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The development of a heme-responsive biosensor for dynamic pathway regulation in eukaryotes has never been reported, posing a challenge for achieving the efficient synthesis of multifunctional hemoproteins and maintaining intracellular heme homeostasis. Herein, a biosensor containing a newly identified heme-responsive promoter, CRISPR/dCas9, and a degradation tag N-degron was designed and optimized to fine-tune heme biosynthesis in the efficient heme-supplying Pichia pastoris P1H9 chassis. After identifying literature-reported promoters insensitive to heme, the endogenous heme-responsive promoters were mined by transcriptomics, and an optimal biosensor was screened from different combinations of regulatory elements. The dynamic regulation pattern of the biosensor was validated by the transcriptional fluctuations of the HEM2 gene involved in heme biosynthesis and the subsequent responsive changes in intracellular heme titers. We demonstrate the efficiency of this regulatory system by improving the production of high-active porcine myoglobin and soy hemoglobin, which can be used to develop artificial meat and artificial metalloenzymes. Moreover, these findings can offer valuable strategies for the synthesis of other hemoproteins. • Endogenous heme-responsive promoters were identified in P. pastoris. • Heme-responsive biosensors were designed and optimized. • Heme biosynthesis was fine-tuned using an optimized heme-responsive biosensor. • Fine-tuned heme biosynthesis enhanced the expression of high-active P-Mb and S-Hb. [ABSTRACT FROM AUTHOR]

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Background: Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are rare disorders of heme biosynthesis characterized by severe cutaneous phototoxicity. Afamelanotide, an α-melanocyte-stimulating hormone analogue, is the only approved treatment for protoporphyria and leads to increased light tolerance and improved quality of life (QoL). However, published experience with afamelanotide in the US is limited. Methods: Here, we report on all adults who received at least one dose of afamelanotide at the Massachusetts General Hospital Porphyria Center from 2021 to 2022. Changes in the time to phototoxic symptom onset, QoL, and laboratory parameters were assessed before and during treatment with afamelanotide. Results: A total of 29 patients with protoporphyria were included, 26 of whom (72.2%) received ≥2 afamelanotide implants. Among the patients who received ≥2 implants, the median time to symptom onset following sunlight exposure was 12.5 min (IQR, 5–20) prior to the initiation of afamelanotide and 120 min (IQR, 60–240) after treatment (p < 0.001). Improvements in QoL during afamelanotide treatment were measured using two QoL tools, with good correlation observed between these two instruments. Finally, we found no improvements in the median levels of metal-free erythrocyte protoporphyrin, plasma protoporphyrin, or liver biochemistries during versus prior to the initiation of afamelanotide treatment. Conclusions: This study highlights a dramatic clinical benefit of afamelanotide in relation to light tolerance and QoL in protoporphyria, albeit without improvement in protoporphyrin levels or measures of liver function. [ABSTRACT FROM AUTHOR]

Heme biosynthesis in the Gram-positive bacteria occurs mostly via a pathway that is distinct from that of eukaryotes and Gram-negative bacteria in the three terminal heme synthesis steps. In many of these bacteria heme is a necessary cofactor that fulfills roles in respiration, gas sensing, and detoxification of reactive oxygen species. These varying roles for heme, the requirement of iron and glutamate, as glutamyl tRNA, for synthesis, and the sharing of intermediates with the synthesis of other porphyrin derivatives necessitates the need for many points of regulation in response to nutrient availability and metabolic state. In this review we examine the regulation of heme biosynthesis in these bacteria via heme, iron, and oxygen species. We also discuss our perspective on emerging roles of protein-protein interactions and post-translational modifications in regulating heme biosynthesis. [ABSTRACT FROM AUTHOR]

δ-Aminolevulinic acid dehydratase (ALAD) is a key enzyme of the cytoplasmic heme biosynthesis pathway. The primary structure of the ALAD gene, the multimeric structure of the ALAD/hemB protein, and ALAD expression during the annual reproductive cycle were studied in the cold-water marine sponge Halisarca dujardinii. The results implicated the GATA-1 transcription factor and DNA methylation in regulating ALAD expression. Re-aggregation of sponge cells was accompanied by a decrease in ALAD expression and a change in the cell content of an active ALAD/hemB form. Further study of heme biosynthesis and the role of ALAD/hemB in morphogenesis of basal animals may provide new opportunities for treating pathologies in higher animals. [ABSTRACT FROM AUTHOR]

Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants. van Loggerenberg et al. functionally assay ∼90% of all possible missense variants in HMBS , which is associated with acute intermittent porphyria. The resulting variant effect maps reveal sequence-structure-function relationships and provide evidence to distinguish pathogenic from benign variants. [ABSTRACT FROM AUTHOR]

The final three steps of heme biogenesis exhibit notable differences between di- and mono-derm bacteria. The former employs the protoporphyrin-dependent (PPD) pathway, while the latter utilizes the more recently uncovered coproporphyrin-dependent (CPD) pathway. In order to devise a rapid screen for potential inhibitors that differentiate the two pathways, the genes associated with the protoporphyrin pathway in an Escherichia coli YFP strain were replaced with those for the CPD pathway from Staphylococcus aureus (SA) through a sliding modular gene replacement recombineering strategy to generate the E. coli strain Sa-CPD-YFP. Potential inhibitors that differentially target the pathways were identified by screening compound libraries against the YFP-producing Sa-CPD-YFP strain in comparison to a CFP-producing E. coli strain. Using a mixed strain assay, inhibitors targeting either the CPD or PPD heme pathways were identified through a decrease in one fluorescent signal but not the other. An initial screen identified both azole and prodigiosin-derived compounds that were shown to specifically target the CPD pathway and which led to the accumulation of coproheme, indicating that the main target of inhibition would appear to be the coproheme decarboxylase (ChdC) enzyme. In silico modeling highlighted that these inhibitors are able to bind within the active site of ChdC. [ABSTRACT FROM AUTHOR]

Heme biosynthesis in the Gram-positive bacteria occurs mostly via a pathway that is distinct from that of eukaryotes and Gram-negative bacteria in the three terminal heme synthesis steps. In many of these bacteria heme is a necessary cofactor that fulfills roles in respiration, gas sensing, and detoxification of reactive oxygen species. These varying roles for heme, the requirement of iron and glutamate, as glutamyl tRNA, for synthesis, and the sharing of intermediates with the synthesis of other porphyrin derivatives necessitates the need for many points of regulation in response to nutrient availability and metabolic state. In this review we examine the regulation of heme biosynthesis in these bacteria via heme, iron, and oxygen species. We also discuss our perspective on emerging roles of protein-protein interactions and post-translational modifications in regulating heme biosynthesis.

Soy leghemoglobin, when bound to heme, imparts a meat-like color and flavor and can serve as a substitute for animal-derived proteins. Enhancing cellular heme synthesis improves the recombinant expression of leghemoglobin in yeast. To achieve high-level expression of leghemoglobin A (LBA) in Kluyveromyces marxianus, a food-safe yeast, large-scale heme synthesis modules were transferred into K. marxianus using yeast artificial chromosomes (KmYACs). These modules contained up to 8 native and heterologous genes to promote the supply of heme precursors and downstream synthesis. Next, eight genes inhibiting heme or LBA synthesis were individually or combinatorially deleted, with the lsc1Δssn3Δ mutant yielding the best results. Subsequently, heme synthesis modules were combined with the lsc1Δssn3Δ mutant. In the resulting strains, the module genes were all actively expressed. Among these module genes, heterologous S. cerevisiae genes in the downstream heme synthesis pathway significantly enhanced the expression of their counterparts in K. marxianus, resulting in high heme content and LBA yield. After optimizing the medium recipe by adjusting the concentrations of glucose, glycine, and FeSO4·7H2O, a heme content of 66.32 mg/L and an intracellular LBA titer of 7.27 g/L were achieved in the engineered strain in a 5 L fermentor. This represents the highest intracellular expression of leghemoglobin in microorganisms to date. The leghemoglobin produced by K. marxianus can be utilized as a safe ingredient for plant-based protein products.

