Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.

Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.

Engineering sub-organelles of a diploid Saccharomyces cerevisiae to enhance the production of 7-dehydrocholesterol.

7-Dehydrocholesterol (7-DHC) is widely present in various organisms and is an important precursor of vitamin D3. Despite significant improvements in the biosynthesis of 7-DHC, it remains insufficient to meet the industrial demands. In this study, we reported high-level production of 7-DHC in an industrial Saccharomyces cerevisiae leveraging subcellular organelles. Initially, the copy numbers of DHCR24 were increased in combination with sterol transcriptional factor engineering and rebalanced the redox power of the strain. Subsequently, the effects of compartmentalizing the post-squalene pathway in peroxisomes were validated by assembling various pathway modules in this organelle. Furthermore, several peroxisomes engineering was conducted to enhance the production of 7-DHC. Utilizing the peroxisome as a vessel for partial post-squalene pathways, the potential of yeast for 7-dehydrocholesterol production was demonstrated by achieving a 26-fold increase over the initial production level. 7-DHC titer reached 640.77 mg/L in shake flasks and 4.28 g/L in a 10 L bench-top fermentor, the highest titer ever reported. The present work lays solid foundation for large-scale and cost-effective production of 7-DHC for practical applications.

Construction of an orthogonal transport system for Saccharomyces cerevisiae peroxisome to efficiently produce sesquiterpenes.

Subcellular compartmentalization is a crucial evolution characteristic of eukaryotic cells, providing inherent advantages for the construction of artificial biological systems to efficiently produce natural products. The establishment of an artificial protein transport system represents a pivotal initial step towards developing efficient artificial biological systems. Peroxisome has been demonstrated as a suitable subcellular compartment for the biosynthesis of terpenes in yeast. In this study, an artificial protein transporter ScPEX5* was firstly constructed by fusing the N-terminal sequence of PEX5 from S. cerevisiae and the C-terminal sequence of PEX5. Subsequently, an artificial protein transport system including the artificial signaling peptide YQSYY and its enhancing upstream 9 amino acid (9AA) residues along with ScPEX5* was demonstrated to exhibit orthogonality to the internal transport system of peroxisomes in S. cerevisiae. Furthermore, a library of 9AA residues was constructed and selected using high throughput pigment screening system to obtain an optimized signaling peptide (oPTS1*). Finally, the ScPEX5*-oPTS1* system was employed to construct yeast cell factories capable of producing the sesquiterpene α-humulene, resulting in an impressive α-humulene titer of 17.33 g/L and a productivity of 0.22 g/L/h achieved through fed-batch fermentation in a 5 L bioreactor. This research presents a valuable tool for the construction of artificial peroxisome cell factories and effective strategies for synthesizing other natural products in yeast.

A novel fluorescence-activated cell sorting (FACS)-based screening identified ATG14, the gene required for pexophagy in the methylotrophic yeast.

Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.

Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on ß-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans.

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes.

A preferred sequence for organelle inheritance during polarized cell growth.

Some organelles cannot be synthesized anew, so they are segregated into daughter cells during cell division. In Saccharomyces cerevisiae, daughter cells bud from mother cells and are populated by organelles inherited from the mothers. To determine whether this organelle inheritance occurs in a stereotyped manner, we tracked organelles using fluorescence microscopy. We describe a program for organelle inheritance in budding yeast. The cortical endoplasmic reticulum (ER) and peroxisomes are inherited concomitantly with bud emergence. Next, vacuoles are inherited in small buds, followed closely by mitochondria. Finally, the nucleus and perinuclear ER are inherited when buds have nearly reached their maximal size. Because organelle inheritance timing correlates with bud morphology, which is coupled to the cell cycle, we tested whether disrupting the cell cycle alters organelle inheritance order. By arresting cell cycle progression but allowing continued bud growth, we determined that organelle inheritance still occurs when DNA replication is blocked, and that the general inheritance order is maintained. Thus, organelle inheritance follows a preferred order during polarized cell division and does not require completion of S-phase.

Acyl CoA oxidase: from its expression, structure, folding, and import to its role in human health and disease.

