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Overexpression of metK shows different effects on avermectin production in various Streptomyces avermitilis strains
Xuejin Zhao1, Qingxin Wang, Weiqun Guo
1Department of Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China.
Abstract:
The S-adenosylmethionine synthetase gene (metK) from Streptomyces avermitilis was cloned into multi-copy vector pIJ653 and integrative vector pSET152 yielding two metK expression plasmids pYJ02 and pYJ03, respectively. When wild-type strain ATCC31267 was transformed with these two plasmids, avermectin production was increased about 2.0-fold and 5.5-fold, respectively. The introduction of integrative expression plasmid pYJ03 into the engineered strain GB-165, which produces only avermectin B, promoted the production of avermectin approximately 2.0-fold. However, introduction of pYJ02 did not influence avermectin accumulation in GB-165. Moreover, transformation of the avermectin-overproducing industry strain 76-05 with these two plasmids did not stimulate avermectin production. These results showed that there were different effects of metK expression levels on avermectin production in various S. avermitilis strains. Additionally, the transcript levels of metK, aveR (the avermectin pathway-specific regulatory gene) and aveA1 (one avermectin biosynthesis gene) meet the expectation of fermentation levels of avermectin in wild-type strain and its recombinant strains. The gene expression levels of metK, aveR and aveA1 in GB-165 and 76-05 were much higher then those in wild-type strain, which probably limited the increasement of avermectin by overexpression of metK.
Insights
Overexpressing the S-adenosylmethionine synthetase gene (metK) in Streptomyces avermitilis enhanced avermectin production in wild-type strains but not in engineered or overproducing strains. This indicates strain-specific effects of metK expression on avermectin biosynthesis.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Avermectin, a crucial antiparasitic drug, is produced by Streptomyces avermitilis.
- Optimizing avermectin production is vital for pharmaceutical applications.
- The S-adenosylmethionine synthetase gene (metK) is a potential target for metabolic engineering.
Purpose of the Study:
- To investigate the effect of overexpressing the metK gene on avermectin production in different Streptomyces avermitilis strains.
- To understand the relationship between metK expression levels and avermectin biosynthesis pathway regulation.
- To identify optimal strategies for enhancing avermectin yield through genetic manipulation.
Main Methods:
- Cloning of the metK gene into multi-copy (pIJ653) and integrative (pSET152) vectors to create expression plasmids.
- Transformation of wild-type, engineered (GB-165), and industrial (76-05) Streptomyces avermitilis strains with metK expression plasmids.
- Quantification of avermectin production and analysis of gene transcript levels (metK, aveR, aveA1) using quantitative PCR.
Main Results:
- Overexpression of metK using pYJ02 and pYJ03 increased avermectin production by 2.0-fold and 5.5-fold, respectively, in the wild-type strain ATCC31267.
- Introduction of pYJ03 into the engineered strain GB-165 resulted in a 2.0-fold increase in avermectin production, while pYJ02 had no effect.
- metK overexpression did not enhance avermectin production in the industrial strain 76-05.
- Transcript levels of metK, aveR, and aveA1 correlated with avermectin fermentation levels in wild-type and recombinant strains, with higher basal levels in GB-165 and 76-05.
Conclusions:
- The impact of metK gene overexpression on avermectin production is strain-dependent in Streptomyces avermitilis.
- High endogenous expression of metK, aveR, and aveA1 in engineered and industrial strains may limit the benefits of further metK overexpression.
- Targeted genetic engineering strategies need to consider the specific genetic background of the production strain for effective yield improvement.
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