acetyl-CoA C-acetyltransferase Research Articles

PLS-based integration of metabolomics and transcriptomics to reveal the effect of different growth years on Eurycoma longifolia roots and hairy root cultures

Growth years are important factors that affect the accumulation of secondary metabolites in Eurycoma longifolia roots. This study aimed to investigate the effect of different growth years on the metabolites and gene expression of E. longifolia roots and hairy root cultures. To achieve this, metabolite profiles and candidate transcripts of terpenoid backbone biosynthesis and sesquiterpenoid and triterpenoid pathways of 1- and 11-year old E. longifolia roots and hairy root cultures were obtained and integrated using partial least squares (PLS). Integration of eighty-two metabolites and forty-five selected candidate genes using PLS successfully differentiated 1-, 11-year-old E. longifolia roots and hairy root cultures, indicating significant metabolite-gene expression differences in response to different growth years and root types. Amino acids, 2-C-methyl-D-erythritol-4-phosphate cytidylyl-transferase (MCT) and acetyl-CoA C-acetyltransferase (AACT) were responsible for the discrimination of 1-year-old E. longifolia root. Quassinoids of eurycomanone and eurycomalactone and genes linked to the upstream synthesis in mevalonate (MVA) pathway contributed to the discrimination of the11-year-old E. longifolia root. 11-hydroxycanthin-6-one and genes that participate in the methylerythritol phosphate (MEP) pathway upstream of terpenoid biosynthesis were responsible for the discrimination of E. longifolia hairy root cultures.

Cereal leaf beetle-associated bacteria enhance the survival of their host upon insecticide treatments and respond differently to insecticides with different modes of action.

The cereal leaf beetle (CLB, Oulema melanopus) is one of the major cereal pests. The effect of insecticides belonging to different chemical classes, with different mechanisms of action and the active substances' concentrations on the CLB bacterial microbiome, was investigated. Targeted metagenomic analysis of the V3-V4 regions of the 16S ribosomal gene was used to determine the composition of the CLB bacterial microbiome. Each of the insecticides caused a decrease in the abundance of bacteria of the genus Pantoea, and an increase in the abundance of bacteria of the genus Stenotrophomonas, Acinetobacter, compared to untreated insects. After cypermethrin application, a decrease in the relative abundance of bacteria of the genus Pseudomonas was noted. The dominant bacterial genera in cypermethrin-treated larvae were Lactococcus, Pantoea, while in insects exposed to chlorpyrifos or flonicamid it was Pseudomonas. Insecticide-treated larvae were characterized, on average, by higher biodiversity and richness of bacterial genera, compared to untreated insects. The depletion of CLB-associated bacteria resulted in a decrease in larval survival, especially after cypermethrin and chlorpyrifos treatments. The use of a metagenome-based functional prediction approach revealed a higher predicted function of bacterial acetyl-CoA C-acetyltransferase in flonicamid and chlorpyrifos-treated larvae and tRNA dimethyltransferase in cypermethrin-treated insects than in untreated insects.

The Ginkgo biloba microRNA160-ERF4 module participates in terpene trilactone biosynthesis.

Terpene trilactones (TTLs) are important secondary metabolites in ginkgo (Ginkgo biloba); however, their biosynthesis gene regulatory network remains unclear. Here, we isolated a G. biloba ethylene response factors 4 (GbERF4) involved in TTL synthesis. Overexpression of GbERF4 in tobacco (Nicotiana tabacum) significantly increased terpenoid content and upregulated the expression of key enzyme genes (3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS), acetyl-CoA C-acetyltransferase (AACT), and geranylgeranyl diphosphate synthase (GGPPS)) in the terpenoid pathway in tobacco, suggesting that GbERF4 functions in regulating the synthesis of terpenoids. The expression pattern analysis and previous microRNA (miRNA) sequencing showed that gb-miR160 negatively regulates the biosynthesis of TTLs. Transgenic experiments showed that overexpression of gb-miR160 could significantly inhibit the accumulation of terpenoids in tobacco. Targeted inhibition and dual-luciferase reporter assays confirmed that gb-miR160 targets and negatively regulates GbERF4. Transient overexpression of GbERF4 increased TTL content in G. biloba, and further transcriptome analysis revealed that DXS, HMGS, CYPs, and transcription factor genes were upregulated. In addition, yeast one-hybrid and dual-luciferase reporter assays showed that GbERF4 could bind to the promoters of the HMGS1, AACT1, DXS1, levopimaradiene synthase (LPS2), and GGPPS2 genes in the TTL biosynthesis pathway and activate their expression. In summary, this study investigated the molecular mechanism of the gb-miR160-GbERF4 regulatory module in regulating the synthesis of TTLs. It provides information for enriching the understanding of the regulatory network of TTL biosynthesis and offers important gene resources for the genetic improvement of G. biloba with high contents of TTLs.

