Zearalenone is a common mycotoxin contaminant in cereals that causes severe economic losses and serious risks to health of human and animals. Many strategies have been devised to degrade ZEN and keep food safe. The hydrolase ZHD101 from Clonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, has been studied widely. In the current research, three new enzymes that have the capacity to detoxify ZEN were identified, namely CLA, EXO, and TRI, showing 61%, 63%, and 97% amino acids identities with ZHD101, respectively. Three coding genes was expressed as heterologous in Escherichia coli BL21. Through biochemical analysis, the purified recombinant CLA, EXO, TRI, and ZHD101 exhibited high activities of degrading ZEN with the specific activity of 114.8 U/mg, 459.0 U/mg, 239.8 U/mg, and 242.8 U/mg. The optimal temperatures of CLA, EXO, TRI, and ZHD101 were 40 °C, 40 °C, 40 °C, and 45 °C, and their optimum pH were 7.0, 9.0, 9.5, and 9.0, respectively. Our study demonstrated that the novel enzymes CLA, EXO, and TRI possessed high ability to degrade ZEN from the model solutions and could be the promising candidates for ZEN detoxification in practical application.

Saccharopolyspora spinosa is a well-known actinomycete for producing the secondary metabolites, spinosad, which is a potent insecticides possessing both efficiency and safety. In the previous researches, great efforts, including physical mutagenesis, fermentation optimization, genetic manipulation and other methods, have been employed to increase the yield of spinosad to hundreds of folds from the low-yield strain. However, the metabolic network in S. spinosa still remained un-revealed. In this study, two S. spinosa strains with different spinosad production capability were fermented and sampled at three fermentation periods. Then the total RNA of these samples was isolated and sequenced to construct the transcriptome libraries. Through transcriptomic analysis, large numbers of differentially expressed genes were identified and classified according to their different functions. According to the results, spnI and spnP were suggested as the bottleneck during spinosad biosynthesis. Primary metabolic pathways such as carbon metabolic pathways exhibited close relationship with spinosad formation, as pyruvate and phosphoenolpyruvic acid were suggested to accumulate in spinosad high-yield strain during fermentation. The addition of soybean oil in the fermentation medium activated the lipid metabolism pathway, enhancing spinosad production. Glutamic acid and aspartic acid were suggested to be the most important amino acids and might participate in spinosad biosynthesis.

Spinosad (spinosyn A and spinosyn D), the secondary metabolite produced by Saccharopolyspora spinosa, is a potent insecticide with low effects on the environment and mammals. Strategies such as metabolic engineering, mutagenesis and fermentation process optimization have been employed for its production enhancement. Quantitative real-time polymerase chain reaction(qRT-PCR) is one of the preferred methods for the evaluation of gene transcript levels, whose accuracy and sensitivity depend on the normalization using optimal reference genes. However, no single reference gene is universally appropriate for all strains under various conditions. In this study, the transcriptional stability of 35 candidate reference genes, including 23 traditionally used reference genes and 12 novel reference genes identified in S. spinosa homologous species, was analysed in S. spinosa ATCC 49460 and spinosad high-yield strain S. spinosa S3-3 under three fermentation phases. The transcriptional stability of these genes was assessed by three statistical algorithms, geNorm, NormFinder and BestKeeper. The overall rankings suggested that rpoB and efp were the most stable candidate reference genes, and they may be the most promising reference genes for further study on the measurement of expression levels of target genes involved in the biosynthetic process of spinosad.