Abstract
BackgroundPolyketides, such as spinosad, are mainly synthesized in the stationary phase of the fermentation. The synthesis of these compounds requires many primary metabolites, such as acetyl-CoA, propinyl-CoA, NADPH, and succinyl-CoA. Their synthesis is also significantly influenced by NADH/NAD+. Rex is the sensor of NADH/NAD+ redox state, whose structure is under the control of NADH/NAD+ ratio. The structure of rex controls the expression of many NADH dehydrogenases genes and cytochrome bd genes. Intracellular redox state can be influenced by adding extracellular electron acceptor H2O2. The effect of extracellular oxidoreduction potential on spinosad production has not been studied. Although extracellular oxidoreduction potential is an important environment effect in polyketides production, it has always been overlooked. Thus, it is important to study the effect of extracellular oxidoreduction potential on Saccharopolyspora spinosa growth and spinosad production.ResultsDuring stationary phase, S. spinosa was cultured under oxidative (H2O2) and reductive (dithiothreitol) conditions. The results show that the yield of spinosad and pseudoaglycone increased 3.11 fold under oxidative condition. As H2O2 can be served as extracellular electron acceptor, the ratios of NADH/NAD+ were measured. We found that the ratio of NADH/NAD+ under oxidative condition was much lower than that in the control group. The expression of cytA and cytB in the rex mutant indicated that the expression of these two genes was controlled by rex, and it was not activated under oxidative condition. Enzyme activities of PFK, ICDH, and G6PDH and metabolites results indicated that more metabolic flux flow through spinosad synthesis.ConclusionThe regulation function of rex was inhibited by adding extracellular electron acceptor-H2O2 in the stationary phase. Under this condition, many NADH dehydrogenases which were used to balance NADH/NAD+ by converting useful metabolites to useless metabolites and unefficient terminal oxidases (cytochrome bd) were not expressed. So lots of metabolites were not waste to balance. As a result, un-wasted metabolites related to spinosad and PSA synthesis resulted in a high production of spinosad and PSA under oxidative condition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0098-z) contains supplementary material, which is available to authorized users.
Highlights
Polyketides, such as spinosad, are mainly synthesized in the stationary phase of the fermentation
Spinosad and PSA production and S. spinosa growth under different extracellular oxidation-reduction potentials Batch cell growth, spinosad production, and fermentation parameters were analyzed for the whole fermentation process under different extracellular oxidoreduction potential
By blasting genes located in the downstream of rex with the genome of Saccharopolyspora erythraea, Streptomyces coelicolor, and Streptomyces avermitilis, we found that genes located in the downstream of rex were cytochrome bd oxidase synthesis gene, cytAB
Summary
Polyketides, such as spinosad, are mainly synthesized in the stationary phase of the fermentation The synthesis of these compounds requires many primary metabolites, such as acetyl-CoA, propinyl-CoA, NADPH, and succinyl-CoA. The effect of extracellular oxidoreduction potential on spinosad production has not been studied. It is important to study the effect of extracellular oxidoreduction potential on Saccharopolyspora spinosa growth and spinosad production. Studies have demonstrated that S. spinosa can synthesize more than 25 spinosyns that vary in structures and functions [2]. Among these spinosyns, spinosyn A and spinosyn D, the mixture of which was called spinosad, are the most two abundant and effective spinosyns [2]. Spinosad-based insect control pesticide was awarded the Presidential Green Chemistry Challenge Award in 1999 [5]
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