Rainfall-induced changes in aquatic microbial communities and stability of dissolved organic matter: Insight from a Fen river analysis

Microbial communities are pivotal in aquatic ecosystems, as they affect water quality, energy dynamics, nutrient cycling, and hydrological stability. This study explored the effects of rainfall on hydrological and photosynthetic parameters, microbial composition, and functional gene profiles in the Fen River. Our results demonstrated that rainfall-induced decreases in stream temperature, dissolved oxygen, pH, total phosphorus, chemical oxygen demand, and dissolved organic carbon concentrations. In contrast, rainfall increased total dissolved solids, salinity, and ammonia-nitrogen concentrations. A detailed microbial community structure analysis revealed that Cyanobacteria was the dominant microbial taxon in the Fen River, accounting for approximately 75% and 25% of the microalgal and bacterial communities, respectively. The abundance of Chlorophyta and Bacillariophyta increased by 47.66% and 29.92%, respectively, whereas the relative abundance of Bacteroidetes decreased by 37.55% under rainfall conditions. Stochastic processes predominantly affected the assembly of the bacterial community on rainy days. Functional gene analysis revealed variations in bacterial functions between sunny (Sun) and rainy (Rain) conditions, particularly in genes associated with the carbon cycle. The 3-oxoacyl-[acyl-carrier-protein] reductase gene was more abundant in the Fen River bacterial community. Particular genes involved in metabolism and environmental information processing, including the acetyl-CoA C-acetyltransferase (atoB), enoyl-CoA hydratase (paaF), and branched-chain amino acid transport system gene (livK), which are integral to environmental information processing, were more abundant in Sun than the Rain conditions. In contrast, the phosphate transport system gene, the galactose metabolic gene, and the pyruvate metabolic gene were more abundant in Rain. The excitation-emission matrix analysis with parallel factor analysis identified four fluorescence components (C1–C4) in the river, which were predominantly protein- (C1) and humic-like (C2–C4) substances. Rainfall affected organic matter production and transport, leading to changes in the degradation and stability of dissolved organic matter. Overall, this study offers insight into how rainfall affects aquatic ecosystems.

Microalgal cycling in the cathode of microbial fuel cells (MFCs) induced oxygen reduction reaction (ORR) and electricity: A biocatalytic process for clean energy

Development in electricity generation technologies is necessary to ensure a safe and sustainable future. Catalytic activity of oxygen reduction reaction (ORR) is still the key challenge in microbial fuel cells (MFCs) which has not improved significantly even upon the application of catalysts. In this study, microalgae as biocatalysts (i.e., availability of electron acceptors and maintain ORR) were applied for spontaneous bioelectricity generation. Microalgal cycling was performed at various concentrations (5, 10, 15, and 20 % vv−1) in the cathode whereas, 2 % fat, oil, and grease (FOG) were added in the anode. Electrochemical and next-generation sequencing (NGS) analyses along with biological parameters were studied to fully understand the anodic and cathodic behavior towards electrogenesis. Continuous microalgal cycling resulted in the highest power density (21.0 mW m−2) and current density (245 mA m−2) in the reactor loaded with 2 % FOG + 15 % vv−1 microalgae. Electrochemical analysis showed the potential of 2 % FOG + 15 % vv−1 microalgae via exhibiting Ohmic resistance of 34. 9 Ω and charge transfer resistance of 100 Ω with improved ORR activity. NGS revealed that Clostridium (7–16.5 %), Bacteroidetes (4.54–12.87 %), and Geobacter (6.5–22.7 %) were the dominant species involved in the electron transport mechanism. CoA ligase, acetyl-CoA C-acetyltransferase, and acetate kinase were the major enzymes involved in β-oxidation and electrogenesis. This study demonstrated the baseline for the use of microalgae as biocatalyst in cathode of MFCs to improve the catalytic efficiency of ORR.

