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The grape hyacinth is renowned for its profuse blue flowers, which confer substantial scientific and ornamental significance as well as considerable potential for industrial applications. The serine carboxypeptidase-like acyltransferases (SCPL-ATs) family is crucial for the blue flower coloration. To elucidate SCPL-ATs involved in anthocyanin modification in grape hyacinth, we performed a transcriptomic analysis of grape hyacinth SCPL-ATs. Through gene expression profiling, we identified a promising candidate gene, MaSCPL1, whose expression patterns corresponded with variations in anthocyanin content throughout petal coloration. Subsequently, the functional role of the MaSCPL1 gene was validated using the native petal regeneration system, and the silencing of MaSCPL1 led to a decreased total anthocyanin content and Dp3MG content in grape hyacinth petals. Furthermore, we employed yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase assays to explore the regulatory interactions between the anthocyanin biosynthesis transcription factor MaMybA and the MaSCPL1 promoter. Our findings indicate that MaMybA can bind to the MaSCPL1 promoter and significantly activate its expression. Furthermore, the MaMybA-RNAi resulted in a substantial multifold reduction in the expression of MaSCPL1, implying that the regulation of MaSCPL1 expression is mediated by MaMybA. This study revealed the MaSCPL1 gene has been associated with anthocyanin acylated modification in grape hyacinth and elucidated the important role of the MaMybA-MaSCPL1 module in colouration grape hyacinth., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Plant chemical diversity is largely owing to a number of enzymes which catalyse reactions involved in the assembly, and in the subsequent chemical modifications, of the core structures of major classes of plant specialized metabolites. One such reaction is acylation. With this in mind, to study the deep evolutionary history of BAHD and the serine-carboxypeptidase-like (SCPL) acyltransferase genes, we assembled phylogenomic synteny networks based on a large-scale inference analysis of orthologues across whole-genome sequences of 126 species spanning Stramenopiles and Archaeplastida, including Arabidopsis thaliana , tomato ( Solanum lycopersicum ) and maize ( Zea mays ). As such, this study combined the study of genomic location with changes in gene sequences. Our analyses revealed that serine-carboxypeptidase (SCP)/serine-carboxypeptidase-like (SCPL) genes had a deeper evolutionary origin than BAHD genes, which expanded massively on the transition to land and with the development of the vascular system. The two gene families additionally display quite distinct patterns of copy number variation across phylogenies as well as differences in cross-phylogenetic syntenic network components. In unlocking the above observations, our analyses demonstrate the possibilities afforded by modern phylogenomic (syntenic) networks, but also highlight their current limitations, as demonstrated by the inability of phylogenetic methods to separate authentic SCPL acyltransferases from standard SCP peptide hydrolases.This article is part of the theme issue 'The evolution of plant metabolism'.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is an immune checkpoint that has critical roles in immune exhaustion. However, little is known about the mechanisms that regulate TIM-3 surface expression and turnover. Here, we report that human TIM-3 is palmitoylated by the palmitoyltransferase DHHC9 at residue cysteine 296 (Cys 296 ). Palmitoylation stabilized TIM-3 by preventing binding to E3 ubiquitin ligase HRD1, thereby suppressing its polyubiquitination and degradation. DHHC9 knockdown attenuated chimeric antigen receptor T (CAR-T) cell exhaustion, and a peptidic inhibitor of TIM-3 palmitoylation accelerated TIM-3 degradation and enhanced antitumor immunity mediated by CAR-T cells and natural killer (NK) cells. In hepatocellular carcinoma, DHHC9 expression correlated with TIM-3 expression in CD8 + T cells and NK cells, and high DHHC9 expression was associated with shorter survival in patients with high TIM-3. These findings demonstrate that palmitoylation of TIM-3 catalyzed by DHHC9 promotes its stability, resulting in immune exhaustion and impaired antitumor immunity.

Background: Cellular carcinogenesis is often marked by the accumulation of lipid droplets (LDs) due to reprogrammed lipid metabolism. LDs are dynamic organelles that primarily store intracellular triacylglycerol (TAG) and cholesteryl esters (CEs). Transmembrane protein 68 (TMEM68), a potential modifier of human breast cancer risk and outcomes, functions as a diacylglycerol acyltransferase, synthesizing TAG. However, the specific roles of TMEM68 in breast cancer cells remain unclear., Methods: Gene expression profiling interactive analysis and survival analysis were conducted. TMEM68 was overexpressed or knockdown in breast cancer cells to assess its impact on cell proliferation, migration and invasion. Targeted quantitative lipidomic analysis and quantitative polymerase chain reaction were used to profile lipid alterations and examine gene expression related to lipid metabolism following changes in TMEM68 levels., Results: TMEM68 gene was upregulated in breast cancer patients and higher TMEM68 levels were associated with poorer survival outcomes. Overexpression of TMEM68 increased breast cancer cell proliferation and invasion, whereas knockdown had minimal or no impact on reducing proliferation and invasion. Altering TMEM68 levels resulted in corresponding changes in TAG levels and cytoplasmic LDs, with overexpression increasing both and knockdown decreasing them. Lipidomic analysis revealed that TMEM68 regulated TAG levels and altered diacylglycerol content in breast cancer cells. Additionally, TMEM68 influenced the metabolism of glycerophospholipids, CEs and acylcarnitines. TMEM68 also modified the expression of key genes encoding enzymes related to neutral lipid metabolism, including TAG and CEs., Conclusions: TMEM68 is highly expressed in breast cancer and negatively correlated with survival. Its overexpression promotes breast cancer cell proliferation while knockdown has varied effects depending on TMEM68 levels. TMEM68 regulates intracellular TAG and LDs contents along with alterations in glycerophospholipids. These findings suggest that TMEM68 may drive breast cancer cells proliferation by modulating TAG and LD content., Competing Interests: Declarations Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: H56:IC31 is a candidate vaccine against tuberculosis (TB) with the potential to reduce TB recurrence rate. It is thus important for future clinical trials to demonstrate safety and immunogenicity of H56:IC31 in individuals treated for TB., Methods: Twenty-two adults confirmed to be Mycobacterium tuberculosis negative (by 2 GeneXpert tests or 2 sputum cultures) after 4-5 months of TB treatment, and not more than 28 days after completion of TB treatment, were randomized to receive 2 doses of H56:IC31 (5 mg H56:500 nmol IC31; n = 16) or placebo (n = 6) 56 days apart. Participants were followed for 420 days for safety and immunogenicity., Results: H56:IC31 vaccination was associated with an acceptable safety profile, consisting mostly of mild self-limited injection site reactions. No serious adverse events or vaccine-related severe adverse events were reported. H56:IC31 induced a CD4+ T-cell response for Ag85B and ESAT-6, with ESAT-6 being immunodominant, which persisted through 6 months after the last vaccination. There was some evidence of CD8+ T-cell responses for both Ag85B and ESAT-6, but to a lesser extent than CD4+ responses., Conclusions: H56:IC31 was associated with an acceptable safety profile, and induced a predominant CD4+ T-cell response, in adults recently treated for drug-susceptible, uncomplicated pulmonary TB., Clinical Trials Registration: NCT02375698., Competing Interests: Potential conflicts of interest. P. L. A., I. K., M. R., and A. H. B. were employed at SSI, the developer of H56:IC31. P. L. A. is the inventor of patents related to H56:IC31. All rights have been assigned to SSI, a Danish not-for-profit institute under the Ministry of Health. D. T. was employed by Aeras (now IAVI) during the conduct of this study and is now an independent consultant. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Members of the Bacteroidota compose a large portion of the human gut microbiota, contributing to overall gut health via the degradation of various polysaccharides. This process is facilitated by lipoproteins, globular proteins anchored to the cell surface by a lipidated N-terminal cysteine. Despite their importance, lipoprotein synthesis by these bacteria is understudied. In Escherichia coli , the α-amino-linked lipid of lipoproteins is added by the l ipoprotein N -acyl t ransferase Lnt. Herein, we have identified a protein distinct from Lnt responsible for the same process in Bacteroides , named l ipoprotein N -acyltransferase in B acteroides (Lnb). Deletion of Lnb yields cells that synthesize diacylated lipoproteins, with impacts on cell viability and morphology, growth on polysaccharides, and protein composition of membranes and outer membrane vesicles (OMVs). Our results not only challenge the accepted paradigms of lipoprotein biosynthesis in gram-negative bacteria but also suggest the existence of a new family of lipoprotein N -acyltransferases., Competing Interests: Competing interests statement:The authors declare no competing interest.

