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Summary Inflammasomes are multiprotein complexes that play a crucial role in regulating immune responses by governing the activation of Caspase‐1, the secretion of pro‐inflammatory cytokines, and the induction of inflammatory cell death, pyroptosis. The inflammasomes are pivotal in effective host defense against a range of pathogens. Yet, overt activation of inflammasome signaling can be detrimental. The most well‐studied NLRP3 inflammasome has the ability to detect a variety of stimuli including pathogen‐associated molecular patterns, environmental irritants, and endogenous stimuli released from dying cells. Additionally, NLRP3 acts as a key sensor of cellular homeostasis and can be activated by disturbances in diverse metabolic pathways. Consequently, NLRP3 is considered a key player linking metabolic dysregulation to numerous inflammatory disorders such as gout, diabetes, and atherosclerosis. Recently, compelling studies have highlighted a connection between lipids and the regulation of NLRP3 inflammasome. Lipids are integral to cellular processes that serve not only in maintaining the structural integrity and subcellular compartmentalization, but also in contributing to physiological equilibrium. Certain lipid species are known to define NLRP3 subcellular localization, therefore directly influencing the site of inflammasome assembly and activation. For instance, phosphatidylinositol 4‐phosphate plays a crucial role in NLRP3 localization to the trans Golgi network. Moreover, new evidence has demonstrated the roles of lipid biosynthesis and trafficking in activation of the NLRP3 inflammasome. This review summarizes and discusses these emerging and varied roles of lipid metabolism in inflammasome activation. A deeper understanding of lipid‐inflammasome interactions may open new avenues for therapeutic interventions to prevent or treat chronic inflammatory and autoimmune conditions. [ABSTRACT FROM AUTHOR]

This study demonstrates the feasibility and effectiveness of utilizing native soils as a resource for inocula to produce n-caproate through the chain elongation (CE) platform, offering new insights into anaerobic soil processes. The results reveal that all five of the tested soil types exhibit CE activity when supplied with high concentrations of ethanol and acetate, highlighting the suitability of soil as an ideal source for n-caproate production. Compared with anaerobic sludge and pit mud, the native soil CE system exhibited higher selectivity (60.53%), specificity (82.32%), carbon distribution (60.00%), electron transfer efficiency (165.00%), and conductivity (0.59 ms∙cm−1). Kinetic analysis further confirmed the superiority of soil in terms of a shorter lag time and higher yield. A microbial community analysis indicated a positive correlation between the relative abundances of Pseudomonas, Azotobacter, and Clostridium and n-caproate production. Moreover, metagenomics analysis revealed a higher abundance of functional genes in key microbial species, providing direct insights into the pathways involved in n-caproate formation, including in situ CO2 utilization, ethanol oxidation, fatty acid biosynthesis (FAB), and reverse beta-oxidation (RBO). The numerous functions in FAB and RBO are primarily associated with Pseudomonas, Clostridium, Rhodococcus, Stenotrophomonas, and Geobacter, suggesting that these genera may play roles that are involved or associated with the CE process. Overall, this innovative inoculation strategy offers an efficient microbial source for n-caproate production, underscoring the importance of considering CE activity in anaerobic soil microbial ecology and holding potential for significant economic and environmental benefits through soil consortia exploration.

Fatty acids produced by the type‐II fatty acid biosynthetic pathway (FAS‐II) are essential biomaterials for bacterial membrane construction and numerous metabolic routes. The β‐ketoacyl‐ACP synthase FabF catalyzes the key C−C bond formation step for fatty acid elongation in FAS‐II. Here, we revealed the substrate recognition and catalytic mechanisms of FabF by determining FabF‐ACP complexes. FabF displays a distinctive bimodal catalytic pattern specifically on C6 and C10 acyl‐ACP substrates. It utilizes positively charged residues located on the η3‐helix and loop1 regions near the catalytic tunnel entrance to bind ACP, and two hydrophobic cavities as well as “front”, “middle”, and “back” door residues to specifically stabilize C6 and C10 acyl substrates for preferential catalysis. Further quantum chemistry calculations suggest that the FabF catalytic residues Lys336 and His304 facilitate proton transfer during condensation catalysis and C−C bond formation. Our results provide key mechanistic insights into the biosynthesis of molecular carbon skeletons based on ketosynthases that are highly conserved through the FAS and polyketide synthase (PKS) analogous biosynthetic routes, broaden the understanding of the tricarboxylic acid cycle that utilizes lipoic acid derived from C8‐ACP accumulated due to the FabF distinctive catalytic pattern for oxidative decarboxylations, and may facilitate the development of narrow‐spectrum antibacterial drugs. [ABSTRACT FROM AUTHOR]

The cGAS-STING innate immunity pathway and the SREBP-activated cholesterol and fatty acid synthesis pathway are abnormally co-regulated in neurodegenerative disease. Activation of STING signaling occurs at the endoplasmic reticulum (ER) membrane with STING anchored by INSIG1 along with SREBP and the sterol-bound SREBP cleavage activating protein (SCAP) when sterols are in abundance. When sterols are low, the INSIG-dependent STING pathway is inactivated and the SREBPSCAP complex is translocated to the Golgi where SREBP is cleaved and translocated to the nucleus to transactivate genes for cholesterol and fatty acid synthesis. Thus, there is inverse activation of STING vs. SREBP: when innate immunity is active, pathways for cholesterol and fatty acid synthesis are suppressed, and vice versa. The STING pathway is stimulated by foreign viral cytoplasmic nucleic acids interacting with the cyclic GMP-AMP synthase (cGAS) DNA sensor or RIG-I and MDA5 dsRNA sensors, but with neurodegeneration innate immunity is also activated by self-DNAs and double-stranded RNAs that accumulate with neuronal death. Downstream, activated STING recruits TBK1 and stimulates the transactivation of interferon stimulated genes and the autophagy pathway, which are both protective. However, chronic activation of innate immunity contributes to microglia activation, neuroinflammation and autophagy failure leading to neurodegeneration. STING is also a proton channel that when activated stimulates proton exit from STING vesicles leading to cell death. Here we review the salient features of the innate immunity and cholesterol and fatty acid synthesis pathways, observations of abnormal STING and SREBP signaling in neurodegenerative disease, and relevant therapeutic approaches. [ABSTRACT FROM AUTHOR]

Purpose: Pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge due to its high heterogeneity and aggressiveness. Recognizing the urgency to delineate molecular subtypes, our study focused on the emerging field of lipid metabolism remodeling in PDAC, particularly exploring the prognostic potential and molecular classification associated with fatty acid biosynthesis. Methods: Gene set variation analysis (GSVA) and single-sample gene set enrichment analysis (ssGSEA) were performed to evaluate the dysregulation of lipid metabolism in PDAC. Univariate cox analysis and the LASSO module were used to build a prognostic risk score signature. The distinction of gene expression in different risk groups was explored by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Weighted Gene Co-expression Network Analysis (WGCNA). The biological function of Acyl-CoA Synthetase Long Chain Family Member 5 (ACSL5), a pivotal gene within 7-hub gene signature panel, was validated through in vitro assays. Results: Our study identified a 7-hub gene signature associated with fatty acid biosynthesis-related genes (FRGs), providing a robust tool for prognosis prediction. The high-FRGs score group displayed a poorer prognosis, decreased immune cell infiltration, and a higher tumor mutation burden. Interestingly, this group exhibited enhanced responsiveness to various compounds according to the Genomics of Drug Sensitivity in Cancer (GDSC) database. Notably, ACSL5 was upregulated in PDAC and essential for tumor progression. Conclusion: In conclusion, our research defined two novel fatty acid biosynthesis-based subtypes in PDAC, characterized by distinct transcriptional profiles. These subtypes not only served as prognostic indicator, but also offered valuable insights into their metastatic propensity and therapeutic potential. [ABSTRACT FROM AUTHOR]