Investigation of sex expression profiles and the cantharidin biosynthesis genes in two blister beetles.

Cantharidin (CTD) is a well-established defensive toxin synthesized by blister beetles, displaying both therapeutic potential and toxicity. Among these beetles, Hycleus cichorii and Hycleus phaleratus are the two most commercially significant species due to their capacity to produce CTD in males. In this investigation, we conducted a gene expression profiling analysis of male and female individuals of these two species, utilizing the Illumina Hiseq4000 platform. We identified 7,983 expressed genes, including 2,823 differentially expressed genes (DEGs) shared by both male and female blister beetles. Nineteen genes related to CTD biosynthesis in the terpenoid backbone biosynthesis pathway were identified, including hydroxymethylglutaryl-CoA reductase (HMGR; EC:1.1.1.34), which demonstrated a significant correlation with CTD content. Furthermore, hydroxymethylglutaryl-CoA synthase (HMGS; EC:2.3.3.10) and isopentenyl-diphosphate Delta-isomerase (IDI; EC:5.3.3.2) were also found to be significantly up-regulated in males. Comparative analysis revealed that NADP+-dependent farnesol dehydrogenase (FOHSDR; EC:1.1.1.216) and farnesyl diphosphate synthase (FDPS; EC:2.5.1.1) had the highest copy number in these beetles, significantly higher than the copy number of the other four non-Meloidae insects. The analysis of the protein-protein interaction network of genes related to CTD biosynthesis revealed that the acetyl-CoA C-acetyltransferase (ACAT; EC:2.3.1.9) gene was the central gene, exhibiting greater expression in male blister beetles than in females. This study offers novel insights into the mechanisms of CTD biosynthesis in blister beetles and enhances our comprehensions of the association between particular genes and CTD content.

Fat, oil, and grease as new feedstock towards bioelectrogenesis in microbial fuel cells: Microbial diversity, metabolic pathways, and key enzymes

Microbial fuel cells (MFCs) are a well-known technology used for bioelectricity production from the decomposition of organic waste via electroactive microbes. Fat, oil, and grease (FOG) as a new substrate in the anode and microalgae in the cathode were added to accelerate the electrogenesis. The effect of FOG concentrations (0.1%, 0.5%, 1%, and 1.5%) on the anode chamber was investigated. The FOG degradation, volatile fatty acid (VFAs) production, and soluble chemical oxygen demand along with voltage output kinetics were analyzed. Moreover, the microbial community analysis and active functional enzymes were also evaluated. The maximum power and current density were observed at 0.5% FOG which accounts for 96 mW m−2 (8-folds enhancement) and 560 mA m−2 (3.7-folds enhancement), respectively. The daily voltage output enhanced upto 2.3-folds with 77.08% coulombic efficiency under 0.5% FOG, which was the highest among all the reactors. The 0.5% FOG was degraded >85%, followed by a 1% FOG-loaded reactor. The chief enzymes in β-oxidation and electrogenesis were acetyl-CoA C-acetyltransferase, riboflavin synthase, and riboflavin kinase. The identified enzymes symbolize the presence of Clostridium sp. (>15%) and Pseudomonas (>10%) which served as electrochemical active bacteria (EAB). The major metabolic pathways involved in electrogenesis and FOG degradation were fatty acid biosynthesis and glycerophospholipid metabolism. Utilization of lipidic-waste (such as FOG) in MFCs could be a potential approach for simultaneous biowaste utilization and bioenergy generation.

