Molecular cloning, characterization, and heterologous expression of an acetyl-CoA acetyl transferase gene from Sanghuangporus baumii.

Acetyl-CoA C-acetyltransferase synthase gene (AACT) cDNA, DNA and promoter were cloned from Sanghuangporus baumii. The gene ORF (1260 bp) encoded 419 amino acids. The AACT DNA includes five exons (1-84 bp, 140-513 bp, 570-1027 bp, 1090-1282 bp, 1344-1494 bp) and four introns (85-139 bp, 514-569 bp, 1028-1089 bp, 1283-1343 bp). The molecular weight of AACT protein is 43.40 kDa, it is hydrophilic with a theoretical isoelectric point of 8.96. Furthermore, The region of the transcription start site is 1997-2047 bp of AACT promoter, and it contained promoter elements (TATA Boxs, CAAT Boxs, CAAT-box, ABRE, G-Boxs, Sp1, MSA-like, LTR). AACT recombinant protein (43.40 KDa + Tag protein 22.68 KDa) was subjected in SDS-PAGE. AACT the transcription levels of in different development stages were investigated. The expression of AACT in primordia (2.4-fold) and 15 d mycelia (2.3- fold) were significantly higher than 9 d mycelia (contral). The expression level of the AACT downstream genes and triterpenoids content were determined at different developmental stages. Triterpenoid content reached its peak on day 15(7.21 mg/g).

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

Platycodon grandiflorus is a well-known and widely distributed traditional herbal medicine and functional food in Asia, with triterpenoids as the main bioactive component in its roots. Acetyl-CoA C-acetyltransferase (AACT) is the initiation enzyme in the mevalonate pathway and plays an important role in the biosynthesis of terpenoids. OBJECTIVE: The objective of this study was to clone and identify the PgAACT function in P. grandiflorus. METHODS: 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. RESULTS: 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. CONCLUSION: Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful in understanding the role of these two genes in terpene biosynthesis.

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

BACKGROUND: 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. OBJECTIVE: The objective of the present work was to clone and identify function of AlAACT from Atractylodes lancea. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Association of polymorphisms of the acetyl-coA acetyltransferase 1 gene and the melatonin receptor 1B gene with the susceptibility to nonalcoholic fatty liver disease / 临床肝胆病杂志

ObjectiveTo investigate the association of the polymorphisms of the acetyl-CoA acetyltransferase 1 （ACAT1） gene and the melatonin receptor 1B （MTNR1B） gene with the susceptibility to nonalcoholic fatty liver disease （NAFLD）. MethodsA total of 164 healthy controls and 228 NAFLD patients were enrolled in this study. PCR and sequencing methods were used to determine the genotypes of the polymorphisms of the ACAT1 gene at the rs1044925 and rs1157651 loci and the MTNR1B gene at the rs10830963 locus， and fasting venous blood samples were collected for biochemical analysis. The t-test was used for comparison of normally distributed continuous data between groups， and the non-parametric Mann-Whitney U test was used for comparison of non-normally distributed continuous data between groups； the chi-square test was used for comparison of categorical data between groups. ResultsThere were no significant differences between the NAFLD group and the healthy control group in the genotype distribution of the ACAT1 gene at the rs1044925 and rs1157651 loci and the MTNR1B gene at the rs10830963 locus （all P>0.05）. The carriers of AA genotype at the rs1044925 locus of the ACAT1 gene had a significantly higher level of low-density lipoprotein than the carriers of C allele （Z=-2.08， P=0.04）， and the carriers of G allele at the rs10830963 locus of the MTNR1B gene had a significantly higher level of fasting blood glucose than the carriers of CC genotype （Z=-3.01， P<0.01）. ConclusionThe polymorphisms of the ACAT1 gene at the rs1044925 and rs1157651 loci and the MTNR1B gene at the rs10830963 locus were not associated with the susceptibility to NAFLD. The rs1044925 locus of the ACAT1 gene and the rs10830963 locus of the MTNR1B gene are associated with the levels of low-density lipoprotein and fasting blood glucose， respectively.

