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Cloning of the Actinorhodin Biosynthesis Up-Regulating Genes and Their Effects on the Production of Avermectins

Author: YuZuoZuo
Tutor: TaoMeiFeng
School: Huazhong Agricultural University
Course: Microbiology
Keywords: secondary metabolites regulatory factor actinorhodin avermectins conjugation heterologous expression
CLC: Q93
Type: Master's thesis
Year: 2010
Downloads: 137
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Abstract


Actinorhodin and avermectins are polyketide antibiotics. Actinorhodin is produced by Streptomyces coelicolor via the typeⅡpolyketide synthase (PKS) pathway. Avermectins are produced by Streptomyces avermitilis via the type I PKS pathway. Genomes of both S. coelicolor A3(2) and S. avermitilis NRRL8165 have been sequenced. It is of great value in terms of both fundamental research and industrial production of relevant antibiotics to comparing the regulations of secondary metabolism in different Streptomyces spp.This study was based on the disclosure of 11 cosmids from an S. coelicolor M600 genomic library which up-regulated the biosynthesis of actinorhodin in a primary screening carried out by my colleagues. In this study, these cosmids were introduced into both S. coelicolor M145 and S.livians 1326. It turned out that 6 of them, ie. SCOL5E7, SCOL6G2, SCOL5C10, SCOL5C12, SCOL5F10 and SCOL5B9, improved the actinorhodin production in both strains. These 6 cosmids were then introduced into S. avermitilis NRRL8165 by conjugation, and the resulting exconjugants were fermented and analyzed by HPLC. SCOL5B9 was found to improve the production of several avermectins components. In particularly, the production of B1a component was increased by 8 fold. The other cosmids did not cause significant increment to the avermectins production. The 6 cosmids were ends sequencing and subcloned to determine the functional regions. The functional regions of SCOL6G2 were localized to SCO0431, a possible secreted protein gene and SCO0432, a probable secreted peptidase gene.Streptomyces avermitilis NRRL8165 could serve as a good host for heterlogous expression of antibiotics biosynthetic gene clusters. However, it needs to improve its conjugation frequency to accept large plasmids. We chose 4 inorganic salts, i.e. MgCl2, NaCl, Ca(NO3)2 and CaCl2, to test whether they affected the conjugation frequency of large plasmids at salt concentration from 0-200 mM. It was found that CaCl2 promoted conjugation dramatically, and MgCl2 did significantly too. Complete random experiments were designed to test the combination of MgCl2 and CaCl2, leading to disclosure of an optimal combination which improved the conjugation frequency by 11 fold. In addition, a supplemented medium was found to lead to successful heterologous expression of actinorhodin in S. avermitilis. The successful heterologous expression of actinorhodin paved the road towards the further study of regulation of actinorhodin production in S. avermitilis.

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