Peroxisomal Pathway Research Articles

Unveiling the proteome of the fasting heart: Insights into HIF-1 pathway regulation.

Fasting is a common dietary intervention known for its protective effects against metabolic and cardiovascular diseases. While its effects are mostly systemic, understanding tissue-specific changes in the heart is crucial for the identification of the mechanisms underlying fasting-induced cardioprotection. In this study, we performed a proteomic analysis of the fasting heart and attempted to clarify the molecular basis of fasting-induced cardioprotection. Our investigation identified a total of 4,652 proteins, with 127 exhibiting downregulation and 118 showing upregulation after fasting. Annotation analysis highlighted significant changes in processes such as lipid metabolism, the peroxisome pathway, and reactive oxygen species metabolism. Notably, the HIF-1 signaling pathway emerged as one of the focal points, with various HIF-1 targets exhibiting differential responses to fasting. Further experiments demonstrated downregulation of HIF-1α at both transcript and protein levels. Intriguingly, while gene expression of Egln3 decreased, its protein product PHD3 remained unaffected by fasting. The unchanged levels of pro-inflammatory cytokines indicated that the observed reduction in Hif1a expression did not stem from a decrease in basal inflammation. These findings underscore the complex regulation of the well-established cardioprotective HIF-1 signaling within the heart during 3-day fasting.

A-066 Does essential fatty acid deficiency alter ethanolamine plasmalogen concentrations in red blood cells? Implications for clinical testing

Abstract Background Plasmalogens are glycerophospholipids characterized by a fatty alcohol at the sn-1 position, a polyunsaturated fatty acid at the sn-2 position, most commonly docosahexaenoic acid (DHA) or arachidonic acid, and a polar head group at the sn-3 position, mainly phosphoethanolamine. Markedly reduced plasmalogens are seen in peroxisome biogenesis defects, including Zellweger spectrum disorders (ZSD), since the first two steps of plasmalogen synthesis occur in peroxisomes. Disruption of the peroxisomal β-oxidation pathway also causes DHA deficiency in ZSD patients. Since plasmalogens are composed of fatty acids, the deficiency of DHA or other essential fatty acids could further decrease the plasmalogen level in ZSD patients and lower the results of plasmalogen testing in patients without ZSD. To test this hypothesis, we evaluated the impact of essential fatty acid (EFA) deficiency on plasmalogens in patients with ZSD and individuals with EFA deficiency due to unrelated causes. Methods Red blood cell (RBC) samples were obtained from individuals without ZSD referred to ARUP laboratories for clinical EFA testing (n = 80; age range: birth - 89 years), and from patients with ZSD (n = 7; age range birth - 9 years). Three ZSD patients were older and with a less severe phenotype. A total of 18 intact ethanolamine plasmalogen species were extracted from packed RBCs and quantified using a XEVO TQ-XS Mass Spectrometer in conjunction with Ultra-High Performance Liquid Chromatography (Waters). Total plasmalogen values were also calculated; plasmalogen deficiency was defined as ≤60% of age-matched control median. Fatty acids (FAs) were quantified by gas chromatography negative chemical ionization-mass spectrometry (GC-NCI-MS). EFA deficiency was diagnosed by increased Triene/Tetraene ratio (Mead acid/Arachidonic acid). Data was analyzed using Prism v.8.3.0 software (La Jolla, CA). The study was approved by the Institutional Review Board of the University of Utah. Results Out of the 80 samples from individuals without ZSD referred for EFA testing, 18% had normal fatty acid profile, 57% were receiving dietary supplements and 25% had elevated Triene/Tetraene ratio suggesting EFA deficiency. EFA deficiency was accompanied by low DHA only in two individuals without ZSD, whereas it was present in most of the samples from ZSD patients evaluated (5/7). None of the individuals without ZSD were deficient in plasmalogens. Moreover, when compared to individuals without ZSD with a normal FA profile, there was no significant difference observed with EFA deficiency or FA supplementation. Although our ZSD cohort is small, no correlation between EFA deficiency and plasmalogen levels was also seen in ZSD: 2 patients with milder phenotype had EFA deficiency but normal plasmalogens, 2 patients had EFA deficiency and low plasmalogens, 2 patients with low plasmalogens had low DHA without EFA deficiency, and 1 patient had low plasmalogens but normal EFA. Conclusions Plasmalogen quantitation in RBCs by LC-MS/MS is not significantly affected by EFA deficiency. Although more data is warranted to elucidate the correlation between plasmalogen levels and ZSD phenotype, plasmalogens can be within normal reference intervals in ZSD regardless of EFA status.