Time-resolved transcriptomic profile of oleaginous yeast Rhodotorula mucilaginosa during lipid and carotenoids accumulation on glycerol

The study reports the exploration of the transcriptome landscape of the red oleaginous yeast Rhodotorula mucilaginosa IIPL32 coinciding with the fermentation kinetics of the yeast cultivated in a two-stage fermentation process to exploit the time-series approach to get the complete transcripts picture and reveal the persuasive genes for fatty acid and terpenoid synthesis. The finding displayed the molecular drivers with more than 2-fold upregulation in the nitrogen-limited stage than in the nitrogen-excess stage. The rate-limiting diphosphomevalonate decarboxylase, acetylCoA-citrate lyase, and acetyl-CoA C-acetyltransferase were significant in controlling the metabolic flux in the synthesis of reduced compounds, and acetoacetyl-CoA synthase, 3-ketoacyl-acyl carrier-protein reductase, and β-subunit enoyl reductase catalyze the key starting steps of lipids or terpenoid synthesis. The last two catalyze essential reduction steps in fatty acid synthesis. These enzymes would be the prime targets for the metabolic engineering of the oleaginous yeast for enhanced fatty acids and terpenoid production.

Physiological and transcriptional studies reveal Cr(VI) reduction mechanisms in the exoelectrogen Cellulomonas fimi Clb-11.

A facultative exoelectrogen, Cellulomonas fimi strain Clb-11, was isolated from polluted river water. This strain could generate electricity in microbial fuel cells (MFCs) with carboxymethyl cellulose (CMC) as the carbon source, and the maximum output power density was 12.17 ± 2.74 mW·m-2. In addition, Clb-11 could secrete extracellular chromate reductase or extracellular electron mediator to reduce Cr(VI) to Cr(III). When the Cr(VI) concentration was less than 0.5 mM in Luria-Bertani (LB) medium, Cr(VI) could be completely reduced by Clb-11. However, the Clb-11 cells swelled significantly in the presence of Cr(VI). We employed transcriptome sequencing analysis to identify genes involved in different Cr(VI) stress responses in Clb-11. The results indicate that 99 genes were continuously upregulated while 78 genes were continuously downregulated as the Cr(VI) concentration increased in the growth medium. These genes were mostly associated with DNA replication and repair, biosynthesis of secondary metabolites, ABC transporters, amino sugar and nucleotide sugar metabolism, and carbon metabolism. The swelling of Clb-11 cells might have been related to the upregulation of the genes atoB, INO1, dhaM, dhal, dhak, and bccA, which encode acetyl-CoA C-acetyltransferase, myo-inositol-1-phosphate synthase, phosphoenolpyruvate-glycerone phosphotransferase, and acetyl-CoA/propionyl-CoA carboxylase, respectively. Interestingly, the genes cydA and cydB related to electron transport were continuously downregulated as the Cr(VI) concentration increased. Our results provide clues to the molecular mechanism of Cr(VI) reduction by microorganisms in MFCs systems.

Effect and mechanism analysis of dual enzyme pretreatment on enhancement of volatile fatty acids production in kitchen waste anaerobic acidification

The high content of cellulose and hemicellulose in kitchen waste (KW) results in a slow hydrolysis rate and subsequently hinders the yields of volatile fatty acids (VFAs) in anaerobic acidification. In this study, cellulase and hemicellulase were used for the pretreatments of KW to tackle the problem of difficult hydrolysis in anaerobic acidification. The results indicated that the joint use of double enzymes significantly improved the hydrolysis of cellulose and hemicellulose during pretreatment, thus enhancing the VFAs production in anaerobic acidification. The highest production of TVFAs was 50.73 ± 2.10 g COD/L and it was improved by 62.77% compared with the unpretreated group. In addition, the 16S rRNA amplicon sequencing analysis showed that the relative abundance of Firmicutes related with the VFAs production increased from 17.77% to 50.55%. The potential metabolism function analysis (PICRUSt2) indicated that the enrichment of glyceraldehyde-3-phosphate dehydrogenase (EC:1.2.1.9) and acetyl-CoA C-acetyltransferase (EC:2.3.1.9) contributed to the production of VFAs in AD. The results suggested that collaborative enzyme pretreatment could be one of the effective methods for the hydrolysis of cellulose and hemicellulose in KW and provided constructive mechanistic insights into recovering valuable products from KW.