Barth syndrome (BTHS) is one of the rare X linked recessive diseases that appear in infancy with a triad of myocardial and skeletal muscle diseases, neutropenia and growth retardation. The pathogenic variant of TAFAZZIN gene leads to BTHS, which encodes the TAFAZZIN protein of the inner membrane of the mitochondria, a phosphatidyltransferase involved in cardiolipin remodelling and functional maturation. We present a case of a neonate presenting with early-onset cardiomyopathy, neutropenia and failure to thrive with no family history of cardiac diseases. Echocardiography suggested a dilated left ventricle with non-compaction and a low ejection fraction. The baby was managed with diuretics and decongestive measures. Clinical exome sequencing detected a hemizygous novel splice site variant c.541+2 T>C in TAFAZZIN , confirming the diagnosis of BTHS., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Biocatalysis can be powerful in organic synthesis but is often limited by enzymes' substrate scope and selectivity. Developing a biocatalytic step involves identifying an initial enzyme for the target reaction followed by optimization through rational design, directed evolution, or both. These steps are time consuming, resource-intensive, and require expertise beyond typical organic chemistry. Thus, an effective strategy for streamlining the process from enzyme identification to implementation is essential to expanding biocatalysis. Here, we present a strategy combining bioinformatics-guided enzyme mining and ancestral sequence reconstruction (ASR) to resurrect enzymes for biocatalytic synthesis. Specifically, we achieve an enantioselective synthesis of azaphilone natural products using two ancestral enzymes: a flavin-dependent monooxygenase (FDMO) for stereodivergent oxidative dearomatization and a substrate-selective acyltransferase (AT) for the acylation of the enzymatically installed hydroxyl group. This cascade, stereocomplementary to established chemoenzymatic routes, expands access to enantiomeric linear tricyclic azaphilones. By leveraging the co-occurrence and coevolution of FDMO and AT in azaphilone biosynthetic pathways, we identified an AT candidate, CazE, and addressed its low solubility and stability through ASR, obtaining a more soluble, stable, promiscuous, and reactive ancestral AT (AncAT). Sequence analysis revealed AncAT as a chimeric composition of its descendants with enhanced reactivity likely due to ancestral promiscuity. Flexible receptor docking and molecular dynamics simulations showed that the most reactive AncAT promotes a reactive geometry between substrates. We anticipate that our bioinformatics-guided, ASR-based approach can be broadly applied in target-oriented synthesis, reducing the time required to develop biocatalytic steps and efficiently access superior biocatalysts.

Endophilin A2, the sole endophilin A family member expressed in hematopoietic cells, regulates various aspects of membrane dynamics, including autophagy and endocytosis. Recent studies in rodents highlight the essential role of endophilin A2 in modulating immune responses. Here we report a homozygous frameshift variant in the SH3GL1 gene (NM_003025.3:c.427delC; p.Leu143Serfs*9), detected by whole exome sequencing in a 14-year-old boy with predominantly antibody deficiency. The patient who is issued from a consanguineous Lebanese family, presents since the age of 18 months with recurrent respiratory tract infections, low peripheral B cell counts and pan-hypogammaglobulinemia, with no history of opportunistic infections. This defect is associated with decrease in switched memory B cells development, impaired in-vitro B cell proliferation and diminished in-vitro IgG production. The detected variant in SH3GL1 segregates with the disease in the family. It significantly decreases the expression of the protein in the patient's peripheral blood compared to healthy controls, thus confirming its pathogenicity. Interestingly, endophilin A2-deficient Sh3gl1 -/- mice have been reported to present defects in germinal center B cell responses and in the production of high-affinity IgG. Our data suggests that endophilin A2 deficiency impairs antibody production in humans. Reporting further cases with mutations in SH3GL1 is needed to better characterize the inborn error of immunity linked to this gene., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Glycine N-acyltransferase (GLYAT), known to influence glycine metabolism, has been implicated in the progression of various malignant tumours. However, its clinical relevance in hepatocellular carcinoma (HCC) remains unexplored. Here, GLYAT expression levels in HCC tissues were significantly reduced compared to normal liver tissues. Similarly, GLYAT expression levels in Huh 7, HepG2, PLC and SK-HEP1 were lower than those in LO2. Receiver operating characteristic curve analysis demonstrated that GLYAT exhibited good diagnostic performance for HCC. Kaplan-Meier analyses suggested that decreased GLYAT expression was correlated with poorer progress in HCC. Low GLYAT expression was significantly associated with gender and histologic grade. Multivariate Cox regression analysis identified low GLYAT expression and T stage as independent prognostic factors. Nomograms based on GLYAT mRNA expression and T stage showed good concordance with actual survival rates at 1, 2, 3 and 5 years. Moreover, GLYAT downregulation in the Huh 7 cell line enhanced cell proliferation, invasion and migration abilities, while GLYAT overexpression in the HepG2 cell line inhibited these abilities. HCC patients with low GLYAT expression exhibited a predisposition to immune escape and poor response to immunotherapy. This research revealed that GLYAT holds promise as both a prognostic biomarker and a potential therapeutic target in HCC., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Exposure to ambient air pollutants has emerged as a risk for metabolic-dysfunction associated steatotic liver disease (MASLD)., Objectives: We sought to examine associations between short-term (prior month) and long-term (prior year) ambient air pollution exposure with hepatic fat fraction (HFF) and liver stiffness in Latino youth with obesity. A secondary aim was to investigate effect modification by patatin-like phospholipase domain-containing protein 3 (PNPLA3) genotype and liver disease severity., Methods: Data was analyzed from 113 Latino youth (age 11-19) with obesity in Southern California. Individual exposure to particulate matter with aerodynamic diameter ≤ 2.5μm (PM 2.5 ), ≤ 10μm (PM 10 ), nitrogen dioxide (NO 2 ), 8-hour maximum ozone (8hrMax-O 3 ), 24-hr O 3 , and redox-weighted oxidative capacity (O x wt ) were estimated using residential address histories and United States Environmental Protection Agency air quality observations. HFF and liver stiffness were measured using magnetic resonance imaging. Linear models were used to determine associations between short-term and long-term exposure to air pollutants with HFF and liver stiffness. Modification by PNPLA3 and liver disease severity was then examined., Results: Short-term exposure to 8hrMax-O 3 was positively associated with HFF. Relationships between air pollution exposure and HFF were not impacted by PNPLA3 genotype or liver disease severity. Long-term exposure to 8hrMax-O 3 and O x wt were positively associated with liver stiffness. Associations between air pollution exposure and liver stiffness depended on PNPLA3 genotype, such that individuals with GG genotypes exhibited stronger, more positive relationships between short-term exposure to PM 10 , 8hrMax-O 3 , 24-hr O 3, and O x wt and liver stiffness than individuals with CC/CG genotypes. In addition, relationships between short-term exposure to NO 2 and liver stiffness were stronger in those with severe liver disease., Discussion: Air pollution exposure may be a risk factor for liver disease among Latino youth with obesity, particularly in those with other preexisting risks for liver damage., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rohit Kohli reports a relationship with Mirum Biopharma that includes: consulting or advisory. Rohit Kohli reports a relationship with Ipsen that includes: consulting or advisory. Michael Goran reports a relationship with YUMI foods that includes: consulting or advisory. Michael Goran reports a relationship with Bobbie Labs that includes: consulting or advisory. Michael Goran receives royalties from Penguin Random House for Sugarproof If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Focal dermal hypoplasia (FDH), also known as Goltz syndrome, is a rare ectodermal dysplasia that primarily affects the skin, skeleton, and eyes. It is an X-linked dominant disorder, predominantly seen in females, caused by pathogenic variants in PORCN., Methods: We characterized a case series of four genetically confirmed FDH patients (three females, one male) at Aarhus University Hospital, Denmark. We estimated the FDH prevalence from our local cohort and nationwide registry data., Results: Three patients had characteristic dermatological findings suspicious for FDH and confirmed by targeted PORCN analysis. One patient had an atypical presentation with several malformations but only subtle skin changes and was diagnosed following trio exome-sequencing analysis. Skin atrophy with fat herniations and telangiectasias were typical cutaneous findings. Limb malformations included oligodactyly (cleft foot), syndactyly, and polydactyly. Eye abnormalities included coloboma and microphthalmos. Facial dysmorphology was defined by asymmetry, thin upper lip, and malformed ears. One patient developed a giant cell bone tumor, which is a rare feature of FDH. Dental findings included enamel hypoplasia with vertical grooving and irregular crowns. Four PORCN variants were identified, including three not previously reported in the literature.We estimated a regional point prevalence in Western Denmark of 1.6 cases per million population (95% confidence intervals (CI): 0.7-3.7 per million) and a nationwide registry-based point prevalence of 1.2 cases per million population (95% CI: 0.6-2.4 per million)., Conclusions: FDH is an extremely rare and complex multisystem disorder of variable presentation, which requires close multidisciplinary collaboration for diagnosis and patient care., (© 2024 The Author(s). Pediatric Dermatology published by Wiley Periodicals LLC.)

