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.

Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli.

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

Exogenous 5-Aminolevulinic acid improved low-temperature tolerance tomato seedling by regulating starch content and phenylalanine metabolism.

Tomato is an important horticultural cash crop, and low-temperature stress has seriously affected the yield and quality of tomato. 5-Aminolevulinic acid (ALA) is widely used in agriculture as an efficient and harmless growth regulator. It is currently unclear whether exogenous ALA can cope with low-temperature stress by regulating tomato starch content and phenylalanine metabolism. In this study, exogenous ALA remarkably improved the low-temperature tolerance of tomato seedlings. RNA-sequencing results showed that exogenous ALA affected starch metabolism and phenylalanine metabolism in tomato seedling leaves under low-temperature stress. Subsequently, we used histochemical staining, observation of chloroplast microstructure, substance content determination, and qRT-PCR analysis to demonstrate that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism (SlPAL, SlPOD1, and SlPOD2). Simultaneously, we found that exogenous ALA induced the expression of SlMYBs and SlWRKYs under low-temperature stress. In addition, dual luciferase, yeast one hybrid, and electrophoretic mobility shift assays indicate that SlMYB4 and SlMYB88 could regulate the expression of SlPOD2 in phenylalanine metabolism. We demonstrated that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism.

Construction of 5-Aminolevulinic Acid Microbial Cell Factories through Identification of Novel Synthases and Metabolic Pathway Screens and Transporters.

5-Aminolevulinic acid (5-ALA) plays a pivotal role in the biosynthesis of heme and chlorophyll and has garnered great attention for its agricultural applications. This study explores the multifaceted construction of 5-ALA microbial cell factories. Evolutionary analysis-guided screening identified a novel 5-ALA synthase from Sphingobium amiense as the best synthase. An sRNA library facilitated global gene screening that demonstrated that trpC and ilvA repression enhanced 5-ALA production by 74.3% and 102%, respectively. Subsequently, efflux of 5-ALA by the transporter Gdx increased 5-ALA biosynthesis by 25.7%. To mitigate oxidative toxicity, DNA-binding proteins from starved cells were employed, enhancing cell density and 5-ALA titer by 21.1 and 4.1%, respectively. Combining these strategies resulted in an Escherichia coli strain that produced 5-ALA to 1.51 g·L-1 in shake flask experiments and 6.19 g·L-1 through fed-batch fermentation. This study broadens the repertoire of available 5-ALA synthases and transporters and provides a new platform for optimizing 5-ALA bioproduction.

5-Aminolevulinic acid treatment mitigates pesticide stress in bean seedlings by regulating stress-related gene expression and retrotransposon movements.

Overdoses of pesticides lead to a decrease in the yield and quality of plants, such as beans. The unconscious use of deltamethrin, one of the synthetic insecticides, increases the amount of reactive oxygen species (ROS) by causing oxidative stress in plants. In this case, plants tolerate stress by activating the antioxidant defense mechanism and many genes. 5-Aminolevulinic acid (ALA) improves tolerance to stress by acting exogenously in low doses. There are many gene families that are effective in the regulation of this mechanism. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. In this study, the expression levels of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and stress-associated protein (SAP) genes were determined by Q-PCR in deltamethrin (0.5 ppm) and various doses (20, 40, and 80 mg/l) of ALA-treated bean seedlings. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. It was determined that deltamethrin increased the expression of SOD (1.8-fold), GPX (1.4-fold), CAT (2.7-fold), and SAP (2.5-fold) genes, while 20 and 40 mg/l ALA gradually increased the expression of these genes at levels close to control, but 80 mg/l ALA increased the expression of these genes almost to the same level as deltamethrin (2.1-fold, 1.4-fold, 2.6-fold, and 2.6-fold in SOD, GPX, CAT, and SAP genes, respectively). In addition, retrotransposon-microsatellite amplified polymorphism (REMAP) was performed to determine the polymorphism caused by retrotransposon movements. While deltamethrin treatment has caused a decrease in genomic template stability (GTS) (27%), ALA treatments have prevented this decline. At doses of 20, 40, and 80 mg/L of ALA treatments, the GTS ratios were determined to be 96.8%, 74.6%, and 58.7%, respectively. Collectively, these findings demonstrated that ALA has the utility of alleviating pesticide stress effects on beans.

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.

Transcription factor MdNAC33 is involved in ALA-induced anthocyanin accumulation in apples.

