mechanisms of leaves color change in cold-acclimated Y-05.

Crosstalk between GABA and ALA to improve antioxidation and cell expansion of tomato seedling under cold stress

Cold stress inhibits plant growth and ultimately affects yield formation. Exogenous 5- aminolevulinic acid (ALA) can improve tomato cold tolerance and promote plant growth. It significantly up-regulated glutamate decarboxylase gene (SlGAD4) expression, which was involved in gamma-aminobutyric acid (GABA) synthesis. Whether the GABA was involved in ALA-regulated tomato cold tolerance and plant growth was unclear. Thus, this study aimed to explore the effects of exogenous ALA and GABA on endogenous ALA and GABA synthesis. The roles of ALA and GABA on regulating the antioxidation and cell morphological changes related to plant growth in tomato leaves were further explored. And the internal relationship between ALA and GABA in increasing tomato cold tolerance was also determined. Results showed that cold stress increased the glutamate (Glu) and GABA contents and reduced the ALA content. The exogenous ALA- or GABA-treated plants demonstrated decreased Glu content and increased GABA or ALA contents. Exogenous ALA or GABA treatment promoted the expansion of upper epidermal (UEP) and palisade parenchymal (PA) cells and up-regulated the xyloglucan endotransglucosylase/hydrolases gene (SlXTH23) expression in tomato leaves. Exogenous ALA or GABA significantly alleviated cold-induced tomato membrane lipid peroxidation and enhanced C-repeat binding factors gene (SlCBF2) expression and superoxide dismutase (SOD), catalase (CAT), APX and GR activities. Inhibiting endogenous GABA with 3-mercaptopropionic (3-mp) dramatically decreased the size of UEP and PA cells and reduced SOD and CAT activities. It also aggravated the membrane peroxidation damage, which notably weakened the alleviated effects of exogenous ALA. Inhibiting endogenous ALA with gabaculine (gaba) partly inhibited the alleviated effects of exogenous GABA. These findings indicated that spray with exogenous ALA or GABA could increase endogenous ALA and GABA levels in tomato leaves. In addition, endogenous GABA and ALA could be mutually converted through Glu. Exogenous ALA and GABA promoted cell expansion, stimulated the antioxidant system and might enhance tomato cold tolerance via endogenous GABA signal and the C-repeat binding factor regulation pathway, thereby alleviating cold inhibited plant growth. In conclusion, GABA crosstalk with ALA improved tomato cold stress tolerance and promoted plant growth by regulating antioxidants and cell expansion.

Transcriptome Analysis of Differentially Expressed Genes in Wild Jujube Seedlings under Salt Stress

Wild jujube (Ziziphus acidojujuba) and cultivated jujube (Ziziphus jujuba) belong to the family Rhamnaceae. Jujubes have marked drought- and salt-tolerant properties. After salt stress, wild jujube seedling growth was inhibited and photosynthetic efficiency was reduced. A bioinformatics approach was used to analyze the transcriptomics data from wild jujube seedlings grown under salt stress, and the genes differentially expressed under the salt stress were identified to provide a theoretical basis for the development and use of wild jujube plantations in salinized soil. The transcriptome sequencing from leaves of wild jujube seedlings was carried out using second-generation sequencing technology. The effects of salt stress on the differential expression of photosynthesis-related genes in wild jujube seedlings were analyzed. Transcriptome sequencing revealed a total of 5269 differentially expressed genes (DEGs), of which 2729 were up-regulated and 2540 were down-regulated. DEGs were mainly enriched with respect to photosynthesis, photosynthetic antenna proteins, glyoxylic acid and dicarboxylic acid metabolism, linolenic acid metabolism, cysteine and methionine metabolism, and porphyrin and chlorophyll metabolism. Among them, the photosynthesis pathway-related DEGs were most highly enriched. Further analysis of porphyrin and chlorophyll synthesis and photosynthesis-related pathways revealed that they were significantly enriched by 97 photosynthesis-related DEGs. The DEGs in the photosynthesis and photosynthetic antenna protein pathways were down-regulated, whereas the DEGs glutamyl-tRNA reductase (HEMA), ferrochelatase (HEMH), and pheophorbide a oxygenase (PAO) in the porphyrin and chlorophyll synthesis pathways were up-regulated, with the remainder being down-regulated. The nuclear gene encoding Rubisco, the key enzyme in the photosynthetic carbon fixation pathway, was also down-regulated. The results showed that the photosynthetic rate of wild jujube seedlings decreased following exposure to salinity stress, an effect that was related to the increased synthesis of 5-aminolevulinic acid and heme, and the up-regulation of expression of a gene encoding a chlorophyll-degrading enzyme, and was related to the down-regulation of gene expression in photosynthesis-related pathways such as light energy capture and carbon fixation. Selection of nine DEGs related to photosynthesis and chlorophyll biosynthesis by quantitative real-time-PCR confirmed that expression changes of these nine DEGs were consistent with the transcriptome sequencing results.

Multi-enzymatic recycling of ATP and NADPH for the synthesis of 5-aminolevulinic acid using a semipermeable reaction system.