We report the development of a metabolically engineered bacterium for the fermentative production of polyesters containing aromatic side chains, serving as sustainable alternatives to petroleum-based plastics. A metabolic pathway was constructed in an Escherichia coli strain to produce poly[d-phenyllactate(PhLA)], followed by three strategies to enhance polymer production. First, polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were introduced to increase the polymer accumulation. Next, metabolic engineering was performed to redirect the metabolic flux toward PhLA. Furthermore, PHA synthase was engineered based on in silico simulation results to enhance the polymerization of PhLA. The final strain was capable of producing 12.3 g/l of poly(PhLA), marking it the first bio-based process for producing an aromatic homopolyester. Additional heterologous gene introductions led to the high level production of poly(3-hydroxybutyrate-co-11.7 mol% PhLA) copolymer (61.4 g/l). The strategies described here will be useful for the bio-based production of aromatic polyesters from renewable resources., Competing Interests: Declaration of interests S.Y.L and J.E.Y. declare that they have competing financial interests because the HadA enzyme described in this paper is of commercial interest and is patent filed including, but not limited to, KR1020368280000, US10961521, and JP6854909. The remaining authors have no interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Glycosylphosphatidylinositol (GPI) acyltransferase is crucial for the synthesis of GPI-anchored proteins. Targeting the fungal glycosylphosphatidylinositol acyltransferase GWT1 by manogepix is a promising antifungal strategy. However, the inhibitory mechanism of manogepix remains unclear. Here, we present cryo-EM structures of yeast GWT1 bound to the substrate (palmitoyl-CoA) and inhibitor (manogepix) at 3.3 Å and 3.5 Å, respectively. GWT1 adopts a unique fold with 13 transmembrane (TM) helixes. The palmitoyl-CoA inserts into the chamber among TM4, 5, 6, 7, and 12. The crucial residues (D145 and K155) located on the loop between TM4 and TM5 potentially bind to the GPI precursor, contributing to substrate recognition and catalysis, respectively. The antifungal drug, manogepix, occupies the hydrophobic cavity of the palmitoyl-CoA binding site, suggesting a competitive inhibitory mechanism. Structural analysis of resistance mutations elucidates the drug specificity and selectivity. These findings pave the way for the development of potent and selective antifungal drugs targeting GWT1., (© 2024. The Author(s).)

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles.IMPORTANCEIn this study, we systematically examined the biochemistry of palmitoylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) E protein and demonstrated that palmitoylation of SARS-CoV-2 E protein is required for virus-like particle (VLP) production and maintaining normal particle density. These results suggest that palmitoylated E protein is central for proper morphogenesis of SARS-CoV-2 VLPs in densities required for viral infectivity. This study presents a significant advancement in the understanding of how palmitoylation of viral proteins is vital for assembling SARS-CoV-2 particles and supports that palmitoyl acyltransferases can be potential therapeutic targets for the development of SARS-CoV-2 inhibitors., Competing Interests: The authors declare no conflict of interest.

The bioconversion of low value-added phytosterols into high value-added 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) in Mycolicibacterium neoaurum is a representative step in the steroid pharmaceutical industry. However, the complex mycobacterial cell walls with extremely low permeability and flowability greatly decrease the overall conversion efficiency. Herein, we preliminarily identified two key acyltransferases encoded by Mn_TmaT and Mn_SucT required for the proper synthesis of cell wall in mycobacteria and achieved a significant increase in cell permeability by disrupting them without affecting the cell wall structural stability. At length, the destruction of Mn_TmaT and Mn_SucT alone increased the conversion rate of 9-OHAD from 45.3% (6.67 ± 0.39 g/L) to 62.4% (9.19 ± 0.58 g/L) and 67.9% (10.02 ± 0.62 g/L) while the continuous destruction of Mn_TmaT and Mn_SucT did not further improve the conversion efficiency of 9-OHAD. Notably, it was investigated that the continuous destruction of Mn_TmaT and Mn_SucT led to alterations in both the covalent and non-covalent binding layers of the cell wall, resulting in excessive changes in cell morphology and structure, which ultimately decreased 9-OHAD production. Therefore, this study deciphered a pivotal biosynthetic path of cell wall and provided an efficient and feasible construction strategy of 9-OHAD synthesis in mycobacteria., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Key Message: The N-terminal transmembrane domain of LPAT1 crosses the inner membrane placing the N terminus in the intermembrane space and the C-terminal enzymatic domain in the stroma. Galactolipids mono- and di-galactosyl diacylglycerol are the major and vital lipids of photosynthetic membranes. They are synthesized by five enzymes hosted at different sub-chloroplast locations. However, localization and topology of the second-acting enzyme, lysophosphatidic acid acyltransferase 1 (LPAT1), which acylates the sn-2 position of glycerol-3-phosphate (G3P) to produce phosphatidic acid (PA), remain unclear. It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. We show that LPAT1 traverses the inner membrane via an N-terminal transmembrane domain, with its N terminus protruding into the intermembrane space and the C-terminal enzymatic domain residing in the stroma, hence displaying a different membrane topology from its bacterial homolog, PlsC., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of Mycobacterium tuberculosis , the bacterial organism causing tuberculosis, catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of M. tuberculosis Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 Å. We observed a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predicted to contain a dimeric domain similar to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of M. smegmatis Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, which combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.

