The Biosynthesis Pattern and Transcriptome Analysis of Sapindus saponaria Oil.

The Sapindus saponaria (soapberry) kernel is rich in oil that has antibacterial, anti-inflammatory, and antioxidant properties, promotes cell proliferation, cell migration, and stimulates skin wound-healing effects. S. saponaria oil has excellent lubricating properties and is a high-quality raw material for biodiesel and premium lubricants, showing great potential in industrial and medical applications. Metabolite and transcriptome analysis revealed patterns of oil accumulation and composition and differentially expressed genes (DEGs) during seed development. Morphological observations of soapberry fruits at different developmental stages were conducted, and the oil content and fatty acid composition of the kernels were determined. Transcriptome sequencing was performed on kernels at 70, 100, and 130 days after flowering (DAF). The oil content of soapberry kernels was lowest at 60 DAF (5%) and peaked at 130 DAF (31%). Following soapberry fruit-ripening, the primary fatty acids in the kernels were C18:1 (oleic acid) and C18:3 (linolenic acid), accounting for an average proportion of 62% and 18%, respectively. The average contents of unsaturated fatty acids and saturated fatty acids in the kernel were 86% and 14%, respectively. Through the dynamic changes in fatty acid composition and DEGs analysis of soapberry kernels, FATA, KCR1, ECR, FAD2 and FAD3 were identified as candidate genes contributing to a high proportion of C18:1 and C18:3, while DGAT3 emerged as a key candidate gene for TAG biosynthesis. The combined analysis of transcriptome and metabolism unveiled the molecular mechanism of oil accumulation, leading to the creation of a metabolic pathway pattern diagram for oil biosynthesis in S. saponaria kernels. The study of soapberry fruit development, kernel oil accumulation, and the molecular mechanism of oil biosynthesis holds great significance in increasing oil yield and improving oil quality.

Methodological and Physiological Study during Seed Dormancy Release of Symplocos paniculata.

Symplocos paniculata are reported to exhibit seed dormancy, which impedes its cultivation and widespread adoption. In this study, a comprehensive method was established to overcome seed dormancy by subjecting seeds to scarification in 98% H2SO4 for 10 min, followed by 1000 mg·L-1 GA3 soaking for 48 h and stratification at 4 °C for 100 days. The seed germination percentage has increased significantly, to a peak of 42.67%, though the seeds could not germinate timely by NaOH scarification. Additionally, the dynamic changes of key stored substances (proteins, soluble sugars, starches, and fats), associated enzyme activities (amylases, peroxidase, and catalase), and endogenous hormones (abscisic acid, gibberellic acid, and indole-3-acetic acid) in seeds were investigated. The results demonstrated a continuous degradation of starch and fat in S. paniculata seeds, while the levels of protein and soluble sugar exhibited fluctuations, which probably facilitated seed dormancy breaking through energy supply and transformation. The enzymatic activities underwent rapid changes, accompanied by a gradual decrease in ABA content within the seeds with increasing stratification time. Notably, GA3, GA3/ABA, and (GA3 + IAA)/ABA showed significant increases, indicating their positive regulatory roles in seed germination. This study clarified the dormancy mechanism and established an effective method for the release dormancy of S. paniculata seeds.

The Impact of Oil Type on the Performance of ß-Amyrin-Based Oleogels: Formation, Physicochemical Properties, and Potential Correlation Analysis.

Pentacyclic triterpenes show potential as oleogelators, but their combination with various vegetable oils has limited research. This study selected linseed, rapeseed, sunflower, coconut, and palm oils to combine with the triterpenoid compound ß-amyrin for the preparation of oleogels. The stability, crystal network structure, and other properties of each oleogel were evaluated. The correlation between different oil types and the properties of corresponding oleogels was explored. The results showed that ß-amyrin formed stable oleogels with five vegetable oils under suitable temperature conditions, wherein especially the LO-based oleogel not only exhibited higher oil-binding capacity and hardness, but also demonstrated excellent stability at the microscopic level and notable rheological properties. Further analysis revealed a close correlation between the physicochemical properties of the oleogels and lipid characteristics, indicating that oleogels prepared from long-chain highly unsaturated fatty acids exhibit high stability. The above results indicate that ß-amyrin can be a novel candidate oleogelator and that the oil type can modify the properties of ß-amyrin-based oleogels. This study provides the latest reference for the application of pentacyclic triterpenoids in food.

Metabolic engineering of Yarrowia lipolytica for high-level production of squalene.

