Bacterioferritin: a key iron storage modulator that affects strain growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona.

BACKGROUND: Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. RESULTS: Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. CONCLUSION: Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes.

Comparative transcriptomic analysis of two Saccharopolyspora spinosa strains reveals the relationships between primary metabolism and spinosad production.

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.

Effects of acuC on the growth development and spinosad biosynthesis of Saccharopolyspora spinosa.

BACKGROUND: Acetoin utilization protein (acuC) is a type I histone deacetylase which is highly conserved in bacteria. The acuC gene is related to the acetylation/deacetylation posttranslational modification (PTM) system in S. spinosa. Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. However, the specific functions and influences of acuC protein in S. spinosa are yet to be characterized. RESULTS: The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The production of spinosyns A and D from S. spinosa-acuC were 105.02 mg/L and 20.63 mg/L, which were 1.82-fold and 1.63-fold higher than those of the wild-type strain (57.76 mg/L and 12.64 mg/L), respectively. The production of spinosyns A and D from S. spinosa-ΔacuC were 32.78 mg/L and 10.89 mg/L, respectively. The qRT-PCR results of three selected genes (bldD, ssgA and whiA) confirmed that the overexpression of acuC affected the capacities of mycelial differentiation and sporulation. Comparative proteomics analysis was performed on these strains to investigate the underlying mechanism leading to the enhancement of spinosad yield. CONCLUSIONS: This study first systematically analysed the effects of overexpression acuC on the growth of S. spinosa and the production of spinosad. The results identify the differentially expressed proteins and provide evidences to understand the acetylation metabolic mechanisms which can lead to the increase of secondary metabolites.

Effect of the TetR family transcriptional regulator Sp1418 on the global metabolic network of Saccharopolyspora pogona.

BACKGROUND: Saccharopolyspora pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. pogona are yet to characterize. RESULTS: In the present study, the overexpression of the tetR-like gene sp1418 in S. pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. CONCLUSION: This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. pogona, and provided insights into the improvement of secondary metabolite production.

Combined available nitrogen resources enhanced erythromycin production and preliminary exploration of metabolic flux analysis under nitrogen perturbations.

In the current study, the effect of different available nitrogen sources on erythromycin fermentation by Saccharopolyspora erythraea No. 8 is evaluated. Three different combinations of corn steep liquor and yeast powder were developed to investigate their impacts on erythromycin production. The results indicate that the optimal combination of available nitrogen sources was 10.0 g/L corn steep liquor and 4.0 g/L yeast power, generating a maximum yield of erythromycin of 13672 U/mL. To explore the effects of nitrogen perturbations on cell metabolism, metabolic flux analyses were performed and compared under different conditions. A high flux pentose phosphate pathway provided more NADPH for erythromycin synthesis via nitrogen optimization. Moreover, high n-propanol specific consumption rate enhanced erythromycin synthesis and n-propanol flowed into the central carbon metabolism by methylmalonyl-CoA node. These results indicate that the selection of an appropriate organic nitrogen source is essential for cell metabolism and erythromycin synthesis, and this is the first report of the successful application of available nitrogen source combinations in industrial erythromycin production.

A novel multidomain acyl-CoA carboxylase in Saccharopolyspora erythraea provides malonyl-CoA for de novo fatty acid biosynthesis.

Acetyl-CoA carboxylases (ACCs) are enzyme complexes generally composed of three catalytic domains and distributed in all organisms. In prokaryotes and plastids of most plants, these domains are encoded in distinct subunits forming heteromeric complexes. Distinctively, cytosolic ACCs from eukaryotes and plastids of graminaceous monocots, are organized in a single multidomain polypeptide. Until now, no multidomain ACCs had been discovered in bacteria. Here, we show that a putative multidomain ACC in Saccharopolyspora erythraea is encoded by the sace_4237 gene, representing the first prokaryotic ACC homodimeric multidomain complex described. The SACE_4237 complex has both acetyl-CoA and propionyl-CoA carboxylase activities. Importantly, we demonstrate that sace_4237 is essential for S. erythraea survival as determined by the construction of a sace_4237 conditional mutant. Altogether, our results show that this prokaryotic homodimeric multidomain ACC provides malonyl-CoA for de novo fatty acid biosynthesis. Furthermore, the data presented here suggests that evolution of these enzyme complexes, from single domain subunits to eukaryotic multidomain ACCs, occurred in bacteria through domain fusion.