Having evolved from a prokaryotic origin, mitochondria retain pathways required for the catabolism of energy‐rich molecules and for the biosynthesis of molecules that aid catabolism and/or participate in other cellular processes essential for life of the cell. Reviewed here are details of the mitochondrial fatty acid biosynthetic pathway (FAS II) and its role in building both the octanoic acid precursor for lipoic acid biosynthesis (LAS) and longer‐chain fatty acids functioning in chaperoning the assembly of mitochondrial multisubunit complexes. Also covered are the details of mitochondrial lipoic acid biosynthesis, which is distinct from that of prokaryotes, and the attachment of lipoic acid to subunits of pyruvate dehydrogenase, α‐ketoglutarate dehydrogenase, and glycine cleavage system complexes. Special emphasis has been placed on presenting what is currently known about the interconnected paths and loops linking the FAS II–LAS pathway and two other mitochondrial realms, the organellar translation machinery and Fe‐S cluster biosynthesis and function. [ABSTRACT FROM AUTHOR]

Camellia oil is valuable as an edible oil and serves as a base material for a range of high-value products. Camellia plants of significant economic importance, such as Camellia sinensis and Camellia oleifera, have been classified into sect. Thea and sect. Oleifera, respectively. Fatty acid desaturases play a crucial role in catalyzing the formation of double bonds at specific positions of fatty acid chains, leading to the production of unsaturated fatty acids and contributing to lipid synthesis. Comparative genomics results have revealed that expanded gene families in oil tea are enriched in functions related to lipid, fatty acid, and seed processes. To explore the function of the FAD gene family, a total of 82 FAD genes were identified in tea and oil tea. Transcriptome data showed the differential expression of the FAD gene family in mature seeds of tea tree and oil tea tree. Furthermore, the structural analysis and clustering of FAD proteins provided insights for the further exploration of the function of the FAD gene family and its role in lipid synthesis. Overall, these findings shed light on the role of the FAD gene family in Camellia plants and their involvement in lipid metabolism, as well as provide a reference for understanding their function in oil synthesis. [ABSTRACT FROM AUTHOR]

Abstract Domesticated safflower (Carthamus tinctorius L.) is a widely cultivated edible oil crop. However, despite its economic importance, the genetic basis underlying key traits such as oil content, resistance to biotic and abiotic stresses, and flowering time remains poorly understood. Here, we present the genome assembly for C. tinctorius variety Jihong01, which was obtained by integrating Oxford Nanopore Technologies (ONT) and BGI-SEQ500 sequencing results. The assembled genome was 1,061.1 Mb, and consisted of 32,379 protein-coding genes, 97.71% of which were functionally annotated. Safflower had a recent whole genome duplication (WGD) event in evolution history and diverged from sunflower approximately 37.3 million years ago. Through comparative genomic analysis at five seed development stages, we unveiled the pivotal roles of fatty acid desaturase 2 (FAD2) and fatty acid desaturase 6 (FAD6) in linoleic acid (LA) biosynthesis. Similarly, the differential gene expression analysis further reinforced the significance of these genes in regulating LA accumulation. Moreover, our investigation of seed fatty acid composition at different seed developmental stages unveiled the crucial roles of FAD2 and FAD6 in LA biosynthesis. These findings offer important insights into enhancing breeding programs for the improvement of quality traits and provide reference resource for further research on the natural properties of safflower.

The synthesis of fatty acids plays a critical role in shaping milk production characteristics in dairy cattle. Thus, identifying effective haplotypes within the fatty acid metabolism pathway will provide novel and robust insights into the genetics of dairy cattle. This study aimed to comprehensively examine the individual and combined impacts of fundamental genes within the fatty acid metabolic process pathway in Jersey cows. A comprehensive phenotypic dataset was compiled, considering milk production traits, to summarize a cow’s productivity across three lactations. Genotyping was conducted through PCR-RFLP and Sanger sequencing, while the association between genotype and phenotype was quantified using linear mixed models. Moderate biodiversity and abundant variation suitable for haplotype analysis were observed across all examined markers. The individual effects of the FABP3, LTF and ANXA9 genes significantly influenced both milk yield and milk fat production. Additionally, this study reveals novel two-way interactions between genes in the fatty acid metabolism pathway that directly affect milk fat properties. Notably, we identified that the GGAAGG haplotype in FABP3×LTF×ANXA9 interaction may be a robust genetic marker concerning both milk fat yield and percentage. Consequently, the genotype combinations highlighted in this study serve as novel and efficient markers for assessing the fat content in cow’s milk.

The utilization of natural products in food preservation represents a promising strategy for the dual benefits of controlling foodborne pathogens and enhancing the nutritional properties of foods. Among the phytonutrients, flavonoids have been shown to exert antibacterial effects by disrupting bacterial cell membrane functionality; however, the underlying molecular mechanisms remain elusive. In this study, we investigated the effect of quercetin on the cell membrane permeability of Staphylococcus aureus ATCC 27217. A combined metabolomic and transcriptomic approach was adopted to examine the regulatory mechanism of quercetin with respect to the fatty acid composition and associated genes. Kinetic analysis and molecular docking simulations were conducted to assess quercetin's inhibition of β-ketoacyl-acyl carrier protein reductase (FabG), a potential target in the bacterial fatty acid biosynthesis pathway. Metabolomic and transcriptomic results showed that quercetin increased the ratio of unsaturated to saturated fatty acids and the levels of membrane phospholipids. The bacteria reacted to quercetin-induced stress by attempting to enhance fatty acid biosynthesis; however, quercetin directly inhibited FabG activity, thereby disrupting bacterial fatty acid biosynthesis. These findings provide new insights into the mechanism of quercetin's effects on bacterial cell membranes and suggest potential applications for quercetin in bacterial inhibition. [ABSTRACT FROM AUTHOR]

The cGAS-STING innate immunity pathway and the SREBP-activated cholesterol and fatty acid synthesis pathway are abnormally co-regulated in neurodegenerative disease. Activation of STING signaling occurs at the endoplasmic reticulum (ER) membrane with STING anchored by INSIG1 along with SREBP and the sterol-bound SREBP cleavage activating protein (SCAP) when sterols are in abundance. When sterols are low, the INSIG-dependent STING pathway is inactivated and the SREBP-SCAP complex is translocated to the Golgi where SREBP is cleaved and translocated to the nucleus to transactivate genes for cholesterol and fatty acid synthesis. Thus, there is inverse activation of STING vs. SREBP: when innate immunity is active, pathways for cholesterol and fatty acid synthesis are suppressed, and vice versa. The STING pathway is stimulated by foreign viral cytoplasmic nucleic acids interacting with the cyclic GMP–AMP synthase (cGAS) DNA sensor or RIG-I and MDA5 dsRNA sensors, but with neurodegeneration innate immunity is also activated by self-DNAs and double-stranded RNAs that accumulate with neuronal death. Downstream, activated STING recruits TBK1 and stimulates the transactivation of interferon stimulated genes and the autophagy pathway, which are both protective. However, chronic activation of innate immunity contributes to microglia activation, neuroinflammation and autophagy failure leading to neurodegeneration. STING is also a proton channel that when activated stimulates proton exit from STING vesicles leading to cell death. Here we review the salient features of the innate immunity and cholesterol and fatty acid synthesis pathways, observations of abnormal STING and SREBP signaling in neurodegenerative disease, and relevant therapeutic approaches.

