Study On Avermectin Precursors Metabolic Engineering

Avermectins comprise a class of macrolide antibiotics, which exhibit high efficiency, broad-spectrum and low toxicity of anthelmintic activity, and are widely used in the fields of agriculture, pharmaceutical industry and animal husbandry. Avermectins include a series of eight major compounds (Ala, A2a, Bla, B2a, Alb, A2b, B1b and B2b), among which the B1a fraction has the most effective antiparasitic activity. How to improve yield, and the ratio of B1a component especially, has become a hot research topic. However, unknown mechanisms are usually involved in the traditional random mutagenesis and screening method. Therefore, we adopted metabolic engineering and synthetic biology approach to improve avermectin production and the ratio of "a" components by rational manipulation of precursor metabolic network.First of all, we studied the influence of precursor availability on the avermectin production in S. avermitilis DJ, a high yield strain, by adding different concentrations of isoleucine and sodium propionate in fermentation broth. After 10 days, we assessed the avermectin production by HPLC. The yield of avermectin in control group were higher than the experimental group. Adding isoleucine and sodium propionate could not improve avermectin production in S. avermitilis DJ.Secondly, the vectors for knocking out the gene ilvBl, ilvB3, ilvB4 and ilvH. related to precursor metabolism were constructed and applied to S. avermitilis WT, 9-39 and DJ. I failed to knock out gene ilvBl and ilvH in these three strains, suggesting that these genes are essential. For the tdh knock-out mutants, the avermectin production increased most significantly in S.avermitilis 9-39 by 75%, followed inWT by 44%, and the yield was slightly higher in DJ. The result indicates that truncation of the tributary pathway can promote the biosynthesis of the precursors isoleucine and valine, which could increase the yield of avermectin. Gene ilvB3 could not be knocked out in S. avermitilis 9-39, suggesting that ilvB3 may be essential in 9-39. But it can be knocked out in S.avermitilis WT and DJ. The avermectin production of the former had no significant change and the later increased 88%. The effect of gene ilvB4 knock-out in S. avermitilis WT and 9-39 were not obvious, but in S. avermitilis DJ the production increased 77%. The influence on avermectin production of S. avermitilis DJ by knocking out ilvB3 or ilvB4 may be due to some metabolic changes in the avermectin biosynthetic pathway. The avermectin component produced by S.avermitilis 9-39 and DJ with knocking-out gene ilvB3 or ilvB4 did not change significantly, probably because acetolactate synthase in high-yielding strains is mainly regulated by gene ilvBl and ilvH, but not by ilvB3 or ilvB4. With the gene ilvB3 or ilvB4 knocking-out in the wild-type strain WT, the ratio of avermectin components varied. The result indicated the two genes in WT would participate in the regulation of isoleucine and valine, with a preference for isoleucine synthesis.At last, the vectors for site-directed mutagenesis of ilvH, N11D and N29D, were constructed and applied to the S. avermitilis WT,9-39 and DJ. The vectors for ilvH overexpression with different promoters were constructed and applied to S. avermitilis WT. Except N11D in S. avermitilis DJ, the production of component "b" of all other site-directed mutant strains increased and the ratio of component "a" to "b" decreased. The effect of the mutation N11D is not as obvious as N29D. This suggested that the mutation in ilvH really relieved the feedback inhibition of valine to some extent. However, the improvement of production was not significant In the case of overexpresion of ilvH with different promoters in S.avermitilis WT, the production of avermectin improved to different extent, and the one with promoter gapdhp(RER) is the best, which improved about 70%. This study will offer a reference for the research of acetolactate synthase and provide the foundation for further improvement of avermectin production and fraction "a" in particular.