Squalene is an important triterpene with a wide range of applications. Given the growing market demand for squalene, the development of microbial cell factories capable of squalene production is considered a sustainable method. This study aimed to investigate the squalene production potential of Yarrowia lipolytica. First, HMG-CoA reductase from Saccharomyces cerevisiae and squalene synthase from Y. lipolytica was co-overexpressed in Y. lipolytica. Second, by enhancing the supply of NADPH in the squalene synthesis pathway, the production of squalene in Y. lipolytica was effectively increased. Furthermore, by constructing an isoprenol utilization pathway and overexpressing YlDGA1, the strain YLSQ9, capable of producing 868.1 mg/L squalene, was obtained. Finally, by optimizing the fermentation conditions, the highest squalene concentration of 1628.2 mg/L (81.0 mg/g DCW) in Y. lipolytica to date was achieved. This study demonstrated the potential for achieving high squalene production using Y. lipolytica.

Transcriptome Analysis Unveiled the Intricate Interplay between Sugar Metabolism and Lipid Biosynthesis in Symplocos paniculate Fruit.

Symplocos paniculate is an oil plant exhibiting tissue-specific variations in oil content and fatty acid composition across the whole fruit (mainly pulp and seed). And its oil synthesis is intricately linked to the accumulation and transformation of sugars. Nevertheless, there remains a dearth of understanding regarding how sugar metabolism impacts oil synthesis in S. paniculate fruit. To unravel the intricate mechanism underlying the impact of sugar metabolism on lipid biosynthesis in S. paniculata fruit, a comparative analysis was conducted on the transcriptome and metabolite content of pulp and seed throughout fruit development. The findings revealed that the impact of sugar metabolism on oil synthesis varied across different stages of fruit development. Notably, during the early fruit developmental stage (from 90 to 120 DAF), pivotal genes involved in sugar metabolism, such as PGK3, PKP1, PDH-E1, MDH, and malQ, along with key genes associated with oil synthesis like KAR, HAD, and PAP were predominantly expressed in the pulp. Consequently, this preferential expression led to earlier accumulation of oil in the pulp tissue compared to the seed. Whereas, during the fruit maturity stage (from 120 DAF to 140 DAF), these genes exhibited a high level of expression in seed, thereby facilitating the rapid and substantial accumulation of seed oil compared to pulp. The sugar metabolism activity in various parts of S. paniculata fruit plays a pivotal role in oil synthesis and is contingent upon the developmental stage. These findings can offer alternative genes for further gene enhancement through molecular biotechnology, thereby augmenting fruit oil yield and altering fatty acid composition.

Elucidation of Geniposide and Crocin Accumulation and Their Biosysnthsis-Related Key Enzymes during Gardenia jasminoides Fruit Growth.

Gardenia jasminoides fruits are extensively grown worldwide, with a large harvest, and its major medicinal ingredients are geniposide and crocins. Research on their accumulation and biosynthsis-related enzymes is rare. In this study, the accumulation of geniposide and crocin of G. jasminoides fruits at different developmental stages were clarified by HPLC. The highest cumulative amount of geniposide was 2.035% during the unripe-fruit period, and the highest content of crocin was 1.098% during the mature-fruit period. Furthermore, transcriptome sequencing was performed. A total of 50 unigenes encoding 4 key enzymes related in geniposide biosynthsis pathways were screened, and 41 unigenes encoding 7 key enzymes in the pathways of crocin were elucidated. It was found that the expression levels of differentially expressed genes of DN67890_c0_g1_i2-encoding GGPS, which is highly related to geniposide biosynthesis, and DN81253_c0_g1_i1-encoding lcyB, DN79477_c0_g1_i2-encoding lcyE, and DN84975_c1_g7_i11-encoding CCD, which are highly related to crocin biosynthesis, were consistent with the accumulation of geniposide and crocin content, respectively. The qRT-PCR results showed that the trends of relative expression were consistent with transcribed genes. This study provides insights for understanding the geniposide and crocin accumulation and biosynthsis during fruit development in G. jasminoides.

Effect and mechanism of edible oil co-digestion on the bioaccessibility and bioavailability of ursolic acid.

Ursolic acid (UA), a pentacyclic triterpenoid, has gained attentions due to its various health-promoting benefits, but exhibits poor bioavailability. This could be enhanced by changing the food matrix of UA in which it is present. In this study, several UA systems were constructed to investigate the bioaccessibility and bioavailability of UA in combination with in vitro simulated digestion and Caco-2 cell models. The results showed that the bioaccessibility of UA was significantly improved after adding rapeseed oil. Caco-2 cell models showed that the UA-oil blend was more advantageous than UA emulsion in total absorption. The results indicate that the location of UA distribution in oil determines the ease of UA release into the mixed micellar phase. This paper brings a new research idea and basis for the design of improving the bioavailability of hydrophobic compounds.