Abstract Background Heme is an iron/porphyrin complex compound, widely used in the health care, food, and pharmaceutical industries. It is more advantageous and attractive to develop microbial cell factories to produce heme by fermentation, with lower production costs and environmentally more friendly procedures than those of the traditional extraction based on animal blood. In this study, Bacillus subtilis, a typical industrial model microorganism of food safety grade, was used for the first time as the host to synthesize heme. Results The heme biosynthetic pathway was engineered as four modules, the endogenous C5 pathway, the heterologous C4 pathway, the uroporphyrinogen (urogen) III synthesis pathway, and the downstream synthesis pathway. Knockout of hemX encoding the negative effector of the concentration of HemA, overexpression of hemA encoding glutamyl-tRNA reductase, and knockout of rocG encoding the major glutamate dehydrogenase in the C5 pathway, resulted in an increase of 427% in heme production. Introduction of the heterologous C4 pathway showed a negligible effect on heme biosynthesis. Overexpression of hemCDB, which encoded hydroxymethylbilane synthase, urogen III synthase, and porphobilinogen synthase participating in the urogen III synthesis pathway, increased heme production by 39%. Knockouts of uroporphyrinogen methyltransferase gene nasF and both heme monooxygenase genes hmoA and hmoB in the downstream synthesis pathway increased heme production by 52%. The engineered B. subtilis produced 248.26 ± 6.97 mg/L of total heme with 221.83 ± 4.71 mg/L of extracellular heme during the fed-batch fermentation in 10 L fermenter. Conclusions Strengthening endogenous C5 pathway, urogen III synthesis pathway and downstream synthesis pathway promoted the biosynthesis of heme in B. subtilis. The engineered B. subtilis strain has great potential as a microbial cell factory for efficient industrial heme production.

ABSTRACT In plant-pathogen interactions, oxidative bursts are crucial for plants to defend themselves against pathogen infections. Rapid production and accumulation of reactive oxygen species kill pathogens directly and cause local cell death, preventing pathogens from spreading to adjacent cells. Meanwhile, the pathogens have developed several mechanisms to tolerate oxidative stress and successfully colonize plant tissues. In this study, we investigated the mechanisms responsible for resistance to oxidative stress by analyzing the transcriptomes of six oxidative stress-sensitive strains of the plant pathogenic fungus Fusarium graminearum. Weighted gene co-expression network analysis identified several pathways related to oxidative stress responses, including the DNA repair system, autophagy, and ubiquitin-mediated proteolysis. We also identified hub genes with high intramodular connectivity in key modules and generated deletion or conditional suppression mutants. Phenotypic characterization of those mutants showed that the deletion of FgHGG4, FgHGG10, and FgHGG13 caused sensitivity to oxidative stress, and further investigation on those genes revealed that transcriptional elongation and DNA damage responses play roles in oxidative stress response and pathogenicity. The suppression of FgHGL7 also led to hypersensitivity to oxidative stress, and we demonstrated that FgHGL7 plays a crucial role in heme biosynthesis and is essential for peroxidase activity. This study increases the understanding of the adaptive mechanisms to cope with oxidative stress in plant pathogenic fungi. IMPORTANCE Fungal pathogens have evolved various mechanisms to overcome host-derived stresses for successful infection. Oxidative stress is a representative defense system induced by the host plant, and fungi have complex response systems to cope with it. Fusarium graminearum is one of the devastating plant pathogenic fungi, and understanding its pathosystem is crucial for disease control. In this study, we investigated adaptive mechanisms for coping with oxidative stress at the transcriptome level using oxidative stress-sensitive strains. In addition, by introducing genetic modification technique such as CRISPR-Cas9 and the conditional gene expression system, we identified pathways/genes required for resistance to oxidative stress and also for virulence. Overall, this study advances the understanding of the oxidative stress response and related mechanisms in plant pathogenic fungi.

Abstract Background Heme has attracted much attention because of its wide applications in medicine and food. The products of genes hemBCDEFY convert 5-aminolevulinic acid to protoporphyrin IX (PPIX; the immediate precursor of heme); protoporphyrin ferrochelatase (FECH) inserts Fe2+ into PPIX to generate heme. Biosynthesis of heme is limited by the need for optimized expression levels of multiple genes, complex regulatory mechanisms, and low enzymatic activity; these problems need to be overcome in metabolic engineering to improve heme synthesis. Results We report a heme biosensor-guided screening strategy using the heme-responsive protein HrtR to regulate tcR expression in Escherichia coli, providing a quantifiable link between the intracellular heme concentration and cell survival in selective conditions (i.e., the presence of tetracycline). This system was used for rapid enrichment screening of heme-producing strains from a library with random ribosome binding site (RBS) variants and from a FECH mutant library. Through up to four rounds of iterative evolution, strains with optimal RBS intensities for the combination of hemBCDEFY were screened; we obtained a PPIX titer of 160.8 mg/L, the highest yield yet reported in shaken-flask fermentation. A high-activity FECH variant was obtained from the saturation mutagenesis library. Fed-batch fermentation of strain SH20C, harboring the optimized hemBCDEFY and the FECH mutant, produced 127.6 mg/L of heme. Conclusion We sequentially improved the multigene biosynthesis pathway of PPIX and performed in vivo directed evolution of FECH, based on a heme biosensor, which demonstrated the effectiveness of the heme biosensor-based pathway optimization strategy and broadens our understanding of the mechanism of heme synthesis.

Background: Heme is an iron/porphyrin complex compound, widely used in the health care, food, and pharmaceutical industries. It is more advantageous and attractive to develop microbial cell factories to produce heme by fermentation, with lower production costs and environmentally more friendly procedures than those of the traditional extraction based on animal blood. In this study, Bacillus subtilis, a typical industrial model microorganism of food safety grade, was used for the first time as the host to synthesize heme. Results: The heme biosynthetic pathway was engineered as four modules, the endogenous C5 pathway, the heterologous C4 pathway, the uroporphyrinogen (urogen) III synthesis pathway, and the downstream synthesis pathway. Knockout of hemX encoding the negative effector of the concentration of HemA, overexpression of hemA encoding glutamyl-tRNA reductase, and knockout of rocG encoding the major glutamate dehydrogenase in the C5 pathway, resulted in an increase of 427% in heme production. Introduction of the heterologous C4 pathway showed a negligible effect on heme biosynthesis. Overexpression of hemCDB, which encoded hydroxymethylbilane synthase, urogen III synthase, and porphobilinogen synthase participating in the urogen III synthesis pathway, increased heme production by 39%. Knockouts of uroporphyrinogen methyltransferase gene nasF and both heme monooxygenase genes hmoA and hmoB in the downstream synthesis pathway increased heme production by 52%. The engineered B. subtilis produced 248.26 ± 6.97 mg/L of total heme with 221.83 ± 4.71 mg/L of extracellular heme during the fed-batch fermentation in 10 L fermenter. Conclusions: Strengthening endogenous C5 pathway, urogen III synthesis pathway and downstream synthesis pathway promoted the biosynthesis of heme in B. subtilis. The engineered B. subtilis strain has great potential as a microbial cell factory for efficient industrial heme production. [ABSTRACT FROM AUTHOR]