Abstract Background Safflower (Carthamus tinctorius L.) is an oilseed crop with substantial medicinal and economic value. However, the methods for constructing safflower core germplasm resources are limited, and the molecular mechanisms of lipid biosynthesis in safflower seeds are not well understood. Results In this study, 11 oil-related quantitative traits and 50 pairs of InDel markers were used to assess the diversity of a collection of 605 safflower germplasms. The original safflower germplasm exhibited rich phenotypic diversity, with high variation for most of the phenotypic traits under investigation. Similarly, high genetic diversity was evaluated in the original germplasm, in which the mean Shannon’s information index (I), observed heterozygosity (H 0 ), and expected heterozygosity (He) were 0.553, 0.182, and 0.374, respectively. Four subgroups with strong genetic structures were identified and a core germplasm of 214 cultivars was constructed, which is well represented in the original germplasm. Meanwhile, differential expression analysis of the transcriptomes of high and low linoleic acid safflower varieties at two stages of seed development identified a total of 47 genes associated with lipid biosynthesis. High expression of the genes KAS II and SAD enhanced the synthesis and accumulation of oleic acid, while FAD genes like FAD2 (Chr8G0104100), FAD3, FAD7 and FAD8 promoted the consumption of oleic acid conversion. The coordinated regulation of these multiple genes ensures the high accumulation of oleic acid in safflower seed oil. Conclusions Based on these findings, a core germplasm of 214 cultivars was constructed and 47 candidate genes related to unsaturated fatty acid biosynthesis and lipid accumulation were identified. These results not only provide guidance for further studies to elucidate the molecular basis of oil lipid accumulation in safflower seeds, but also contribute to safflower cultivar improvements.

Yellowhorn (Xanthoceras sorbifolium Bunge) is an oil-bearing tree species in northern China. In this study, we used yellowhorn from Heilongjiang to analyze the morphological and physiological changes of fruit development and conducted transcriptome sequencing. The results showed that the fruit experienced relatively slow growth from fertilization to DAF20 (20 days after flowering). From DAF40 to DAF60, the fruit entered an accelerated development stage, with a rapid increase in both transverse and longitudinal diameters, and the kernel contour developed completely at DAF40. From DAF60 to DAF80, the transverse and vertical diameters of the fruit developed slowly, and the overall measures remained stable until maturity. The soluble sugar, starch, and anthocyanin content gradually accumulated until reaching a peak at DAF80 and then rapidly decreased. RNA-seq analysis revealed differentially expressed genes (DEGs) in the seed coat and kernel, implying that seed components have different metabolite accumulation mechanisms. During the stages of seed kernel development, k-means clustering separated the DEGs into eight sub-classes, indicating gene expression shifts during the fruit ripening process. In subclass 8, the fatty acid biosynthesis pathway was enriched, suggesting that this class was responsible for lipid accumulation in the kernel. WGCNA revealed ten tissue-specific modules for the 12 samples among 20 modules. We identified 54 fatty acid biosynthesis pathway genes across the genome, of which 14 was quantified and confirmed by RT-qPCR. Most genes in the plastid synthesis stage showed high expression during the DAF40-DAF60 period, while genes in the endoplasmic reticulum synthesis stage showed diverse expression patterns. EVM0012847 (KCS) and EVM0002968 (HCD) showed similar high expression in the early stages and low expression in the late stages. EVM0022385 (HCD) exhibited decreased expression from DAF40 to DAF60 and then increased from DAF60 to DAF100. EVM0000575 (KCS) was increasingly expressed from DAF40 to DAF60 and then decreased from DAF60 to DAF100. Finally, we identified transcription factors (TFs) (HB-other, bHLH and ARF) that were predicted to bind to fatty acid biosynthesis pathway genes with significant correlations. These results are conducive to promoting the transcriptional regulation of lipid metabolism and the genetic improvement in terms of high lipid content of yellowhorn. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae, a bacterial pathogen infamous for antibiotic resistance, is included in the priority list of pathogens by various public health organizations due to its extraordinary ability to develop multidrug resistance. Bacterial fatty acid biosynthesis pathway-II (FAS-II) has been considered a therapeutic drug target for antibacterial drug discovery. Inhibition of FAS-II enzyme, enoyl-acyl carrier protein reductase, FabI, not only inhibits bacterial infections but also reverses antibiotic resistance. Here, we characterized Klebsiella pneumoniae FabI (KpFabI) using complementary experimental approaches including, biochemical, x-ray crystallography, and molecular dynamics simulation studies. Biophysical studies shows that KpFabI organizes as a tetramer molecular assembly in solution as well as in the crystal structure. Enzyme kinetics studies reveal a distinct catalytic property towards crotonyl CoA and reducing cofactor NADH. Michaelis-Menten constant (Km) values of substrates show that KpFabI has higher preference towards NADH as compared to crotonyl CoA. The crystal structure of tetrameric apo KpFabI folds into a classic Rossman fold in which β-strands are sandwiched between α-helices. A highly flexible substrate binding region is located toward the interior of the tetrameric assembly. Thermal stability assay on KpFabI with its substrate shows that the flexibility is primarily stabilized by cofactor NADH. Moreover, the molecular dynamics further supports that KpFabI has highly flexible regions at the substrate binding site. Together, these findings provide evidence for highly dynamic substrate binding sites in KpFabI, therefore, this information will be vital for specific inhibitors discovery targeting Klebsiella pneumoniae. [ABSTRACT FROM AUTHOR]

Xanthoceras sorbifolium (yellowhorn) is a woody oil plant with super stress resistance and excellent oil characteristics. The yellowhorn oil can be used as biofuel and edible oil with high nutritional and medicinal value. However, genetic studies on yellowhorn are just in the beginning, and fundamental biological questions regarding its very long-chain fatty acid (VLCFA) biosynthesis pathway remain largely unknown. In this study, we reconstructed the VLCFA biosynthesis pathway and annotated 137 genes encoding relevant enzymes. We identified four oleosin genes that package triacylglycerols (TAGs) and are specifically expressed in fruits, likely playing key roles in yellowhorn oil production. Especially, by examining time-ordered gene co-expression network (TO-GCN) constructed from fruit and leaf developments, we identified key enzymatic genes and potential regulatory transcription factors involved in VLCFA synthesis. In fruits, we further inferred a hierarchical regulatory network with MYB-related (XS03G0296800) and B3 (XS02G0057600) transcription factors as top-tier regulators, providing clues into factors controlling carbon flux into fatty acids. Our results offer new insights into key genes and transcriptional regulators governing fatty acid production in yellowhorn, laying the foundation for efforts to optimize oil content and fatty acid composition. Moreover, the gene expression patterns and putative regulatory relationships identified here will inform metabolic engineering and molecular breeding approaches tailored to meet biofuel and bioproduct demands. [ABSTRACT FROM AUTHOR]

The Leach Bed Reactor (LBR) application is a promising alternative for the anaerobic digestion of solid waste and wastewater. In this study, the LBR was continuously operated for 160 days in four different phases, for co-fermentation of solid and liquid waste from the citrus agroindustry, at 37 °C, evaluating the effect of hydraulic retention time (HRT) and organic loading rate (OLR). The maximum volumetric hydrogen production (VHPR) was observed at an HRT of 12h (958.3 mLH 2.L-1.d-1); however, the highest hydrogen yield (HY) was identified at an HRT of 24h (104.9 mLH 2.g-1CHO). Based on the metabolic characteristics of microorganisms identified in higher relative abundance, by 16S rRNA sequencing, such as Lactobacillus, Caproicoproducens, Bifidobacterium, Olsenella, and Solobacterium, an occurrence of chain elongation pathways in the production of butyric and caproic acids was inferred, with hydrogen production resulting from the lactic acid oxidation. The maximum electricity production potential of 16.3 kWh.m-3 liquor was estimated. [Display omitted] • The leach bed reactor was applied to the dark fermentation of citrus waste. • The highest H 2 yield was obtained in a hydraulic retention time (HRT) of 24h. • The highest volumetric H 2 production rate was verified in HRT of 12h. • Lactobacillus was predominant in the fixed bed and leachate tank at HRT of 12h. • Caproiciproducens were identified with high relative abundance in HRT of 36h. [ABSTRACT FROM AUTHOR]