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has a significant function in promoting anthocyanin accumulation in many species of fruits. However, the mechanisms underlying remain obscure. In a transcriptome study of our group, it was found that many transcription factors (TFs) including NACs responsive to ALA treatment during anthocyanin accumulation. In the present study, we found a NAC of apple, MdNAC33 was coordinatively expressed with anthocyanin accumulation after ALA treatment in the apple fruits and leaves, suggesting that this TF may be involved in anthocyanin accumulation induced by ALA. We found that the MdNAC33 protein was localized in the nucleus and exhibited strong transcriptional activity in both yeast cells and plants, where its C-terminal contributed to the transcriptional activity. Functional analysis showed that overexpression of MdNAC33 promoted the accumulation of anthocyanin, while the silencing vector of MdNAC33 (RNAi) significantly impaired the anthocyanin accumulation induced by ALA. Yeast one-hybrid (Y1H), luciferase assay and electrophoretic mobility shift assay (EMSA) indicated that MdNAC33 could bind to promoters of MdbHLH3, MdDFR and MdANS to activate the gene expressions. In addition, MdNAC33 specifically interacts with MdMYB1, a positive regulator of anthocyanin biosynthesis, which was then in turn binding to its target genes MdUFGT and MdGSTF12, to promote anthocyanin accumulation in apples. Taken together, our data indicate that MdNAC33 plays multiple roles in ALA-induced anthocyanin biosynthesis. It provides new insights into the mechanisms of anthocyanin accumulation induced by ALA.

Construction of the Rhodobacter sphaeroides strain overproducing 5-aminolevulinic acid by insertion of endogenous promoter.

5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (PrrnB or Prsp_7571) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where PrrnB was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where PrrnB was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where PrrnB was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.

Single-cell analysis of 5-aminolevulinic acid intraoperative labeling specificity for glioblastoma.

OBJECTIVE: Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and resection is a key part of the standard of care. In fluorescence-guided surgery (FGS), fluorophores differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate used for FGS, to fluorescent protoporphyrin IX (PpIX). The resulting fluorescence is believed to be specific to neoplastic glioma cells, but this specificity has not been examined at a single-cell level. The objective of this study was to determine the specificity with which 5-ALA labels the diversity of cell types in GBM. METHODS: The authors performed single-cell optical phenotyping and expression sequencing-version 2 (SCOPE-seq2), a paired single-cell imaging and RNA sequencing method, of individual cells on human GBM surgical specimens with macroscopically visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allowed the authors to simultaneously image PpIX fluorescence and unambiguously identify neoplastic cells from single-cell RNA sequencing. Experiments were also conducted in cell culture and co-culture models of glioma and in acute slice cultures from a mouse glioma model to investigate cell- and tissue-specific uptake and secretion of 5-ALA and PpIX. RESULTS: SCOPE-seq2 analysis of human GBM surgical specimens revealed that 5-ALA treatment resulted in labeling that was not specific to neoplastic glioma cells. The cell culture further demonstrated that nonneoplastic cells could be labeled by 5-ALA directly or by PpIX secreted from surrounding neoplastic cells. Acute slice cultures from mouse glioma models showed that 5-ALA preferentially labeled GBM tumor tissue over nonneoplastic brain tissue with significant labeling in the tumor margins, and that this contrast was not due to blood-brain barrier disruption. CONCLUSIONS: Together, these findings support the use of 5-ALA as an indicator of GBM tissue but question the main advantage of 5-ALA for specific intracellular labeling of neoplastic glioma cells in FGS. Further studies are needed to systematically compare the performance of 5-ALA to that of potential alternatives for FGS.

ALA-PDT promotes the death and contractile capacity of hypertrophic scar fibroblasts through inhibiting the TGF-ß1/Smad2/3/4 signaling pathway.