ß-oxidation of fatty acids is an important metabolic pathway and is a shared function between mitochondria and peroxisomes in mammalian cells. On the other hand, peroxisomes are the sole site for the degradation of fatty acids in yeast. The first reaction of this pathway is catalyzed by the enzyme acyl CoA oxidase housed in the matrix of peroxisomes. Studies in various model organisms have reported the conserved function of the protein in fatty acid oxidation. The importance of this enzyme is highlighted by the lethal conditions caused in humans due to its altered function. In this review, we discuss various aspects ranging from gene expression, structure, folding, and import of the protein in both yeast and human cells. Further, we highlight recent findings on the role of the protein in human health and aging, and discuss the identified mutations in the protein associated with debilitating conditions in patients.

Comparison of human PEX knockout cell lines suggests a dual role of PEX1 in peroxisome biogenesis.

For the biogenesis and maintenance of peroxisomes several proteins, called peroxins, are essential. Malfunctions of these proteins lead to severe diseases summarized as peroxisome biogenesis disorders. The different genetic background of patient-derived cell lines and the residual expression of mutated PEX genes impede analysis of the whole spectrum of cellular functions of affected peroxins. To overcome these difficulties, we have generated a selected PEX knockout resource of HEK T-REx293 cells using the CRISPR/Cas9 technique. Comparative analyses of whole cell lysates revealed PEX-KO specific alterations in the steady-state level of peroxins and variations in the import efficacy of matrix proteins with a Type 2 peroxisomal targeting signal. One of the observed differences concerned PEX1 as in the complete absence of the protein, the number of peroxisomal ghosts is significantly increased. Upon expression of PEX1, import competence and abundance of peroxisomes was adjusted to the level of normal HEK cells. In contrast, expression of an alternatively spliced PEX1 isoform lacking 321 amino acids of the N-terminal region failed to rescue the peroxisomal import defects but reduced the number of peroxisomal vesicles. All in all, the data suggest a novel 'moonlighting' function of human PEX1 in the regulation of pre-peroxisomal vesicles.

Autosomal dominant Zellweger spectrum disorder caused by de novo variants in PEX14 gene.

PURPOSE: Zellweger spectrum disorders (ZSDs) are known as autosomal recessive disorders caused by defective peroxisome biogenesis due to bi-allelic pathogenic variants in any of at least 13 different PEX genes. Here, we report 2 unrelated patients who present with an autosomal dominant ZSD. METHODS: We performed biochemical and genetic studies in blood and skin fibroblasts of the patients and demonstrated the pathogenicity of the identified PEX14 variants by functional cell studies. RESULTS: We identified 2 different single heterozygous de novo variants in the PEX14 genes of 2 patients diagnosed with ZSD. Both variants cause messenger RNA mis-splicing, leading to stable expression of similar C-terminally truncated PEX14 proteins. Functional studies indicated that the truncated PEX14 proteins lost their function in peroxisomal matrix protein import and cause increased degradation of peroxisomes, ie, pexophagy, thus exerting a dominant-negative effect on peroxisome functioning. Inhibition of pexophagy by different autophagy inhibitors or genetic knockdown of the peroxisomal autophagy receptor NBR1 resulted in restoration of peroxisomal functions in the patients' fibroblasts. CONCLUSION: Our finding of an autosomal dominant ZSD expands the genetic repertoire of ZSDs. Our study underscores that single heterozygous variants should not be ignored as possible genetic cause of diseases with an established autosomal recessive mode of inheritance.

The AAA+ ATPase Msp1 is a processive protein translocase with robust unfoldase activity.

Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1's ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.

Anaerobic peroxisomes in Mastigamoeba balamuthi.

The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either ß-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named "anaerobic" peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.

Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids.

Current approaches for the production of high-value compounds in microorganisms mostly use the cytosol as a general reaction vessel. However, competing pathways and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Eukaryotic cells control the complexity of their metabolism by harnessing organelles to insulate biochemical pathways. Inspired by this concept, herein we transform yeast peroxisomes into microfactories for geranyl diphosphate-derived compounds, focusing on monoterpenoids, monoterpene indole alkaloids, and cannabinoids. We introduce a complete mevalonate pathway in the peroxisome to convert acetyl-CoA to several commercially important monoterpenes and achieve up to 125-fold increase over cytosolic production. Furthermore, peroxisomal production improves subsequent decoration by cytochrome P450s, supporting efficient conversion of (S)-(-)-limonene to the menthol precursor trans-isopiperitenol. We also establish synthesis of 8-hydroxygeraniol, the precursor of monoterpene indole alkaloids, and cannabigerolic acid, the cannabinoid precursor. Our findings establish peroxisomal engineering as an efficient strategy for the production of isoprenoids.