Pharmacological targeting of AMPK to restore glucose and fatty acid metabolism homeostasis attenuates transplanted kidney fibrosis

Chronic fibrosis often occurs in transplanted kidneys, leading to progressive functional decline. The underlying mechanisms may involve disruption in the metabolism of renal tubular epithelial cells. The liver kinase B1 (LKB1)-AMPK pathway is a pivotal regulatory hub for glucose and fatty acid metabolism and may play a role in transplanted kidney fibrosis, but it has not been reported. In this study we administered fenofibrate, 2-deoxyglucose, or metformin to modulate metabolism in Brown Norway rat kidney transplants and investigated pathways involved in fibrosis using various assays. We identified an impaired LKB1-AMPK pathway within epithelial cells, resulting in perturbed glucose and fatty acid metabolism, collagen secretion, extracellular matrix remodeling, and epithelial-mesenchymal transition. ACOX1, a pivotal enzyme in the fatty acid peroxisomal β-oxidation pathway, played an important role in transplanted renal fibrosis. Furthermore, several metabolism-targeting drugs, particularly metformin, emerged as potent fibrosis inhibitors. Metformin attenuated fibrosis, improved renal function, and reduced inflammation and macrophage infiltration in the transplanted kidneys. These results provide new perspectives for understanding the complex molecular basis underlying transplanted renal fibrosis and developing novel therapeutic strategies.

Antifungal metabolites of biocontrol stain LB-1 and their inhibition mechanism against Botrytis cinerea.

Chaetomium subaffine LB-1 is a novel biocontrol strain that produces non-volatile metabolites that inhibit the growth of Botrytis cinerea. However, the specific metabolites and antimicrobial mechanism of the strain LB-1 remains unclear. In this study, the antifungal substances produced by strain LB-1, as well as the underlying mechanism of its inhibitory effect against B. cinerea, were explored using metabolomic and transcriptomic analysis. The results found that 45 metabolites might be the key antifungal substances, such as ouabain, ferulic acid, chlorogenic acid, spermidine, stachydrine, and stearic acid. The transcriptomic analysis indicated that the inhibition effect of LB-1 on B. cinerea resulted in the upregulation of genes related to adenosine triphosphate (ATP)-binding cassette (ABC) transporters, peroxisome, ER stress, and multiple metabolic pathways, and in downregulation of many genes associated with the synthesis of cell walls/membranes, carbohydrate metabolism, cell cycle, meiosis, and DNA replication. These results suggested that the inhibitory effect of strain LB-1 against B. cinerea might be due to the destroyed cell wall and membrane integrity exerted by antimicrobial substances, which affect cell metabolism and inhibit cell proliferation.

Engineering high production of fatty alcohols from methanol by constructing coordinated dual biosynthetic pathways

Microbial cell factories provide an efficient approach for the green manufacturing of chemicals. However, the excessive use of sugars increases the potential risk of food crisis. Methanol, an abundant feedstock, holds promise in facilitating low-carbon production processes. However, the current methanol bioconversion is hindered by limited regulatory strategies and relatively low conversion efficiency. Here, a yeast biocatalyst was extensively engineered for efficient biosynthesis of fatty alcohols through reinforcement of precursor supply and methanol assimilation in Pichia pastoris. Furthermore, the dual cytoplasmic and peroxisomal biosynthetic pathways were constructed by mating and exhibited robust production of 5.6 g/L fatty alcohols by using methanol as the sole carbon source. This study provides a heterozygous diploid P. pastoris strain with dual cytoplasmic and peroxisomal biosynthetic pathways, which achieved the highest fatty alcohol production from one-carbon feedstocks to date.