5‐Aminolevulinic acid synthase (ALAS) is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that catalyzes the first and rate‐limiting step of heme biosynthesis in α‐proteobacteria and several non‐plant eukaryotes. All ALAS homologs contain a highly conserved catalytic core, but eukaryotes also have a unique C‐terminal extension that plays a role in enzyme regulation. Several mutations in this region are implicated in multiple blood disorders in humans. In Saccharomyces cerevisiae ALAS (Hem1), the C‐terminal extension wraps around the homodimer core to contact conserved ALAS motifs proximal to the opposite active site. To determine the importance of these Hem1 C‐terminal interactions, we determined the crystal structure of S. cerevisiae Hem1 lacking the terminal 14 amino acids (Hem1 ΔCT). With truncation of the C‐terminal extension, we show structurally and biochemically that multiple catalytic motifs become flexible, including an antiparallel β‐sheet important to Fold‐Type I PLP‐dependent enzymes. The changes in protein conformation result in an altered cofactor microenvironment, decreased enzyme activity and catalytic efficiency, and ablation of subunit cooperativity. These findings suggest that the eukaryotic ALAS C‐terminus has a homolog‐specific role in mediating heme biosynthesis, indicating a mechanism for autoregulation that can be exploited to allosterically modulate heme biosynthesis in different organisms. PDB Code(s): 8EIM; [ABSTRACT FROM AUTHOR]

Heme is an essential cofactor for multiple cellular processes in most organisms. In developing erythroid cells, the demand for heme synthesis is high, but is significantly lower in non-erythroid cells. While the biosynthesis of heme in metazoans is well understood, the tissue-specific regulation of the pathway is less explored. To better understand this, we analyzed the mitochondrial heme metabolon in erythroid and non-erythroid cell lines from the perspective of ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Affinity purification of FLAG-tagged-FECH, together with mass spectrometric analysis, was carried out to identify putative protein partners in human and murine cell lines. Proteins involved in the heme biosynthetic process and mitochondrial organization were identified as the core components of the FECH interactome. Interestingly, in non-erythroid cell lines, the FECH interactome is highly enriched with proteins associated with the tricarboxylic acid (TCA) cycle. Overall, our study shows that the mitochondrial heme metabolon in erythroid and non-erythroid cells has similarities and differences, and suggests new roles for the mitochondrial heme metabolon and heme in regulating metabolic flux and key cellular processes. [ABSTRACT FROM AUTHOR]

(1) Background: Human frataxin is an iron binding protein that participates in the biogenesis of iron sulfur clusters and enhances ferrochelatase activity. While frataxin association to other proteins has been extensively characterized up to the structural level, much less is known about the putative capacity of frataxin to interact with functionally related metabolites. In turn, current knowledge about frataxin's capacity to coordinate metal ions is limited to iron (II and III); (2) Methods: here, we used NMR spectroscopy, Molecular Dynamics, and Docking approaches to demonstrate new roles of frataxin; (3) Results: We demonstrate that frataxin also binds Zn2+ in a structurally similar way to Fe2+, but with lower affinity. In turn, both Fe2+-loaded and Zn2+-loaded frataxins specifically associate to protoporphyrin IX with micromolar affinity, while apo-frataxin does not bind to the porphyrin. Protoporphyrin IX association to metal-loaded frataxin shares the binding epitope with ferrochelatase; and (4) Conclusions: these findings expand the plethora of relevant molecular targets for frataxin and may help to elucidate the yet unknown different roles that this protein exerts in iron regulation and metabolism. [ABSTRACT FROM AUTHOR]

Sideroblastic anemia is a heterogeneous group of disorders typified by the presence of ring sideroblasts in the bone marrow and has congenital and acquired types. Sideroblastic anemia is a rare event in pregnancy. We report a case of a 32‐year‐old female patient, gravida 4 para 3, 27th weeks pregnant, who presented to the emergency department complaining of palpitation and generalized weakness for 2 weeks. She was found to have severe normochromic normocytic anemia, with hemoglobin of 4.2 g/dl, and low reticulocytes count of 13 × 103/μl. She gave a history of recurrent anemia, which had only occurred during pregnancy. Her bone marrow aspirate showed many ring sideroblasts concluding the diagnosis of sideroblastic anemia (SA). Further investigation revealed a significantly low pyridoxine level (vitamin B6) of (8 nmol/L). The Hb level improved with vitamin B6 replacement, without any transfusion support. [ABSTRACT FROM AUTHOR]

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin‐dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII‐ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in‐solution data obtained by resonance Raman and UV–vis spectroscopy, for wild‐type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in‐depth characterization of the catalytically‐relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin‐enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies. PDB Code(s): 8AT8 and 8AW7; [ABSTRACT FROM AUTHOR]

The final three steps of heme biogenesis exhibit notable differences between di- and mono-derm bacteria. The former employs the protoporphyrin-dependent (PPD) pathway, while the latter utilizes the more recently uncovered coproporphyrin-dependent (CPD) pathway. In order to devise a rapid screen for potential inhibitors that differentiate the two pathways, the genes associated with the protoporphyrin pathway in an Escherichia coli YFP strain were replaced with those for the CPD pathway from Staphylococcus aureus (SA) through a sliding modular gene replacement recombineering strategy to generate the E. coli strain Sa-CPD-YFP. Potential inhibitors that differentially target the pathways were identified by screening compound libraries against the YFP-producing Sa-CPD-YFP strain in comparison to a CFP-producing E. coli strain. Using a mixed strain assay, inhibitors targeting either the CPD or PPD heme pathways were identified through a decrease in one fluorescent signal but not the other. An initial screen identified both azole and prodigiosin-derived compounds that were shown to specifically target the CPD pathway and which led to the accumulation of coproheme, indicating that the main target of inhibition would appear to be the coproheme decarboxylase (ChdC) enzyme. In silico modeling highlighted that these inhibitors are able to bind within the active site of ChdC.

Environmental pollutants including halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, including benzo[a]pyrene, exert their deleterious effects through the activation of the aryl hydrocarbon receptor (AHR) and by the resulting transcription of genes not yet fully identified. Ligand-bound AHR translocates from cytoplasm to nucleus, where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The AHR/ARNT dimer binds to enhancer regions of responsive genes to activate transcription. AHR also mediates carcinogenesis caused by PAHs, likely via CYP1A1, CYP1A2, and CYP1B1, which are massively induced by activated AHR in many tissues and generate carcinogenic electrophilic derivatives of PAHs. In the current study, we have used the mouse GeCKOv2 genome-wide CRISPR/Cas9 library to identify novel genes in the AHR pathway by taking advantage of a B[a]P selection assay that we previously used to identify core AHR pathway genes in Hepa-1c1c7 murine hepatoma cells. Besides Ahr, Arnt, and Cyp1a1, we report the identification of multiple additional putative AHR pathway genes including several that we validated. These include cytochrome P450 reductase (Por), which mediates redox regeneration of cytochromes P450, and 5 genes of the heme biosynthesis pathway: delta-aminolevulinate synthase 1 (Alas1), porphobilinogen deaminase (Hmbs), uroporphyrinogen decarboxylase (Urod), coproporphyrinogen oxidase (Cpox), and ferrochelatase (Fech): heme being an essential prosthetic group of cytochrome P450 proteins. Notably, several of these genes were identified by GeCKO screening, despite not being identifiable by reverse genetics approaches. This indicates the power of high-sensitivity genome-wide genetic screening for identifying genes in the AHR pathway.

Abstract Sideroblastic anemia is a heterogeneous group of disorders typified by the presence of ring sideroblasts in the bone marrow and has congenital and acquired types. Sideroblastic anemia is a rare event in pregnancy. We report a case of a 32‐year‐old female patient, gravida 4 para 3, 27th weeks pregnant, who presented to the emergency department complaining of palpitation and generalized weakness for 2 weeks. She was found to have severe normochromic normocytic anemia, with hemoglobin of 4.2 g/dl, and low reticulocytes count of 13 × 103/μl. She gave a history of recurrent anemia, which had only occurred during pregnancy. Her bone marrow aspirate showed many ring sideroblasts concluding the diagnosis of sideroblastic anemia (SA). Further investigation revealed a significantly low pyridoxine level (vitamin B6) of (8 nmol/L). The Hb level improved with vitamin B6 replacement, without any transfusion support.