Acquired deficiency of peroxisomal dicarboxylic acid catabolism is a metabolic vulnerability in hepatoblastoma.

Metabolic reprogramming provides transformed cells with proliferative and/or survival advantages. Capitalizing on this therapeutically, however, has been only moderately successful because of the relatively small magnitude of these differences and because cancers may further adapt their metabolism to evade metabolic pathway inhibition. Mice lacking the peroxisomal bifunctional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) and supplemented with the 12-carbon fatty acid lauric acid (C12) accumulate the toxic metabolite dodecanedioic acid (DDDA), which causes acute hepatocyte necrosis and liver failure. We noted that, in a murine model of pediatric hepatoblastoma (HB) and in primary human HBs, downregulation of Ehhadh occurs in association with the suppression of mitochondrial ß- and endosomal/peroxisomal ω-fatty acid oxidation pathways. This suggested that HBs might be more susceptible than normal liver tissue to C12 dietary intervention. Indeed, HB-bearing mice provided with C12- and/or DDDA-supplemented diets survived significantly longer than those on standard diets. In addition, larger tumors developed massive necrosis following short-term DDDA administration. In some HBs, the eventual development of DDDA resistance was associated with 129 transcript differences, ∼90% of which were downregulated, and approximately two-thirds of which correlated with survival in numerous human cancers. These transcripts often encoded extracellular matrix components, suggesting that DDDA resistance arises from reduced Ehhadh uptake. Lower Ehhadh expression was also noted in murine hepatocellular carcinomas and in subsets of certain human cancers, supporting the likely generality of these results. Our results demonstrate the feasibility of C12 or DDDA dietary supplementation that is nontoxic, inexpensive, and likely compatible with more standard chemotherapies.

Peroxisome biogenesis and inter-organelle communication: an indispensable role for Pex11 and Pex30 family proteins in yeast.

Peroxisomes are highly dynamic organelles present in most eukaryotic cells. They also play an important role in human health and the optimum functioning of cells. An extensive repertoire of proteins is associated with the biogenesis and function of these organelles. Two protein families that are involved in regulating peroxisome number in a cell directly or indirectly are Pex11 and Pex30. Interestingly, these proteins are also reported to regulate the contact sites between peroxisomes and other cell organelles such as mitochondria, endoplasmic reticulum and lipid droplets. In this manuscript, we review our current knowledge of the role of these proteins in peroxisome biogenesis in various yeast species. Further, we also discuss in detail the role of these protein families in the regulation of inter-organelle contacts in yeast.

Increased peroxisome proliferation is associated with early yeast replicative ageing.

Peroxisomes are single membrane-bound organelles ubiquitously present in several cell types and are associated with cell and tissue-specific functions. Their role in cellular ageing is under investigation in various model systems. Metabolism of cellular reactive oxygen species is a universal function performed by these organelles. In this study, we investigated alterations in peroxisome number upon early replicative ageing of yeast cells. Increase in the number of peroxisomes in replicatively aged mother cells of wild-type yeast was observed when cultured in both peroxisome-inducing and non-inducing medium. Further, we investigated if this increase in peroxisome number in replicatively aged cells is due to enhanced peroxisome proliferation. For this, the number of peroxisomes in replicatively aged mother cells of pex11, pex25 and pex11pex25 was analysed. Increased percentage of aged cells was observed in pex25 and pex11pex25 cells cultured in peroxisome-inducing oleic acid medium. Interestingly, when cultured in oleic acid, young mother cells devoid of Pex11 showed reduced peroxisome proliferation compared to old mother cells. Induced activity of the antioxidant enzyme catalase and reduced accumulation of reactive oxygen species were reported in all studied strains when cultured in oleic acid medium. Further, our data also suggest that replicatively aged cells with increased peroxisome number also display mitochondrial dysfunction and fragmentation in all the strains studied. In conclusion, our data suggests a correlation between increase in peroxisome number and replicative age of yeast cells and interestingly this increase seems to be partly dependent on the fission proteins.

Pex30 undergoes phosphorylation and regulates peroxisome number in Saccharomyces cerevisiae.