Metal cofactor regulation combined with rational genetic engineering of Schizochytrium sp. for high-yield production of squalene.

The increasing market demand for squalene requires novel biotechnological production platforms. Schizochytrium sp. is an industrial oleaginous host with a high potential for squalene production due to its abundant native acetyl-CoA pool. We first found that iron starvation led to the accumulation of 1.5 g/L of squalene by Schizochytrium sp., which was 40-fold higher than in the control. Subsequent transcriptomic and lipidomic analyses showed that the high squalene titer is due to the diversion of precursors from lipid biosynthesis and increased triglycerides (TAG) content for squalene storage. Furthermore, we constructed the engineered acetyl-CoA C-acetyltransferase (ACAT)-overexpressing strain 18S::ACAT, which produced 2.79 g/L of squalene, representing an 86% increase over the original strain. Finally, a nitrogen-rich feeding strategy was developed to further increase the squalene titer of the engineered strain, which reached 10.78 g/L in fed-batch fermentation, a remarkable 161-fold increase over the control. To our best knowledge, this is the highest squalene yield in thraustochytrids reported to date.

Responses of microRNA in digestive glands of mussel Mytilus galloprovincialis exposed to polystyrene nanoplastics

Polystyrene nanoplastics (PS-NPs) are typical accumulated nanoplastics in the marine environment and organisms, and have strong potential risks to marine ecological environment and human health. MiRNAs could respond to and participate in the response process of environmental stressors. However, the response of miRNAs to nanoplastics has not been fully explored. In this study, miRNA responses of digestive glands in mussels Mytilus galloprovincialis treated by 200 nm PS-NPs (20, 200, 2000 μg/L) for 7 days were characterized by BGISEQ-500 deep sequencing and bioinformatics analysis, along with histopathological quantification with planimetric parameters on hematoxylin and eosin (H&E) staining. Results showed that one novel miRNA (novel_mir63) and seven known miRNAs (miR-34_2, miR-34_5, miR-281_8, let-7–5p_6, miR-10, miR-124, miR-29b-3p) were significantly (adjusted P-value < 0.05) differentially expressed after PS-NPs treatments, and most of them were down-regulated expect for novel_mir63 and miR-34_2. Function analysis of target genes corresponding to these differentially expressed miRNAs indicated that PS-NPs disturbed the process related to metabolism, aging, cardiac function, neural excitation, and repairment. Among them, acetyl-CoA C-acetyltransferase and purine metabolism pathway played vital connection roles. Meanwhile, significantly morphology changes of digestive tubes obtained from H&E stained sections also implied severely disrupted metabolic capability in digestive glands, reflected by significantly increased mean diverticular radius (MDR) and mean luminal radius (MLR) values and the ratio of MLR to mean epithelial thickness (MET), and significantly decreased MET value and MET/MDR. Overall, these findings have revealed new characterization of miRNAs and their target genes in mussel M. galloprovincialis under PS-NPs stress, and provide important clues to further elucidate the toxicity mechanisms of PS-NPs.

Cloning, Expression, and Functional Analysis of the Full-Length cDNA of Acetyl-CoA C-acetyltransferase (AACT) Genes Related to Terpenoid Synthesis in Platycodon grandiflorus.

