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Construction of Expression Vectors for the High Level Expression in Actinomycetes
Lamichhane, Tek Narayan,Lamichhane, Rajan,Lee, Hei Chan,Liou, Kwangkyoung,Sohng, Jae Kyung 한국공업화학회 2003 응용화학 Vol.7 No.1
Two expression vectors pIBR100 and pIBR20 were constructed for the high level expression in actinomycetes. The pathway specific gene (actII-ORF4) present in pIBR100 along with bi-directional Pact promoter helps to regulate the expression of different genes in actinomycetes. The act promoter, associated RBS, and His-tag were kept at the upstream of the multiple cloning sitcs. Similarly in pIBR20, a strong constitutive promoter ermE* was cloned along with suitable multiple cloning sites with His-tag. It is a bi-replicate origin vector, which can be used for the expression in both E. coli and actinomycetes. The use of the vector system provides the means for overexpression of several Streptomyces genes and provides a ground for the development of several bioactive materials.
The Biosynthesis of Necarzinostatin Produced by S. carzinostaticus ATCC15944
Sthapi, Basundharat,Oh, Tae-Jin,Lamichhane, Rajan,Liou, Kwangkyoung,Lee, Hei-Chan,Kim, Chun-Gyu,Sohng, Jae Kyung 한국공업화학회 2004 응용화학 Vol.8 No.1
Enediyne antibiotics are known for their potent anti tumor activities. Neocarzinostatin (NCS) is produces by Streptomyces carzinostaticus and comprised 1:1 complex of non-peptide chromophore and peptide apoperotein (NcsA). Adapting a strategy for cloning NCS biosynthetic genes, the naphthoate synthase (NS) gene was localized in a cosmid that expanded 33.0 kb. One of the components of NCS, naphthoic acid moiety is produced via PKS pathway by the six intact acetate units. The 5.45 bk iterative type I PKS, NS responsible for its biosynthesis shares sequence homology with 6-MSAS of fungi origin as well as with AviM and CalO5 of Streptomyces origins. It was heterologously expressed in Streptomyces hosts and the major isolated product identified was 2-hydroxy-5-methyl-1-naphthoic acid. This result shows the involvement of this NS gene in NCS biosynthesis. To date iterative type I PKS for monocyclic aromatic compound have been found in limited Streptomyces. This is the first report of similar bacterial PKS from enediyne producer that enables the biosynthesis of bicyclic aromatic compound.