Pex30 is a dysferlin domain-containing protein whose role in peroxisome biogenesis has been studied by several research groups. Notably, recent studies have linked this protein to peroxisomes, endoplasmic reticulum and lipid bodies in Saccharomyces cerevisiae. Phosphoproteome studies of S. cerevisiae have identified several phosphorylation sites in Pex30. In this study we expressed and purified Pex30 from its native host. Analysis of the purified protein by circular dichroism spectroscopy showed that it retained its secondary structure and revealed primarily a helical structure. Further phosphorylation of Pex30 at three residues, Threonine 60, Serine 61 and Serine 511 was identified by mass spectrometry in this study. To understand the importance of this post-translational modification in peroxisome biogenesis, the identified residues were mutated to both non-phosphorylatable (alanine) and phosphomimetic (aspartic acid) variants. Upon analysis of the mutant variants by fluorescence microscopy, no alteration in the localization of the protein to ER and peroxisomes was observed. Interestingly, reduced number of peroxisomes were observed in cells expressing phosphomimetic mutations when cultured in peroxisome-inducing conditions. Our data suggest that phosphorylation and dephosphorylation of Pex30 may promote distinct interactions essential in regulating peroxisome number in a cell.

Genetic variants in DDO and PEX5L in peroxisome-related pathways predict non-small cell lung cancer survival.

Peroxisomes play a role in lipid metabolism and regulation of reactive oxygen species, but its role in development and progression of non-small cell lung cancer (NSCLC) is not well understood. Here, we investigated the associations between 9708 single-nucleotide polymorphisms (SNPs) in 113 genes in the peroxisome-related pathways and survival of NSCLC patients from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) and the Harvard Lung Cancer Susceptibility (HLCS) study. In 1185 NSCLC patients from the PLCO trial, we found that 213 SNPs were significantly associated with NSCLC overall survival (OS) (p ≤ 0.05, Bayesian false discovery probability [BFDP] ≤ 0.80), of which eight SNPs were validated in the HLCS data set. In a multivariate Cox proportional hazards regression model, two independent SNPs (rs9384742 DDO and rs9825224 PEX5L) were significantly associated with NSCLC survival (hazards ratios [HR] of 1.17 with 95% CI [confidence interval] of 1.06-1.28 and 0.86 with 95% CI of 0.77-0.96, respectively). Patients with one or two protective genotypes had a significantly higher OS (HR: 0.787 [95% CI: 0.620-0.998] and 0.691 [95% CI: 0.543-0.879], respectively). Further expression quantitative trait loci analysis using whole blood and lung tissue showed that the minor allele of rs9384742 DDO was significantly associated with decreased messenger RNA (mRNA) expression levels and that DDO expression was also decreased in NSCLC tumor tissue. Additionally, high PEX5L expression levels were significantly associated with lower survival of NSCLC. Our data suggest that variants in these peroxisome-related genes may influence gene regulation and are potential predictors of NSCLC OS, once validated by additional studies.

Hypomorphic mutation of PEX3 with peroxisomal mosaicism reveals the oscillating nature of peroxisome biogenesis coupled with differential metabolic activities.

Impaired peroxisome assembly caused by mutations in PEX genes results in a human congenital metabolic disease called Zellweger spectrum disorder (ZSD), which impacts the development and physiological function of multiple organs. In this study, we revealed a long-standing problem of heterogeneous peroxisome distribution among cell population, so called "peroxisomal mosaicism", which appears in patients with mild form of ZSD. We mutated PEX3 gene in HEK293 cells and obtained a mutant clone with peroxisomal mosaicism. We found that peroxisomal mosaicism can be reproducibly arise from a single cell, even if the cell has many or no peroxisomes. Using time-lapse imaging and a long-term culture experiment, we revealed that peroxisome biogenesis oscillates over a span of days; this was also confirmed in the patient's fibroblasts. During the oscillation, the metabolic activity of peroxisomes was maintained in the cells with many peroxisomes while depleted in the cells without peroxisomes. Our results indicate that ZSD patients with peroxisomal mosaicism have a cell population whose number and metabolic activities of peroxisomes can be recovered. This finding opens the way to develop novel treatment strategy for ZSD patients with peroxisomal mosaicism, who currently have very limited treatment options.