BACKGROUND: Hypertrophic scars, an abnormal wound-healing response to burn injuries, are characterized by massive fibroblast proliferation and excessive deposition of extracellular matrix and collagen. 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is a promising therapy for hypertrophic scar, details of the mechanisms remain to be elucidated. In this study, we aimed to investigate the molecular mechanisms involved in ALA-PDT against hypertrophic scar fibroblasts. METHODS: The morphologies of hypertrophic scar fibroblasts (HSFs) treated with ALA-PDT were observed under a light microscopy. The viability of HSFs was detected using the CCK-8 assay. HSFs-populated collagen gel contraction assays were conducted to examine the fibroblast contractility and the cytotoxicity of HSFs in 3D collagen tissues were observed using confocal microscopy. The effect of ALA-PDT on TGF-ß1/Smad2/3/4 signaling pathway activation and effector gene expression were verified by immunoprecipitation, western blot and real-time quantitative PCR analysis. RESULTS: We observed significant changes in cell morphology after ALA-PDT treatment of HSFs. As ALA concentration and light dose increased, the viability of HSFs significantly decreased. ALA-PDT can significantly alleviate the contractile capacity and promote the death of HSFs induced by TGF-ß1 treatment in a three-dimensional collagen culture model. TGF-ß1 treatment of HSFs can significantly induce phosphorylation of Smad2/3 (p-Smad2/3) in whole cells, as well as p-Smad2/3 and Smad4 proteins into the nucleus and increase the mRNA levels of collagen 1/3 and α-SMA. ALA-PDT hampers the TGF-ß1-Smad2/3/4 signaling pathway activation by inducing K48-linked ubiquitination and degradation of Smad4. CONCLUSIONS: Our results provide evidence that ALA-PDT can inhibit fibroblast contraction and promote cell death by inhibiting the activation of the TGF-ß1 signaling pathway that mediates hypertrophic scar formation, which may be the basis for the efficacy of ALA-PDT in the treatment of hypertrophic scars.

Modified 5-aminolevulinic acid photodynamic therapy induces cutaneous squamous cell carcinoma cell pyroptosis via the JNK signaling pathway.

Modified 5-aminolevulinic acid photodynamic therapy (M-PDT) is a novel therapeutic modality for cutaneous squamous cell carcinoma (cSCC) that is reported to be effective and well tolerated. However, the mechanisms underlying its antitumor effects are not fully understood. In this research, we investigated the effects of M-PDT on pyroptosis, a form of programmed cell death characterized by cell swelling, ruptures of cell membrane, and inflammatory cytokine release, in two human cSCC cell lines, SCL-1 and HSC-5. We found that M-PDT triggered pyroptosis in a dose-dependent manner, as evidenced by increased lactate dehydrogenase release, propidium iodide staining, and expression of pyroptosis-related proteins, such as NLR family pyrin domain containing 3 (NLRP3), N-terminal of gasdermin D (N-GSDMD), cleaved caspase-1, and mature interleukin 1 beta (IL-1B) in both cell lines. This process was inhibited by treatment with MCC950, an NLRP3-specific inhibitor, suggesting the involvement of the NLRP3 inflammasome in M-PDT-induced pyroptosis. We also demonstrated that M-PDT activated c-Jun N-terminal kinase (JNK) signaling, which is required for pyroptosis induction, as treatment with SP600125, a JNK inhibitor, suppressed the expression of pyroptosis-related proteins after M-PDT. JNK activation enhanced M-PDT-induced pyroptosis, highlighting the significance of the JNK pathway in M-PDT. Moreover, M-PDT increased intracellular reactive oxygen species (ROS) levels, which are responsible for JNK activation and pyroptosis induction. In summary, our results revealed that M-PDT triggers pyroptosis through ROS-mediated JNK activation and subsequent NLRP3 inflammasome activation in cSCC cells, providing a better understanding of the molecular mechanism of M-PDT and promoting its clinical application.

Exogenously applied 5-aminolevulinic acid modulates growth, yield, and physiological parameters in lentil (Lens culinaris Medik.) under rain-fed and supplemental irrigation conditions.

