ABSTRACT
AIMS: To investigate the effect of the yeast-conform variant of the Artemisia annua gene encoding for amorpha-4,11-diene synthase (ADS) on the production of amorpha-4,11-diene in a transformed yeast. METHODS AND RESULTS: The ADS gene was mutated to the yeast-conform variant ADSm. The ADSm synthesis was performed based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial ADSm gene contained codons preferred by the yeast translation machinery. The sequence was then integrated into a yeast expression vector pYeDP60. The fusion construct was active and the transformed yeast cells produced higher level of amorpha-4,11-diene compared with the plant gene-transformed yeast cells. CONCLUSIONS: Strains transformed with the yeast-conform allele (ADSm) were more efficient in terms of production of amorpha-4,11-diene than those transformed with the plant gene. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated that yeast-conform allele of foreign genes by serial PCR reactions can be a solution to low efficiency of heterologous gene expression in Saccharomyces cerevisiae cells.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Artemisia annua/enzymology , Genetic Engineering/methods , Saccharomyces cerevisiae/genetics , Sesquiterpenes/metabolism , Alkyl and Aryl Transferases/genetics , Artemisia annua/genetics , Artemisia annua/metabolism , Gas Chromatography-Mass Spectrometry , Genome, Fungal/genetics , Molecular Sequence Data , Plasmids/genetics , Polycyclic Sesquiterpenes , Polymerase Chain Reaction , Recombination, Genetic , Saccharomyces cerevisiae/metabolism , Transformation, GeneticABSTRACT
Cytochalasin D which shows marked cytotoxic effects on multi-tumor cells was newly isolated at high content(5.28 mg/g, dry weight) from Fungus Hypocrella bambusae(B.et Br.) Sacc. Its structure was elucidated by spectroscopic methods. Two-dimensional NMR techniques were applied to make complete assignment for the 1H- and 13C-NMR chemical shifts of this compound.
Subject(s)
Ascomycota/chemistry , Cytochalasin D/isolation & purification , Animals , China , Cytochalasin D/chemistry , Magnetic Resonance Spectroscopy , Molecular Structure , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/isolation & purificationABSTRACT
A new sesquiterpenoid, named gloesteretriol, C15H26O3, mp 205-206 degrees C, [alpha]22D + 5.6 degrees (c 0.115, EtOH), was isolated from the fermentation products of Gloeostereum incarnatum S. Ito et Imai.. On the basis of spectral analysis its structure was deduced as I and confirmed by single-crystal X-ray analysis.
Subject(s)
Anti-Infective Agents/isolation & purification , Basidiomycota/chemistry , Drugs, Chinese Herbal/chemistry , Sesquiterpenes/isolation & purification , Anti-Infective Agents/chemistry , Fermentation , Molecular Conformation , Sesquiterpenes/chemistryABSTRACT
A method for analyzing belladonna alkaloids--hyoscyamine, scopolamine, anisodamine and anisodine, by means of capillary GC and GC-MSD was described. The retention data and characteristic ions of the parent and TMS derivatives of these alkaloids are given and will be very useful for identification of unknown alkaloids. The derivatization of different silylation reagents was compared, and the derivatization with MSTFA was found to be better than with BSA and BSTFA. The method has been used for studying biotransformation of these alkaloids in biosynthesis. Analysis of anisodamine and anisodine by CGC and GC-MS has not been found in the literature. In addition, the advantages of TMS derivatization are also discussed.