Abstract 5-aminolevulinic acid (ALA) has broad potential applications in the medical, agricultural and food industries. Several strategies have been implemented successfully to try to improve ALA synthesis. Nonetheless, the low yield has got in the way of large-scale bio-manufacture of 5-ALA. In this study, we explored strain engineering strategies for high‐level 5‐ALA production in Corynebacterium glutamicum F343 using the C4 pathway. Initially, the glutamate dehydrogenase-encoding gene gdhA was deleted to reduce glutamate yield. Then the C4 pathway was introduced in the gdhA mutant strain F2-A (∆gdhA + hemA), resulting in a 5-ALA yield of up to 3.2 g/L. Furthermore, the accumulations of downstream metabolites such as heme, porphobilinogen, and protoporphyrin IX, were decreased. After evaluating the mechanisms of this synthetic pathway by RNA-Seq, the results showed that genes involved in both the C5 pathway and heme pathways were down-regulated in strain F2-A (∆gdhA + hemA). Interestingly, upstream genes of succinyl-CoA in the tricarboxylic acid (TCA) cycle, such as icd, lpdA, were up-regulated, while its downstream genes, including sucC, sucD, sdhB, sdhA, sdhCD, were down-regulated. These changes amplify the sources of succinyl-CoA and reduce its expenditure, before pulling the carbon flux to produce 5-ALA. Furthermore, the down-regulation of most genes of the heme pathway could reduce the drainage of 5‐ALA, which further enhance its accumulation. To alleviate competition between glyoxylate and the TCA cycle, the isocitrate dehydrogenase-encoding gene aceA was also knocked out, resulting in 3.86 g/L of 5‐ALA. Finally, the fermentation conditions were optimized, resulting in a maximum 5-ALA yield of 5.6 g/L. Overall, the blocking of the glutamate synthesis pathway could be a powerful strategy to re-allocate the carbon flux to produce 5-ALA. It could also enable the efficient synthesis of other TCA derivatives in C. glutamicum.

Background: The prognosis of diffusely infiltrating glioma patients is dismal but varies greatly between individuals. While characterization of gliomas primarily relied on histopathological features, molecular markers increasingly gained importance and play a key role in the recently published 5th edition of the World Health Organization (WHO) classification. Heme biosynthesis represents a crucial pathway due to its paramount importance in oxygen transport, energy production and drug metabolism. Recently, we described a "heme biosynthesis mRNA expression signature" that correlates with histopathological glioma grade and survival. The aim of the current study was to correlate this heme biosynthesis mRNA expression signature with diagnostic molecular markers and investigate its continued prognostic relevance. Materials and methods: In this study, patient data were derived from the "The Cancer Genome Atlas" (TCGA) lower-grade glioma and glioblastoma cohorts. We identified diffusely infiltrating gliomas correlating molecular tumor diagnosis according to the most recent WHO classification with heme biosynthesis mRNA expression. The following molecular markers were analyzed: EGFR amplification, TERT promoter mutation, CDKN2A/B homozygous loss, chromosome 7 + /10- aneuploidy, MGMT methylation, IDH mutation, ATRX loss, p53 mutation and 1p19q codeletion. Subsequently, we calculated the heme biosynthesis mRNA expression signature for correlation with distinct molecular glioma markers/molecular subgroups and performed survival analyses. Results: A total of 649 patients with available data on up-to-date molecular markers and heme biosynthesis mRNA expression were included. According to analysis of individual molecular markers, we found a significantly higher heme biosynthesis mRNA expression signature in gliomas with IDH wildtype (p < 0.0005), without 1p19q codeletion (p < 0.0005), with homozygous CDKN2A/B loss (p < 0.0005) and with EGFR amplification (p = 0.001). Furthermore, we observed that the heme biosynthesis mRNA expression signature increased with molecular subgroup aggressiveness (p < 0.0005), being lowest in WHO grade 2 oligodendrogliomas and highest in WHO grade 4 glioblastomas. Finally, the heme biosynthesis mRNA expression signature was a statistically significant survival predictor after multivariate correction for all molecular markers (p < 0.0005). Conclusion: Our data demonstrate a significant correlation between heme biosynthesis regulation and diagnostic molecular markers and a prognostic relevance independent of these established markers. Consequently, heme biosynthesis expression is a promising biomarker for glioma aggressiveness and might constitute a potential target for novel therapeutic approaches. [ABSTRACT FROM AUTHOR]

Background: The heme biosynthesis (HB) involves eight subsequent enzymatic steps. Erythropoietic protoporphyria (EPP) is caused by loss-of-function mutations in the ferrochelatase (FECH) gene, which in the last HB step inserts ferrous iron into protoporphyrin IX (PPIX) to form heme. Aim and method: The aim of this work was to for the first time analyze the mRNA expression of all HB genes in peripheral blood samples of patients with EPP having the same genotype FECH c.[215dupT]; [315-48T > C] as compared to healthy controls by highly sensitive and specific digital PCR assays (dPCR). Results: We confirmed a decreased FECH mRNA expression in patients with EPP. Further, we found increased ALAS2 and decreased ALAS1, CPOX, PPOX and HMBS mRNA expression in patients with EPP compared to healthy controls. ALAS2 correlated with FECH mRNA expression (EPP: r = 0.63, p = 0.03 and controls: r = 0.68, p = 0.02) and blood parameters like PPIX (EPP: r = 0.58 p = 0.06). Conclusion: Our method is the first that accurately quantifies HB mRNA from blood samples with potential applications in the monitoring of treatment effects of mRNA modifying therapies in vivo, or investigation of the HB pathway and its regulation. However, our findings should be studied in separated blood cell fractions and on the enzymatic level. [ABSTRACT FROM AUTHOR]

Bilirubin (BR) is an important ingredient of a valuable Chinese medicine, Calculus bovis. Over recent decades, increasing evidence has confirmed that BR offers health benefits in cardiovascular health, stroke, diabetes, and metabolic syndrome. However, BR is mainly produced by extraction from pig bile. In this study, we assembled an efficient pathway for BR production by metabolic engineering of Escherichia coli . First, heme oxygenase (HO1) and biliverdin reductase were co-expressed in E. coli . HPLC and LC-MS confirmed the accumulation of BR in the recombinant E. coli cells. To improve BR production, the catalytic abilities of HO1 from different species were investigated. In addition, the outermembrane-bound heme receptor (ChuA) and the enzymes involved in heme biosynthesis were overexpressed among which ChuA, 5-aminolevulinic acid dehydratase (HemB), protoporphyrin oxidase (HemG), and ferrochelatase (HemH) were found to enhance BR accumulation in E. coli . In addition, expression of ferredoxin (Fd) was shown to contribute to efficient conversion of heme to BR in E. coli . To increase supply of NADPH, isocitrate dehydrogenase (IDH), NAD kinase (nadK), NADP-specific glutamate dehydrogenase (gdhA), and glucose-6-phosphate 1-dehydrogenase (ZWF) were overexpressed and were found to enhance BR accumulation when these proteins were expressed with a low-copy plasmid pACYCduet-1. Modular optimization of the committed genes led to a titer of 17.2 mg/L in strain M1BHG. Finally, fed-batch fermentation was performed for the strains M1BHG and M1, resulting in accumulation of 75.5 mg/L and 25.8 mg/L of BR, respectively. This is the first report on biosynthesis of BR through metabolic engineering in a heterologous host.