Two amino acids missing of MtrA resulted in increased erythromycin level and altered phenotypes in Saccharopolyspora erythraea.

The MtrA-MtrB two-component regulatory system is highly conserved in Actinobacteria and plays crucial roles in cell cycle progression, cell morphology, antibiotic resistance, and osmoprotection. Previously, we revealed that the MtrA protein of Saccharopolyspora erythraea E3 strain (a high erythromycin-producing strain) had a two amino acid (H197 and V198) deletion in the DNA recognition helices of the C-terminal domain compared to the wild type S. erythraea strain NRRL2338. Here, we identified mepA (encoding a membrane protein related to metalloendopeptidases) as an MtrA target gene, and found that deleting the two amino acids in MtrA (MtrAdel) resulted in the loss of its DNA-binding activity for the mepA gene. The mutant MtrAdel lost its regulatory activity and affected various physiological functions consistent with mtrA deletion, including increased erythromycin biosynthesis, enhanced antibiotic resistance, deregulated osmoprotection, and improved transport of substances. The introduction of the wild type mtrA gene into the S. erythraea E3 strain with the mtrAdel gene decreased the erythromycin yield by approximately 50%, confirming that MtrA repressed erythromycin production. These findings demonstrate that MtrA is an important pleiotropic regulator of erythromycin biosynthesis, antibiotic resistance, osmoprotection, and substance transport in S. erythraea and provide new insights for improving erythromycin production. Future studies linking the molecular effects of MtrA to these phenotypes will improve our understanding of the MtrA-MtrB two-component regulatory system in Actinobacteria.

Impact on strain growth and butenyl-spinosyn biosynthesis by overexpression of polynucleotide phosphorylase gene in Saccharopolyspora pogona.

Polynucleotide phosphorylase is a highly conserved protein found in bacteria and fungi that can regulate the transcription of related enzymes involved in amino acid metabolism, organic acid metabolism, and cell biosynthesis. We studied the effect of polynucleotide phosphorylase on Saccharopolyspora pogona (S. pogona) growth and the synthesis of secondary metabolites. First, we generated the overexpression vector pOJ260-PermE-pnp via overlap extension PCR. The vector pOJ260-PermE-pnp was then introduced into S. pogona by conjugal transfer, thereby generating the recombination strain S. pogona-Pnp. Results showed that engineering strains possessed higher biomass than those of the wild-type strains. Moreover, the ability of these strains to produce spores on solid medium was stronger than that of the wild-type strains. HPLC results revealed that the butenyl-spinosyn yield in S. pogona-Pnp increased by 1.92-fold compared with that of S. pogona alone. These findings revealed that overexpression of polynucleotide phosphorylase effectively promoted butenyl-spinosyn biosynthesis in S. pogona. This result may be extended to other Streptomyces for strain improvement.

High-throughput optimization of the chemically defined synthetic medium for the production of erythromycin A.

Erythromycin A is an important antibiotic. A chemically defined synthetic medium for erythromycin production was systematically optimized in this study. A high-throughput method was employed to reduce the number of components and optimize the concentration of each component. After two round single composition deletion experiment, only 19 components were remained in the medium, and then the concentration of each component was optimized through PB experiment. The optimal medium from the PB experiment was further optimized according to the nitrogen and phosphate metabolic consumption in 5 L bioreactor. It was observed that among the 8 amino acids concluded in the media, 4 amino acids were first consumed, when they are almost depleted, the other 4 amino acids were initiated their consumption afterwards in 5 L bioreactor. The decrease of phosphate concentration would increase qglc and qery. However, when phosphate concentration was too low, the production of erythromycin was hindered. The positive correlation between intracellular metabolite pools and Yery/glc indicated that low phosphate concentration in the medium can promote cell metabolism especially secondary metabolism during the stationary phase; however, if it was too low (5 mmol/L), the cell metabolism and secondary metabolism would both slow down. The erythromycin titer in the optimized medium (medium V) reached 1380 mg/L, which was 17 times higher than the previously used synthetic medium in our lab. The optimized medium can facilitate the metabolomics study or metabolic flux analysis of the erythromycin fermentation process, which laid a solid foundation for further study of erythromycin fermentation process.