The objective of this study was to clone and identify the PgAACT function in P. grandiflorus. The full-length sequence of PgAACT genes was isolated and cloned from P. grandiflorus by polymerase chain reaction (PCR). The recombinant plasmid was constructed using the pET-32a vector and expressed in E. coli Transetta (DE3) cells. Subcellular localization of AACT was observed in the epidermal cells of N. tabacum. Quantitative reverse transcription-PCR (qRT-PCR) was used to identify the PgAACT gene transcription levels. After MeJA treatment, the changes in AACT gene expression were observed, and UHPLC-Q-Exactive Orbitrap MS/MS was used to detect the changes in P. grandiflorus saponins. In this study, two full-length cDNAs encoding AACT1 (PgAACT1) and AACT2 (PgAACT2) were isolated and cloned from P. grandiflorus. The deduced PgAACT1 and PgAACT2 proteins contain 408 and 416 amino acids, respectively. The recombinant vectors were constructed, and the protein expression was improved by optimizing the reaction conditions. Sodium dodecyl sulphate-polycrylamide gel electrophloresis and western blot analysis showed that the PgAACT genes were successfully expressed, with molecular weights of the recombinant proteins of 61 and 63 kDa, respectively. Subcellular localization showed that the PgAACT genes were localized in the cytoplasm. Tissue specificity analysis of P. grandiflorus from different habitats showed that PgAACT genes were expressed in the roots, stems, and leaves. After MeJA treatment, the expression level of PgAACT genes and the content of total saponins of P. grandiflorus were significantly increased, suggesting that PgAACT genes play an important role in regulating plant defense systems. Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful in understanding the role of these two genes in terpene biosynthesis.

Waste cooking oils (WCOs) to biogas nexus: Kinetics, active microbes, and functional enzymes

The efficiency of anaerobic digestion (AD) chiefly relies on substrate composition and microbial structure. The lipidic substrates such as waste cooking oils (WCOs) have high biogas potential. The biogas production from WCOs can be increased by active microbes with simultaneous alleviation of environmental contamination. Thus, this study explored the biogas potential of two different WCOs including duck cooking oil (DCO) and fat, oil, and grease (FOG) in AD. The effects on biogas kinetics, oils degradation, microbial community dynamics, and variations in bacterial and archaeal functional enzymes were investigated. The modified Gompertz and logistic growth models showed a curve-fit within a range of 0.98–0.99 R2 value for biogas production in DCO and FOG-loaded reactors. The biogas production was > 5-folds higher in WCOs compared to control. The addition of DCO and FOG showed 76.87 % and 69.76 % of lipid degradation, respectively. The major bacterial enzymes involved in β-oxidation (based on Kyoto Encyclopaedia of Genes and Genomes, KEGG map 00071) of DCO and FOG were acetyl-CoA C-acetyltransferase and enoyl-CoA hydratase. These enzymes correspond to the dominance of Clostridium (>90 %) and might be involved in the formation of biomethane precursors. While the abundance of Methanosphaera (>60 %) and Methanobrevibacter (>30 %) increased the archaeal CoB-CoM reductases and methyltransferase involved in biomethanation. This study demonstrated that the WCOs could be used as potential substrates for biogas production.

Formation mechanisms of ethyl acetate and organic acids in Kluyveromyces marxianus L1-1 in Chinese acid rice soup

This study aims to explore the formation mechanism of ethyl acetate and organic acids in acid rice soup (rice-acid soup) inoculated with Kluyveromyces marxianus L1-1 through the complementary analysis of transcriptome and proteome. The quantity of K. marxianus L1-1 varied significantly in the fermentation process of rice-acid soup and the first and third days were the two key turning points in the growth phase of K. marxianus L1-1. Importantly, the concentrations of ethyl acetate, ethanol, acetic acid, and L-lactic acid increased from day 1 to day 3. At least 4231 genes and 2937 proteins were identified and 610 differentially expressed proteins were annotated to 30 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways based on the analysis results of transcriptome and proteome. The key genes and proteins including up-regulated alcohol dehydrogenase family, alcohol O-acetyltransferase, acetyl-CoA C-acetyltransferase, acyl-coenzyme A thioester hydrolase, and down-regulated aldehyde dehydrogenase family were involved in glycolysis/gluconeogenesis pathways, starch and sucrose metabolism pathways, amino sugar and nucleotide sugar metabolism pathways, tricarboxylic acid (TCA) cycle, and pyruvate metabolism pathways, thus promoting the formation of ethyl acetate, organic acids, alcohols, and other esters. Our results revealed the formation mechanisms of ethyl acetate and organic acids in rice-acid soup inoculated with K. marxianus L1-1.