Heme is a primordial macrocycle upon which most aerobic life on Earth depends. It is essential to the survival and health of nearly all cells, functioning as a prosthetic group for oxygen-carrying proteins and enzymes involved in oxidation/reduction and electron transport reactions. Heme is essential for the function of numerous hemoproteins and has numerous other roles in the biochemistry of life. In mammals, heme is synthesised from glycine, succinyl-CoA, and ferrous iron in a series of eight steps. The first and normally rate-controlling step is catalysed by 5-aminolevulinate synthase (ALAS), which has two forms: ALAS1 is the housekeeping form with highly variable expression, depending upon the supply of the end-product heme, which acts to repress its activity; ALAS2 is the erythroid form, which is regulated chiefly by the adequacy of iron for erythroid haemoglobin synthesis. Abnormalities in the several enzymes of the heme synthetic pathway, most of which are inherited partial enzyme deficiencies, give rise to rare diseases called porphyrias. The existence and role of heme importers and exporters in mammals have been debated. Recent evidence established the presence of heme transporters. Such transporters are important for the transfer of heme from mitochondria, where the penultimate and ultimate steps of heme synthesis occur, and for the transfer of heme from cytoplasm to other cellular organelles. Several chaperones of heme and iron are known and important for cell health. Heme and iron, although promoters of oxidative stress and potentially toxic, are essential cofactors for cellular energy production and oxygenation., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Heme has attracted considerable attention due to its indispensable biological roles and applications in healthcare and artificial foods. The development and utilization of edible microorganisms instead of animals to produce heme is the most promising method to promote the large-scale industrial production and safe application of heme. However, the cytotoxicity of heme severely restricts its efficient synthesis by microorganisms, and the cytotoxic mechanism is not fully understood. In this study, the effect of heme toxicity on Saccharomyces cerevisiae was evaluated by enhancing its synthesis using metabolic engineering. The results showed that the accumulation of heme after the disruption of heme homeostasis caused serious impairments in cell growth and metabolism, as demonstrated by significantly poor growth, mitochondrial damage, cell deformations, and chapped cell surfaces, and these features which were further associated with substantially elevated reactive oxygen species (ROS) levels within the cell (mainly H 2 O 2 and superoxide anion radicals). To improve cellular tolerance to heme, 5 rounds of laboratory evolution were performed, increasing heme production by 7.3-fold and 4.2-fold in terms of the titer (38.9 mg/L) and specific production capacity (1.4 mg/L/OD 600 ), respectively. Based on comparative transcriptomic analyses, 32 genes were identified as candidates that can be modified to enhance heme production by more than 20% in S. cerevisiae. The combined overexpression of 5 genes (SPS22, REE1, PHO84, HEM4 and CLB2) was shown to be an optimal method to enhance heme production. Therefore, a strain with enhanced heme tolerance and ROS quenching ability (R5-M) was developed that could generate 380.5 mg/L heme with a productivity of 4.2 mg/L/h in fed-batch fermentation, with S. cerevisiae strains being the highest producers reported to date. These findings highlight the importance of improving heme tolerance for the microbial production of heme and provide a solution for efficient heme production by engineered yeasts., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Transcriptional mechanisms establish and maintain complex genetic and protein networks to control cell state transitions. The hematopoietic transcription factor GATA1 is a master regulator of erythropoiesis and megakaryopoiesis, and human GATA1 genetic variants cause anemia and megakaryoblastic leukemia. Multiomic analyses revealed that GATA1 controls expression of transporters and metabolic enzymes that dictate intracellular levels of endogenous small molecules, including heme, metal ions, and sphingolipids. Besides its canonical function as a hemoglobin component, heme facilitates or antagonizes GATA1 function to regulate erythropoiesis via mechanisms dependent or independent of the heme-binding transcription factor BTB domain and CNC homology 1 (BACH1). GATA1 regulates the expression of genes encoding heme biosynthetic enzymes and BACH1. GATA1 maintains homeostasis of bioactive ceramides during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Disrupting ceramide homeostasis impairs critical cytokine signaling and is detrimental to erythroid cells. During erythroid maturation, GATA1 induces a zinc transporter switch that favors export versus import, thus dictating the intracellular zinc level, erythroblast survival, and differentiation. In aggregate, these studies support an emerging paradigm in which GATA factor-dependent transcriptional mechanisms control the intracellular levels of endogenous small molecules and small molecule-dependent feedback loops that serve as vital effectors of transcription factor activity, genome function, and cell state transitions., Competing Interests: Conflict of Interest Disclosure The authors do not have any conflicts of interest to declare in relation to this work., (Copyright © 2024 International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Porphyrias are rare, mostly inherited disorders resulting from altered activity of specific enzymes in the haem synthesis pathway that lead to accumulation of pathway intermediates. Photocutaneous symptoms occur when excess amounts of photoreactive porphyrins circulate in the blood to the skin, whereas increases in potentially neurotoxic porphyrin precursors are associated with neurovisceral symptoms. Current therapies are suboptimal and their mechanisms are not well established. As described here, emerging therapies address underlying disease mechanisms by introducing a gene, RNA or other specific molecule with the potential to cure or slow progression of the disease. Recent progress in nanotechnology and nanoscience, particularly regarding particle design and formulation, is expanding disease targets. More secure and efficient drug delivery systems have extended our toolbox for transferring specific molecules, especially into hepatocytes, and led to proof-of-concept studies in animal models. Repurposing existing drugs as molecular chaperones or haem synthesis inhibitors is also promising. This review summarizes key examples of these emerging therapeutic approaches and their application for hepatic and erythropoietic porphyrias., (© 2024 The Authors. Liver International published by John Wiley & Sons Ltd.)

We describe developments in understanding of the porphyrias associated with each step in the haem biosynthesis pathway and the role of individuals whose contributions led to major advances over the past 150 years. The first case of erythropoietic porphyria was reported in 1870, and the first with acute porphyria in 1889. Photosensitisation by porphyrin was confirmed by Meyer-Betz, who self-injected haematoporphyrin. Günther classified porphyrias into haematoporphyria acuta, acuta toxica, congenita and chronica. This was revised by Waldenström into porphyria congenita, acuta and cutanea tarda, with the latter describing those with late-onset skin lesions. Waldenström was the first to recognise porphobilinogen's association with acute porphyria, although its structure was not solved until 1953. Hans Fischer was awarded the Nobel prize in 1930 for solving the structure of porphyrins and the synthesis of haemin. After 1945, research by several groups elucidated the pathway of haem biosynthesis and its negative feedback regulation by haem. By 1961, following the work of Watson, Schmid, Rimington, Goldberg, Dean, Magnus and others, aided by the availability of modern techniques of porphyrin separation, six of the porphyrias were identified and classified as erythropoietic or hepatic. The seventh, 5-aminolaevulinate dehydratase deficiency porphyria, was described by Doss in 1979. The discovery of increased hepatic 5-aminolaevulinate synthase activity in acute porphyria led to development of haematin as a treatment for acute attacks. By 2000, all the haem biosynthesis genes were cloned, sequenced and assigned to chromosomes and disease-specific mutations identified in all inherited porphyrias. These advances have allowed definitive family studies and development of new treatments., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hemoglobins, with heme as a cofactor, are functional proteins that have extensive applications in the fields of artificial oxygen carriers and foods. Although Saccharomyces cerevisiae is an ideal host for hemoglobin synthesis, it lacks a suitable transport system to utilize additional heme for active expression of hemoglobins, resulting in the cellular aggregation and degradation of the latter. Here, an effective heme importer, heme-responsive gene 4 (Hrg-4), was selected from six candidates through the comparison of effects on the growth rates of Δhem1 S. cerevisiae strain and the activities of various hemoglobins when supplemented with 5 mg·L -1 exogenous heme. Additionally, to counter the instability of plasmid-based expression and the metabolic burden introduced from overexpressing Hrg-4, a series of hrg-4 integrated strains were constructed and the best engineered strain with five copies of hrg-4 was chosen. We found that this engineered strain was associated with an increased binding rate of heme in monomeric leghemoglobin and multimeric human hemoglobin (76.3% and 16.5%, respectively), as well as an enhanced expression of both hemoglobins (52.8% and 17.0%, respectively). Thus, the engineered strain with improved heme uptake can be used to efficiently synthesize other heme-binding proteins and enzymes in S. cerevisiae., (© 2024 Federation of European Biochemical Societies.)

The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology., (© 2024. The Author(s).)