The Chinese gentian, Gentiana sino-ornata produces brilliant blue flowers. To investigate the biological function and transcriptional regulation mechanism of the anthocyanin 5-O-acyltransferase gene (Gs5AT ) in the corolla, it is beneficial to analyse the mechanism of blue flower colour presentation. In this investigation, we obtained the CDS and promoter sequences of the gene Gs5AT . Yeast one-hybrid experiments were used to identify the transcription factor GsbHLH7 that activates the gene Gs5AT . According to quantitive reverse transcription polymerase chain reaction analysis, the expression of the gene Gs5AT was significantly and positively correlated with the gene GsbHLH7 . The colour phenotype of the flowers was significantly altered by the virus-induced gene silencing transduction of Gs5AT and GsbHLH7 , with GsbHLH7 silencing producing more pronounced changes in the corolla colour than Gs5AT . The expression of GsF3'5'H , GsDFR , GsANS , Gs3GT , and Gs5GT all fell to varying degrees after GsbHLH7 silencing, indicating that GsbHLH7 may regulate transcription of these genes as well as Gs5AT . The results of this study indicate that Gs5AT was positively regulated by the GsbHLH7 , and thus affects the colour presentation of the blue corolla.

Autosomal recessive coding variants are well-known causes of rare disorders. We quantified the contribution of these variants to developmental disorders in a large, ancestrally diverse cohort comprising 29,745 trios, of whom 20.4% had genetically inferred non-European ancestries. The estimated fraction of patients attributable to exome-wide autosomal recessive coding variants ranged from ~2-19% across genetically inferred ancestry groups and was significantly correlated with average autozygosity. Established autosomal recessive developmental disorder-associated (ARDD) genes explained 84.0% of the total autosomal recessive coding burden, and 34.4% of the burden in these established genes was explained by variants not already reported as pathogenic in ClinVar. Statistical analyses identified two novel ARDD genes: KBTBD2 and ZDHHC16. This study expands our understanding of the genetic architecture of developmental disorders across diverse genetically inferred ancestry groups and suggests that improving strategies for interpreting missense variants in known ARDD genes may help diagnose more patients than discovering the remaining genes., (© 2024. The Author(s).)

Plasmodium vivax is a major cause of malaria, which poses an increased health burden on approximately one third of the world's population due to climate change. Primaquine, the preferred treatment for P. vivax malaria, is contraindicated in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common genetic cause of hemolytic anemia, that affects ∼2.5% of the world's population and ∼8% of the population in areas of the world where P. vivax malaria is endemic. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) conducted a structure-function analysis of P. vivax N-myristoyltransferase (PvNMT) as part of efforts to develop alternative malaria drugs. PvNMT catalyzes the attachment of myristate to the N-terminal glycine of many proteins, and this critical post-translational modification is required for the survival of P. vivax. The first step is the formation of a PvNMT-myristoyl-CoA binary complex that can bind to peptides. Understanding how inhibitors prevent protein binding will facilitate the development of PvNMT as a viable drug target. NMTs are secreted in all life stages of malarial parasites, making them attractive targets, unlike current antimalarials that are only effective during the plasmodial erythrocytic stages. The 2.3 Å resolution crystal structure of the ternary complex of PvNMT with myristoyl-CoA and a novel inhibitor is reported. One asymmetric unit contains two monomers. The structure reveals notable differences between the PvNMT and human enzymes and similarities to other plasmodial NMTs that can be exploited to develop new antimalarials., (open access.)

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases. Cardiolipin, the signature phospholipid of the mitochondrion, affects proper cristae morphology, bioenergetic functions, and metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in tafazzin are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impacts metabolic flux through the TCA cycle and associated yeast pathways. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13 C 3 -pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a WT strain to mitochondria from a Δtaz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin and mitochondria from a Δcrd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13 C-label from the pyruvate substrate was distributed through twelve metabolites. Several of the metabolites were specific to yeast pathways including branched chain amino acids and fusel alcohol synthesis. While most metabolites showed similar kinetics among the different strains, mevalonate concentrations were significantly increased in Δtaz1 mitochondria. Additionally, the kinetic profiles of α-ketoglutarate, as well as NAD + and NADH measured in separate experiments, displayed significantly lower concentrations for Δtaz1 and Δcrd1 mitochondria at most time points. Taken together, the results show how cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides., Competing Interests: Conflicts of interest The author declares that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The detection of pathogenicity and immunogenicity in Vibrio parahaemolyticus poses a significant challenge due to its threat to human health and food safety, which is strongly correlated with lipid A. Lipid A, a critical component found in most Gram-negative bacteria, functions as a hydrophobic anchor for lipopolysaccharide. V. parahaemolyticus synthesizes multiple lipid A species with various secondary acyl chains. In this study, a secondary acyltransferase of lipid A encoded by VP_RS08405 in V. parahaemolyticus was identified. Based on sequence alignment analysis, V. parahaemolyticus VP_RS08405 has high homology to E. coli lpxL, lpxM and lpxP which encode the three secondary acyltransferases of lipid A. Therefore, V. parahaemolyticus VP_RS08405 was cloned into pBAD33, and the resulting pB08405 was introduced in E. coli mutants WHL00 in which lpxL was deleted, WHM00 in which lpxM was deleted, WHP00 in which lpxP was deleted, and WH300 in which lpxL, lpxM and lpxP were deleted. The recombinant strains WHL00/pB08405, WHM00/pB08405, WHP00/pB08405, WH300/pB08405, as well as their vector controls, were grown at normal and low temperatures. Lipid A species were isolated from the above strains and analyzed by using high-performance liquid chromatography-tandem mass spectrometry and thin-layer chromatography. After comparing the secondary acyl alterations of lipid A from different recombinant strains, it is concluded that VP_RS08405 specifically catalyzed the addition of a palmitoleate to the 2'-position of lipid A and its activity is not temperature-sensitive. In addition, to determine the dependence of VP_RS08405 on Kdo, VP_RS08405 was overexpressed in E. coli mutants WH001 in which waaA was deleted, and WH400 in which waaA, lpxL, lpxM and lpxP were deleted. Lipid A species were isolated from WH001/pB08405 and WH400/pB08405, and analyzed. The results show that the function of VP_RS08405 is Kdo-dependent. These findings provide a better understanding of the structural diversity of lipid A in V. parahaemolyticus., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms., (© 2024. The Author(s).)

The gaseous ethylene (ET) and the oxylipin-derived jasmonic acid (JA) in plants jointly regulate an arsenal of pathogen responsive genes involved in defending against necrotrophic pathogens. The APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factor ORA59 is a major positive regulator of the ET/JA-mediated defense pathway in Arabidopsis thaliana. The Arabidopsis agmatine coumaroyltransferase (AtACT) catalyzes the formation of hydroxycinnamic acid amides (HCAAs) which are effective toxic antimicrobial substances known as phytoalexins and play an important role in plant defense response. However, induction and regulation of AtACT gene expression and HCAAs synthesis in plants remain less understood. Through gene coexpression network analysis, we identified a list of GCC-box cis-element containing genes that were coexpressed with ORA59 under diverse biotic stress conditions and might be potential downstream targets of this AP2/ERF-domain transcription factor. Particularly, ORA59 directly binds to AtACT gene promoter via the GCC-boxes and activates AtACT gene expression. The ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC)-treatment significantly induces AtACT gene expression. Both ORA59 and members of the class II TGA transcription factors are indispensable for ACC-induced AtACT expression. Interestingly, the expression of AtACT is also subject to the signaling crosstalk of the salicylic acid- and ET/JA-mediated defense response pathways. In addition, we found that genes of the phenylpropanoid metabolism pathway were specifically induced by Botrytis cinerea. Taking together, these evidence suggest that the ET/JA signaling pathway activate the expression of AtACT to increase antimicrobial HCAAs production through the transcription factor ORA59 in response to the infection of necrotrophic plant pathogens., (© 2022 Wiley Periodicals LLC.)