Differential expression analysis of sugar accumulation-related genes during chestnut nut development.

The chestnuts of Castanea mollissima Bl. are an important food crop in China, and have high nutritional content. To understand the pattern of sugar accumulation during chestnut nut development, the related enzyme gene regulatory pathways, and the molecular regulatory mechanisms of chestnut sugar biosynthesis metabolism, two chestnut varieties with different sugar content, namely Chengbu Youzhu (hereinafter referred to as CBYZ) and AnYou No. 1 (hereinafter referred to as AY01), were selected for investigation. Total sugar and starch content, and the activity of enzymes related to sugar accumulation, were measured in the nuts of the two chestnut varieties 10 days after flowering (DAF), 20 DAF, 30 DAF, 40 DAF, 50 DAF, 60 DAF, 70 DAF, 80 DAF, and 90 DAF. Changes in starch, straight-chain starch, and branched-chain starch content, and sucrose-phosphate synthase, soluble starch synthase, and granule-bound starch synthase enzyme activities were consistent with one-another. A total of 24 differentially expressed genes between the two varieties were associated with sugar biosynthesis and metabolism at three key stages (30, 60, and 90 DAF) of sugar accumulation. Further analysis showing upregulation of the expression of starch-related genes, such as ß-amylase, GYS, and INV indicated that these genes were not actively expressed in AY01, resulting in slow accumulation of starch and reduced sugar content. By contrast, the downregulation of the expression of genes, such as PGK and MDH1, indicated that these genes were actively expressed in low-sugar chestnuts, resulting in the rapid fermentation of sugars. A link between gene up- or down-regulation during different developmental stages of chestnut and the effect of their expression on sugar content were established by KEGG pathway enrichment analysis. These findings provide further insights into the mechanism of sugar biosynthesis in chestnuts.

Preparation of Aliphatic Hydroxamic Acid from Litsea cubeba Kernel Oil and Its Application to Flotation of Fe(III)-Activated Wolframite.

Litsea cubeba is a characteristic woody oil resource in Hunan. As a solid waste of woody oil resources, Litsea cubeba kernels are rich in Litsea cubeba kernel oil with a carbon chain length of C10-12 fatty acid. In this work, aliphatic hydroxamic acids (AHAs) with carbon chain lengths of C10-12 were prepared from Litsea cubeba kernel oil via methylation and hydroximation reactions. The adsorption and hydrophobicity mechanism of AHA towards wolframite was explored by contact angle, zeta potential, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The flotation results demonstrated that AHA was a superior collector than the traditional collector such as benzoyl hydroxamic acid (BHA). Zeta potential and contact angle results have shown that AHA was adsorbed on the surface of the Fe(III)-activated wolframite in its anionic form, which significantly improved the surface hydrophobicity of wolframite. FTIR and XPS revealed that AHA was chemically adsorbed on the surface of Fe(III)-activated wolframite in the form of a five-member ring, which made the hydrophobic chain reach into the solution, come in contact with bubbles, and achieve flotation separation.

Characterization and mechanism of seed dormancy in Symplocos paniculata.

Symplocos paniculata is a highly desirable oil species for biodiesel and premium edible oil feedstock. While germplasm preservation and breeding are crucial, the severity of seed dormancy poses a challenge to successful germination. We employed S. paniculata seeds as experimental materials and conducted an investigation into the types and causes of seed dormancy by analyzing the morphology and developmental characteristics of its embryo, exploring the water permeability property of the endocarp, and examining the presence of endogenous inhibitors, aiming to establish a theoretical foundation for overcoming seed dormancy and maximizing germplasm resource utilization. The findings revealed that the seed embryo had matured into a fully developed embryo, and no dormancy in terms of embryo morphology was observed. Upon reaching maturity, the endocarp of seeds undergoes significant lignification, resulting in notable differences in water absorption between cracked and intact seeds. The impermeability of the endocarp is one of the factors contributing to mechanical restriction. The different phases of endosperm extraction exerted varying effects on the germination of Chinese cabbage seeds, with the methanol phase exhibiting the most potent inhibitory effect. The presence of endogenous inhibitors emerged as the primary factor contributing to physiological dormancy in seeds. GC-MS analysis and validation trials revealed that fatty acids and phenolics, including hexadecanoic acid, oxadecanoic acid, and m-cresol, constituted the main types of endogenous inhibitory compounds found within the endosperm. These findings suggest that the seed dormancy in S. paniculata seeds has endocarp mechanical restriction, and the presence of endogenous inhibitors causes physiological dormancy.