Lentils are a significant source of plant protein and are cultivated across Asia, Europe, and North Africa. Plants are subjected to various environmental stresses, which can hinder growth, yield, and productivity. 5-aminolevulinic acid (ALA) is a compound that acts as a precursor in the biosynthesis of tetrapyrroles and can increase plant tolerance to different abiotic stressors. However, the effects of exogenously applied ALA on lentil growth, yield, and physiological parameters under rain-fed and supplemental irrigation conditions are not well-known. In this study, a split plot experiment was conducted to investigate the impact of ALA foliar application and supplemental irrigation on lentil (Lens culinaris Medik.). The experiment was designed based on a randomized complete block with three replications. The main plot included four levels of supplemental irrigation [(supplementary irrigation in the flowering and early seed-filling stages, supplementary irrigation in the flowering stage, supplementary irrigation in the early seed-filling along with rain-fed conditions (no irrigation)]. The subplot considered foliar application of ALA at varying levels [(0 (control), 50 and 100 ppm)]. The results showed that water regimes and foliar spray with ALA significantly (P Ë‚ 0.01) affected plant height, number of pods per plant, pod weight, number of seeds per pod and weight of 1000 seeds, biological yield, seed yield, and harvest index. The highest total chlorophyll content was observed in plants that were subjected to supplementary irrigation in flowering and early seed filling stages and foliar sprayed with 100 ppm ALA. The study also found that exogenous ALA improved drought tolerance in lentil plants under rain-fed conditions mainly by regulating antioxidant enzymes, which ultimately protected the cellular membranes against overproduction of H2O2. Furthermore, ALA application increased total carbohydrate contents at all supplemental irrigation levels, but the rate was higher in complementary irrigation conditions during flowering and early seed-filling stages. Malondialdehyde (MDA), H2O2, and proline contents were increased in field-grown plants under rain-fed conditions without exogenous ALA application. In conclusion, this study sheds light on the effects of ALA foliar spray and supplemental irrigation on lentil growth, yield, and physiological parameters. The findings suggest that exogenous ALA can improve plant tolerance to various abiotic stressors and enhance plant growth, yield, and physiological parameters.

Oral 5-aminolevulinic acid combined with sodium ferrous citrate prevents blood-aqueous barrier breakdown after anterior chamber paracentesis in healthy beagle dogs.

This study investigated the preventive effect of 5-aminolevulinic acid combined with sodium ferrous citrate (5-ALA/SFC) on blood-aqueous barrier (BAB) breakdown induced after anterior chamber paracentesis (ACP) in beagles. 5-ALA/SFC (1/0.64 mg/kg or 3/1.92 mg/kg) or carprofen (4.0 mg/kg) was orally administered daily for 7 days prior to ACP. Then, a sample of the aqueous humor (AH) was collected from one eye via ACP (first sample) and again 60 min later (second sample). The protein and prostaglandin E2 (PGE2) concentrations in both samples were measured. Compared with the control group, high-dose 5-ALA/SFC and carprofen significantly reduced the AH protein and PGE2 concentrations in the second sample. Our findings suggest that 5-ALA/SFC suppresses BAB breakdown in dogs.

Crosstalk between 5-Aminolevulinic Acid and Abscisic Acid Adjusted Leaf Iron Accumulation and Chlorophyll Synthesis to Enhance the Cold Tolerance in Solanum lycopersicum Seedlings.

Previous studies found that 5-aminolevulinic acid (ALA) and abscisic acid (ABA) can mitigate damage from adversity by enhancing photosynthesis. However, it is not clear whether they have positive effects on iron utilization and chlorophyll synthesis of tomato seedlings under low-temperature stress. To investigate the possible functional relationship between ABA and ALA and elucidate the possible mechanisms of action of ALA to alleviate low-temperature stress in tomato seedlings, this experiment analyzed the effects of ALA and ABA on chlorophyll synthesis in tomato seedling leaves sprayed with exogenous of ALA (25 mg·L-1) or ABA (100 µM) under low-temperature stress (8-18 °C/8-12 °C, day/night). The results show that exogenous ALA increased the Fv/Fm of tomato leaves by 5.31% and increased the accumulation of iron and chlorophyll by 101.15% and 15.18%, respectively, compared to the low-temperature treatment alone, and tomato resistance of low-temperature stress was enhanced. Meanwhile, exogenous application of ALA increased the ABA content by 39.43%, and subsequent application of exogenous ABA revealed that tomato seedlings showed similar effects to exogenous ALA under low-temperature stress, with increased accumulation of iron and chlorophyll in tomato seedlings, which eventually increased the maximum photochemical efficiency of PS II. Under low-temperature stress, application of exogenous ABA significantly reduced ALA content, but the expression of key enzyme genes (PPGD, HEMB1, HEME1, and HEMF1), precursors of chlorophyll synthesis by ALA, was significantly elevated, presumably because the increased activity of these enzymes after external application of ABA accelerated ALA consumption. In conclusion, ABA may crosstalk with ALA to improve the photochemical efficiency and low temperature resistance of tomatoes by regulating chlorophyll synthesis and iron accumulation.

Regulating Photosensitizer Metabolism with DNAzyme-Loaded Nanoparticles for Amplified Mitochondria-Targeting Photodynamic Immunotherapy.