Molecular Mechanism of Salvia miltiorrhiza Quality Formation Based on Single Nucleotide Polymorphism of Functional Genes / 中国实验方剂学杂志

Objective:To investigate the relationship between the single nucleotide polymorphism（SNP）of function genes and effective components of <italic>Salvia miltiorrhiza</italic> and the molecular mechanism of specific quality formation of <italic>S. miltiorrhiza</italic>. Method:The fingerprints of components in <italic>S. miltiorrhiza</italic> from eight different habitats and varieties were obtained by high-performance liquid chromatography （HPLC）. The full-length cDNA of three functional genes<italic> </italic>acetyl-CoA C-acetyltransferase（<italic>SmAACT</italic>），4-diphosphocytidyl-2-C-methyl-<italic>D</italic>-erythritol kinase（<italic>SmCMK</italic>） and isopentenyl diphosphate isomerase（<italic>SmIPPI</italic>） in tanshinone metabolic pathway were amplified by polymerase chain reaction（PCR），cloned， and sequenced，followed by bioinformatics analysis. Result:The full-length cDNA sequences of three functional genes <italic>SmAACT</italic>，<italic>SmCMK</italic>， and <italic>SmIPPI</italic> in tanshinone metabolic pathway were obtained from 23 strains of <italic>S. miltiorrhiza</italic> from eight different habitats and varieties. As revealed by the analysis of SNP and amino acid polymorphisms of three functional genes，18，16， and 14 SNP sites were found respectively. HPLC results showed the samples from Beijing，Hubei，Shandong （No. SDB），Shanxi，Henan， and Shandong （No. SDZ） were clustered into one branch，and those from Hebei and Inner Mongolia were clustered into another branch， which suggested that the variation trend of <italic>S. miltiorrhiza</italic> components had little correlation with geographical distance，but the variety was a critical factor for the quality. Conclusion:There was an obvious genetic differentiation trend in <italic>S. miltiorrhiza</italic> from different habitats，and different origin-specific genotypes were formed. The molecular mechanism of the formation of the specific quality of <italic>S. miltiorrhiza</italic> from different habitats was discussed，which laid a foundation for the stability and effectiveness of clinical medication，and guided the breeding of excellent varieties of <italic>S. miltiorrhiza</italic>.

Production of (S)-3-hydroxybutyrate by metabolically engineered Saccharomyces cerevisiae.

(S)-3-Hydroxybutyrate (S-3HB) can be used as a precursor for the synthesis of biodegradable polymers such as polyhydroxyalkanoate and stereo-specific fine chemicals such as antibiotics, pheromones, and drugs. For the production of S-3HB in yeast, the biosynthetic pathway of S-3HB from acetyl-CoA, consisting of the three enzymes, acetyl-CoA C-acetyltransferase (ACCT), acetoacetyl-CoA reductase (ACR), and 3-hydroxybutyryl-CoA thioesterase (HBT), was introduced into Saccharomyces cerevisiae. An engineered yeast strain overexpressing ERG10, hbd, and tesB genes not only exhibited enzyme activities of AACT, ACR, and HBT, but also produced S-3HB from ethanol. In order to increase the titer of S-3HB, a fed-batch fermentation based on pulse feeding of ethanol as a carbon source was performed, and a final S-3HB titer of 12.0g/L was achieved. This is the first report on the production of 3HB by engineered yeast, utilizing ethanol as the carbon source, suggesting that the industrially preferred S. cerevisiae can be a promising host for producing S-3HB.

Cloning, expression, and bioinformatics analysis of acetyl-CoA C-acetyltransferase gene in Houttuynia cordata / 中草药

Objective: Acetyl-CoA C-acetyltransferase (AACT) is the initial enzyme in the terpenoid biosynthesis pathway of mevalonate (MVA), two units of acetyl-CoA were catalyzed to acetoacetyl-CoA. To clone the full length cDNA of AACT gene and carry out the bioinformatics analysis and expression analysis in order to provide the basis on resolving the mechanism of biosynthesis for terpenoid secondary metabolites from Houttuynia cordata. Methods: The cDNA sequence of AACT gene was obtained from H. cordata by using RT-PCR strategy. And the different expression of AACT gene in the rhizomes, stems, leaves, and flowers of H. cordata was analyzed by fluorescent quantitative PCR. Results: The cDNA contains a 1 218 bp open reading frame and encodes a predicted protein of 405 amino acids. No transmembrane region and signal peptide were present in AACT protein by bioinformatics prediction. Relative real-time PCR analysis indicated that AACT gene showed the highest transcript abundance in the stems and rhizomes of H. cordata lower levels in the flowers and leaves, the values of them were 1.49, 0.96, 0.20, and 0.10, respectively. Conclusion: This AACT gene is cloned from H. cordata for the first time. The results will provide a foundation for exploring the mechanism of the gene in terpenoid biosynthesis and metabolism in H. cordata.

Anti-TNF-α and hydralazine drug-induced lupus

Drug-induced lupus is a rare drug reaction featuring the same symptoms as idiopathic lupus erythematosus. Recently, with the introduction of new medicines in clinical practice, an increase in the number of illness-triggering implicated drugs has been reported, with special emphasis on anti-TNF-α drugs. In the up-to-date list, almost one hundred medications have been associated with the occurrence of drug-induced lupus. The authors present two case reports of the illness induced respectively by hydralazine and infliximab, addressing the clinical and laboratorial characteristics, diagnosis, and treatment.