Background: Safflower (Carthamus tinctorius L.) is an oilseed crop with substantial medicinal and economic value. However, the methods for constructing safflower core germplasm resources are limited, and the molecular mechanisms of lipid biosynthesis in safflower seeds are not well understood. Results: In this study, 11 oil-related quantitative traits and 50 pairs of InDel markers were used to assess the diversity of a collection of 605 safflower germplasms. The original safflower germplasm exhibited rich phenotypic diversity, with high variation for most of the phenotypic traits under investigation. Similarly, high genetic diversity was evaluated in the original germplasm, in which the mean Shannon's information index (I), observed heterozygosity (H0), and expected heterozygosity (He) were 0.553, 0.182, and 0.374, respectively. Four subgroups with strong genetic structures were identified and a core germplasm of 214 cultivars was constructed, which is well represented in the original germplasm. Meanwhile, differential expression analysis of the transcriptomes of high and low linoleic acid safflower varieties at two stages of seed development identified a total of 47 genes associated with lipid biosynthesis. High expression of the genes KAS II and SAD enhanced the synthesis and accumulation of oleic acid, while FAD genes like FAD2 (Chr8G0104100), FAD3, FAD7 and FAD8 promoted the consumption of oleic acid conversion. The coordinated regulation of these multiple genes ensures the high accumulation of oleic acid in safflower seed oil. Conclusions: Based on these findings, a core germplasm of 214 cultivars was constructed and 47 candidate genes related to unsaturated fatty acid biosynthesis and lipid accumulation were identified. These results not only provide guidance for further studies to elucidate the molecular basis of oil lipid accumulation in safflower seeds, but also contribute to safflower cultivar improvements. [ABSTRACT FROM AUTHOR]

Abstract Background Cottonseed oil is a promising edible plant oil with abundant unsaturated fatty acids. However, few studies have been conducted to explore the characteristics of cottonseed oil. The molecular mechanism of cottonseed oil accumulation remains unclear. Results In the present study, we conducted comparative transcriptome and weighted gene co-expression network (WGCNA) analysis for two G. hirsutum materials with significant difference in cottonseed oil content. Results showed that, between the high oil genotype 6053 (H6053) and the low oil genotype 2052 (L2052), a total of 412, 507, 1,121, 1,953, and 2,019 differentially expressed genes (DEGs) were detected at 10, 15, 20, 25, and 30 DPA, respectively. Remarkably, a large number of the down-regulated DEGs were enriched in the phenylalanine metabolic processes. Investigation into the dynamic changes of expression profiling of genes associated with both phenylalanine metabolism and oil biosynthesis has shed light on a significant competitive relationship in substrate allocation during cottonseed development. Additionally, the WGCNA analysis of all DEGs identified eight distinct modules, one of which includes GhPXN1, a gene closely associated with oil accumulation. Through phylogenetic analysis, we hypothesized that GhPXN1 in G. hirsutum might have been introgressed from G. arboreum. Overexpression of the GhPXN1 gene in tobacco leaf suggested a significant reduction in oil content compared to the empty-vector transformants. Furthermore, ten other crucial oil candidate genes identified in this study were also validated using quantitative real-time PCR (qRT-PCR). Conclusions Overall, this study enhances our comprehension of the molecular mechanisms underlying cottonseed oil accumulation.

Abstract Background Fatty acid (FA) is an important platform compound for the further synthesis of high‐value biofuels and oleochemicals, but chemical synthesis of FA has many limitations. One way to meet the future demand for FA could be to use microbial cell factories for FA biosynthesis. Results Thioesterase (TE; TesA, TesB, and TE9) of Corynebacterium glutamicum (CG) can potentially improve FA biosynthesis, and tesA, tesB, and te9 were overexpressed in C. glutamicum and Escherichia coli (EC), respectively, in this study. The results showed that the total fatty acid (TFA) production of CGtesB and ECtesB significantly increased to 180.52 mg/g dry cell weight (DCW) and 123.52 mg/g DCW, respectively (P

Abstract The 3-ketoacyl-ACP synthase III (FabH), a key enzyme for bacteria growth, catalyses the last step of the initiation of bacterial fatty acid synthesis. Rice bacterial blight is caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo), which is widely studied as a model bacterium. Bioinformatics analysis showed that the X. oryzae pv. oryzae PXO99A genome encodes three FabH homologous proteins with unknown functions. In this study, we found that only PXO_02706 (fabH1) encodes a functional FabH, the key enzyme in the production of branched-chain fatty acid, which is essential for the branched-chain diffusible signal factor family signals in Xoo. Interestingly, we found that FabH1 is not essential for fatty acid biosynthesis in Xoo. Pathogenicity analysis showed that loss of fabH1 caused a significant decrease in virulence of Xoo. Genetic and phenotypic analyses revealed that fabH1 plays a key role in multiple Xoo virulence-related activities, including exopolysaccharide (EPS) production, biofilm formation, motility, and resistance to environmental stresses.

Abstract Background Sapria himalayana (Rafflesiaceae) is an endoparasitic plant characterized by a greatly reduced vegetative body and giant flowers; however, the mechanisms underlying its special lifestyle and greatly altered plant form remain unknown. To illustrate the evolution and adaptation of S. himalayasna, we report its de novo assembled genome and key insights into the molecular basis of its floral development, flowering time, fatty acid biosynthesis, and defense responses. Results The genome of S. himalayana is ~ 1.92 Gb with 13,670 protein-coding genes, indicating remarkable gene loss (~ 54%), especially genes involved in photosynthesis, plant body, nutrients, and defense response. Genes specifying floral organ identity and controlling organ size were identified in S. himalayana and Rafflesia cantleyi, and showed analogous spatiotemporal expression patterns in both plant species. Although the plastid genome had been lost, plastids likely biosynthesize essential fatty acids and amino acids (aromatic amino acids and lysine). A set of credible and functional horizontal gene transfer (HGT) events (involving genes and mRNAs) were identified in the nuclear and mitochondrial genomes of S. himalayana, most of which were under purifying selection. Convergent HGTs in Cuscuta, Orobanchaceae, and S. himalayana were mainly expressed at the parasite–host interface. Together, these results suggest that HGTs act as a bridge between the parasite and host, assisting the parasite in acquiring nutrients from the host. Conclusions Our results provide new insights into the flower development process and endoparasitic lifestyle of Rafflesiaceae plants. The amount of gene loss in S. himalayana is consistent with the degree of reduction in its body plan. HGT events are common among endoparasites and play an important role in their lifestyle adaptation.