Lecithin:retinol acyltransferase (LRAT) is the main enzyme producing retinyl esters (REs) in quiescent hepatic stellate cells (HSCs). When cultured on stiff plastic culture plates, quiescent HSCs activate and lose their RE stores in a process similar to that in the liver following tissue damage, leading to fibrosis. Here we validated HSC cultures in soft gels to study RE metabolism in stable quiescent HSCs and investigated RE synthesis and breakdown in activating HSCs. HSCs cultured in a soft gel maintained characteristics of quiescent HSCs, including the size, amount and composition of their characteristic large lipid droplets. Quiescent gel-cultured HSCs maintained high expression levels of Lrat and a RE storing phenotype with low levels of RE breakdown. Newly formed REs are highly enriched in retinyl palmitate (RP), similar to freshly isolated quiescent HSCs, which is associated with high LRAT activity. Comparison of these quiescent gel-cultured HSCs with activated plastic-cultured HSCs showed that although during early activation the total RE levels and RP-enrichment are reduced, levels of RE formation are maintained and mediated by LRAT. Loss of REs was caused by enhanced RE breakdown in activating HSCs. Upon prolonged culturing, activated HSCs have lost their LRAT activity and produce small amounts of REs by DGAT1. This study reveals unexpected dynamics in RE metabolism during early HSC activation, which might be important in liver disease as early stages are reversible. Soft gel cultures provide a promising model to study RE metabolism in quiescent HSCs, allowing detailed molecular investigations on the mechanisms for storage and release., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Solanaceae (nightshade family) species synthesize a remarkable array of clade- and tissue-specific specialized metabolites. Protective acylsugars, one such class of structurally diverse metabolites, are produced by ACYLSUGAR ACYLTRANSFERASE (ASAT) enzymes from sugars and acyl-coenzyme A esters. Published research has revealed trichome acylsugars composed of glucose and sucrose cores in species across the family. In addition, acylsugars have been analyzed across a small fraction of the >1,200 species in the phenotypically megadiverse Solanum genus, with a handful containing inositol and glycosylated inositol cores. The current study sampled several dozen species across subclades of Solanum to get a more detailed view of acylsugar chemodiversity. In depth characterization of acylsugars from the clade II species brinjal eggplant (Solanum melongena) led to the identification of eight unusual structures with inositol or inositol glycoside cores and hydroxyacyl chains. Liquid chromatography-mass spectrometry analysis of 31 additional species in the Solanum genus revealed striking acylsugar diversity, with some traits restricted to specific clades and species. Acylinositols and inositol-based acyldisaccharides were detected throughout much of the genus. In contrast, acylglucoses and acylsucroses were more restricted in distribution. Analysis of tissue-specific transcriptomes and interspecific acylsugar acetylation differences led to the identification of the brinjal eggplant ASAT 3-LIKE 1 (SmASAT3-L1; SMEL4.1_12g015780) enzyme. This enzyme is distinct from previously characterized acylsugar acetyltransferases, which are in the ASAT4 clade, and appears to be a functionally divergent ASAT3. This study provides a foundation for investigating the evolution and function of diverse Solanum acylsugar structures and harnessing this diversity in breeding and synthetic biology., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Dysregulation of energy balance leading to obesity is a significant risk factor for cardiometabolic diseases such as diabetes, non-alcoholic fatty liver disease and atherosclerosis. In rodents and several other vertebrates, feeding has been shown to induce a rapid rise in the intestinal levels of N-acyl-ethanolamines (NAEs) and the chronic consumption of a high fat diet abolishes this rise. Administering NAEs to rodents consuming a high fat diet reduces their adiposity, in part by reducing food intake and enhancing fat oxidation, so that feeding-induced intestinal NAE biosynthesis appears to be critical to appropriate regulation of energy balance. However, the contribution of feeding-induced intestinal NAE biosynthesis to appropriate energy balance remains poorly understood in part because there are multiple enzymes that can contribute to NAE biosynthesis and the specific enzyme(s) that are responsible for feeding-induced intestinal NAE biosynthesis have not been identified. The rate-limiting step in the intestinal biosynthesis of NAEs is formation of their immediate precursors, the N-acyl-phosphatidylethanolamines (NAPEs), by phosphatidylethanolamine N-acyltransferases (NATs). At least six NATs are found in humans and multiple homologs of these NATs are found in most vertebrate species. In recent years, the fecundity and small size of zebrafish (Danio rerio), as well as their similarities in feeding behavior and energy balance regulation with mammals, have led to their use to model key features of cardiometabolic disease. We therefore searched the Danio rerio genome to identify all NAT homologs and found two additional NAT homologs besides the previously reported plaat1, rarres3, and rarres3l, and used CRISPR/cas9 to delete these two NAT homologs (plaat1l1 and plaat1l2). While wild-type fish markedly increased their intestinal NAPE levels in response to a meal after fasting, this response was completely ablated in plaat1l1 -/- fish. Furthermore, plaat1l1 -/- fish fed a standard flake diet had increased weight gain and glucose intolerance compared to wild-type fish. The results support a critical role for feeding-induced NAPE and NAE biosynthesis in regulating energy balance and suggest that restoring this response in obese animals could potentially be used to treat obesity and cardiometabolic disease., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phenolamides are specialized metabolites widely distributed in the plant kingdom. Their structure is composed by the association of hydroxycinnamic acid derivatives to mono-/poly-amine through an amination catalyzed by N-hydroxycinnamoyltransferases enzymes. Tomato plants accumulate putrescine-derived phenolamides in their vegetative parts. Recently, two first genes coding for putrescine-hydroxycinnamoyltransferase (PHT, Solyc11g071470 and Solyc11g071480) were identified in tomato and demonstrated to control the leaf accumulation of caffeoylputrescine in response to leafminer infestation. In this study, two additional genes (Solyc06g074710 and Solyc11g066640) were functionally characterized as new tomato PHT. The substrate specificity and the expression pattern in planta were determined for the four tomato PHT. Taken together the results give a comprehensive view of the control of the putrescine-derived phenolamide accumulation in tomato plant through the biochemical specificity and the spatial expression of this small family of PHT., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Romain Larbat reports financial support was provided by Regional Council of Lorraine. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The downregulation of Cadm4 (Cell adhesion molecular 4) is a prominent feature in demyelination diseases, yet, the underlying molecular mechanism remains elusive. Here, we reveal that Cadm4 undergoes specific palmitoylation at cysteine-347 (C347), which is crucial for its stable localization on the plasma membrane (PM). Mutation of C347 to alanine (C347A), blocking palmitoylation, causes Cadm4 internalization from the PM and subsequent degradation. In vivo experiments introducing the C347A mutation (Cadm4-KI) lead to severe myelin abnormalities in the central nervous system (CNS), characterized by loss, demyelination, and hypermyelination. We further identify ZDHHC3 (Zinc finger DHHC-type palmitoyltransferase 3) as the enzyme responsible for catalyzing Cadm4 palmitoylation. Depletion of ZDHHC3 reduces Cadm4 palmitoylation and diminishes its PM localization. Remarkably, genetic deletion of ZDHHC3 results in decreased Cadm4 palmitoylation and defects in CNS myelination, phenocopying the Cadm4-KI mouse model. Consequently, altered Cadm4 palmitoylation impairs neuronal transmission and cognitive behaviors in both Cadm4-KI and ZDHHC3 knockout mice. Importantly, attenuated ZDHHC3-Cadm4 signaling significantly influences neuroinflammation in diverse demyelination diseases. Mechanistically, we demonstrate the predominant expression of Cadm4 in the oligodendrocyte lineage and its potential role in modulating cell differentiation via the WNT-β-Catenin pathway. Together, our findings propose that dysregulated ZDHHC3-Cadm4 signaling contributes to myelin abnormalities, suggesting a common pathological mechanism underlying demyelination diseases associated with neuroinflammation., (© 2024. The Author(s).)