Chronic effects of benzalkonium chlorides on short chain fatty acids and methane production in semi-continuous anaerobic digestion of waste activated sludge

As an emerging pollutant, benzalkonium chlorides (BACs) potentially enriched in waste activated sludge (WAS). However, the microbial response mechanism under chronic effects of BACs on acidogenesis and methanogenesis in anaerobic digestion (AD) has not been clearly disclosed. This study investigated the AD (by-)products and microbial evolution under low to high BACs concentrations from bioreactor startup to steady running. It was found that BACs can lead to an increase of WAS hydrolysis and fermentation, but a disturbance to acidogenic bacteria also occurred at low BACs concentration. A noticeable inhibition to methanogenesis occurred when BAC concentration was up to 15 mg/g TSS. Metagenomic analysis revealed the key genes involved in acetic acid (HAc) biosynthesis (i.e. phosphate acetyltransferase, PTA), β-oxidation pathway (acetyl-CoA C-acetyltransferase) and propionic acid (HPr) conversion was slightly promoted compared with control. Furthermore, BACs inhibited the acetotrophic methanogenesis (i.e. acetyl-CoA synthetase), especially BAC concentration was up to 15 mg/g TSS, thereby enhanced short chain fatty acids (SCFAs) accumulation. Overall, chronic stimulation of functional microorganisms with increasing concentrations of BACs impact WAS fermentation.

Quantitative analysis and screening for key genes related to tea saponin in Camellia Oleifera Abel. Seeds

Tea saponin is an important factor affecting the taste of oil-tea camellia seed oil. The content of tea saponin varied in different plant parts and different oil-tea camellia varieties. The tea saponin content was the highest in oil-tea camellia seed oil of Liping 2, 3.392 mg/mL, and the lowest in that from Changlin 53, 1.016 mg/mL. With increased storage time, the content of tea saponin first increased, peaked at 21 days, and then decreased. Transcriptome sequencing was performed to identify genes related to Camellia Oleifera tea saponin metabolism. A total of 8107 differentially expressed genes were identified, and these genes were classified into 49 Gene Ontology (GO) functional categories and involved 129 Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways. Genes highly correlated with tea saponin metabolism showed significant variation, including c96515.graph_c0 (annotated as acetyl-CoA C-acetyltransferase, AACT), c92925.graph_c0 (annotated as hydroxymethyl glutaryl-CoA synthase, HMGS), c95088.graph_c4, c92367.graph_c0 (annotated as hydroxymethyl glutaryl-CoA reductase, HMGR), c100030.graph_c3 (annotated as 1-deoxy-D-xylulose-5-phosphate synthase, DXS), c86734.graph_c0, c108153.graph_c1 (annotated as β-amyrin synthase), c92020.graph_c0, c105492.graph_c1, c92290.graph_c1 (annotated as cytochrome P450, CYP450), c102136.graph_c0, c95951.graph_c1 (annotated as UDP-Glycosyltransferase, UGT). This research provides theoretical reference for improved processing and utilization of Camellia Oleifera.

Molecular Cloning and Analysis of an Acetyl-CoA C-acetyltransferase Gene (EkAACT) from Euphorbia kansui Liou

Terpenoids are the largest class of natural products and are essential for cell functions in plants and their interactions with the environment. Acetyl-CoA acetyltransferase (AACT, EC2.3.1.9) can catalyze a key initiation step of the mevalonate pathway (MVA) for terpenoid biosynthesis and is modulated by many endogenous and external stimuli. Here, the function and expression regulation activities of AACT in Euphorbia kansui Liou (EkAACT) were reported. Compared with wild-type Arabidopsis, the root length, whole seedling fresh weight and growth morphology of EkAACT-overexpressing plants were slightly improved. The transcription levels of AtAACT, AtMDC, AtMK, AtHMGR, and AtHMGS in the MVA pathway and total triterpenoid accumulation increased significantly in transgenic Arabidopsis. Under NaCl and PEG treatment, EkAACT-overexpressing Arabidopsis showed a higher accumulation of total triterpenoids, higher enzyme activity of peroxidase (POD) and superoxide dismutase (SOD), increased root length and whole seedling fresh weight, and a decrease in the proline content, which indicated that plant tolerance to abiotic stress was enhanced. Thus, AACT, as the first crucial enzyme, plays a major role in the overall regulation of the MVA pathway.