Mitochondria-specific photosensitizer accumulation is highly recommended for photodynamic therapy and mitochondrial DNA (mtDNA) oxidative damage-based innate immunotherapy but remains challenging. 5-Aminolevulinic acid (ALA), precursor of photosensitizer protoporphyrin IX (PpIX), can induce the exclusive biosynthesis of PpIX in mitochondria. Nevertheless, its photodynamic effect is limited by the intracellular biotransformation of ALA in tumors. Here, we report a photosensitizer metabolism-regulating strategy using ALA/DNAzyme-co-loaded nanoparticles (ALA&Dz@ZIF-PEG) for mitochondria-targeting photodynamic immunotherapy. The zeolitic imidazolate framework (ZIF-8) nanoparticles can be disassembled and release large amounts of zinc ions (Zn2+) within tumor cells. Notably, Zn2+ can relieve tumor hypoxia for promoting the conversion of ALA to PpIX. Moreover, Zn2+ acts as a cofactor of rationally designed DNAzyme for silencing excessive ferrochelatase (FECH; which catalyzes PpIX into photoinactive Heme), cooperatively promoting the exclusive accumulation of PpIX in mitochondria via the "open source and reduced expenditure" manner. Subsequently, the photodynamic effects derived from PpIX lead to the damage and release of mtDNA and activate the innate immune response. In addition, the released Zn2+ further enhances the mtDNA/cGAS-STING pathway mediated innate immunity. The ALA&Dz@ZIF-PEG system induced 3 times more PpIX accumulation than ALA-loaded liposome, significantly enhancing tumor regression in xenograft tumor models.

A PPIX-binding probe facilitates discovery of PPIX-induced cell death modulation by peroxiredoxin.

While heme synthesis requires the formation of a potentially lethal intermediate, protoporphyrin IX (PPIX), surprisingly little is known about the mechanism of its toxicity, aside from its phototoxicity. The cellular protein interactions of PPIX might provide insight into modulators of PPIX-induced cell death. Here we report the development of PPB, a biotin-conjugated, PPIX-probe that captures proteins capable of interacting with PPIX. Quantitative proteomics in a diverse panel of mammalian cell lines reveal a high degree of concordance for PPB-interacting proteins identified for each cell line. Most differences are quantitative, despite marked differences in PPIX formation and sensitivity. Pathway and quantitative difference analysis indicate that iron and heme metabolism proteins are prominent among PPB-bound proteins in fibroblasts, which undergo PPIX-mediated death determined to occur through ferroptosis. PPB proteomic data (available at PRIDE ProteomeXchange # PXD042631) reveal that redox proteins from PRDX family of glutathione peroxidases interact with PPIX. Targeted gene knockdown of the mitochondrial PRDX3, but not PRDX1 or 2, enhance PPIX-induced death in fibroblasts, an effect blocked by the radical-trapping antioxidant, ferrostatin-1. Increased PPIX formation and death was also observed in a T-lymphoblastoid ferrochelatase-deficient leukemia cell line, suggesting that PPIX elevation might serve as a potential strategy for killing certain leukemias.

FC2 stabilizes POR and suppresses ALA formation in the tetrapyrrole biosynthesis pathway.

During photoperiodic growth, the light-dependent nature of chlorophyll synthesis in angiosperms necessitates robust control of the production of 5-aminolevulinic acid (ALA), the rate-limiting step in the initial stage of tetrapyrrole biosynthesis (TBS). We are interested in dissecting the post-translational control of this process, which suppresses ALA synthesis for chlorophyll synthesis in dark-grown plants. Using biochemical approaches for analysis of Arabidopsis wild-type (WT) and mutant lines as well as complementation lines, we show that the heme-synthesizing ferrochelatase 2 (FC2) interacts with protochlorophyllide oxidoreductase and the regulator FLU which both promote the feedback-controlled suppression of ALA synthesis by inactivation of glutamyl-tRNA reductase, thus preventing excessive accumulation of potentially deleterious tetrapyrrole intermediates. Thereby, FC2 stabilizes POR by physical interaction. When the interaction between FC2 and POR is perturbed, suppression of ALA synthesis is attenuated and photoreactive protochlorophyllide accumulates. FC2 is anchored in the thylakoid membrane via its membrane-spanning CAB (chlorophyll-a-binding) domain. FC2 is one of the two isoforms of ferrochelatase catalyzing the last step of heme synthesis. Although FC2 belongs to the heme-synthesizing branch of TBS, its interaction with POR potentiates the effects of the GluTR-inactivation complex on the chlorophyll-synthesizing branch and ensures reciprocal control of chlorophyll and heme synthesis.