Background: Cottonseed oil is a promising edible plant oil with abundant unsaturated fatty acids. However, few studies have been conducted to explore the characteristics of cottonseed oil. The molecular mechanism of cottonseed oil accumulation remains unclear. Results: In the present study, we conducted comparative transcriptome and weighted gene co-expression network (WGCNA) analysis for two G. hirsutum materials with significant difference in cottonseed oil content. Results showed that, between the high oil genotype 6053 (H6053) and the low oil genotype 2052 (L2052), a total of 412, 507, 1,121, 1,953, and 2,019 differentially expressed genes (DEGs) were detected at 10, 15, 20, 25, and 30 DPA, respectively. Remarkably, a large number of the down-regulated DEGs were enriched in the phenylalanine metabolic processes. Investigation into the dynamic changes of expression profiling of genes associated with both phenylalanine metabolism and oil biosynthesis has shed light on a significant competitive relationship in substrate allocation during cottonseed development. Additionally, the WGCNA analysis of all DEGs identified eight distinct modules, one of which includes GhPXN1, a gene closely associated with oil accumulation. Through phylogenetic analysis, we hypothesized that GhPXN1 in G. hirsutum might have been introgressed from G. arboreum. Overexpression of the GhPXN1 gene in tobacco leaf suggested a significant reduction in oil content compared to the empty-vector transformants. Furthermore, ten other crucial oil candidate genes identified in this study were also validated using quantitative real-time PCR (qRT-PCR). Conclusions: Overall, this study enhances our comprehension of the molecular mechanisms underlying cottonseed oil accumulation. [ABSTRACT FROM AUTHOR]

Linolenic acid (LA) has poor oxidative stability since it is a polyunsaturated fatty acid. Soybean oil has a high LA content and thus has poor oxidative stability. To identify candidate genes that affect the linolenic acid (LA) content in soybean seeds, a genome-wide association study (GWAS) was performed with 1,060 soybean cultivars collected in China between 2019-2021 and which LA content was measured using matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF IMS). A candidate gene, GmWRI14, encoding an APETALA2 (AP2)-type transcription factor, was detected by GWAS in cultivars from all three study years. Multiple sequence alignments showed that GmWRI14 belongs to the plant WRI1 family. The fatty acid contents of different soybean lines were evaluated in transgenic lines with a copy of GmWRI14, control lines without GmWRI14, and the gmwri14 mutant. MALDI-TOF IMS revealed that GmWRI14 transgenic soybeans had a lower LA content with a significant effect on seed size and shape, whereas gmwri14 mutants had a higher LA content. compared to control. The RNA-seq results showed that GmWRI14 suppresses GmFAD3s (GmFAD3B and GmFAD3C) and GmbZIP54 expression in soybean seeds, leading to decreased LA content. Based on the RNA-seq data, yeast one-hybrid (Y1H) and qRT-PCR were performed to confirm the transcriptional regulation of FAD3s by GmWRI14. Our results suggest that FAD3 is indirectly regulated by GmWRI14, representing a new molecular mechanism of fatty acid biosynthesis, in which GmWRI14 regulates LA content in soybean seeds. [ABSTRACT FROM AUTHOR]

Perilla is a key component of Korean food. It contains several plant-specialized metabolites that provide medical benefits. In response to an increased interest in healthy supplement food from the public, people are focusing on the properties of Perilla. Nevertheless, unlike rice and soybeans, there are few studies based on molecular genetics on Perilla, so it is difficult to systematically study the molecular breed. The wild Perilla, Perilla citriodora 'Jeju17', was identified a decade ago on the Korean island of Jeju. Using short-reads, long-reads, and Hi-C, a chromosome-scale genome spanning 676 Mbp, with high contiguity, was assembled. Aligning the 'Jeju17' genome to the 'PC002' Chinese species revealed significant collinearity with respect to the total length. A total of 31,769 coding sequences were predicted, among which 3331 were 'Jeju17'-specific. Gene enrichment of the species-specific gene repertoire highlighted environment adaptation, fatty acid metabolism, and plant-specialized metabolite biosynthesis. Using a homology-based approach, genes involved in fatty acid and lipid triacylglycerol biosynthesis were identified. A total of 22 fatty acid desaturases were found and comprehensively characterized. Expression of the FAD genes in 'Jeju17' was examined at the seed level, and hormone signaling factors were identified. The results showed that the expression of FAD genes in 'Jeju17' at the seed level was high 25 days after flowering, and their responses of hormones and stress were mainly associated with hormone signal transduction and abiotic stress via cis-elements patterns. This study presents a chromosome-level genome assembly of P. citriodora 'Jeju17', the first wild Perilla to be sequenced from the Korean island of Jeju. The analyses provided can be useful in designing ALA-enhanced Perilla genotypes in the future. [ABSTRACT FROM AUTHOR]

Background: Fatty acid (FA) is an important platform compound for the further synthesis of high‐value biofuels and oleochemicals, but chemical synthesis of FA has many limitations. One way to meet the future demand for FA could be to use microbial cell factories for FA biosynthesis. Results: Thioesterase (TE; TesA, TesB, and TE9) of Corynebacterium glutamicum (CG) can potentially improve FA biosynthesis, and tesA, tesB, and te9 were overexpressed in C. glutamicum and Escherichia coli (EC), respectively, in this study. The results showed that the total fatty acid (TFA) production of CGtesB and ECtesB significantly increased to 180.52 mg/g dry cell weight (DCW) and 123.52 mg/g DCW, respectively (P < 0.05). Overexpression strains CG and EC could increase the production of C16:0, C18:1(t), C18:2, C20:1, C16:1, C18:0, and C18:1(c) (P < 0.05), respectively, and the changes of long-chain FA resulted in the enhancement of TFA production. The enzymatic properties of TesA, TesB, and TE9 in vitro were determined: they were specific for long-, broad and short-chain substrates, respectively; the optimal temperature was 30.0 °C and the optimal acid–base (pH) were 8.0, 8.0, and 9.0, respectively; they were inhibited by Fe2+, Cu2+, Zn2+, Mg2+, and K+. Conclusion: Overexpression TE enhances and modifies FA biosynthesis with multiple productive applications, and the enzyme properties provided useful clues for optimizing FA synthesis. [ABSTRACT FROM AUTHOR]

Pecan (Carya illinoinensis (Wagenh.) K. Koch) is an important oilseed nut and is rich in fatty acids (FAs) and flavonols. Pecan FA has significantly edible, industrial and clinical value. To investigate the dynamic patterns and compositions of FA, and the molecular mechanism that controls FA accumulation in pecan, lipidomic and transcriptomic analyses were performed to determine lipid profiles and gene expression in pecan's FA biosynthesis pathway. In the present study, compared with cultivars 'Caddo' and 'Y-01', 'Mahan' formed larger and heavier embryos and accumulated higher oil content. Lipidomic analysis showed that FA and (O-acyl)-1-hydroxy FA contents were higher in 'Mahan' at the mature stage. Based on full-length and comparative RNA-Seq, differential expression gene enrichment analysis revealed that many functional genes participated in the pathways of 'fatty acid biosynthesis', 'fatty acid metabolism' and 'linoleic acid metabolism'. High FA accumulation model from 'Mahan' demonstrated that key enzyme-encoding genes played an important role in regulating FA biosynthesis. Co-expression module analysis indicated that several transcription factors (TFs; MYB, TCP, bHLH, Dof, ERF, NAC) were involved in FA accumulation by regulating the expression of functional genes, and real-time quantitative PCR verification proved that these TFs had a high correlation with the pecan FA accumulation pattern. These findings provided an insight into the molecular mechanism of FA accumulation in C. illinoinensis embryo, which contributes to pecan oil yielding and pecan molecular breeding. [ABSTRACT FROM AUTHOR]