5-Aminolevulinic acid (ALA) is an important cellular metabolic intermediate that has broad agricultural and medical applications. Previously, attempts have been made to synthesize ALA by multiple enzymes in cell free systems. Here we report the development of a semi-permeable system for ALA production using stable enzymes. Glucose, sodium polyphosphate, ATP, tRNA, glutamate and NADPH were used as substrates for ALA synthesis by a total of nine enzymes: adenylate kinase, polyphosphate kinase, glucose-6-phosphate dehydrogenase, phosphogluconolactonase, 6-phosphogluconate dehydrogenase, glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase from E. coli, hexokinase from yeast, as well as glutamyl-tRNA reductase and its stimulator protein glutamyl-tRNA reductase binding protein (GBP) from Arabidopsis in a semi-permeable system. After reaction for 48h, the glutamate conversion reached about 95%. This semi-permeable system facilitated the reuse of enzymes, and was helpful for the separation and purification of the product. The ALA production could be further improved by process optimization and enzyme engineering.Abbreviations: PPK: polyphosphate kinase; ADK: adenylate kinase; ALA: 5-Aminolevulinic acid; HK: hexokinase; ZWF: glucose-6-phosphatedehydrogenase; PGL: phosphogluconolactonase; GND: 6-phosphogluconate dehydrogenase; GTS: glutamyl-tRNA synthetase; GTR: glutamyl-tRNA reductase; GBP: GTR binding protein; GSAAT: glutamate-1-semialdehyde aminotransferase.

The GluTR-binding protein is the heme-binding factor for feedback control of glutamyl-tRNA reductase.

Synthesis of 5-aminolevulinic acid (ALA) is the rate-limiting step in tetrapyrrole biosynthesis in land plants. In photosynthetic eukaryotes and many bacteria, glutamyl-tRNA reductase (GluTR) is the most tightly controlled enzyme upstream of ALA. Higher plants possess two GluTR isoforms: GluTR1 is predominantly expressed in green tissue, and GluTR2 is constitutively expressed in all organs. Although proposed long time ago, the molecular mechanism of heme-dependent inhibition of GluTR in planta has remained elusive. Here, we report that accumulation of heme, induced by feeding with ALA, stimulates Clp-protease-dependent degradation of Arabidopsis GluTR1. We demonstrate that binding of heme to the GluTR-binding protein (GBP) inhibits interaction of GBP with the N-terminal regulatory domain of GluTR1, thus making it accessible to the Clp protease. The results presented uncover a functional link between heme content and the post-translational control of GluTR stability, which helps to ensure adequate availability of chlorophyll and heme.

Synthesis of 5-aminolevulinic acid with nontoxic regents and renewable methyl levulinate.

Synthesis of 5-aminolevulinic acid (5-ALA) was presented with novel bromination of biobased methyl levulinate (ML), followed by ammoniation and hydrolysis. Copper bromide (CuBr2) was employed as the bromination reagent with higher selectivity and activity instead of the conventional liquid bromine (Br2). 5-ALA was obtained in a high yield (64%) and purity (>95%) by optimum design, which is of great potential in industrialization.

Fluorescence in blue light (FLU) is involved in inactivation and localization of glutamyl-tRNA reductase during light exposure.

Fluorescent in blue light (FLU) is a negative regulator involved in dark repression of 5-aminolevulinic acid (ALA) synthesis and interacts with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme of tetrapyrrole biosynthesis. In this study, we investigated FLU's regulatory function in light-exposed FLU-overexpressing (FLUOE) Arabidopsis lines and under fluctuating light intensities in wild-type (WT) and flu seedlings. FLUOE lines suppress ALA synthesis in the light, resulting in reduced chlorophyll content, but more strongly in low and high light than in medium growth light. This situation indicates that FLU's impact on chlorophyll biosynthesis depends on light intensity. FLU overexpressors contain strongly increased amounts of mainly membrane-associated GluTR. These findings correlate with FLU-dependent localization of GluTR to plastidic membranes and concomitant inhibition, such that only the soluble GluTR fraction is active. The overaccumulation of membrane-associated GluTR indicates that FLU binding enhances GluTR stability. Interestingly, under fluctuating light, the leaves of flu mutants contain less chlorophyll compared with WT and become necrotic. We propose that FLU is basically required for fine-tuned ALA synthesis. FLU not only mediates dark repression of ALA synthesis, but functions also to control balanced ALA synthesis under variable light intensities to ensure the adequate supply of chlorophyll.

Controlled Partitioning of Glutamyl-tRNA Reductase in Stroma- and Membrane-Associated Fractions Affects the Synthesis of 5-Aminolevulinic Acid.

The synthesis of 5-aminolevulinic acid (ALA) determines adequate amounts of metabolites for the tetrapyrrole biosynthetic pathway. Glutamyl-tRNA reductase (GluTR) catalyzes the rate-limiting step of ALA synthesis and was previously considered to be exclusively localized in the chloroplast stroma of light-exposed plants. To assess the intraplastidic localization of GluTR, we developed a fast separation protocol of soluble and membrane-bound proteins and reassessed the subplastidal allocation of GluTR in stroma and membrane fractions of Arabidopsis plants grown under different light regimes as well as during de-etiolation and dark incubations. Under the examined conditions, the amount of stroma-localized GluTR correlated with the ALA synthesis rate. The transfer to dark repression of ALA synthesis resulted in a loss of soluble GluTR. Arabidopsis mutants lacking one of the GluTR-interacting factors FLUORESCENT (FLU), the GluTR-binding protein (GBP) or ClpC, a chaperone of the Clp protease system, were applied to examine the amount of GluTR and its distribution to the stroma or membrane in darkness and light. Taking into consideration the different compartmental allocation of GluTR, its stability and ALA synthesis rates, the post-translational impact of these regulatory factors on GluTR activity and plastidic sublocalization is discussed.