Innate immunity serves as the primary defense against viral and microbial infections in humans. The precise influence of cellular metabolites, especially fatty acids, on antiviral innate immunity remains largely elusive. Here, through screening a metabolite library, palmitic acid (PA) has been identified as a key modulator of antiviral infections in human cells. Mechanistically, PA induces mitochondrial antiviral signaling protein (MAVS) palmitoylation, aggregation, and subsequent activation, thereby enhancing the innate immune response. The palmitoyl-transferase ZDHHC24 catalyzes MAVS palmitoylation, thereby boosting the TBK1-IRF3-interferon (IFN) pathway, particularly under conditions of PA stimulation or high-fat-diet-fed mouse models, leading to antiviral immune responses. Additionally, APT2 de-palmitoylates MAVS, thus inhibiting antiviral signaling, suggesting that its inhibitors, such as ML349, effectively reverse MAVS activation in response to antiviral infections. These findings underscore the critical role of PA in regulating antiviral innate immunity through MAVS palmitoylation and provide strategies for enhancing PA intake or targeting APT2 for combating viral infections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Adult mammary stem cells (aMaSCs) are vital to tissue expansion and remodeling during the process of postnatal mammary development. The protein C receptor (Procr) is one of the well-identified surface markers of multipotent aMaSCs. However, an understanding of the regulatory mechanisms governing Procr's protein stability remains incomplete. In this study, we identified Glycoprotein m6a (Gpm6a) as a critical protein for aMaSC activity modulation by using the Gpm6a knockout mouse model. Interestingly, we determined that Gpm6a depletion results in a reduction of Procr protein stability. Mechanistically, Gpm6a regulates Procr protein stability by mediating the formation of lipid rafts, a process requiring Zdhhc1 and Zdhhc2 to palmitate Gpm6a at Cys 17,18 and Cys 246 sites. Our findings highlight an important mechanism involving Zdhhc1- and Zdhhc2-mediated Gpm6a palmitoylation for the regulation of Procr stability, aMaSC activity, and postnatal mammary development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens, thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in development. One of the primary challenges associated with mucosal vaccines has been identifying and characterizing safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. We formulated a vaccine based on the H1 antigen (fusion protein of Ag85b and ESAT-6) adjuvanted with NanoSTING. Intranasal immunization of NanoSTING-H1 elicited a strong T-cell response in the lung of vaccinated animals characterized by (a) CXCR3 + KLRG1 - lung resident T cells that are known to be essential for controlling bacterial infection, (b) IFNγ-secreting CD4 + T cells which is necessary for intracellular bactericidal activity, and (c) IL17-secreting CD4 + T cells that can confer protective immunity against multiple clinically relevant strains of Mtb. Upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain, intranasal NanoSTING-H1 provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille-Calmette-Guérin (BCG). Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that protects from infection by M. tuberculosis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Navin Varadarajan reports financial support was provided by National Institutes of Health. Navin Varadarajan reports financial support was provided by AuraVax Therapeutics. Navin Varadarajan reports a relationship with AuraVax Therapeutics and CellChorus that includes: equity or stocks., (Copyright © 2024. Published by Elsevier Ltd.)

Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Rationale: Skeletal dysplasias are a complex series of rare genetic disorders that cause irregular development of bones, joints, and cartilages in children. A total of 770 disorders associated with 41 groups of skeletal dysplasia have been documented, demonstrating a wide range of clinical manifestations and varying levels of severity. In addition to conventional methods, whole genome sequencing has emerged as a useful approach to pinpointing the underlying etiology of skeletal dysplasias., Patient Concerns: A 13-month-old female was admitted to the hospital due to the symptoms of jaundice and failure to thrive., Diagnoses: The child was subjected to blood tests and a radiographic assessment. The blood chemistries revealed elevated levels of total bilirubin (178 µmol/L), bile acids (198 µmol/L), and low levels of serum calcium (1.69 mmol/L) and phosphate (0.8 mmol/L), along with irregular skeletal development in the forearms and legs, considering rickets and cholestasis., Interventions: Whole exome sequencing data of the proband revealed a homozygous mutation of c.388dupA in the BAAT (bile acid-CoA: amino acid N-acyltransferase) gene sequence. This mutation caused a frameshift in the amino acid of the BAAT protein, resulting in the pR130Kfs*12 variant. This mutation has been identified as the underlying cause of skeletal dysplasia in the proband., Outcomes: A novel frameshift mutation in the BAAT gene of a Vietnamese female child diagnosed with skeletal dysplasia has been studied by whole exome sequencing analysis., Lessons: This research reported a case of skeletal dysplasia caused by a frameshift mutation in the BAAT gene. The results of this study contribute to our understanding of the diverse factors that influence irregular skeletal development in children and provide genetic data to support clinical practice., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Docking domains (DDs) located at the C- and N-termini of polypeptides play a crucial role in directing the assembly of polyketide synthases (PKSs), which are multienzyme complexes. Here, we determined the crystal structure of a complex comprising the C-terminal DD ( C DD MlnB ) and N-terminal DD ( N DD MlnC ) of macrolactin trans-acyltransferase (AT) PKS that were fused to a functional enzyme, AmpC EC2 β-lactamase. Interface analyses of the C DD MlnB / N DD MlnC complex revealed the molecular intricacies in the core section underpinning the precise DD assembly. Additionally, circular dichroism and steady-state kinetics demonstrated that the formation of the C DD MlnB / N DD MlnC complex had no influence on the structural and functional fidelity of the fusion partner, AmpC EC2. This inspired us to apply the C DD MlnB / N DD MlnC assembly to metabolon engineering. Indeed, DD assembly induced the formation of a complex between 4-coumarate-CoA ligase and chalcone synthase both involved in flavonoid biosynthesis, leading to a remarkable increase in naringenin production in vitro., Competing Interests: Declaration of interests D.-W.B., E.K., B.-G.J., S.-J.Y., N.K.L., J.-S.H., S.-S.C., and Y.J.Y. have filed a patent for employing docking domains to assemble an artificial metabolon, as described in this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