Identification of microRNA and their potential role in regulation diapause termination in seven spot ladybird beetle, Coccinella septempunctata

Diapause is an alternative development stage in seven spot ladybird beetle, Coccinella septempunctata. However, the regulatory mechanism governing the initiation, maintenance, and termination of diapause in the seven-spot ladybird have not been extensively studied. MicroRNAs (miRNAs), a type of non-coding RNA, might be involved in diapause regulation and related physiological processes. The objective of this study is to investigate the potential involvement of miRNAs in diapause termination in C. septempunctata. High-throughput sequencing was used to identify miRNAs associated with diapause termination in C. septempunctata. A total of 769 miRNAs were identified, potentially implicated in diapause termination, including 673 evolutionarily conserved miRNA and 96 putatively novel-miRNAs. Among these, two evolutionarily conserved miRNAs, aae-miR-305-5P and tca-miR-277-5P, exhibited differential abundance during diapause termination compared to diapause. aae-miR-305-5P was overexpressed in diapause termination ladybird beetle and may be responsible for silencing the expression of candidate genes in peroxisome pathway associated with diapause termination. Conversely, tca-miR-277-5P was under-expressed in diapause termination and may promote the expression of genes related to the longevity regulating pathway, thereby increase the lifetime, a characteristic feature of diapause termination. In addition, a putatively novel-miRNA (unconservative_c62764) was overexpressed in diapause termination ladybird beetle, potentially contributing to the decreased expression of genes related to Wnt signaling pathway during diapause termination. These findings highlighting the significant roles of microRNAs in pathway such as longevity regulation, perisome function, and Wnt signaling, which may regulate diapause termination in C. septempunctata. This study might help us to unveil the miRNA involvement in gene expression regulation of diapause termination in insects.

N-glycosylation of SnRK2s affects NADPH maintenance in peroxisomes during prolonged ABA signalling

Unfavourable conditions, such as prolonged drought and high salinity, pose a threat to the survival and agricultural yield of plants. The phytohormone ABA plays a key role in the regulation of plant stress adaptation and is often maintained at high levels for extended periods. While much is known about ABA signal perception and activation in the early signalling stage, the molecular mechanism underlying desensitization of ABA signalling remains largely unknown. Here we demonstrate that in the endoplasmic reticulum (ER)-Golgi network, the key regulators of ABA signalling, SnRK2.2/2.3, undergo N-glycosylation, which promotes their redistribution from the nucleus to the peroxisomes in Arabidopsis roots and influences the transcriptional response in the nucleus during prolonged ABA signalling. On the peroxisomal membrane, SnRK2s can interact with glucose-6-phosphate (G6P)/phosphate translocator 1 (GPT1) to maintain NADPH homeostasis through increased activity of the peroxisomal oxidative pentose phosphate pathway (OPPP). The resulting maintenance of NADPH is essential for the modulation of hydrogen peroxide (H2O2) accumulation, thereby relieving ABA-induced root growth inhibition. The subcellular dynamics of SnRK2s, mediated by N-glycosylation suggest that ABA responses transition from transcriptional regulation in the nucleus to metabolic processes in the peroxisomes, aiding plants in adapting to long-term environmental stress.

Sublethal effects of nitenpyram on the development of silkworm

The utilization of nitenpyram for aphid and whitefly control may induce environmental contamination and negative repercussions on non-target organisms. Formerly, we found that nitenpyram would pollute the peripheral and sub-peripheral areas of the adjacent mulberry orchard. Under acute toxicity conditions, nitenpyram induced oxidative damage in silkworms, affected biological metabolism, synthesis, immunity, and signal transduction. Considering the impact of nitenpyram mist drift on mulberry leaves, we investigated the effects of low concentrations of nitenpyram on silkworms. The results showed that silkworms exposed to 0.17 mg/L, 0.35 mg/L and 0.70 mg/L of nitenpyram (1/40 LC50, 1/20 LC50 and 1/10 LC50) showed obvious poisoning symptoms. The cocoon weight and cocoon shell weight decreased gradually with increases in the concentration, and these decreases prolonged the growth and development time of silkworms and induced the detoxification enzymes carboxylesterase (CarE) and glutathione-S-transferase (GST) to cope with the stress damage caused by nitenpyram. Exposure to low concentrations of nitenpyram downregulates genes involved in the drug metabolism-other enzymes and peroxisome pathway in silkworms. Additionally, through injection of miRNA mimics and inhibitors, we discovered that detoxifying enzyme pathway genes are influenced by bmo-miR-3382-3P, bmo-miR-3213-5P and bmo-miR-133, regulating the immune response of silkworms. This study provides an overall view of the toxicity and detoxification metabolism of nitenpyram in silkworm, and provides a reference for environmental assessment.

