Binhui Guo

Bioactive natural products from endophytes: A review

Endophytes, microorganisms that reside in the internal tissues of living plants without causing any immediate overt negative effects, have been found in every plant species examined to date and recognized as potential sources of novel natural products for exploitation in medicine, agriculture, and industry with more and more bioactive natural products isolated from the microorganisms. In this review, we focus mainly on bioactive natural products from endophytic microorganisms by their different functional roles. The prospect and facing problems of isolating natural products from endophytes are also discussed.

A new endophytic taxane production fungus from Taxus chinensis

More than 50 kinds of endophytic fungi associated with Taxus chinensis were isolated and examined as a potential source of the imposing anticancer drug taxol. Of these, four isolates show ability to produce taxane when measured with the competitive inhibition enzyme immunoassay method. The most promising clone, DA10, identified as Mucor rouxianus sp., is the first rouxianus reported as taxol production fungus. The presence of taxol and its important precursors, such as 10-diacetyl baccatinIII (10-DAB) and baccatinIII, in theculture of this fungus was confirmed by reactivity with a taxane-specific monoclonal antibody, comparative chromatographic and mass spectrometric behavior, cytotoxity to liver carcinoma 7402, and molecular cloning of kernel fragment of taxadiene synthase gene.

[Isolation and characterization of a 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene from Taxus media].

Abstract2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) synthase (MECS, EC: 4.6.1.12) is the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and further Taxol biosynthesis. The full-length MECS cDNA sequence (GenBank accession number DQ286391) was cloned and characterized for the first time from Taxus media, using the Rapid Amplification of cDNA Ends (RACE) technique. The full-length cDNA of Tmmecs was 1081 bp containing a 741 bp open reading frame (ORF) encoding a peptide of 247 amino acids with a calculated molecular mass of 26.1 kDa and an isoelectric point of 8.97. Comparative and bioinformatic analyses revealed that TmMECS had extensive homology with MECSs from other plant species. Phylogenetic analysis indicated that TmMECS was more ancient than other plant MECSs. Southern blot analysis revealed that Tmmecs belonged to a small gene family. Tissue expression pattern analysis indicated that Tmmecs expressed constitutively in all tissues including roots, stems and leaves. The cloning and characterization of Tmmecs will be helpful to understand more about the role of MECS involved in the Taxol biosynthesis at the molecular level.

Molecular cloning, expression profiling and functional analyses of a cDNA encoding isopentenyl diphosphate isomerase from Gossypium barbadense.

Gossypol, a type of plant defence sesquiterpenoid phytoalexin, is synthesized from the MEP (2C-methyl-D-erythritol 4-phosphate) and MVA (mevalonate) pathway in the isoprenoid biosynthetic system. The key step is the isomerization of IPP (isopentenyl diphosphate) to DMAPP (dimethylallyl diphosphate), which is catalysed by IPI (IPP isomerase; EC 5.3.3.2). A full-length cDNA encoding IPI (designated GbIPI) was cloned from Gossypium barbadense by RACE (rapid amplification of cDNA ends). The full-length cDNA of GbIPI was 1205 bp and contained a 906 bp ORF (open reading frame) encoding a protein of 302 amino acids, with a predicted molecular mass of 34.39 kDa and an isoelectric point of 6.07. Amino acid sequence analysis revealed that the GbIPI has a high level of similarity to other IPIs. Southern-blot analysis revealed that GbIPI belongs to a small gene family. Expression analysis indicated that GbIPI expression is highest in stems, followed by leaves, and is lowest in roots, and that the expression of GbIPI could be induced by Verticillium dahliae Kleb, MeJA (methyl jasmonate) and SA (salicylic acid). The functional colour assay indicated that GbIPI could accelerate the accumulation of beta-carotene in Escherichia coli transformants. The cloning and functional analysis of GbIPI will be useful in increasing understanding of the role of IPI in isoprenoid biosynthesis at the molecular level.

Characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from Camptotheca acuminata

Abstract5-enolpyruvylshikimate 3-phosphate synthase (EPSPS; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a critical enzyme in the shikimate pathway. The full-length EPSPS cDNA sequence (CaEPSPS, GenBank accession number: AY639815) was cloned and characterized for the first time from woody plant, Camptotheca acuminata, using rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of CaEPSPS was 1778 bp containing a 1557 bp ORF (open reading frame) encoding a polypeptide of 519 amino acids with a calculated molecular mass of 55.6 kDa and an isoelectric point of 8.22. Comparative and bioinformatic analyses revealed that CaEPSPS showed extensive homology with EPSPSs from other plant species. CaEPSPS contained two highly conserved motifs owned by plant and most bacteria EPSPSs in its N-terminal region. Phylogenetic analysis revealed that CaEPSPS belonged to dicotyledonous plant EPSPS group. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in leaves, stems and roots, with the lower expression being found in roots. The coding sequence of CaEPSPS gene was successfully subcloned in a plasmid-Escherichia coli system (pET-32a), and the cells containing the plasmid carrying the CaEPSPS gene exhibited enhanced tolerance to herbicide glyphosate, compared to the control.

Molecular cloning and heterologous expression of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus x media

A full-length cDNA encoding 10-deacetylbaccatin III-10-O-acetyl transferase (designated as TmDBAT), which catalyzes the acetylation of the C-10 hydroxyl group of the advanced metabolite 10-deacetylbaccatin III (10-DAB) to yield baccatin III, the immediate diterpenoid precursor of Taxol, was isolated from Taxus x media. Heterologous expression of TmDBAT in E. coli demonstrated that TmDBAT was a functional gene. Tissue expression pattern analysis revealed that TmDBAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, implying that TmDBAT was tissue-specific. Expression profiling analysis of TmDBAT under different elicitor treatments including silver nitrate, ammonium ceric sulphate and methyl jasmonate indicated that TmDBAT was an elicitor-responsive gene. Southern blot analysis suggested that TmDBAT belonged to a small multigene family.

The development of biotechnology education in China

From the middle of the 20th century, Chinese scientists have been actively involved in biotechnology. However, biotechnology education in China is a relatively recent phenomenon. This subject has not been addressed at the undergraduate level in a serious way until recently. In the last decade, biotechnology education developed rapidly and reached a new level in Chinese universities. The Chinese scientific establishment is very much aware of the importance of biotechnology and has identified this subject as one of the priority areas. Some universities are taking positive steps toward enhancing biotechnology education. This article focuses on the emergence, as well as the problems and prospects, of biotechnology education in China.