Metabolomics for industrial fermentation.

Metabolomics is essential to understand the metabolism and identify engineering targets to improve the performances of strains and bioprocesses. Although numerous metabolomics techniques have been developed and applied to various organisms, the metabolome of Saccharopolyspora erythraea, a native producer of erythromycin, had never been studied. The 2017 best paper of Bioprocess and Biosystems Engineering reports examination of three methods for quenching and extraction to analyze the intracellular metabolome of S. erythraea, and identified the most reliable methods for studying different groups of the metabolites. Subsequent studies on the dynamics of the intracellular metabolome of S. erythraea during the fed-batch fermentation identified a positive correlation between the specific erythromycin production rate and the pool size of intracellular propionyl-CoA and other precursors of erythromycin. A series of follow-up studies, such as demonstrating the applicability of the quenching/extraction methods in other related antibiotic producers, demonstrating the generality of the best matches between the quenching/extraction methods and the metabolite groups, and combining metabolomics approaches with the fluxomics and systems metabolic engineering approaches, will facilitate the metabolomics studies on important antibiotic producers, enable standardization of the quenching/extraction protocols, and improve the performance of the antibiotic production with deeper insight into their metabolism.

GlnR and PhoP regulate ß-glucosidases involved in cellulose digestion in response to nitrogen and phosphate availability.

The limited catalytic efficiency of cellulose-degrading enzymes restricts cellulose digestion. We investigated the transcriptional regulation of genes encoding key cellulose degrading enzymes, namely ß-glucosidases, in the industrial actinobacterium Saccharopolyspora erythraea. We observed that the expression of most ß-glucosidase-encoding genes was controlled by the availability of nitrogen and phosphate via their respective global regulators, namely GlnR and PhoP. Electrophoretic mobility shift assay demonstrated that GlnR and PhoP bound directly to the promoters of ß-glucosidase-encoding genes. Deletion of glnR resulted in lower transcript levels and activity of ß-glucosidases, leading to decreased bacterial growth on cellulose. Overexpression of glnR and phoP or nitrogen/phosphate starvation increased the transcript levels and total activity of ß-glucosidases. Moreover, GlnR/PhoP-mediated cellobiose utilization was also observed in Streptomyces coelicolor A3(2). These findings provide insights into the regulatory roles played by GlnR and PhoP in coordinating nitrogen/phosphate metabolism and carbohydrate utilization, and indicate potential strategies for cellulose fermentation in the production of bio-based chemicals by actinobacteria.

Saccharopolyspora Species: Laboratory Maintenance and Enhanced Production of Secondary Metabolites.

Saccharopolyspora spp. are aerobic, Gram-positive, non-acid-fast, and non-motile actinomycetes. Various species of the genus Saccharopolyspora have been reported with an ability to produce various bioactive compounds for pharmaceutical and agricultural uses. This unit includes general protocols for the laboratory maintenance of Saccharopolyspora species, including growth in liquid medium, growth on solid agar, long-term storage, and generation of a higher producer strain by mutagenesis. Saccharopolyspora spinosa ATCC 49460 is used as a prototype for explaining the considerations for efficient laboratory maintenance of Saccharopolyspora spp. Saccharopolyspora spinosa is a producer of spinosad, a prominent insecticide with selective activity against various insects. © 2017 by John Wiley & Sons, Inc.

Reciprocal Regulation of GlnR and PhoP in Response to Nitrogen and Phosphate Limitations in Saccharopolyspora erythraea.