NLRP3 inflammasome activation, essential for cytokine secretion and pyroptosis in response to diverse stimuli, is closely associated with various diseases. Upon stimulation, NLRP3 undergoes subcellular membrane trafficking and conformational rearrangements, preparing itself for inflammasome assembly at the microtubule-organizing center (MTOC). Here, we elucidate an orchestrated mechanism underlying these ordered processes using human and murine cells. Specifically, NLRP3 undergoes palmitoylation at two sites by palmitoyl transferase zDHHC1, facilitating its trafficking between subcellular membranes, including the mitochondria, trans-Golgi network (TGN), and endosome. This dynamic trafficking culminates in the localization of NLRP3 to the MTOC, where LATS1/2, pre-recruited to MTOC during priming, phosphorylates NLRP3 to further facilitate its interaction with NIMA-related kinase 7 (NEK7), ultimately leading to full NLRP3 activation. Consistently, Zdhhc1-deficiency mitigated LPS-induced inflammation and conferred protection against mortality in mice. Altogether, our findings provide valuable insights into the regulation of NLRP3 membrane trafficking and inflammasome activation, governed by palmitoylation and phosphorylation events., Competing Interests: Declaration of interests W.W. is a co-founder and consultant for ReKindle Therapeutics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Protein S-acylation catalyzed by protein S-acyl transferases (PATs) is a reversible lipid modification regulating protein targeting, stability, and interaction profiles. PATs are encoded by large gene families in plants, and many proteins including receptor-like cytoplasmic kinases (RLCKs) and receptor-like kinases (RLKs) are subject to S-acylation. However, few PATs have been assigned substrates, and few S-acylated proteins have known upstream enzymes. We report that Arabidopsis (Arabidopsis thaliana) class A PATs redundantly mediate pollen tube guidance and participate in the S-acylation of POLLEN RECEPTOR KINASE1 (PRK1) and LOST IN POLLEN TUBE GUIDANCE1 (LIP1), a critical RLK or RLCK for pollen tube guidance, respectively. PAT1, PAT2, PAT3, PAT4, and PAT8, collectively named PENTAPAT for simplicity, are enriched in pollen and show similar subcellular distribution. Functional loss of PENTAPAT reduces seed set due to male gametophytic defects. Specifically, pentapat pollen tubes are compromised in directional growth. We determine that PRK1 and LIP1 interact with PENTAPAT, and their S-acylation is reduced in pentapat pollen. The plasma membrane (PM) association of LIP1 is reduced in pentapat pollen, whereas point mutations reducing PRK1 S-acylation affect its affinity with its interacting proteins. Our results suggest a key role of S-acylation in pollen tube guidance through modulating PM receptor complexes., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Over the past three decades, researchers have isolated plant mutants that show constitutively activated defence responses in the absence of pathogen infection. These mutants are called autoimmune mutants and are typically dwarf and/or bearing chlorotic/necrotic lesions. Here, from a genetic screen for Arabidopsis genes involved in maintaining a normal leaf microbiota, we identified TIP GROWTH DEFECTIVE 1 (TIP1), which encodes an S-acyltransferase, as a key player in guarding leaves against abnormal microbiota level and composition under high-humidity conditions. The tip1 mutant has several characteristic phenotypes of classical autoimmune mutants, including a dwarf stature, showing lesions, and having a high basal level of defence gene expression. Gnotobiotic experiments revealed that the autoimmune phenotypes of the tip1 mutant are largely dependent on the presence of microbiota as axenic tip1 plants have markedly reduced autoimmune phenotypes. We found that the microbiota dependency of autoimmune phenotypes is shared by several 'lesion mimic'-type autoimmune mutants in Arabidopsis. It is worth noting that autoimmune phenotypes caused by mutations in two Nucleotide-Binding, Leucine-Rich Repeat (NLR) genes do not require the presence of microbiota and can even be partially alleviated by microbiota. Our results therefore suggest the existence of at least two classes of autoimmunity (microbiota-dependent versus microbiota-independent) in plants. The observed interplay between autoimmunity and microbiota in the lesion mimic class of autoimmunity is reminiscent of the interactions between autoimmunity and dysbiosis in the animal kingdom. These parallels highlight the intricate relationship between host immunity and microbial communities across various biological systems., (© 2024. The Author(s).)

Cuproptosis, a cell death process caused by copper ions, is mediated by protein lipidation related to lipoic acid metabolism. There is a close connection between cuproptosis and the progression and prognosis of various tumors. Here, we identified lipoyltransferase 1 (LIPT1), a key gene related to cuproptosis, was downregulated in bladder cancer (BLCA) and was associated with unfavorable patient prognosis. Restoring the LIPT1 expression in BLCA cells suppressed the proliferation and promoted cuproptosis. Moreover, the consequences of RNA sequencing and Bodipy staining showed that the metabolic pathway mediated by LIPT1 inhibited the accumulation of lipid droplets in cells, disrupted endoplasmic reticulum (ER) homeostasis, and promoted cell apoptosis. Additionally, overexpression of LIPT1 not only repressed the proliferation rate of BLCA cells in vitro but also in vivo. Mechanistically, YTH N6-Methyladenosine RNA Binding Protein F2 (YTHDF2) promoted the degradation of LIPT1 mRNA in a m 6 A-dependent manner. In summary, these conclusions reveal that LIPT1 promotes cuprotosis and ER stress to inhibit the progression of BLCA, indicating that LIPT1 will provide a powerful treatment direction and drug target for treating BLCA., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Polyketides are natural products synthesized by polyketide synthases (PKSs), where acyltransferase (AT) domains play a crucial role in selection of extender units. Engineering of AT domains enables the site-specific incorporation of non-natural extender units, leading to generation of novel derivatives. Here, we determined the crystal structures of AT domains from the fifth module of tylosin PKS (TylAT5) derived from Streptomyces fradiae and the eighth module of spinosad PKS (SpnAT8) derived from Saccharopolyspora spinosa, and combined them with molecular dynamics simulations and enzyme kinetic studies to elucidate the molecular basis of substrate selection. The ethylmalonyl-CoA-specific conserved motif TAGH of TylAT5 and the MMCoA-specific conserved motif YASH of SpnAT8 were identified within the substrate-binding pocket, and several key residues close to the substrate acyl moiety were located. Through site-directed mutagenesis of four residues near the active site, we successfully reprogrammed the specificity of these two AT domains toward malonyl-CoA. Mutations in TylAT5 enhanced its catalytic activity 2.6-fold toward malonyl-CoA, and mutations in SpnAT8 eliminated the substrate promiscuity. These results extend our understanding of AT substrate specificity and would benefit the engineering of PKSs., (© 2024 Federation of European Biochemical Societies.)

Although oxaliplatin (OXA) is widely used in the frontline treatment of colorectal cancer (CRC), CRC recurrence is commonly observed due to OXA resistance. OXA resistance is associated with a number of factors, including abnormal regulation of pyroptosis. It is therefore important to elucidate the abnormal regulatory mechanism underlying pyroptosis. Here, we identified that the circular RNA circPDIA3 played an important role in chemoresistance in CRC. CircPDIA3 could induce chemoresistance in CRC by inhibiting pyroptosis both in vitro and in vivo. Mechanistically, RIP, RNA pull-down and co-IP assays revealed that circPDIA3 directly bonded to the GSDME-C domain, subsequently enhanced the autoinhibitory effect of the GSDME-C domain through blocking the GSDME-C domain palmitoylation by ZDHHC3 and ZDHHC17, thereby restraining pyroptosis. Additionally, it was found that the circPDIA3/miR-449a/XBP1 positive feedback loop increased the expression of circPDIA3 to induce chemoresistance. Furthermore, our clinical data and patient-derived tumor xenograft (PDX) models supported the positive association of circPDIA3 with development of chemoresistance in CRC patients. Taken together, our findings demonstrated that circPDIA3 could promote chemoresistance by amplifying the autoinhibitory effect of the GSDME-C domain through inhibition of the GSDME-C domain palmitoylation in CRC. This study provides novel insights into the mechanism of circRNA in regulating pyroptosis and providing a potential therapeutic target for reversing chemoresistance of CRC., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Alteration in lipid metabolism is recognized as a hallmark feature of colorectal cancer (CRC). Protein S-palmitoylation plays a critical role in many different cellular processes including protein-lipid interaction. Zinc Finger DHHC-Type Containing 1 (ZDHHC1, also known as ZNF377) belongs to the palmitoyl-transferase ZDHHC family, and is a potential tumor suppressor. However, our knowledge of the functional roles of ZDHHC1 in CRC is limited. We discovered that ZDHHC1 expression was downregulated in CRC tissues and that low levels of ZDHHC1 were associated with unfavorable prognosis. Functional studies showed that ZDHHC1 inhibited CRC cell proliferation and invasion in vitro and in vivo. We also found that lipase G (LIPG) is negatively regulated by ZDHHC1 and plays a key role in CRC cell growth through lipid storage. Additionally, we demonstrated that ZDHHC1 functions as a IGF2BP1-palmitoylating enzyme that induces S-palmitoylation at IGF2BP1-C337, which results in downregulated LIPG expression via m6A modification. Mechanistic investigations revealed that the ZDHHC1/IGF2BP1/LIPG signaling axis is associated with inhibition of CRC cell growth. Our study uncovers the potential role of ZDHHC1 in CRC, including inhibition of CRC growth by reducing the stability of LIPG mRNA in an m6A dependent-manner by palmitoylation of IGF2BP1., (© 2024. The Author(s).)