GSK1702934A and M085 directly activate TRPC6 via a mechanism of stimulating the extracellular cavity formed by the pore helix and transmembrane helix S6.

Transient receptor potential canonical (TRPC) channels, as important membrane proteins regulating intracellular calcium (Ca2+i) signaling, are involved in a variety of physiological and pathological processes. Activation and regulation of TRPC are more dependent on membrane or intracellular signals. However, how extracellular signals regulate TRPC6 function remains to be further investigated. Here, we suggest that two distinct small molecules, M085 and GSK1702934A, directly activate TRPC6, both through a mechanism of stimulation of extracellular sites formed by the pore helix (PH) and transmembrane (TM) helix S6. In silico docking scanning of TRPC6 identified three extracellular sites that can bind small molecules, of which only mutations on residues of PH and S6 helix significantly reduced the apparent affinity of M085 and GSK1702934A and attenuated the maximal response of TRPC6 to these two chemicals by altering channel gating of TRPC6. Combing metadynamics, molecular dynamics simulations, and mutagenesis, we revealed that W679, E671, E672, and K675 in the PH and N701 and Y704 in the S6 helix constitute an orthosteric site for the recognition of these two agonists. The importance of this site was further confirmed by covalent modification of amino acid residing at the interface of the PH and S6 helix. Given that three structurally distinct agonists M085, GSK1702934A, and AM-0883, act at this site, as well as the occupancy of lipid molecules at this position found in other TRP subfamilies, it is suggested that the cavity formed by the PH and S6 has an important role in the regulation of TRP channel function by extracellular signals.

Rhamnolipid Enhances the Nitrogen Fixation Activity of Azotobacter chroococcum by Influencing Lysine Succinylation.

The enhancement of nitrogen fixation activity of diazotrophs is essential for safe crop production. Lysine succinylation (KSuc) is widely present in eukaryotes and prokaryotes and regulates various biological process. However, knowledge of the extent of KSuc in nitrogen fixation of Azotobacter chroococcum is scarce. In this study, we found that 250 mg/l of rhamnolipid (RL) significantly increased the nitrogen fixation activity of A. chroococcum by 39%, as compared with the control. Real-time quantitative reverse transcription PCR (qRT-PCR) confirmed that RL could remarkably increase the transcript levels of nifA and nifHDK genes. In addition, a global KSuc of A. chroococcum was profiled using a 4D label-free quantitative proteomic approach. In total, 5,008 KSuc sites were identified on 1,376 succinylated proteins. Bioinformatics analysis showed that the addition of RL influence on the KSuc level, and the succinylated proteins were involved in various metabolic processes, particularly enriched in oxidative phosphorylation, tricarboxylic acid cycle (TCA) cycle, and nitrogen metabolism. Meanwhile, multiple succinylation sites on MoFe protein (NifDK) may influence nitrogenase activity. These results would provide an experimental basis for the regulation of biological nitrogen fixation with KSuc and shed new light on the mechanistic study of nitrogen fixation.

A conserved residue in the P2X4 receptor has a nonconserved function in ATP recognition.

Highly conserved amino acids are generally anticipated to have similar functions across a protein superfamily, including that of the P2X ion channels, which are gated by extracellular ATP. However, whether and how these functions are conserved becomes less clear when neighboring amino acids are not conserved. Here, we investigate one such case, focused on the highly conserved residue from P2X4, E118 (rat P2X4 numbering, rP2X4), a P2X subtype associated with human neuropathic pain. When we compared the crystal structures of P2X4 with those of other P2X subtypes, including P2X3, P2X7, and AmP2X, we observed a slightly altered side-chain orientation of E118. We used protein chimeras, double-mutant cycle analysis, and molecular modeling to reveal that E118 forms specific contacts with amino acids in the "beak" region, which facilitates ATP binding to rP2X4. These contacts are not present in other subtypes because of sequence variance in the beak region, resulting in decoupling of this conserved residue from ATP recognition and/or channel gating of P2X receptors. Our study provides an example of a conserved residue with a specific role in functional proteins enabled by adjacent nonconserved residues. The unique role established by the E118-beak region contact provides a blueprint for the development of subtype-specific inhibitors of P2X4.

Metabolic engineering of Yarrowia lipolytica for improving squalene production.