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.

Metabolic engineering of Pichia pastoris for overproduction of cis-trans nepetalactol

Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical application is limited due to challenges in plant extraction. Microbial production has emerged as a promising strategy to meet the clinical demands for MIAs. The biosynthetic pathway of cis-trans nepetalactol, which serves as the universal iridoid scaffold for all MIAs, has been successfully identified and reconstituted. However, bottlenecks and challenges remain to construct a high-yielding platform strain for cis-trans nepetalactol production, which is vital for subsequent MIAs biosynthesis. In the present study, we focused on engineering of Pichia pastoris cell factories to enhance the production of geraniol, 8-hydroxygeraniol, and cis-trans nepetalactol. By targeting the biosynthetic pathway from acetyl-CoA to geraniol in both peroxisomes and cytoplasm, we achieved comparable geraniol titers in both compartments. Through protein engineering, we found that either G8H or CPR truncation increased the production of 8-hydroxygeraniol, with a 47.8-fold and 14.0-fold increase in the peroxisomal and cytosolic pathway strain, respectively. Furthermore, through a combination of dynamical control of ERG20, precursor and cofactor supply engineering, diploid engineering, and dual subcellular compartmentalization engineering, we achieved the highest ever reported production of cis-trans nepetalactol, with a titer of 4429.4 mg/L using fed-batch fermentation in a 5-L bioreactor. We anticipate our systematic metabolic engineering strategies to facilitate the development of P. pastoris cell factories for sustainable production of MIAs and other plant natural products.

EHHADH deficiency regulates pexophagy and accelerates tubulointerstitial injury in diabetic kidney disease

Peroxisomal l-bifunctional enzyme (EHHADH) plays a role in the classic peroxisomal fatty acid β-oxidation pathway; however, the relationship between EHHADH expression and diabetic kidney disease has not been well understood. Here, we found that endogenous EHHADH levels were strongly correlated with the progression and severity of diabetic nephropathy in T2D patients. EHHADH knockout mice exhibited worsened renal tubular injury in diabetic mice. Furthermore, EHHADH is a modulator of pexophagy. In renal tubular epithelial cells (RTECs) in vitro, the knockdown of EHHADH induced a dramatic loss of peroxisomes. The loss of peroxisomes in EHHADH-deficient RTECs was restored by either an autophagic inhibitor 3-methyladenine or bafilomycin A1 both in vitro and in vivo. NBR1 was required for pexophagy in EHHADH-knockdown cells, where the level of reactive oxygen species (ROS) was increased, while inhibition of ROS blocked pexophagy. In summary, our findings revealed EHHADH deficiency accelerated renal injury in DKD as a modulator of pexophagy.

Molecular mechanism of delayed development by interfering RNA targeting the phenylalanine ammonia lyase gene (pal1) in Pleurotus ostreatus