A biosurfactant-producing yeast Rhodotorula sp.CC01 utilizing landfill leachate as nitrogen source and its broad degradation spectra of petroleum hydrocarbons.

Biosurfactants (BSs) are known for their remarkable properties, however, their commercial applications are hampered partly by the high production cost. To overcome this issue, a biosurfactant producing strain, Rhodotorula sp.CC01 was isolated using landfill leachate as nitrogen source, while olive oil was determined as the best sole carbon source. The BS produced by Rhodotorula sp.CC01 had oil displacement diameter of 19.90 ± 0.10cm and could reduce the surface tension of water to 34.77 ± 0.63 mN/m. It was characterized as glycolipids by thin layer chromatography, FTIR spectra, and GC-MS analysis, with the critical micelle concentration of 70mg/L. Meanwhile, the BS showed stability over a wide range of pH (2-12), salinity (0-100g/L), and temperature (20-100°C). During the cultivation process, BS was produced with a maximum rate of 163.33mgL-1h-1 and a maximum yield of 1360mg/L at 50h. In addition, the removal efficiency of NH4+-N reached 84.2% after 75h cultivation with a maximum NH4+-N removal rate of 3.92mgL-1h-1. Moreover, Rhodotorula sp.CC01 has proven to be of great potential in remediating petroleum hydrocarbons, as revealed by chromogenic assays. Furthermore, genes related to nitrogen metabolism and glycolipid metabolism were found in this strain CC01 after annotating the genome data with KEGG database, such as narB, glycoprotein glucosyltransferase, acetyl-CoA C-acetyltransferase, LRA1, LRA3, and LRA4. The findings of this study prove a cost-effective strategy for the production of BS by yeast through the utilization of landfill leachate.

Cloning, Prokaryotic Expression, and Purification of Acetyl-CoA C-Acetyltransferase from Atractylodes lancea.

Cangzhu (Atractylodes lancea), a valuable and common traditional Chinese medicinal herb, is primarily used as an effective medicine with various health-promoting effects. The main pharmacological bioactive ingredients in the rhizome of A. lancea are terpenoids. Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpenoid synthesis pathway and catalyzes two units of acetyl-CoA into acetoacetyl-CoA. The objective of the present work was to clone and identify function of AlAACT from Atractylodes lancea. A full-length cDNA clone of AlAACT was isolated using PCR and expressed in Escherichia coli. The expressed protein was purified using Ni-NTA agarose column using standard protocols. AlAACT was transiently expressed in N. benthamiana leaves to determine their subcellular location. The difference in growth between recombinant bacteria and control bacteria under different stresses was observed using the droplet plate experiment. In this study, a full-length cDNA of AACT (AlAACT) was cloned from A. lancea, which contains a 1,227 bp open reading frame and encodes a protein with 409 amino acids. Bioinformatic and phylogenetic analysis clearly suggested that AlAACT shared high similarity with AACTs from other plants. The recombinant protein pET32a(+)/AlAACT was successfully expressed in Escherichia coli BL21 (DE3) cells induced with 0.4 mM IPTG at 30°C as the optimized condition. The recombinant enzyme pET-32a-AlAACT was purified using the Ni-NTA column based on the His-tag, and the molecular weight was determined to be 62 kDa through SDS-PAGE and Western Blot analysis. The recombinant protein was eluted with 100, 300, and 500 mM imidazole; most of the protein was eluted with 300 mM imidazole. Under mannitol stress, the recombinant pET-32a- AlAACT protein showed a substantial advantage in terms of growth rates compared to the control. However, this phenomenon was directly opposite under NaCl abiotic stress. Subcellular localization showed that AlAACT localizes to the nucleus and cytoplasm. The expression and purification of recombinant enzyme pET-32a-AlAACT were successful, and the recombinant strain pET-32a-AlAACT in showed better growth in a drought stress. The expression of AlAACT-EGFP fusion protein revealed its localization in both nuclear and cytoplasm compartments. This study provides an important foundation for further research into the effects of terpenoid biosynthesis in A. lancea.