Generation of taxol susceptible yeast strain by inducing mutation TUB2 -Asp26
Subject Areas : Microbial Biotechnology
Hamed Ashourion
1
,
Shamsozoha Abolmaali
2
,
Ziba Fooladvand
3
,
Fatemeh Rezagholi
4
1 - M.Sc. student, Department of Biotechology, Faculty of Energy Engineering and New Technologies, Shahid Beheshti University, Tehran, Iran.
2 - Assistant Professor, Department of Biology, Faculty of Basic Sciences, Semnan University, Semnan, Iran.
3 - M.Sc. student, Department of Biotechology, Faculty of Energy Engineering and New Technologies, Shahid Beheshti University, Tehran, Iran.
4 - M.Sc. student, Department of Biotechology, Faculty of Energy Engineering and New Technologies, Shahid Beheshti University, Tehran, Iran.
Keywords: Paclitaxel, Saccharomyces cerevisiae, Tubulin,
Abstract :
Background & Objectives: Paclitaxel is an effective anti-microtubule agent against lung, ovarian and breast cancers. The low production of taxol in the yew, as well as its resistance to chemotherapy, is limiting the use of paclitaxel. This study aimed to create a screening system for new sources of taxol using the taxol sensitive mutant strain of Saccharomyces cerevisiae. Materials & Methods: To mutate the yeast chromosome TUB2 gene, primers containing a change in codon 26 from glycine to aspartic acid (GGT to GAT) were designed and TUB2-Asp26 gene amplified by three-step PCR method. The TUB2 gene was then cloned from the haploid strain into the yeast expression vector in the galactose promoter. After transplantation of haploid yeast with Gal-TUB2 construct (pZF58), transgenic screening was carried out for the second time by PCR product of the TUB2-Assp26 gene and in the medium containing galactose and against 40 μM taxol. Results: Of the 84 transgenes, the yeast strain YNAH1 was selected from the first screening and the integration of mutation in the chromosome was confirmed by the MBC value of 50 μM taxol. The formerly generated membrane-defective yeast strain with amino acid sequences similar to the brain beta-tubulin with an exception for amino acid 26, tolerated 25 µM taxol. However, in the present study, tolerance increased by up to 2-fold due to the use of yeast hosts that had no defects in membrane transporters. Conclusion: The mutant yeast strain can be used in screening to find new sources of taxol producers with production capacity up to 50 µM.
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