The blockage of the development of edible mushrooms will affect the production cycle and yield of fruiting bodies. Phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is an enzyme that catalyses the deamination of phenylalanine to form trans-cinnamic acid. Previous results have shown that a decrease in pal1 gene transcription delays fruiting body development in Pleurotus ostreatus. Herein, we used wild type (WT) and RNA interference (RNAi) strains to study the molecular regulation of pal1 by RNA sequencing and agrobacterium-mediated genetic transformation. Our results showed that interference with the pal1 gene resulted in a decrease in the total PAL enzyme activity and the total phenol content, as well as an increase in the intracellular H2O2 content. RNA-Seq data demonstrated that the significantly enrichment KEGG terms were mainly related to the peroxisome pathway, MAPK signalling pathway-yeast and three other pathways, moreover, the catalase gene cat1 involved in multiple pathways that were enriched above. Exogenous H2O2 significantly enhanced the transcription of the cat1 gene and elevated total CAT enzyme activity. Moreover, the levels of cat1 gene transcription and the total CAT enzyme activity in the RNAi-pal1 strains were gradually closed to those in the WT strain through the removal of H2O2, which indicated that pal1 regulated the expression of cat1 by affecting the intracellular H2O2 content. Finally, the overexpression of the cat1 gene in P. ostreatus caused growth retardation, especially during the process of primordia formation. In conclusion, this study demonstrated that PAL1 affects cat1 gene expression through the signalling molecule H2O2 and regulates the development of P. ostreatus. Our findings have enhanced the understanding of the molecular developmental mechanism of edible mushrooms.

Transcriptome RNA-Seq Analysis of Normal and Keratoconus Corneal Epithelium

Background: Keratoconus (KTCN) is a progressive eye condition characterised by thin, misshapen corneas arising in the teens or early twenties, and can cause visual disability. While this can be corrected with glasses or contact lenses, collagen cross-linking of the cornea slows disease progression. In advanced disease, patients undergo transplantation of the corneal epithelial layer with normal donor tissue. The cause of KTCN is unknown, but there is evidence for both environmental and genetic bases. Material and Methods: RNA-Seq data from published work by Kabza et al., 2017, was used to investigate the pathology of cornea tissue, analysing twenty-five KTCN and twenty-five non-KTCN samples from RNA-seq experiments. Principal component analysis plots were used to identify how characteristics impact development of keratoconus. Results: The findings show that KTCN is more common in teenagers and young adults, and all 25 KTCN samples were for individuals under 30 years old. Principal component analysis also showed that approximately 35% of samples were from individuals who rubbed their eyes. It also showed that KTCN is more common in males, representing over 65% of samples. Differential expression analysis of RNA-sequencing data was used to identify differentially expressed genes, downregulated genes were found to be enriched in many pathways, including cytokinecytokine receptor interaction pathways, and played a role in keratoconus development. Conclusion: Upregulated genes were enhanced in six pathways, including the peroxisome pathway. A potential relation was found between defects in peroxisome and keratoconus development. Overexpression of peroxisome genes increases phytanic acid levels, and causes Zellweger spectrum, affecting the cornea and potentially leading to keratoconus.

Transcriptome and physiological analyses reveal the response of Arabidopsis thaliana to poly(aspartic acid)

Poly(aspartic acid) (PASP) is an environmentally friendly biopolymer used as a fertilizer synergist and known to increase agricultural yields. The mechanism of PASP enhancement has, however, not been established, although the general hypothesis is that the polymer functions to hold nutrients closer to the root zone. The objective of this study was to determine the physiological and molecular changes that occur when plants are exposed to PASP, with future directions leading to a proposed mode of action. A whole genome transcriptome study was conducted. Arabidopsis thaliana seeds were germinated and grown in sterile plates treated with 250 ppm PASP under continuous light. Total RNA was extracted from whole seedlings and sequenced. The results revealed 462 differentially expressed genes (DEGs), 245 of which were upregulated and 217 downregulated. Gene Ontology, KEGG and MAPman analyses revealed DEGs involved in photosynthesis with 11 light harvesting complexes upregulated (e.g. LHCB1.1, LHCB2.2, LHCA1, LHCB4.2, LHCB2.1, LHCA4, LHCB1.1, LHCB3, LHCA3); the peroxisome pathway had 6 DEGs (CAT1, KAT1 and XDH2) upregulated (CSD1, CSD2 and FSD2) downregulated, the phenylpropanoid biosynthesis pathway had 7 DEGs upregulated. Other key DEGs were associated with the amino acid (e.g. ASN1) and nitrogen metabolism pathways. Physiology assessment results showed significant differences between control and treated plants with a 33 % increase in leaf area, 25 % increase chlorophyll content (p ≤ 0.05) and a 4-fold increase in photosynthetic rate (p ≤ 0.001). This information helps to increase our understanding of the key genes and metabolic pathways associated with plant response to PASP.