Lysophospholipid transporter LplT and acyltransferase Aas consist of a lysophospholipid-remodeling system ubiquitously found in gram-negative microorganisms. LplT flips lysophospholipid across the inner membrane which is subsequently acylated by Aas on the cytoplasmic membrane surface. Our previous study showed that the proper functioning of this system is important to protecting Escherichia coli from phospholipase-mediated host attack by maintaining the integrity of the bacterial cell envelope. However, the working mechanism of this system is still unclear. Herein, we report that LplT and Aas form a membrane protein complex in E. coli which allows these two enzymes to cooperate efficiently to move lysophospholipids across the bacterial membrane and catalyze their acylation. The direct interaction of LplT and Aas was demonstrated both in vivo and in vitro with a binding affinity of 2.3 μM. We found that a cytoplasmic loop of LplT adjacent to the exit of the substrate translocation pathway plays an important role in maintaining its interaction with Aas. Aas contains an acyl-acyl carrier protein synthase domain and an acyl-transferase domain. Its interaction with LplT is mediated exclusively by its transferase domain. Mutations within the three loops near the putative catalytic site of the transferase domain, respectively, disrupt its interaction with LplT and lysophospholipid acylation activity. These results support a hypothesis of the functional coupling mechanism, in which LplT directly interacts with the transferase domain of Aas for specific substrate membrane migration, providing synchronization of substrate translocation and biosynthetic events., Competing Interests: Conflict of interest The authors (L. Z. and M. B.) are Editorial Board Members for the Journal of Biological Chemistry and were not involved in the editorial review or the decision to publish this article. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent studies have highlighted palmitoylation, a novel protein post-translational modification, as a key player in various signaling pathways that contribute to tumorigenesis and drug resistance. Despite this, its role in bladder cancer (BCa) development remains inadequately understood. In this study, ZDHHC9 emerged as a significantly upregulated oncogene in BCa. Functionally, ZDHHC9 knockdown markedly inhibited tumor proliferation, promoted tumor cell apoptosis, and enhanced the efficacy of gemcitabine (GEM) and cisplatin (CDDP). Mechanistically, SP1 was found to transcriptionally activate ZDHHC9 expression. ZDHHC9 subsequently bound to and palmitoylated the Bip protein at cysteine 420 (Cys420), thereby inhibiting the unfolded protein response (UPR). This palmitoylation at Cys420 enhanced Bip's protein stability and preserved its localization within the endoplasmic reticulum (ER). ZDHHC9 might become a novel therapeutic target for BCa and could also contribute to combination therapy with GEM and CDDP., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The mammarenavirus matrix Z protein plays critical roles in virus assembly and cell egress. Meanwhile, heterotrimer complexes of a stable signal peptide (SSP) together with glycoprotein subunits GP1 and GP2, generated via co-and post-translational processing of the surface glycoprotein precursor GPC, form the spikes that decorate the virion surface and mediate virus cell entry via receptor-mediated endocytosis. The Z protein and the SSP undergo N-terminal myristoylation by host cell N-myristoyltransferases (NMT1 and NMT2), and G2A mutations that prevent myristoylation of Z or SSP have been shown to affect the Z-mediated virus budding and GP2-mediated fusion activity that is required to complete the virus cell entry process. In the present work, we present evidence that the validated on-target specific pan-NMT inhibitor DDD85646 exerts a potent antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) that correlates with reduced Z budding activity and GP2-mediated fusion activity as well as with proteasome-mediated degradation of the Z protein. The potent anti-mammarenaviral activity of DDD85646 was also observed with the hemorrhagic-fever-causing Junin (JUNV) and Lassa (LASV) mammarenaviruses. Our results support the exploration of NMT inhibition as a broad-spectrum antiviral against human pathogenic mammarenaviruses.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy., (© 2024. The Author(s).)

Background: The failure of proper recognition of the intricate nature of pathophysiology in colorectal cancer (CRC) has a substantial effect on the progress of developing novel medications and targeted therapy approaches. Imbalances in the processes of lipid oxidation and biosynthesis of fatty acids are significant risk factors for the development of CRC. Therapeutic intervention that specifically targets the peroxisome proliferator-activated receptor gamma (PPARγ) and its downstream response element, in response to lipid metabolism, has been found to promote the growth of tumors and has shown significant clinical advantages in cancer patients., Methods: Clinical CRC samples and extensive in vitro and in vivo experiments were carried out to determine the role of ZDHHC6 and its downstream targets via a series of biochemical assays, molecular analysis approaches and lipid metabolomics assay, etc. RESULTS: To study the effect of ZDHHC6 on the progression of CRC and identify whether ZDHHC6 is a palmitoyltransferase that regulates fatty acid synthesis, which directly palmitoylates and stabilizes PPARγ, and this stabilization in turn activates the ACLY transcription-related metabolic pathway. In this study, we demonstrate that PPARγ undergoes palmitoylation in its DNA binding domain (DBD) section. This lipid-related modification enhances the stability of PPARγ protein by preventing its destabilization. As a result, palmitoylated PPARγ inhibits its degradation induced by the lysosome and facilitates its translocation into the nucleus. In addition, we have identified zinc finger-aspartate-histidine-cysteine 6 (ZDHHC6) as a crucial controller of fatty acid biosynthesis. ZDHHC6 directly interacts with and adds palmitoyl groups to stabilize PPARγ at the Cys-313 site within the DBD domain of PPARγ. Consequently, this palmitoylation leads to an increase in the expression of ATP citrate lyase (ACLY). Furthermore, our findings reveals that ZDHHC6 actively stimulates the production of fatty acids and plays a role in the development of colorectal cancer. However, we have observed a significant reduction in the cancer-causing effects when the expression of ZDHHC6 is inhibited in in vivo trials. Significantly, in CRC, there is a strong positive correlation between the high expression of ZDHHC6 and the expression of PPARγ. Moreover, this high expression of ZDHHC6 is connected with the severity of CRC and is indicative of a poor prognosis., Conclusions: We have discovered a mechanism in which lipid biosynthesis is controlled by ZDHHC6 and includes the signaling of PPARγ-ACLY in the advancement of CRC. This finding provides a justification for targeting lipid synthesis by blocking ZDHHC6 as a potential therapeutic approach., (© 2024. The Author(s).)

Background: Avocado fruit is rich in xanthophylls, which have been related to positive effects on human health. Xanthophyl acetyltransferases (XATs) are enzymes catalyzing the esterification of carboxylic acids to the hydroxyl group of the xanthophyll molecule. This esterification is thought to increase the lipophilic nature of the xanthophyll and its stability in a lipophilic environment. Studies on XATs in fruits are very scarce, and no studies had been carried out in avocado fruit during postharvest. The objective of this work was to investigate the changes in the expression of genes encoding XAT, during avocado fruit ripening., Results: Avocado fruits were obtained from a local market and stored at 15 °C for 8 days. The fruit respiration rate, ethylene production, and fruit peel's color space parameters (L*, a*, b*) were measured during storage. Fruit mesocarp samples were taken after 1, 3, 5, and 7 days of storage and frozen with liquid nitrogen. Total RNA was extracted from fruit mesocarp, and the quantification of the two genes designated as COGE_ID: 936743791 and COGE_ID: 936800185 encoding XATs was performed with real-time quantitative reverse transcription polymerase chain reaction using actin as a reference gene. The presence of a climacteric peak and large changes in color were recorded during postharvest. The two genes studied showed a large expression after 3 days of fruit storage., Conclusions: We conclude that during the last stages of ripening in avocado fruit there was an active esterification of xanthophylls with carboxylic acids, which suggests the presence of esterified xanthophylls in the fruit mesocarp. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. KEY POINTS: • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli., (© 2024. The Author(s).)