The aim of this present research is to enhance the squalene production in Yarrowia lipolytica using pathway engineering and bioprocess engineering. Firstly, to improve the production of squalene, the endogenous HMG-CoA reductase (HMG1) was overexpressed in Y. lipolytica to yield 208.88 mg/L squalene. Secondly, the HMG1 and diacylglycerol acyltranferase (DGA1) were co-overexpressed, the derived recombinant Y. lipolytica SQ-1 strain produced 439.14 mg/L of squalene. Thirdly, by optimizing the fermentation medium, the improved titer of squalene with 514.34 mg/L was obtained by the engineered strain SQ-1 grown on YPD-80 medium. Finally, by optimizing the addition concentrations of acetate, citrate and terbinafine, the 731.18 mg/L squalene was produced in the engineered strain SQ-1 with the addition of 0.5 mg/L terbinafine. This work describes the highest reported squalene titer in Y. lipolytica to date. This study will provide the foundation for further engineering Y. lipolytica capable of cost-efficiently producing squalene.

Essential Oil Extracted from Cymbopogon citronella Leaves by Supercritical Carbon Dioxide: Antioxidant and Antimicrobial Activities.

To improve essential oil quality, especially to reserve the thermal instability of compounds, supercritical CO2 extraction (SFE) was applied to recover essential oil from Cymbopogon citronella leaves. A response surface methodology was applied to optimize the extraction process. The highest essential oil yield was predicted at extraction time 120 min, extraction pressure 25 MPa, extraction temperature 35°C, and CO2 flow 18 L/h for the SFE processing. Under these experimental conditions, the mean essential oil yield is 4.40%. In addition, the chemical compositions of SFE were compared with those obtained by hydrodistillation extraction (HD). There were 41 compounds obtained of SFE, while 35 compounds of HD. Alcohols and aldehydes were the main compositions in the essential oils. Furthermore, the antioxidant activities and antimicrobial of essential oils obtained by HD and the evaluated condition of SFE were compared. Results showed that the antioxidant activities of SFE oil are better than those of HD. Minimum inhibitory concentrations (MICs) were determined by the microdilution method. Essential oil obtained from SFE and HD exhibited a significant antimicrobial activity against all tested microorganisms. It is confirmed that the SFE method can be an alternative processing method to extract essential oils from Cymbopogon citronella leaves.

Complete Genome Sequence of Enterobacter sp. Strain ODB01, a Bacterium That Degrades Crude Oil.

Enterobacter sp. strain ODB01, which was isolated from the Changqing oil field, can degrade crude oil efficiently and use crude oil as its sole source of carbon and energy. We report the complete genome sequence of ODB01. The results promote its application in the remediation of petroleum contaminants.

Transcriptome analysis revealed the dynamic oil accumulation in Symplocos paniculata fruit.

BACKGROUND: Symplocos paniculata, asiatic sweetleaf or sapphire berry, is a widespread shrub or small tree from Symplocaceae with high oil content and excellent fatty acid composition in fruit. It has been used as feedstocks for biodiesel and cooking oil production in China. Little transcriptome information is available on the regulatory molecular mechanism of oil accumulation at different fruit development stages. RESULTS: The transcriptome at four different stages of fruit development (10, 80,140, and 170 days after flowering) of S. paniculata were analyzed. Approximately 28 million high quality clean reads were generated. These reads were trimmed and assembled into 182,904 non-redundant putative transcripts with a mean length of 592.91 bp and N50 length of 785 bp, respectively. Based on the functional annotation through Basic Local Alignment Search Tool (BLAST) with public protein database, the key enzymes involved in lipid metabolism were identified, and a schematic diagram of the pathway and temporal expression patterns of lipid metabolism was established. About 13,939 differentially expressed unigenes (DEGs) were screened out using differentially expressed sequencing (DESeq) method. The transcriptional regulatory patterns of the identified enzymes were highly related to the dynamic oil accumulation along with the fruit development of S. paniculata. In addition, quantitative real-time PCR (qRT-PCR) of six vital genes was significantly correlated with DESeq data. CONCLUSIONS: The transcriptome sequences obtained and deposited in NCBI would enrich the public database and provide an unprecedented resource for the discovery of the genes associated with lipid metabolism pathway in S. paniculata. Results in this study will lay the foundation for exploring transcriptional regulatory profiles, elucidating molecular regulatory mechanisms, and accelerating genetic engineering process to improve the yield and quality of seed oil of S. paniculata.

[Advances in the study of plant microRNAs].

Plant microRNAs (miRNAs) are single-stranded RNA molecules of around 22 nucleotides (nt) in length that are associated with the RNA-induced silencing complex (RISC). They act as post-transcriptional negative regulators of gene expression mainly by guiding cleavage or attenuating the translation of target transcripts. The targets of plant miRNAs often belong to transcription factors families involved in the control of developmental processes and defense responses. In the present paper, we reviewed the recent advances in our understanding of the biogenesis and mechanism of action of plant miRNAs, as well as the regulatory roles in plants.