SUMMARY: Increasing concentration of CO2 has significant impacts on many biological processes in plants, and its impact is closely associated with changes in the ratio of photosynthesis to photorespiration. Studies have reported that high CO2 can promote carbon fixing and alleviate plant oxidative damage in response to environmental stresses. However, the effect of high CO2 on fatty acid (FA) metabolism and cellular redox balance in FA‐deficient plants is rarely reported. In this study, we identified a high‐CO2‐requiring mutant cac2 through forward genetic screening. CAC2 encodes biotin carboxylase, which is one of the subunits of plastid acetyl‐CoA carboxylase and participates in de novo FA biosynthesis. Null mutation of CAC2 is embryonic lethal. A point mutation of CAC2 in cac2 mutants produces severe defects in chloroplast development, plant growth and photosynthetic performance. These morphological and physiological defects were largely absent under high CO2 conditions. Metabolite analyses showed that FA contents in cac2‐1 leaves were decreased, while photorespiratory metabolites, such as glycine and glycolate, did not significantly change. Meanwhile, cac2 exhibited higher reactive oxygen species (ROS) levels and mRNA expression of stress‐responsive genes than the wild‐type, indicating that cac2 plants may suffer oxidative stress under ambient CO2 conditions. Elevated CO2 significantly increased FA contents, especially C18:3‐FA, and reduced ROS accumulation in cac2‐1 leaves. We propose that stress mitigation by high CO2 in cac2 could be due to increased FA levels by promoting carbon assimilation, and the prevention of over‐reduction due to decreased photorespiration. Significance Statement: Two novel mutant alleles of CAC2 were identified in this study, which reduced FA levels and increased ROS accumulation, and required high concentrations of CO2 to maintain normal growth. The results demonstrate that high CO2 can facilitate FA biosynthesis and cellular redox homeostasis in FA‐deficient plants. [ABSTRACT FROM AUTHOR]

Abstract Background WRINKLED1 (WRI1) encodes a transcription factor, belonging to the APETALA2 (AP2) family, and plays a key role in regulating plant oil biosynthesis. As a newly woody oil crop, tree peony (Paeonia rockii) was notable for the abundant unsaturated fatty acids in its seed oil. However, the role of WRI1 during the accumulation of P. rockii seeds oil remains largely unknown. Results In this study, a new member of the WRI1 family was isolated from P. rockii and was named PrWRI1. The ORF of PrWRI1 consisted of 1269 nucleotides, encoding a putative protein of 422 amino acids, and was highly expressed in immature seeds. Subcellular localization analysis in onion inner epidermal cells showed that PrWRI1 was located at the nucleolus. Ectopic overexpression of PrWRI1 could significantly increase the total fatty acid content in Nicotiana benthamiana leaf tissue and even PUFAs in transgenic Arabidopsis thaliana seeds. Furthermore, the transcript levels of most genes related to fatty acids (FA) synthesis and triacylglycerol (TAG) assembly were also up-regulated in transgenic Arabidopsis seeds. Conclusions Together, PrWRI1 could push carbon flow to FA biosynthesis and further enhance the TAG amount in seeds with a high proportion of PUFAs.

Camellia oil is valuable as an edible oil and serves as a base material for a range of high-value products. Camellia plants of significant economic importance, such as Camellia sinensis and Camellia oleifera, have been classified into sect. Thea and sect. Oleifera, respectively. Fatty acid desaturases play a crucial role in catalyzing the formation of double bonds at specific positions of fatty acid chains, leading to the production of unsaturated fatty acids and contributing to lipid synthesis. Comparative genomics results have revealed that expanded gene families in oil tea are enriched in functions related to lipid, fatty acid, and seed processes. To explore the function of the FAD gene family, a total of 82 FAD genes were identified in tea and oil tea. Transcriptome data showed the differential expression of the FAD gene family in mature seeds of tea tree and oil tea tree. Furthermore, the structural analysis and clustering of FAD proteins provided insights for the further exploration of the function of the FAD gene family and its role in lipid synthesis. Overall, these findings shed light on the role of the FAD gene family in Camellia plants and their involvement in lipid metabolism, as well as provide a reference for understanding their function in oil synthesis.

The utilization of natural products in food preservation represents a promising strategy for the dual benefits of controlling foodborne pathogens and enhancing the nutritional properties of foods. Among the phytonutrients, flavonoids have been shown to exert antibacterial effects by disrupting bacterial cell membrane functionality; however, the underlying molecular mechanisms remain elusive. In this study, we investigated the effect of quercetin on the cell membrane permeability of Staphylococcus aureus ATCC 27217. A combined metabolomic and transcriptomic approach was adopted to examine the regulatory mechanism of quercetin with respect to the fatty acid composition and associated genes. Kinetic analysis and molecular docking simulations were conducted to assess quercetin’s inhibition of β-ketoacyl-acyl carrier protein reductase (FabG), a potential target in the bacterial fatty acid biosynthesis pathway. Metabolomic and transcriptomic results showed that quercetin increased the ratio of unsaturated to saturated fatty acids and the levels of membrane phospholipids. The bacteria reacted to quercetin-induced stress by attempting to enhance fatty acid biosynthesis; however, quercetin directly inhibited FabG activity, thereby disrupting bacterial fatty acid biosynthesis. These findings provide new insights into the mechanism of quercetin’s effects on bacterial cell membranes and suggest potential applications for quercetin in bacterial inhibition.

Drought stress is one of the most important environmental stresses that severely limits the growth and yield of Canola. The re-watering can compensate for the damage caused by drought stress. Investigation of protein's interaction of genes involved in important drought-responsive pathways and their regulatory network by microRNAs (miRNAs) under drought and re-watering conditions are helpful approaches to discovering drought-stress tolerance and recovery mechanisms. In this study, the protein's interaction and functional enrichment analyses of glycolysis, pentose phosphate, glyoxylate cycle, fatty acid biosynthesis, heat shock factor main genes, and the regulatory network of key genes by miRNAs were investigated by in silico analysis. Then, the relative expression of key genes and their related miRNAs were investigated in tolerant and susceptible genotypes of Canola under drought and re-watering conditions by Real-time PCR technique. The bna-miR156b/c/g, bna-miR395d/e/f, bna-miR396a, and all the studied key genes except HSFA1E and PK showed changes in expression levels in one or both genotypes after re-watering. The PPC1 and HSFB2B expression decreased, whereas the MLS and CAC3 expression increased in both genotypes under re-watering treatment after drought stress. It could cause the regulation of oxaloacetate production, the increase of the glyoxylate cycle, lipid biosynthesis, and the reduction of the negative regulation of HSFs under re-watering conditions. It seems that PPC1, G6PD2, MLS, CAC3, and HSFB2B were involved in the recovery mechanisms after drought stress of Canola. They were regulated by drought-responsive miRNAs to respond appropriately to drought stress. Therefore, regulating these genes could be important in plant recovery mechanisms. [ABSTRACT FROM AUTHOR]

Pepper is an important condiment, and its aroma affects its commercial value. In this study, transcriptome sequencing and combined headspace solid-phase microextraction and gas chromatography–mass spectrometry (HS-SPME-GC-MS) were used to analyze the differentially expressed genes and volatile organic compounds in spicy and non-spicy pepper fruits. Compared with non-spicy fruits, there were 27 up-regulated volatile organic compounds (VOCs) and 3353 up-regulated genes (Up-DEGs) in spicy fruits. The results of KEGG enrichment analysis of the Up-DEGs combined with differential VOCs analysis showed that fatty acid biosynthesis and terpenoid biosynthesis may be the main metabolic pathways for aroma differences between non-spicy and spicy pepper fruits. The expression levels of the fatty acid biosynthesis-related genes FAD, LOX1, LOX5, HPL, and ADH and the key terpene synthesis gene TPS in spicy pepper fruits were significantly higher than those in non-spicy pepper fruits. The differential expression of these genes may be the reason for the different aroma. The results can provide reference for the development and utilization of high-aroma pepper germplasm resources and the breeding of new varieties. [ABSTRACT FROM AUTHOR]