Porphyrias are predominantly genetic metabolic disorders caused by dysregulation of specific enzymes in porphyrin-heme biosynthesis. The enzymatic dysfunction leads to formation and excretion of intermediate metabolic products in the form of porphyrins and/or their precursors δ‑aminolevulinic acid and porphobilinogen, which have cyto- and tissue-toxic properties. Clinically, porphyrias are extremely diverse, with symptoms ranging from skin changes on light-exposed areas of the body to potentially life-threatening neurovisceral attacks. Biochemical tests in urine, blood and stool are used for diagnosis, which can be supplemented by molecular genetic analyses. Treatment of the various forms of porphyria is complex and often requires close interdisciplinary cooperation between different medical specialties., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

BackgroundThe prognosis of diffusely infiltrating glioma patients is dismal but varies greatly between individuals. While characterization of gliomas primarily relied on histopathological features, molecular markers increasingly gained importance and play a key role in the recently published 5th edition of the World Health Organization (WHO) classification. Heme biosynthesis represents a crucial pathway due to its paramount importance in oxygen transport, energy production and drug metabolism. Recently, we described a “heme biosynthesis mRNA expression signature” that correlates with histopathological glioma grade and survival. The aim of the current study was to correlate this heme biosynthesis mRNA expression signature with diagnostic molecular markers and investigate its continued prognostic relevance.Materials and methodsIn this study, patient data were derived from the “The Cancer Genome Atlas” (TCGA) lower-grade glioma and glioblastoma cohorts. We identified diffusely infiltrating gliomas correlating molecular tumor diagnosis according to the most recent WHO classification with heme biosynthesis mRNA expression. The following molecular markers were analyzed: EGFR amplification, TERT promoter mutation, CDKN2A/B homozygous loss, chromosome 7 + /10- aneuploidy, MGMT methylation, IDH mutation, ATRX loss, p53 mutation and 1p19q codeletion. Subsequently, we calculated the heme biosynthesis mRNA expression signature for correlation with distinct molecular glioma markers/molecular subgroups and performed survival analyses.ResultsA total of 649 patients with available data on up-to-date molecular markers and heme biosynthesis mRNA expression were included. According to analysis of individual molecular markers, we found a significantly higher heme biosynthesis mRNA expression signature in gliomas with IDH wildtype (p < 0.0005), without 1p19q codeletion (p < 0.0005), with homozygous CDKN2A/B loss (p < 0.0005) and with EGFR amplification (p = 0.001). Furthermore, we observed that the heme biosynthesis mRNA expression signature increased with molecular subgroup aggressiveness (p < 0.0005), being lowest in WHO grade 2 oligodendrogliomas and highest in WHO grade 4 glioblastomas. Finally, the heme biosynthesis mRNA expression signature was a statistically significant survival predictor after multivariate correction for all molecular markers (p < 0.0005).ConclusionOur data demonstrate a significant correlation between heme biosynthesis regulation and diagnostic molecular markers and a prognostic relevance independent of these established markers. Consequently, heme biosynthesis expression is a promising biomarker for glioma aggressiveness and might constitute a potential target for novel therapeutic approaches.

Background: The heme biosynthesis (HB) involves eight subsequent enzymatic steps. Erythropoietic protoporphyria (EPP) is caused by loss-of-function mutations in the ferrochelatase (FECH) gene, which in the last HB step inserts ferrous iron into protoporphyrin IX (PPIX) to form heme.Aim and method: The aim of this work was to for the first time analyze the mRNA expression of all HB genes in peripheral blood samples of patients with EPP having the same genotype FECH c.[215dupT]; [315-48T > C] as compared to healthy controls by highly sensitive and specific digital PCR assays (dPCR).Results: We confirmed a decreased FECH mRNA expression in patients with EPP. Further, we found increased ALAS2 and decreased ALAS1, CPOX, PPOX and HMBS mRNA expression in patients with EPP compared to healthy controls. ALAS2 correlated with FECH mRNA expression (EPP: r = 0.63, p = 0.03 and controls: r = 0.68, p = 0.02) and blood parameters like PPIX (EPP: r = 0.58 p = 0.06).Conclusion: Our method is the first that accurately quantifies HB mRNA from blood samples with potential applications in the monitoring of treatment effects of mRNA modifying therapies in vivo, or investigation of the HB pathway and its regulation. However, our findings should be studied in separated blood cell fractions and on the enzymatic level.

Porphyrias are a group of rare, mostly inherited metabolic disorders of heme biosynthesis. Each type of porphyria results from a specific deficiency of one of the pathway enzymes, causing a characteristic accumulation and excretion of heme precursors. Diagnosis is confirmed by the biochemical detection of these porphyrins and the precursors in urine, feces and blood. Porphyrias can be classified into acute and non-acute forms. The clinical presentation is unspecific and includes acute neurovisceral and/or cutaneous symptoms. The latent phase can evolve into a potentially life-threatening acute crisis, which is often misdiagnosed. The four acute hepatic porphyrias are relevant for anesthesiologists as precipitating factors are commonly found in the perioperative setting. Safe anesthetic management in cases of known porphyria is possible by adherence to current recommendations. The immediate administration of heme arginate as specific treatment for acute attacks is decisive for the outcome. [ABSTRACT FROM AUTHOR]

ObjectiveThe intraoperative visualization of adult-type diffuse gliomas with 5-aminolevulinic acid (5-ALA) induced fluorescence is widely used in the neurosurgical field. While visible 5-ALA induced fluorescence is found in the majority of high-grade gliomas, most low-grade gliomas lack visible fluorescence during surgery. Recently, the heme biosynthesis pathway was identified as crucial influencing factor for presence of visible fluorescence since it metabolizes 5-ALA to fluorescing Protoporphyrin IX (PpIX). However, the exact alterations within the heme biosynthesis pathway resulting in visible 5-ALA induced fluorescence in gliomas are still unclear. The aim of the present study was thus to compare the mRNA and protein expression of promising intramitochondrial heme biosynthesis enzymes/transporters in glioma tissue samples of different fluorescence behavior.MethodsA total of 19 strongly fluorescing and 21 non-fluorescing tissue samples from neurosurgical adult-type diffuse gliomas (WHO grades II-IV) were included in the current analysis. In these samples, we investigated the mRNA expression by quantitative real time PCR and protein expression using immunohistochemistry of the intramitochondrial heme biosynthesis enzymes Coproporphyrinogen Oxidase (CPOX), Protoporphyrinogen Oxidase (PPOX), Ferrochelatase (FECH), and the transporter ATP-binding Cassette Subfamily B Member 2 (ABCG2).ResultsRegarding mRNA expression analysis, we found a significantly decreased ABCG2 expression in fluorescing specimens compared to non-fluorescing samples (p = 0.001), whereas no difference in CPOX, PPOX and FECH was present. With respect to protein expression, significantly higher levels of CPOX (p = 0.005), PPOX (p < 0.01) and FECH (p = 0.003) were detected in fluorescing samples. Similar to mRNA expression analysis, the protein expression of ABCG2 (p = 0.001) was significantly lower in fluorescing samples.ConclusionDistinct alterations of the analyzed heme biosynthesis factors were found primarily on protein level. Our data indicate that heme biosynthesis pathway activity in general is enhanced in fluorescing gliomas with upregulation of PpIX generating enzymes and decreased ABCG2 mediated PpIX efflux outweighing the also increased further metabolization of PpIX to heme. Intramitochondrial heme biosynthesis factors thus constitute promising pharmacological targets to optimize intraoperative 5-ALA fluorescence visualization of usually non-fluorescing tumors such as low-grade gliomas.