Discovering predisposing genes for hereditary breast cancer using deep learning.

Breast cancer (BC) is the most common malignancy affecting Western women today. It is estimated that as many as 10% of BC cases can be attributed to germline variants. However, the genetic basis of the majority of familial BC cases has yet to be identified. Discovering predisposing genes contributing to familial BC is challenging due to their presumed rarity, low penetrance, and complex biological mechanisms. Here, we focused on an analysis of rare missense variants in a cohort of 12 families of Middle Eastern origins characterized by a high incidence of BC cases. We devised a novel, high-throughput, variant analysis pipeline adapted for family studies, which aims to analyze variants at the protein level by employing state-of-the-art machine learning models and three-dimensional protein structural analysis. Using our pipeline, we analyzed 1218 rare missense variants that are shared between affected family members and classified 80 genes as candidate pathogenic. Among these genes, we found significant functional enrichment in peroxisomal and mitochondrial biological pathways which segregated across seven families in the study and covered diverse ethnic groups. We present multiple evidence that peroxisomal and mitochondrial pathways play an important, yet underappreciated, role in both germline BC predisposition and BC survival.

Transcriptome analysis of antioxidant system response in Styrax tonkinensis seedlings under flood-drought abrupt alternation

BackgroundStyrax tonkinensis (Pierre) Craib ex Hartwich faces challenges in expanding in the south provinces of Yangtze River region due to climate extremes like flood-drought abrupt alternation (FDAA) caused by global warming. Low tolerance to waterlogging and drought restricts its growth in this area. To study its antioxidant system and molecular response related to the peroxisome pathway under FDAA, we conducted experiments on two-year-old seedlings, measuring growth indexes, reactive oxygen species content, antioxidant enzyme activity, and analyzing transcriptomes under FDAA and drought (DT) conditions.ResultsThe physiological results indicated a reduction in water content in roots, stems, and leaves under FDAA conditions. The most significant water loss, amounting to 15.53% was observed in the leaves. Also, ROS accumulation was predominantly observed in leaves rather than roots. Through transcriptome analysis, we assembled a total of 1,111,088 unigenes (with a total length of 1,111,628,179 bp). Generally, SOD1 and CAT genes in S. tonkinensis seedlings were up-regulated to scavenge ROS. Conversely, the MPV17 gene exhibited contrasting reaction with up-regulation in leaves and down-regulation in roots, leading to increased ROS accumulation in leaves. CHS and F3H were down-regulated, which did not play an essential role in scavenging ROS. Moreover, the down-regulation of PYL, CPK and CALM genes in leaves may not contribute to stomatal closure, thereby causing continuous water loss through transpiration. Whereas, the decreased root vigor during the waterlogging phase and up-regulated CPK and CALM in roots posed obstacles to water absorption by roots. Additionally, the DEGs related to energy metabolism, including LHCA and LHCB, were negatively regulated.ConclusionsThe ROS generation triggered by MPV17 genes was not the main reason for the eventual mortality of the plant. Instead, plant mortality may be attributed to water loss during the waterlogging phase, decreased root water uptake capacity, and continued water loss during the subsequent drought period. This study establishes a scientific foundation for comprehending the morphological, physiological, and molecular facts of S. tonkinensis under FDAA conditions.

Investigation of the Alternations in the Muscle Quality of Swimming Crab (Ovalipes punctatus) during Cold-Chain Transportation Using Physicochemical and TMT-Based Quantitative Proteomic Analysis.

