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.

UNASSIGNED: Traumatic and thermal injuries result in a state of systemic immune suppression, yet the mechanisms that underlie its development are poorly understood. Released from injured muscle and lysed red blood cells, heme is a damage associated molecular pattern with potent immune modulatory properties. Here, we measured plasma concentrations of total heme in over 200 traumatic and thermally-injured patients in order to examine its relationship with clinical outcomes and post-injury immune suppression.
UNASSIGNED: Blood samples were collected from 98 burns (≥15% total body surface area) and 147 traumatically-injured (injury severity score ≥8) patients across the ultra-early (≤1 hour) and acute (4-72 hours) post-injury settings. Pro-inflammatory cytokine production by lipopolysaccharide (LPS) challenged whole blood leukocytes was studied, and plasma concentrations of total heme, and its scavengers haptoglobin, hemopexin and albumin measured, alongside the expression of heme-oxygenase-1 (HO-1) in peripheral blood mononuclear cells (PBMCs). LPS-induced tumour necrosis factor-alpha (TNF-α) production by THP-1 cells and monocytes following in vitro heme treatment was also examined.
UNASSIGNED: Burns and traumatic injury resulted in significantly elevated plasma concentrations of heme, which coincided with reduced levels of hemopexin and albumin, and correlated positively with circulating levels of pro and anti-inflammatory cytokines. PBMCs isolated from trauma patients 4-12 and 48-72 hours post-injury exhibited increased HO-1 gene expression. Non-survivors of burn injury and patients who developed sepsis, presented on day 1 with significantly elevated heme levels, with a difference of 6.5 µM in heme concentrations corresponding to a relative 52% increase in the odds of post-burn mortality. On day 1 post-burn, heme levels were negatively associated with ex vivo LPS-induced TNF-α and interleukin-6 production by whole blood leukocytes. THP-1 cells and monocytes pre-treated with heme exhibited significantly reduced TNF-α production following LPS stimulation. This impairment was associated with decreased gene transcription, reduced activation of extracellular signal-regulated kinase 1/2 and an impaired glycolytic response.
UNASSIGNED: Major injury results in elevated plasma concentrations of total heme that may contribute to the development of endotoxin tolerance and increase the risk of poor clinical outcomes. Restoration of the heme scavenging system could be a therapeutic approach by which to improve immune function post-injury.

Cytochrome bd-I from Escherichia coli belongs to the superfamily of prokaryotic bd-type oxygen reductases. It contains three hemes, b558, b595 and d, and couples oxidation of quinol by dioxygen with the generation of a proton-motive force. The enzyme exhibits resistance to various stressors and is considered as a target protein for next-generation antimicrobials. By using electronic absorption and MCD spectroscopy, this work shows that cyanide binds to heme d2+ in the isolated fully reduced cytochrome bd-I. Cyanide-induced difference absorption spectra display changes near the heme d2+ α-band, a minimum at 633 nm and a maximum around 600 nm, and a W-shaped response in the Soret region. Apparent dissociation constant (Kd) of the cyanide complex of heme d2+ is ∼0.052 M. Kinetics of cyanide binding is monophasic, indicating the presence of a single ligand binding site in the enzyme. Consistently, MCD data show that cyanide binds to heme d2+ but not to b5582+ or b5952+. This agrees with the published structural data that the enzyme\'s active site is not a di-heme site. The observed rate of binding (kobs) increases as the concentration of cyanide is increased, giving a second-order rate constant (kon) of ∼0.1 M-1 s-1.

UNASSIGNED: Porphyromonas gingivalis and Porphyromonas endodontalis belong to the Bacteroidota phylum. Both species inhabit the oral cavity and can be associated with periodontal diseases. To survive, they must uptake heme from the host as an iron and protoporphyrin IX source. Among the best-characterized heme acquisition systems identified in members of the Bacteroidota phylum is the P. gingivalis Hmu system, with a leading role played by the hemophore-like HmuY (HmuYPg) protein.
UNASSIGNED: Theoretical analysis of selected HmuY proteins and spectrophotometric methods were employed to determine the heme-binding mode of the P. endodontalis HmuY homolog (HmuYPe) and its ability to sequester heme. Growth phenotype and gene expression analysis of P. endodontalis were employed to reveal the importance of the HmuYPe and Hmu system for this bacterium.
UNASSIGNED: Unlike in P. gingivalis, where HmuYPg uses two histidines for heme-iron coordination, other known HmuY homologs use two methionines in this process. P. endodontalis HmuYPe is the first characterized representative of the HmuY family that binds heme using a histidine-methionine pair. It allows HmuYPe to sequester heme directly from serum albumin and Tannerella forsythia HmuYTf, the HmuY homolog which uses two methionines for heme-iron coordination. In contrast to HmuYPg, which sequesters heme directly from methemoglobin, HmuYPe may bind heme only after the proteolytic digestion of hemoglobin.
UNASSIGNED: We hypothesize that differences in components of the Hmu system and structure-based properties of HmuY proteins may evolved allowing different adaptations of Porphyromonas species to the changing host environment. This may add to the superior virulence potential of P. gingivalis over other members of the Bacteroidota phylum.