[Display omitted] • Ferrochelatases insert ferrous iron in porphyrins in the heme biosynthesis pathway. • The porphyrin proximal ligand is not conserved in the active site of ferrochelatases. • Proximal mutations alter the H-bonds between the porphyrin and the protein. • Effects on the activity are induced by the polarity of the mutated proximal ligand. • The weak coordination by the proximal ligand is essential for substrate and product. Ferrochelatases catalyze the insertion of ferrous iron into the porphyrin during the heme b biosynthesis pathway, which is fundamental for both prokaryotes and eukaryotes. Interestingly, in the active site of ferrochelatases, the proximal ligand coordinating the porphyrin iron of the product is not conserved, and its catalytic role is still unclear. Here we compare the L. monocytogenes bacterial coproporphyrin ferrochelatase native enzyme together with selected variants, where the proximal Tyr residue was replaced by a His (i.e. the most common ligand in heme proteins), a Met or a Phe (as in human and actinobacterial ferrochelatases, respectively), in their Fe(III), Fe(II) and Fe(II)–CO adduct forms. The study of the active site structure and the activity of the proteins in solution has been performed by UV–vis electronic absorption and resonance Raman spectroscopies, biochemical characterization, and classical MD simulations. All the mutations alter the H-bond interactions between the iron porphyrin propionate groups and the protein, and induce effects on the activity, depending on the polarity of the proximal ligand. The overall results confirm that the weak or non-existing coordination of the porphyrin iron by the proximal residue is essential for the binding of the substrate and the release of the final product. [ABSTRACT FROM AUTHOR]

Heme is an essential cofactor for multiple cellular processes in most organisms. In developing erythroid cells, the demand for heme synthesis is high, but is significantly lower in non-erythroid cells. While the biosynthesis of heme in metazoans is well understood, the tissue-specific regulation of the pathway is less explored. To better understand this, we analyzed the mitochondrial heme metabolon in erythroid and non-erythroid cell lines from the perspective of ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Affinity purification of FLAG-tagged-FECH, together with mass spectrometric analysis, was carried out to identify putative protein partners in human and murine cell lines. Proteins involved in the heme biosynthetic process and mitochondrial organization were identified as the core components of the FECH interactome. Interestingly, in non-erythroid cell lines, the FECH interactome is highly enriched with proteins associated with the tricarboxylic acid (TCA) cycle. Overall, our study shows that the mitochondrial heme metabolon in erythroid and non-erythroid cells has similarities and differences, and suggests new roles for the mitochondrial heme metabolon and heme in regulating metabolic flux and key cellular processes.

(1) Background: Human frataxin is an iron binding protein that participates in the biogenesis of iron sulfur clusters and enhances ferrochelatase activity. While frataxin association to other proteins has been extensively characterized up to the structural level, much less is known about the putative capacity of frataxin to interact with functionally related metabolites. In turn, current knowledge about frataxin’s capacity to coordinate metal ions is limited to iron (II and III); (2) Methods: here, we used NMR spectroscopy, Molecular Dynamics, and Docking approaches to demonstrate new roles of frataxin; (3) Results: We demonstrate that frataxin also binds Zn2+ in a structurally similar way to Fe2+, but with lower affinity. In turn, both Fe2+-loaded and Zn2+-loaded frataxins specifically associate to protoporphyrin IX with micromolar affinity, while apo-frataxin does not bind to the porphyrin. Protoporphyrin IX association to metal-loaded frataxin shares the binding epitope with ferrochelatase; and (4) Conclusions: these findings expand the plethora of relevant molecular targets for frataxin and may help to elucidate the yet unknown different roles that this protein exerts in iron regulation and metabolism.

5-aminolevulinic acid (ALA) has broad potential applications in the medical, agricultural and food industries. Several strategies have been implemented successfully to try to improve ALA synthesis. Nonetheless, the low yield has got in the way of large-scale bio-manufacture of 5-ALA. In this study, we explored strain engineering strategies for high‐level 5‐ALA production in Corynebacterium glutamicum F343 using the C4 pathway. Initially, the glutamate dehydrogenase-encoding gene gdhA was deleted to reduce glutamate yield. Then the C4 pathway was introduced in the gdhA mutant strain F2-A (∆gdhA + hemA), resulting in a 5-ALA yield of up to 3.2 g/L. Furthermore, the accumulations of downstream metabolites such as heme, porphobilinogen, and protoporphyrin IX, were decreased. After evaluating the mechanisms of this synthetic pathway by RNA-Seq, the results showed that genes involved in both the C5 pathway and heme pathways were down-regulated in strain F2-A (∆gdhA + hemA). Interestingly, upstream genes of succinyl-CoA in the tricarboxylic acid (TCA) cycle, such as icd, lpdA, were up-regulated, while its downstream genes, including sucC, sucD, sdhB, sdhA, sdhCD, were down-regulated. These changes amplify the sources of succinyl-CoA and reduce its expenditure, before pulling the carbon flux to produce 5-ALA. Furthermore, the down-regulation of most genes of the heme pathway could reduce the drainage of 5‐ALA, which further enhance its accumulation. To alleviate competition between glyoxylate and the TCA cycle, the isocitrate dehydrogenase-encoding gene aceA was also knocked out, resulting in 3.86 g/L of 5‐ALA. Finally, the fermentation conditions were optimized, resulting in a maximum 5-ALA yield of 5.6 g/L. Overall, the blocking of the glutamate synthesis pathway could be a powerful strategy to re-allocate the carbon flux to produce 5-ALA. It could also enable the efficient synthesis of other TCA derivatives in C. glutamicum. [ABSTRACT FROM AUTHOR]

The eastern fox squirrel, Sciurus niger, exhibits marked geographic variation in size and coat color, is a model organism for studies of behavior and ecology, and a potential model for investigating physiological solutions to human porphyrias. We assembled a genome using Illumina HiSeq, PacBio SMRT, and Oxford Nanopore MinION sequencing platforms. Together, the sequencing data resulted in a draft genome of 2.99Gb, containing 32,830 scaffolds with an average size of 90.9 Kb and N50 of 183.8 Kb. Genome completeness was estimated to be 93.78%. A total of 24,443 protein-encoding genes were predicted from the assembly and 23,079 (94.42%) were annotated. Repeat elements comprised an estimated 38.49% of the genome, with the majority being LINEs (13.92%), SINEs (6.04%), and LTR elements. The topology of the species tree reconstructed using maximum-likelihood phylogenetic analysis was congruent with those of previous studies. This genome assembly can prove useful for comparative studies of genome structure and function in this rapidly diversifying lineage of mammals, for studies of population genomics and adaptation, and for biomedical research. Predicted amino acid sequence alignments for genes affecting heme biosynthesis, color vision, and hibernation showed point mutations and indels that may affect protein function and ecological adaptation. [ABSTRACT FROM AUTHOR]

Ghaith Abu-Zeinah, Maria T DeSancho Division of Hematology and Oncology, Department of Medicine, Weill Cornell Medicine, New York Presbyterian Hospital, New York, NY, USACorrespondence: Ghaith Abu-ZeinahWeill Cornell Medicine, New York Presbyterian Hospital, 1300 York Ave. C610-D, New York, NY 10065, USATel +1 646 962 2700Fax +1 646 962 0115Email gfa2001@med.cornell.eduAbstract: Sideroblastic anemia (SA) consists of a group of inherited and acquired anemias of ineffective erythropoiesis characterized by the accumulation of ring sideroblasts in the bone marrow due to disrupted heme biosynthesis. Congenital sideroblastic anemia (CSA) is rare and has three modes of inheritance: X-linked (XLSA), autosomal recessive (ARCSA), and maternal. Acquired SA is more common and can be a result of myelodysplastic syndromes (MDS) or other, generally reversible causes. The diagnostic approach to SA includes a work-up for reversible causes and genetic testing for CSA based on clinical suspicion, family history and genetic pedigree. The treatment of SA depends on the underlying etiology but remains primarily supportive with vitamin B6 supplementation for select cases of XLSA, thiamine for thiamine-responsive megaloblastic anemia subtype, red blood cell transfusions for symptomatic patients and iron chelation therapy for iron overload. The management of anemia in MDS subtypes with ring sideroblasts remains unique and includes the recently approved erythroid maturation agent, Luspatercept. Although there is currently no curative therapy for CSA, anecdotal reports of hematopoietic stem cell transplant demonstrate remissions in selective, non-syndromic cases. This review summarizes the genetics, pathophysiology, diagnosis and treatment of SA for general practitioners and clinical hematologists.Keywords: ring sideroblasts, heme biosynthesis, congenital sideroblastic anemia, acquired sideroblastic anemia, vitamin B6, iron chelation

Heme biosynthesis is a highly conserved pathway from bacteria to higher animals. Heme, which serves as a prosthetic group for various enzymes involved in multiple biochemical processes, is essential in almost all species, making heme homeostasis vital for life. However, studies on the biological functions of heme in filamentous fungi are scarce. In this study, we investigated the role of heme in Fusarium graminearum . A mutant lacking the rate-limiting enzymes in heme synthesis, coproporphyrinogen III oxidase (Cpo) or ferrochelatase (Fc), was constructed using a homologous recombination strategy. The results showed that the absence of these enzymes was lethal to F. graminearum , but the growth defect could be rescued by the addition of hemin, so we carried out further studies with the help of hemin. The results demonstrated that heme was required for the activity of FgCyp51, and its absence increased the sensitivity to tebuconazole and led to the upregulation of FgCYP51 in F. graminearum . Additionally, heme plays an indispensable role in the life cycle of F. graminearum , which is essential for vegetative growth, conidiation, external stress response (especially oxidative stress), lipid accumulation, fatty acid β-oxidation, autophagy, and virulence.

