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- Identification and heterologous reconstitution of a 5-alk(en)ylresorcinol synthase from endophytic fungus Shiraia sp. Slf14
- Huiwen Yan , Lei Sun , Jinge Huang , Yixing Qiu , Fuchao Xu , Riming Yan , Du Zhu , Wei Wang , Jixun Zhan
- J. Microbiol. 2018;56(11):805-812. Published online October 24, 2018
- DOI: https://doi.org/10.1007/s12275-018-8278-x
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Abstract
- A new type III polyketide synthase gene (Ssars) was discovered from the genome of Shiraia sp. Slf14, an endophytic fungal strain from Huperzia serrata. The intron-free gene was cloned from the cDNA and ligated to two expression vectors pET28a and YEpADH2p-URA3 for expression in Escherichia coli BL21(DE3) and Saccharomyces cerevisiae BJ5464, respectively. SsARS was efficiently expressed in E. coli BL21(DE3), leading to the synthesis of a series of polyketide products. Six major products were isolated from the engineered E. coli and characterized as 1,3-dihydroxyphenyl- 5-undecane, 1,3-dihydroxyphenyl-5-cis-6-tridecene,1,3-dihydroxyphenyl- 5-tridecane, 1,3-dihydroxyphenyl-5-cis-8- pentadecene, 1,3-dihydroxyphenyl-5-pentadecane, and 1,3- dihydroxyphenyl-5-cis-10-heptadecene, respectively, based on the spectral data and biosynthetic origin. Expression of SsARS in the yeast also led to the synthesis of the same polyketide products, indicating that this enzyme can be reconstituted in both heterologous hosts. Supplementation of soybean oil into the culture of E. coli BL21(DE3)/SsARS increased the production titers of 1–6 and led to the synthesis of an additional product, which was identified as 5-(8Z,11Z-heptadecadienyl) resorcinol. This work thus allowed the identification of SsARS as a 5-alk(en)ylresorcinol synthase with flexible substrate specificity toward endogenous and exogenous fatty acids. Desired resorcinol derivatives may be synthesized by supplying corresponding fatty acids into the culture medium.
Research Support, Non-U.S. Gov't
- Effect of Mycelial Extract of Clavicorona pyxidata on the Production of Amyloid β-Peptide and the Inhibition of Endogenous β-Secretase Activity in vitro
- Tae-Hee Lee , Young-Il Park , Yeong-Hwan Han
- J. Microbiol. 2006;44(6):665-670.
- DOI: https://doi.org/2459 [pii]
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Abstract
- Amyloid β-peptide (Aβ), which is a product of the proteolytic effect of β-secretase (BACE) on an amyloid precursor protein, is closely associated with Alzheimer’s disease (AD) pathogenesis. There is sufficient evidence to suggest that a BACE inhibitor may reduce Aβ levels, thus decreasing the risk of AD. In a previous study, an extract of Clavicorona pyxidata DGUM 29005 mycelia was found to inhibit the production of a soluble β-amyloid precursor protein (sβAPP), Aβ, and BACE in neuronal cell lines. We sought to determine whether this mycelial extract exerts the same effect in human rhabdomyosarcoma A-204 and rat pheochromocytoma PC-12 cells. We found that the production of Aβ decreased in a dose-dependent manner in the presence of the mycelial extract and that the concentration of Aβ never exceeded 50 μg/ml. The presence of sAPP was detected in every culture medium to which the mycelial extract had been added and its concentration remained the same, regardless of the concentration of the extract used. Endogenous β-secretase <br>activity in A-204 and PC-12 cellular homogenates also decreased in the presence of this extract. These cells, in culture, were not susceptible to the cytotoxic activity of the mycelial extract.
- Expression of immunologically active porcine recombinant TGF-β1 precursor protein in baculovirus system
- Lim, Hyun , Kim, Pyeung Hyeun , Chun, Gie Taek , Choi, Eui Yul , Yie, Se Won
- J. Microbiol. 1997;35(4):341-346.
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Abstract
- In order to express recombinant porcine TGF-β1 protein in a baculovirus expression system the entire TGF-β1 gene containing extra amino acids at the N terminus was cloned into pFBa and pFBb of the Bac-To-Bac^TM baculovirus expression system. One of the clones contained 106 extra amino acids and was designated pFBa-106 TGF-β1, and the other had 28 extra amino acids and was designated pFBb-28 TGF-β1. The orientation of the gene was identified with restriction enzyme mapping and PCR with internal TGF-β1 primers. Sf-9 cells were infected at a m.o.i. of 10 by the recombinant viruses generated from the two expected sizes of 55 kD and 46.4kD. these precursor forms of TGF-β1 with a polyclonal antibody against human TGF-β1. No mature form of TGF-β1 protein was detected on SDS gels and an immunoblot indicated that TGF-β1 precursor is not properlu processed in insect cells.
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