The 3-ketoacyl-ACP synthase III (FabH), a key enzyme for bacteria growth, catalyses the last step of the initiation of bacterial fatty acid synthesis. Rice bacterial blight is caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo), which is widely studied as a model bacterium. Bioinformatics analysis showed that the X. oryzae pv. oryzae PXO99A genome encodes three FabH homologous proteins with unknown functions. In this study, we found that only PXO_02706 (fabH1) encodes a functional FabH, the key enzyme in the production of branched-chain fatty acid, which is essential for the branched-chain diffusible signal factor family signals in Xoo. Interestingly, we found that FabH1 is not essential for fatty acid biosynthesis in Xoo. Pathogenicity analysis showed that loss of fabH1 caused a significant decrease in virulence of Xoo. Genetic and phenotypic analyses revealed that fabH1 plays a key role in multiple Xoo virulence-related activities, including exopolysaccharide (EPS) production, biofilm formation, motility, and resistance to environmental stresses. [ABSTRACT FROM AUTHOR]

Background: Sapria himalayana (Rafflesiaceae) is an endoparasitic plant characterized by a greatly reduced vegetative body and giant flowers; however, the mechanisms underlying its special lifestyle and greatly altered plant form remain unknown. To illustrate the evolution and adaptation of S. himalayasna, we report its de novo assembled genome and key insights into the molecular basis of its floral development, flowering time, fatty acid biosynthesis, and defense responses. Results: The genome of S. himalayana is ~ 1.92 Gb with 13,670 protein-coding genes, indicating remarkable gene loss (~ 54%), especially genes involved in photosynthesis, plant body, nutrients, and defense response. Genes specifying floral organ identity and controlling organ size were identified in S. himalayana and Rafflesia cantleyi, and showed analogous spatiotemporal expression patterns in both plant species. Although the plastid genome had been lost, plastids likely biosynthesize essential fatty acids and amino acids (aromatic amino acids and lysine). A set of credible and functional horizontal gene transfer (HGT) events (involving genes and mRNAs) were identified in the nuclear and mitochondrial genomes of S. himalayana, most of which were under purifying selection. Convergent HGTs in Cuscuta, Orobanchaceae, and S. himalayana were mainly expressed at the parasite–host interface. Together, these results suggest that HGTs act as a bridge between the parasite and host, assisting the parasite in acquiring nutrients from the host. Conclusions: Our results provide new insights into the flower development process and endoparasitic lifestyle of Rafflesiaceae plants. The amount of gene loss in S. himalayana is consistent with the degree of reduction in its body plan. HGT events are common among endoparasites and play an important role in their lifestyle adaptation. [ABSTRACT FROM AUTHOR]

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.

The mitochondrial isoforms VA/VB of metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) are involved in metabolic processes, such as de novo lipogenesis and fatty acid biosynthesis. We review the drug design landscape for obtaining CA VA/VB-selective/effective inhibitors, starting from the clinical observations that CA inhibitory drugs, such as the antiepileptics topiramate and zonisamide, or the diuretic acetazolamide induce a significant weight loss. The main approaches for designing such compounds consisted in drug repurposing of already known CA inhibitors (CAIs); screening of synthetic/natural products libraries both in the classical and virtual modes, and de novo drug design using the tail approach. A number of such studies allowed the identification of lead compounds diverse from sulphonamides, such as tropolones, phenols, polyphenols, flavones, glycosides, fludarabine, lenvatinib, rufinamide, etc., for which the binding mode to the enzyme is not always well understood. Classical drug design studies of sulphonamides, sulfamates and sulfamides afforded low nanomolar mitochondrial CA-selective inhibitors, but detailed antiobesity studies were poorly performed with most of them. A breakthrough in the field may be constituted by the design of hybrids incorporating CAIs and other antiobesity chemotypes.

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L−1 resveratrol from supplemented p -coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas. [Display omitted] • Pseudomonas taiwanensis has a high tolerance towards the stilbenoid pinosylvin. • Heterologous synthesis of the polyketides pinosylvin, resveratrol and flaviolin. • Drain in fatty acid biosynthesis limits malonyl-CoA availability in Pseudomonas. • Engineered P. taiwanensis GRC3Δ6 MC-III platform for secondary metabolite synthesis. [ABSTRACT FROM AUTHOR]

The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by heterologous hosts is expression of a selective acyl-acyl carrier protein (ACP) thioesterase. However, few of these enzymes can generate a precise (greater than 90% of a desired chain-length) product distribution when expressed in a microbial or plant host. The presence of alternative chain-lengths can complicate purification in situations where blends of fatty acids are not desired. We report the assessment of several strategies for improving the dodecanoyl-ACP thioesterase from the California bay laurel to exhibit more selective production of medium-chain free fatty acids to near exclusivity. We demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was an effective library screening technique for identification of thioesterase variants with favorable shifts in chain-length specificity. This strategy proved to be a more effective screening technique than several rational approaches discussed herein. With this data, we isolated four thioesterase variants which exhibited a more selective FFA distribution over wildtype when expressed in the fatty acid accumulating E. coli strain, RL08. We then combined mutations from the MALDI isolates to generate BTE-MMD19, a thioesterase variant capable of producing free fatty acids consisting of 90% of C 12 products. Of the four mutations which conferred a specificity shift, we noted that three affected the shape of the binding pocket, while one occurred on the positively charged acyl carrier protein landing pad. Finally, we fused the maltose binding protein (MBP) from E. coli to the N – terminus of BTE-MMD19 to improve enzyme solubility and achieve a titer of 1.9 g per L of twelve-carbon fatty acids in a shake flask. Graphical abstract. [Display omitted] • Applied MALDI-ToF screening to identify 4 mutations beneficial to enhancing the 12-carbon selectivity of BTE in E. coli. • Met goal of 90% selectivity toward C 12 products in FFA pool via expression of M133L/M222K/R227H variant in E. coli. • Demonstrated a 31% improvement in titer via fusion of solubility tag to engineered BTE variant (1.9 g per L of C 12 FFA). [ABSTRACT FROM AUTHOR]

Background: WRINKLED1 (WRI1) encodes a transcription factor, belonging to the APETALA2 (AP2) family, and plays a key role in regulating plant oil biosynthesis. As a newly woody oil crop, tree peony (Paeonia rockii) was notable for the abundant unsaturated fatty acids in its seed oil. However, the role of WRI1 during the accumulation of P. rockii seeds oil remains largely unknown. Results: In this study, a new member of the WRI1 family was isolated from P. rockii and was named PrWRI1. The ORF of PrWRI1 consisted of 1269 nucleotides, encoding a putative protein of 422 amino acids, and was highly expressed in immature seeds. Subcellular localization analysis in onion inner epidermal cells showed that PrWRI1 was located at the nucleolus. Ectopic overexpression of PrWRI1 could significantly increase the total fatty acid content in Nicotiana benthamiana leaf tissue and even PUFAs in transgenic Arabidopsis thaliana seeds. Furthermore, the transcript levels of most genes related to fatty acids (FA) synthesis and triacylglycerol (TAG) assembly were also up-regulated in transgenic Arabidopsis seeds. Conclusions: Together, PrWRI1 could push carbon flow to FA biosynthesis and further enhance the TAG amount in seeds with a high proportion of PUFAs. [ABSTRACT FROM AUTHOR]

Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a m alonyl- A CP d ecarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications. • P. putida KT2440 harbors three pathways to initiate fatty acid biosynthesis. • FabH1 and FabH2, are KASIII enzymes with differing specificity to acyl-CoAs. • The third pathway proceeds through MadB-catalyzed decarboxylation of malonyl-ACP. • MadB catalyzes its reaction by stabilizing the C3-carbonyl moiety of the substrate. • Functional homologs of MadB are prevalent in the domain Bacteria. [ABSTRACT FROM AUTHOR]

Background: Sustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotrichosporon oleaginosus showed the highest lipid productivity. Moreover, a new lipid production process for C. oleaginosus with minimal waste generation and energy consumption resulted in the highest lipid productivity in the history of oleaginous yeasts. However, productivity and product diversity are restricted because of the genetic intractability of this yeast. To date, successful targeted genetic engineering of C. oleaginosus has not yet been reported. Results: The targeted gene editing was successfully carried out in C. oleaginosus using CRISPR/Cas system. A tailored enzyme system isolated to degrade the C. oleaginosus cell wall enabled the isolation of viable spheroplasts that are amenable to in-cell delivery of nucleic acids and proteins. The employment of both Cas9 protein and Cas mRNA was effective in obtaining strains with URA5 knockout that did not exhibit growth in the absence of uracil. Subsequently, we successfully created several strains with enhanced lipid yield (54% increase compared to that in wild type) or modified fatty acid profiles comparable with those of cocoa butter or sunflower oil compositions. Conclusion: This study establishes the first targeted engineering technique for C. oleaginosus using the CRISPR/Cas system. The current study creates the foundation for flexible and targeted strain optimizations towards building a robust platform for sustainable microbial lipid production. Moreover, the genetic transformation of eukaryotic microbial cells using Cas9 mRNA was successfully achieved. [ABSTRACT FROM AUTHOR]

Clustering enzymes in the same metabolic pathway is a natural strategy to enhance productivity. Synthetic protein, RNA and DNA scaffolds have been designed to artificially cluster multiple enzymes in the cell, which require complex construction processes and possess limited slots for target enzymes. We utilized the Escherichia coli inner cell membrane as a native scaffold to cluster four fatty acid synthases (FAS) and achieved to improve the efficiency of fatty acid synthesis in vivo. The construction strategy is as simple as fusing target enzymes to the N‐terminus or C‐terminus of the membrane anchor protein (Lgt), and the number of anchored enzymes is not restricted. This novel device not only presents a similar efficiency in clustering multiple enzymes to that of other artificial scaffolds but also promotes the product secretion, driving the entire metabolic flux forward and further increasing the gross yield compared with that in a cytoplasmic scaffold system. [ABSTRACT FROM AUTHOR]

Introduction: Potato tubers have the healing capacity to prevent surface water transpiration and pathogen invasion after mechanical damage. Previous research has shown the inability to form healing periderm in potatoes under low temperatures, but the potential mechanism is still unclear. Methods: To explore the effects and mechanisms of low-temperature potato healing, wounded potatoes were stored at low temperature (4°C) and room temperature (22°C), respectively. Results: In this study, compared with 22°C healing, low temperature reduced the content of hydrogen peroxide, and the down-regulation of StAMY23 inhibited the conversion of starch to sugar, alleviated the degradation of starch, and reduced the content of soluble sugars and sucrose. Meanwhile, inhibition of phenylalanine metabolism by suppression of StPAL1 and St4CL expression reduced lignin accumulation. Low temperature also down-regulated the expression of StKCS6, StFAOH, StGPAT5, and StPrx, causing the lower deposition amount of suberin in wounds of potato tubers. Discussion: The above results suggested that low temperature led to less wound tissue deposition at the wound surfaces via suppressing phenylpropanoid metabolism and fatty acid biosynthesis in potato tubers. [ABSTRACT FROM AUTHOR]

Ladderane lipids (found in the membranes of anaerobic ammonium-oxidizing [anammox] bacteria) have unique ladder-like hydrophobic groups, and their highly strained exotic structure has attracted the attention of scientists. Although enzymes encoded in type II fatty acid biosynthesis (FASII) gene clusters in anammox bacteria, such as S-adenosyl-l-methionine (SAM)-dependent enzymes, have been proposed to construct a ladder-like structure using a substrate connected to acyl carrier protein from anammox bacteria (AmxACP), no experimental evidence to support this hypothesis was reported to date. Here, we report the crystal structure of a SAM-dependent methyltransferase from anammox bacteria (AmxMT1) that has a substrate and active site pocket between a class I SAM methyltransferase-like core domain and an additional α-helix inserted into the core domain. Structural comparisons with homologous SAM-dependent C-methyltransferases in polyketide synthase, AmxACP pull-down assays, AmxACP/AmxMT1 complex structure predictions by AlphaFold, and a substrate docking simulation suggested that a small compound connected to AmxACP could be inserted into the pocket of AmxMT1, and then the enzyme transfers a methyl group from SAM to the substrate to produce branched lipids. Although the enzymes responsible for constructing the ladder-like structure remain unknown, our study, for the first time, supports the hypothesis that biosynthetic intermediates connected to AmxACP are processed by SAM-dependent enzymes, which are not typically involved in the FASII system, to produce the ladder-like structure of ladderane lipids in anammox bacteria. [ABSTRACT FROM AUTHOR]

The oleaginous marine microalga Nannochloropsis oceanica strain IMET1 has attracted increasing attention as a promising photosynthetic cell factory due to its unique excellent capacity to accumulate large amounts of triacylglycerols and eicosapentaenoic acid. To complete the genomic annotation for genes in the fatty acid biosynthesis pathway of N. oceanica, we conducted the present study to identify a novel candidate gene encoding the archetypical chloroplast stromal acyl-acyl carrier protein Δ9 desaturase. The full-length cDNA was generated using rapid-amplification of cDNA ends, and the structure of the coding region interrupted by four introns was determined. The RT-qPCR results demonstrated the upregulated transcriptional abundance of this gene under nitrogen starvation condition. Fluorescence localization studies using EGFP-fused protein revealed that the translated protein was localized in chloroplast stroma. The catalytic activity of the translated protein was characterized by inducible expression in Escherichia coli and a mutant yeast strain BY4389, indicating its potential desaturated capacity for palmitoyl-ACP (C16:0-ACP) and stearoyl-ACP (C18:0-ACP). Further functional complementation assay using BY4839 on plate demonstrated that the expressed enzyme restored the biosynthesis of oleic acid. These results support the desaturated activity of the expressed protein in chloroplast stroma to fulfill the biosynthesis and accumulation of monounsaturated fatty acids in N. oceanica strain IMET1. [ABSTRACT FROM AUTHOR]

The mitochondrial isoforms VA/VB of metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) are involved in metabolic processes, such as de novo lipogenesis and fatty acid biosynthesis. We review the drug design landscape for obtaining CA VA/VB-selective/effective inhibitors, starting from the clinical observations that CA inhibitory drugs, such as the antiepileptics topiramate and zonisamide, or the diuretic acetazolamide induce a significant weight loss. The main approaches for designing such compounds consisted in drug repurposing of already known CA inhibitors (CAIs); screening of synthetic/natural products libraries both in the classical and virtual modes, and de novo drug design using the tail approach. A number of such studies allowed the identification of lead compounds diverse from sulphonamides, such as tropolones, phenols, polyphenols, flavones, glycosides, fludarabine, lenvatinib, rufinamide, etc., for which the binding mode to the enzyme is not always well understood. Classical drug design studies of sulphonamides, sulfamates and sulfamides afforded low nanomolar mitochondrial CA-selective inhibitors, but detailed antiobesity studies were poorly performed with most of them. A breakthrough in the field may be constituted by the design of hybrids incorporating CAIs and other antiobesity chemotypes. [ABSTRACT FROM AUTHOR]