Nitrogen and phosphate source sensing, uptake, and assimilation are essential for the growth and development of microorganisms. In this study, we demonstrated that SACE_6965 encodes the phosphate regulator PhoP, which controls the transcription of genes involved in phosphate metabolism in the erythromycin-producing Saccharopolyspora erythraea. We found that PhoP and the nitrogen regulator GlnR both regulate the transcription of glnR as well as other nitrogen metabolism-related genes. Interestingly, both GlnR- and PhoP-binding sites were identified in the phoP promoter region. Unlike the nonreciprocal regulation of GlnR and PhoP observed in Streptomyces coelicolor and Streptomyces lividans, GlnR negatively controls the transcription of the phoP gene in S. erythraea. This suggests that GlnR directly affects phosphate metabolism and demonstrates that the cross talk between GlnR and PhoP is reciprocal. Although GlnR and PhoP sites in the glnR and phoP promoter regions are located in close proximity to one another (separated by only 2 to 4 bp), the binding of both regulators to their respective region was independent and noninterfering. These results indicate that two regulators could separately bind to their respective binding sites and control nitrogen and phosphate metabolism in response to environmental changes. The reciprocal cross talk observed between GlnR and PhoP serves as a foundation for understanding the regulation of complex primary and secondary metabolism in antibiotic-producing actinomycetes.

Controlling the feed rate of propanol to optimize erythromycin fermentation by on-line capacitance and oxygen uptake rate measurement.

The aim of the present study was to optimize the feeding proportion of glucose and propanol for erythromycin biosynthesis by real-time monitoring and exploring its limited ratio by the on-line multi-frequency permittivity measurement. It was found that the capacitance values were sensitive to the variation of biomass concentration and microbial morphology as well as the true state of cell growth. It was most favorable to both cell growth and secondary metabolism to keep the ratio of glucose to propanol at 4.3 (g/g). The specific growth rate calculated by the capacitance measurement correctly and accurately reflected the cell physiological state. An appropriate feed rate of propanol was crucial for cell growth and secondary metabolism, as well as to improve the quality of erythromycin-A. In addition, the erythromycin production titer (10,950 U/mL) was further enhanced by 4 % when the propanol feed was regulated by step-down strategy based on both OUR (oxygen uptake rate) and the on-line monitoring capacitance.

[Disruption of leucyl aminopeptidase gene affects phenotypes and second metabolite production of Saccharopolyspora spinosa].

Objective: In order to investigate effects of leucyl aminopeptidase on mycelia morphology, growth rate, spinosad yield and protein expression in Saccharopolyspora spinosa by disrupting its encoding gene pepA and analyzing the characteristics of engineered S. spinosa. Methods: The pepA gene of S. spinosa was amplified based on the conserved sequence and cloned into Escherichia coli-Streptomyces shuttle vector pOJ260 to generate pOJ260- pepA, which was transformed into S. spinosa by conjugation. Mycelium observation, SDS-PAGE and HPLC were used to analyze the engineered strain. Results: Mycelia in S. sp-ΔpepA displayed a much higher degree of fragmentation and fewer branches compared to that of parental strain. Meanwhile, the growth rate of S. sp-ΔpepA was retarded and its biomass was reduced. Shake-flask fermentation demonstrated that spinosad yield increased by 122% in S. sp-ΔpepA strain compared to that of parental strain. SDS-PAGE analysis showed that protein expression profile of the engineered strain significantly changed. Conclusion: The pepA gene negatively regulates the biosynthesis of spinosad and disruption of pepA gene could affect the mycelial morphology and growth of S. spinosa.

Proteomic insights into metabolic adaptation to deletion of metE in Saccharopolyspora spinosa.

Saccharopolyspora spinosa can produce spinosad as a major secondary metabolite, which is an environmentally friendly agent for insect control. Cobalamin-independent methionine synthase (MetE) is an important enzyme in methionine biosynthesis, and this enzyme is probably closely related to spinosad production. In this study, its corresponding gene metE was inactivated, which resulted in a rapid growth and glucose utilisation rate and almost loss of spinosad production. A label-free quantitative proteomics-based approach was employed to obtain insights into the mechanism by which the metabolic network adapts to the absence of MetE. A total of 1440 proteins were detected from wild-type and ΔmetE mutant strains at three time points: stationary phase of ΔmetE mutant strain (S1ΔmetE , 67 h), first stationary phase of wild-type strain (S1WT, 67 h) and second stationary phase of wild-type strain (S2WT, 100 h). Protein expression patterns were determined using an exponentially modified protein abundance index (emPAI) and analysed by comparing S1ΔmetE /S1WT and S1ΔmetE /S2WT. Results showed that differentially expressed enzymes were mainly involved in primary metabolism and genetic information processing. This study demonstrated that the role of MetE is not restricted to methionine biosynthesis but rather is involved in global metabolic regulation in S. spinosa.