Blood clams differ from their molluscan kins by exhibiting a unique red-blood (RB) phenotype; however, the genetic basis and biochemical machinery subserving this evolutionary innovation remain unclear. As a fundamental step toward resolving this mystery, we presented the first chromosome-level genome and comprehensive transcriptomes of the blood clam Tegillarca granosa for an integrated genomic, evolutionary, and functional analyses of clam RB phenotype. We identified blood clam-specific and expanded gene families, as well as gene pathways that are of RB relevant. Clam-specific RB-related hemoglobins (Hbs) showed close phylogenetic relationships with myoglobins (Mbs) of blood clam and other molluscs without the RB phenotype, indicating that clam-specific Hbs were likely evolutionarily derived from the Mb lineage. Strikingly, similar to vertebrate Hbs, blood clam Hbs were present in a form of gene cluster. Despite the convergent evolution of Hb clusters in blood clam and vertebrates, their Hb clusters may have originated from a single ancestral Mb-like gene as evidenced by gene phylogeny and synteny analysis. A full suite of enzyme-encoding genes for heme synthesis was identified in blood clam, with prominent expression in hemolymph and resembling those in vertebrates, suggesting a convergence of both RB-related Hb and heme functions in vertebrates and blood clam. RNA interference experiments confirmed the functional roles of Hbs and key enzyme of heme synthesis in the maintenance of clam RB phenotype. The high-quality genome assembly and comprehensive transcriptomes presented herein serve new genomic resources for the super-diverse phylum Mollusca, and provide deep insights into the origin and evolution of invertebrate RB. [ABSTRACT FROM AUTHOR]

ABSTRACT Heme is an essential metabolite for most life on earth. Bacterial pathogens almost universally require iron to infect a host, often acquiring this nutrient in the form of heme. The Gram-negative pathogen Pseudomonas aeruginosa is no exception, where heme acquisition and metabolism are known to be crucial for both chronic and acute infections. To unveil unknown genes and pathways that could play a role with heme metabolic flux in this pathogen, we devised an omic-based approach we dubbed “Met-Seq,” for metabolite-coupled transposon sequencing. Met-Seq couples a biosensor with fluorescence-activated cell sorting (FACS) and massively parallel sequencing, allowing for direct identification of genes associated with metabolic changes. In this work, we first construct and validate a heme biosensor for use with P. aeruginosa and exploit Met-Seq to identify 188 genes that potentially influence intracellular heme levels. Identified genes largely consisted of metabolic pathways not previously associated with heme, including many secreted virulence effectors, as well as 11 predicted small RNAs (sRNAs) and riboswitches whose functions are not currently understood. We verify that five Met-Seq hits affect intracellular heme levels; a predicted extracytoplasmic function (ECF) factor, a phospholipid acquisition system, heme biosynthesis regulator Dnr, and two predicted antibiotic monooxygenase (ABM) domains of unknown function (PA0709 and PA3390). Finally, we demonstrate that PA0709 and PA3390 are novel heme-binding proteins. Our data suggest that Met-Seq could be extrapolated to other biological systems and metabolites for which there is an available biosensor, and provides a new template for further exploration of iron/heme regulation and metabolism in P. aeruginosa and other pathogens. IMPORTANCE The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub “Met-Seq” (metabolite-coupled Tn sequencing). Met-Seq uses the powerful combination of fluorescent biosensors, fluorescence-activated cell sorting (FACS), and next-generation sequencing (NGS) to rapidly identify genes that influence the levels of specific intracellular metabolites. For proof of concept, we create and test a heme biosensor and then exploit Met-Seq to identify novel genes involved in the regulation of heme in the pathogen Pseudomonas aeruginosa. Met-Seq-generated data were largely comprised of genes which have not previously been reported to influence heme levels in this pathogen, two of which we verify as novel heme-binding proteins. As heme is a required metabolite for host infection in P. aeruginosa and most other pathogens, our studies provide a new list of targets for potential antimicrobial therapies and shed additional light on the balance between infection, heme uptake, and heme biosynthesis.

Organisms that have lost their photosynthetic capabilities are present in a variety of eukaryotic lineages, such as plants and disparate algal groups. Most of such non-photosynthetic eukaryotes still carry plastids, as these organelles retain essential biological functions. Most non-photosynthetic plastids possess genomes with varied protein-coding contents. Such remnant plastids are known to be present in the non-photosynthetic, bacteriovorous alga Pteridomonas danica (Dictyochophyceae, Ochrophyta), which, regardless of its obligatory heterotrophic lifestyle, has been reported to retain the typically plastid-encoded gene for ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (rbcL). The presence of rbcL without photosynthetic activity suggests that investigating the function of plastids in Pteridomonas spp. would likely bring unique insights into understanding the reductive evolution of plastids, their genomes, and plastid functions retained after the loss of photosynthesis. In this study, we demonstrate that two newly established strains of the non-photosynthetic genus Pteridomonas possess highly reduced plastid genomes lacking rbcL gene, in contrast to the previous report. Interestingly, we discovered that all plastid-encoded proteins in Pteridomonas spp. are involved only in housekeeping processes (e.g., transcription, translation and protein degradation), indicating that all metabolite synthesis pathways in their plastids are supported fully by nuclear genome-encoded proteins. Moreover, through an in-depth survey of the available transcriptomic data of another strain of the genus, we detected no candidate sequences for nuclear-encoded, plastid-directed Fe–S cluster assembly pathway proteins, suggesting complete loss of this pathway in the organelle, despite its widespread conservation in non-photosynthetic plastids. Instead, the transcriptome contains plastid-targeted components of heme biosynthesis, glycolysis, and pentose phosphate pathways. The retention of the plastid genomes in Pteridomonas spp. is not explained by the Suf-mediated constraint against loss of plastid genomes, previously proposed for Alveolates, as they lack Suf genes. Bearing all these findings in mind, we propose the hypothesis that plastid DNA is retained in Pteridomonas spp. for the purpose of providing glutamyl-tRNA, encoded by trnE gene, as a substrate for the heme biosynthesis pathway.

Heme is an iron-containing molecule essential for virtually all living organisms. However, excessive heme is cytotoxic, necessitating tight regulation of intracellular heme concentration. The acute hepatic porphyrias (AHPs) are a group of rare inborn errors of heme biosynthesis that are characterized by episodic acute neurovisceral attacks that are precipitated by various factors. The AHPs are often misdiagnosed, as the acute attack symptom are non-specific and can be attributed to other more common causes. Understanding how heme biosynthesis is dysregulated in AHP patients and the mechanism by which acute attacks are precipitated will aid in accurate and rapid diagnoses, and subsequently, appropriate treatment of these disorders. Therefore, this review article will focus on the biochemical and molecular changes that occur during an acute attack and present what is currently known regarding the underlying pathogenesis of acute attacks.