Physicochemical properties and protein alterations in Ovalipes punctatus during cold-chain transportation were examined via sensory scores, water-holding capacity (WHC), glucose (GLU) content, catalase (CAT) activity, urea nitrogen (UN) content, and tandem mass tag (TMT)-based proteomic analysis. The results revealed that sensory characteristics and texture of crab muscle deteriorated during transportation. Proteomic analysis revealed 442 and 470 different expressed proteins (DEPs) in crabs after 18 h (FC) and 36 h (DC) of transportation compared with live crabs (LC). Proteins related to muscle structure and amino acid metabolism significantly changed, as evidenced by the decreased WHC and sensory scores of crab muscle. Glycolysis, calcium signaling, and peroxisome pathways were upregulated in the FC/LC comparison, aligning with the changes in GLU content and CAT activity, revealing the stress response of energy metabolism and immune response in crabs during 0-18 h of transportation. The downregulated tricarboxylic acid (TCA) cycle and carcinogenesis-reactive oxygen species pathways were correlated with the decreasing trend in CAT activity, suggesting a gradual retardation in both energy and antioxidant metabolism in crabs during 18-36 h of transportation. Furthermore, the regulated purine nucleoside metabolic and nucleoside diphosphate-related processes, with the increasing changes in UN content, revealed the accumulation of metabolites in crabs.

Engineering Yeast Peroxisomes for α-Bisabolene Production from Sole Methanol with the Aid of Proteomic Analysis.

Microbial metabolic engineering provides a feasible approach to sustainably produce advanced biofuels and biochemicals from renewable feedstocks. Methanol is an ideal feedstock since it can be massively produced from CO2 through green energy, such as solar energy. However, engineering microbes to transform methanol and overproduce chemicals is challenging. Notably, the microbial production of isoprenoids from methanol is still rarely reported. Here, we extensively engineered Pichia pastoris (syn. Komagataella phaffii) for the overproduction of sesquiterpene α-bisabolene from sole methanol by optimizing the mevalonate pathway and peroxisomal compartmentalization. Furthermore, through label-free quantification (LFQ) proteomic analysis of the engineered strains, we identified the key bottlenecks in the peroxisomal targeting pathway, and overexpressing the limiting enzyme EfmvaE significantly improved α-bisabolene production to 212 mg/L with the peroxisomal pathway. The engineered strain LH122 with the optimized peroxisomal pathway produced 1.1 g/L α-bisabolene under fed-batch fermentation in shake flasks, achieving a 69% increase over that of the cytosolic pathway. This study provides a viable approach for overproducing isoprenoid from sole methanol in engineered yeast cell factories and shows that proteomic analysis can help optimize the organelle compartmentalized pathways to enhance chemical production.

Physiological, anatomical and transcriptome analyses reveal ‘Huaizhi’ as widely compatible rootstock in Litchi chinensis Sonn. grafting

Litchi chinensis Sonn. cv. ‘Huaizhi’ (HZ) is an ancient cultivar of litchi. It has been used as widely compatible rootstock for litchi grafting for a long time. However, the mechanism of HZ as widely compatible rootstock is still unclear. In the present study, physiology, morphological anatomy, as well as transcriptomics were conducted to reveal the reasons for HZ with wide compatibility in litchi grafting. Field observation were conducted on the grafting combinations of HZ/HZ (homografting as the control), FZX/HZ and MGL/HZ (the scion with good grafting compatibility in production), SYH/HZ, HHDHL/HZ and WHL/HZ (the scion with weak grafting compatibility in production), embodying that HZ as rootstock did show a wide compatibility. The physiological and biochemical indicators of scion new shoot leaves and scion mother tree indicated that nutrients contents (dry matter weight, total soluble sugar, and starch), mineral nutrition (N and P) levels, and antioxidant enzyme (superoxide dismutase and peroxidase) activities, might be related to the wide compatibility of HZ as rootstock. Microscopic observation of the paraffin sections of healing sites at different stages showed that the necrotic layer of three representative litchi grafting combinations of HZ/HZ, WHL/HZ and MGL/HZ basically disappeared from 14 d to 21 d after grafting, and the callus differentiated into a large number of protoxylem cells, indicating a compatible healing dynamics. Subsequently, a total of 14,384 differentially expressed genes (DEGs) were identified by RNA-seq, of which 2,831 DEGs exhibited the same expression trend within three grafting combinations, enriching into phenylpropanoid biosynthesis, plant hormone biosynthesis and signal transduction, sucrose and starch metabolism, MAPK signaling pathway, peroxisome and other metabolic pathways. These results provide a theoretical basis for the adjustment of cultivar structure in litchi production.

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 β-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.