DasR is a pleiotropic regulator required for antibiotic production, pigment biosynthesis, and morphological development in Saccharopolyspora erythraea.

The GntR-family transcription regulator, DasR, was previously identified as pleiotropic, controlling the primary amino sugar N-acetylglucosamine (GlcNAc) and chitin metabolism in Saccharopolyspora erythraea and Streptomyces coelicolor. Due to the remarkable regulatory impact of DasR on antibiotic production and development in the model strain of S. coelicolor, we here identified and characterized the role of DasR to secondary metabolite production and morphological development in industrial erythromycin-producing S. erythraea. The physiological studies have shown that a constructed deletion of dasR in S. erythraea resulted in antibiotic, pigment, and aerial hyphae production deficit in a nutrient-rich condition. DNA microarray assay, combined with quantitative real-time reverse transcription PCR (qRT-PCR), confirmed these results by showing the downregulation of the genes relating to secondary metabolite production in the dasR null mutant. Notably, electrophoretic mobility shift assays (EMSA) showed DasR as being the first identified regulator that directly regulates the pigment biosynthesis rpp gene cluster. In addition, further studies indicated that GlcNAc, the major nutrient signal of DasR-responsed regulation, blocked secondary metabolite production and morphological development. The effects of GlcNAc were shown to be caused by DasR mediation. These findings demonstrated that DasR is an important pleiotropic regulator for both secondary metabolism and morphological development in S. erythraea, providing new insights for the genetic engineering of S. erythraea with increased erythromycin production.

Four-stage dissolved oxygen strategy based on multi-scale analysis for improving spinosad yield by Saccharopolyspora spinosa†ATCC49460.

Dissolved oxygen (DO) is an important influencing factor in the process of aerobic microbial fermentation. Spinosad is an aerobic microbial-derived secondary metabolite. In our study, spinosad was used as an example to establish a DO strategy by multi-scale analysis, which included a reactor, cell and gene scales. We changed DO conditions that are related to the characteristics of cell metabolism (glucose consumption rate, biomass accumulation and spinosad production). Consequently, cell growth was promoted by maintaining DO at 40% in the first 24 h and subsequently increasing DO to 50% in 24 h to 96 h. In an in-depth analysis of the key enzyme genes (gtt, spn A, spn K and spn O), expression of spinosad and specific Adenosine Triphosphate (ATP), the spinosad yield was increased by regulating DO to 30% within 96 h to 192 h and then changing it to 25% in 192 h to 240 h. Under the four-phase DO strategy, spinosad yield increased by 652.1%, 326.1%, 546.8%, and 781.4% compared with the yield obtained under constant DO control at 50%, 40%, 30%, and 20% respectively. The proposed method provides a novel way to develop a precise DO strategy for fermentation.

Enhanced oxygen transfer rate and bioprocess yield by using magnetite nanoparticles in fermentation media of erythromycin.

BACKGROUND: Magnetite nanoparticles have widespread biomedical applications. In the aerobic bioprocesses, oxygen is a limiting factor for the microbial metabolic rate; hence a high availability of oxygen in the medium is crucial for high fermentation productivity. This study aimed to examine the effect of using magnetite nanoparticles on oxygen transfer rate in erythromycin fermentation culture. METHODS: Magnetite nanoparticles were synthetized through co-precipitation method. After observing the enhanced oxygen transfer rate in deionized water enriched with magnetite nanoparticles, these nanoparticles were used in the media of by Saccharopolyspora erythraea growth to explore their impact on erythromycin fermentation titer. Treatments comprised different concentrations of magnetite nanoparticles, (0, 0.005, 0.02 v/v). RESULTS: In the medium containing 0.02 v/v magnetite nanoparticles, KLa was determined to be 1.89 time higher than that in magnetite nanoparticle-free broth. An improved 2.25 time higher erythromycin titer was obtained in presence of 0.02 v/v nanoparticles. CONCLUSIONS: Our results, demonstrate the potential of magnetite nanoparticles for enhancing the productivity of aerobic pharmaceutical bioprocesses.

Suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in Saccharopolyspora spinosa.

BACKGROUND: 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. 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. RESULTS: During 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. CONCLUSION: The 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.