Hell\'s Gate globin-I (HGb-I) is a thermally stable globin from the aerobic methanotroph Methylacidiphilium infernorum. Here we report that HGb-I interacts with lipids stoichiometrically to induce structural changes in the heme pocket, changing the heme iron distal ligation coordination from hexacoordinate to pentacoordinate. Such changes in heme geometry have only been previously reported for cytochrome c and cytoglobin, linked to apoptosis regulation and enhanced lipid peroxidation activity, respectively. However, unlike cytoglobin and cytochrome c, the heme iron of HGb-I is altered by lipids in ferrous as well as ferric oxidation states. The apparent affinity for lipids in this thermally stable globin is highly pH-dependent but essentially temperature-independent within the range of 20-60 °C. We propose a mechanism to explain these observations, in which lipid binding and stability of the distal endogenous ligand are juxtaposed as a function of temperature. Additionally, we propose that these coupled equilibria may constitute a mechanism through which this acidophilic thermophile senses the pH of its environment.

Ferrochelatase (FECH) is the terminal enzyme in human heme biosynthesis, catalyzing the insertion of ferrous iron into protoporphyrin IX (PPIX) to form protoheme IX (Heme). Phosphorylation increases the activity of FECH, and it has been confirmed that the activity of FECH phosphorylated at T116 increases. However, it remains unclear whether the T116 site and other potential phosphorylation modification sites collaboratively regulate the activity of FECH. In this study, we identified a new phosphorylation site, T218, and explored the allosteric effects of unphosphorylated (UP), PT116, PT218, and PT116 + PT218 states on FECH in the presence and absence of substrates (PPIX and Heme) using molecular dynamics (MD) simulations. Binding free energies were evaluated with the MM/PBSA method. Our findings indicate that the PT116 + PT218 state exhibits the lowest binding free energy with PPIX, suggesting the strongest binding affinity. Additionally, this state showed a higher binding free energy with Heme compared to UP, which facilitates Heme release. Moreover, employing multiple analysis methods, including free energy landscape (FEL), principal component analysis (PCA), dynamic cross-correlation matrix (DCCM), and hydrogen bond interaction analysis, we demonstrated that phosphorylation significantly affects the dynamic behavior and binding patterns of substrates to FECH. Insights from this study provide valuable theoretical guidance for treating conditions related to disrupted heme metabolism, such as various porphyrias and iron-related disorders.

LONP1 is the principal AAA+ unfoldase and bulk protease in the mitochondrial matrix, so its deletion causes embryonic lethality. The AAA+ unfoldase CLPX and the peptidase CLPP also act in the matrix, especially during stress periods, but their substrates are poorly defined. Mammalian CLPP deletion triggers infertility, deafness, growth retardation, and cGAS-STING-activated cytosolic innate immunity. CLPX mutations impair heme biosynthesis and heavy metal homeostasis. CLPP and CLPX are conserved from bacteria to humans, despite their secondary role in proteolysis. Based on recent proteomic-metabolomic evidence from knockout mice and patient cells, we propose that CLPP acts on phase-separated ribonucleoprotein granules and CLPX on multi-enzyme condensates as first-aid systems near the inner mitochondrial membrane. Trimming within assemblies, CLPP rescues stalled processes in mitoribosomes, mitochondrial RNA granules and nucleoids, and the D-foci-mediated degradation of toxic double-stranded mtRNA/mtDNA. Unfolding multi-enzyme condensates, CLPX maximizes PLP-dependent delta-transamination and rescues malformed nascent peptides. Overall, their actions occur in granules with multivalent or hydrophobic interactions, separated from the aqueous phase. Thus, the role of CLPXP in the matrix is compartment-selective, as other mitochondrial peptidases: MPPs at precursor import pores, m-AAA and i-AAA at either IMM face, PARL within the IMM, and OMA1/HTRA2 in the intermembrane space.

Cytochrome P450s (P450s), a superfamily of heme-containing enzymes, existed in animals, plants, and microorganisms. P450s can catalyze various regional and stereoselective oxidation reactions, which are widely used in natural product biosynthesis, drug metabolism, and biotechnology. In a typical catalytic cycle, P450s use redox proteins or domains to mediate electron transfer from NAD(P)H to heme iron. Therefore, the main factors determining the catalytic efficiency of P450s include not only the P450s themselves but also their redox-partners and electron transfer pathways. In this review, the electron transfer pathway engineering strategies of the P450s catalytic system are reviewed from four aspects: cofactor regeneration, selection of redox-partners, P450s and redox-partner engineering, and electrochemically or photochemically driven electron transfer.

A key element for the cost-effective development of cultured meat is a cell line culturable in serum-free conditions to reduce production costs. Heme supplementation in cultured meat mimics the original meat flavor and color. This study introduced a bacterial extract generated from Corynebacterium that was selected for high-heme expression by directed evolution. A normal porcine cell line, PK15, was used to apply the bacterial heme extract as a supplement. Consistent with prior research, we observed the cytotoxicity of PK15 to the heme extract at 10 mM or higher. However, after long-term exposure, PK15 adapted to tolerate up to 40 mM of heme. An RNA-seq analysis of these heme-adapted PK15 cells (PK15H) revealed a set of altered genes, mainly involved in cell proliferation, metabolism, and inflammation. We found that cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), lactoperoxidase (LPO), and glutathione peroxidase 5 (GPX5) were upregulated in the PK15H heme dose dependently. When we reduced serum serially from 2% to serum free, we derived the PK15H subpopulation that was transiently maintained with 5-10 mM heme extract. Altogether, our study reports a porcine cell culturable in high-heme media that can be maintained in serum-free conditions and proposes a marker gene that plays a critical role in this adaptation process.

The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5\'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme in vitro, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.