L-amino acid oxidase-1 is involved in the gut-liver axis by regulating 5-aminolevulinic acid production in mice.

L-amino acid oxidase (LAAO) is a metabolic enzyme that converts L-amino acids into ketoacids, ammonia, and hydrogen peroxide (H2O2). The generated H2O2 has previously been shown to have antibacterial and gut microbiota-modulatory properties in LAO1 knock-out (KO) mice. Since most microbial metabolites reach the liver through the portal vein, we examined gut-liver interactions in LAO1 KO mice. We found lower total cholesterol levels, higher glutamic pyruvic transaminase (GPT) levels in the serum, and higher pro-inflammatory cytokine mRNA expression in the liver tissue. In wild-type (WT) mice, LAO1 was expressed in gut tissues (ileum and colon). Microbiome analysis revealed that the abundance of some bacteria was altered in LAO1 KO mice. However, short-chain fatty acid (SCFAs) levels in cecal feces and gut permeability did not change. Fecal microbiota transplantation (FMT) revealed that feces from LAO1 KO mice slightly stimulated pro-inflammatory cytokine expression in the liver. During metabolomic analysis, 5-aminolevulinic acid (5-ALA) was the only metabolite found to be significantly upregulated in the portal and abdominal veins of the LAO1 KO mice. Intraperitoneal administration of 5-ALA to WT mice significantly increased IL-6 mRNA expression in the liver. These observations suggest that gut LAO1 plays a role in regulating 5-ALA production and that a high level of 5-ALA stimulates the liver to increase pro-inflammatory cytokine expression by disrupting LAO1 in mice.

Applications of the Whole-Cell System in the Efficient Biosynthesis of Heme.

Heme has a variety of functions, from electronic reactions to binding gases, which makes it useful in medical treatments, dietary supplements, and food processing. In recent years, whole-cell system-based heme biosynthesis methods have been continuously explored and optimized as an alternative to the low-yield, lasting, and adverse ecological environment of chemical synthesis methods. This method relies on two biosynthetic pathways of microbial precursor 5-aminolevulinic acid (C4, C5) and three known downstream biosynthetic pathways of heme. This paper reviews the genetic and metabolic engineering strategies for heme production in recent years by optimizing culture conditions and techniques from different microorganisms. Specifically, we summarized and analyzed the possibility of using biosensors to explore new strategies for the biosynthesis of heme from the perspective of synthetic biology, providing a new direction for future exploration.

RNA interference therapy in acute hepatic porphyrias.

The acute hepatic porphyrias (AHPs) are inherited disorders of heme biosynthesis characterized by life-threatening acute neurovisceral attacks precipitated by factors that upregulate hepatic 5-aminolevulinic acid synthase 1 (ALAS1) activity. Induction of hepatic ALAS1 leads to the accumulation of porphyrin precursors, in particular 5-aminolevulinic acid (ALA), which is thought to be the neurotoxic mediator leading to acute attack symptoms such as severe abdominal pain and autonomic dysfunction. Patients may also develop debilitating chronic symptoms and long-term medical complications, including kidney disease and an increased risk of hepatocellular carcinoma. Exogenous heme is the historical treatment for attacks and exerts its therapeutic effect by inhibiting hepatic ALAS1 activity. The pathophysiology of acute attacks provided the rationale to develop an RNA interference therapeutic that suppresses hepatic ALAS1 expression. Givosiran is a subcutaneously administered N-acetylgalactosamine-conjugated small interfering RNA against ALAS1 that is taken up nearly exclusively by hepatocytes via the asialoglycoprotein receptor. Clinical trials established that the continuous suppression of hepatic ALAS1 mRNA via monthly givosiran administration effectively reduced urinary ALA and porphobilinogen levels and acute attack rates and improved quality of life. Common side effects include injection site reactions and increases in liver enzymes and creatinine. Givosiran was approved by the US Food and Drug Administration and European Medicines Agency in 2019 and 2020, respectively, for the treatment of patients with AHP. Although givosiran has the potential to decrease the risk of chronic complications, long-term data on the safety and effects of sustained ALAS1 suppression in patients with AHP are lacking.