The Experts below are selected from a list of 4521 Experts worldwide ranked by ideXlab platform
Wenyu Lu - One of the best experts on this subject based on the ideXlab platform.
-
Fed-Batch Fermentation for Spinosad Production in an Improved Reactor
Transactions of Tianjin University, 2017Co-Authors: Chunzhe Lu, Chuanbo Zhang, Wenyu LuAbstract:As a kind of aerobic bacteria, Saccharopolyspora spinosa exhibits a high demand for oxygen. In the fermentation process, the methods of increasing ventilation and improving agitation speed are usually adopted to achieve higher values of dissolved oxygen. These methods decrease the efficiency of Spinosad biosynthesis. In this study, an improved reactor was designed to solve these problems. The exhaust gas reflux device, impellers, and baffles were improved. Furthermore, we established the kinetic models for the cell growth, substrate consumption and Spinosad generation in batch fermentation process. The simulation results were in good agreement with the experimental data. Spinosad production reached 583.86 mg/L after employing the suitable feeding strategy by fed-batch fermentation in the improved reactor, whereas it was only 157.01 mg/L before optimization. The method described can provide insight to strengthen Spinosad production and can be extended to the culturing process of filamentous aerobic bacteria.
-
metabolomics analysis of the effect of dissolved oxygen on Spinosad production by saccharopolyspora spinosa
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2017Co-Authors: Chunzhe Lu, Fanglong Zhao, Feng Li, Wenyu LuAbstract:Spinosad, a universal bio-pesticide, is obtained from the soil actinomycete Saccharopolyspora spinosa. Dissolved oxygen, an important contributing factor in aerobic microbial fermentation, however, is not always available in sufficient amounts. To alleviate oxygen limitation in Spinosad production, three different oxygen vectors, namely oleic acid, toluene, and n-dodecane, were added into early fermentation. Results indicated that n-dodecane was the optimal oxygen vector. Spinosad yield was increased by 44.2% compared to that in the control group in the presence of 0.5% n-dodecane, added after 120 h of incubation. Yields of the test group reached 6.52 mg/g dry cell weight (DCW), while that of the control group was limited to 4.52 mg/g DCW. Metabolomics analysis by gas chromatography coupled to mass spectrometry was performed to demonstrate the metabolism mechanism in the presence and absence of oxygen vector. In total, 78 principal intracellular metabolites in S. spinosa were detected and quantified in the presence and absence of n-dodecane. Levels of some metabolites that were related to the tricarboxylic acid cycle and pentose phosphate pathway varied significantly. Aspartic acid and glucose-1-phosphate levels varied significantly and contributed most in the distinction of the fermentation conditions and phases. The above findings give new insights into the improvement and the metabolomic characteristics of industrial Spinosad production.
-
suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in saccharopolyspora spinosa
Microbial Cell Factories, 2014Co-Authors: Xiangmei Zhang, Chuanbo Zhang, Fanglong Zhao, Qinggele Caiyin, Dashuai Li, Wenyu LuAbstract: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.
-
genome scale metabolic network reconstruction of saccharopolyspora spinosa for Spinosad production improvement
Microbial Cell Factories, 2014Co-Authors: Xiaoyang Wang, Chuanbo Zhang, Meiling Wang, Wenyu LuAbstract:Spinosad is a macrolide antibiotic produced by Saccharopolyspora spinosa with aerobic fermentation. However, the wild strain has a low productivity. In this article, a computational guided engineering approach was adopted in order to improve the yield of Spinosad in S. spinosa. Firstly, a genome-scale metabolic network reconstruction (GSMR) for S.spinosa based on its genome information, literature data and experimental data was extablished. The model was consists of 1,577 reactions, 1,726 metabolites, and 733 enzymes after manually refined. Then, amino acids supplying experiments were performed in order to test the capabilities of the model, and the results showed a high consistency. Subsequently, transhydrogenase (PntAB, EC 1.6.1.2) was chosen as the potential target for Spinosad yield improvement based on the in silico metabolic network models. Furthermore, the target gene was manipulated in the parent strain in order to validate the model predictions. At last, shake flask fermentation was carried out which led to Spinosad production of 75.32 mg/L, 86.5% higher than the parent strain (40.39 mg/L). Results confirmed the model had a high potential in engineering S. spinosa for Spinosad production. It is the first GSMM for S.spinosa, it has significance for a better understanding of the comprehensive metabolism and guiding strain designing of Saccharopolyspora spinosa in the future.
-
stepwise increase of Spinosad production in saccharopolyspora spinosa by metabolic engineering
Biochemical Engineering Journal, 2013Co-Authors: Yuejiao Duan, Fanglong Zhao, Wenyu LuAbstract:Abstract Rational metabolic and cellular engineering approaches are useful in improving strain performance. In the last years, several studies not only clarified the biosynthetic pathway of Spinosad, but also provided useful information of metabolic restrictions in the spinosyn biosynthetic pathway. However, these studies overlooked the problem that the spinosyn biosynthetic pathway was unbalanced: the expression of six genes in the spinosyn biosynthetic pathway was insufficient; Saccharopolyspora spinosa accumulated useless compounds because of insufficient expression of SpnK . So a rational strain improvement strategy was developed to tune the unbalanced spinosyn biosynthetic pathway. First, we overexpressed spnK to increase the amount of the flux from rhamnosylated aglycone to pseudoaglycones (PSA). Then six genes ( spnP , spnO , spnN , spnQ , spnR , and spnS ) involved in forosamine biosynthesis and spnK were co-expressed in S. spinosa LU102 to convert the accumulated PSA to Spinosad. The yield of Spinosad in S. spinosa LU102 was 214 mg/L, which was 2.6-fold higher than that in the wild-type S. spinosa (82 mg/L). Finally, Spinosad production in the tuned S. spinosa LU104 was further increased to 405 mg/L, which was a 5.0-fold enhancement compared with the wild-type S. spinosa , by duplicating spnP , spnO , spnN , spnQ , spnR , spnS , spnK , gtt , gdh and kre genes.
Qinggele Caiyin - One of the best experts on this subject based on the ideXlab platform.
-
suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in saccharopolyspora spinosa
Microbial Cell Factories, 2014Co-Authors: Xiangmei Zhang, Chuanbo Zhang, Fanglong Zhao, Qinggele Caiyin, Dashuai Li, Wenyu LuAbstract: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.
-
Suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in Saccharopolyspora spinosa.
Microbial cell factories, 2014Co-Authors: Xiangmei Zhang, Chuanbo Zhang, Fanglong Zhao, Chaoyou Xue, Jing Yin, Qinggele CaiyinAbstract: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. 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. 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.
Xiangmei Zhang - One of the best experts on this subject based on the ideXlab platform.
-
suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in saccharopolyspora spinosa
Microbial Cell Factories, 2014Co-Authors: Xiangmei Zhang, Chuanbo Zhang, Fanglong Zhao, Qinggele Caiyin, Dashuai Li, Wenyu LuAbstract: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.
-
Suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in Saccharopolyspora spinosa.
Microbial cell factories, 2014Co-Authors: Xiangmei Zhang, Chuanbo Zhang, Fanglong Zhao, Chaoyou Xue, Jing Yin, Qinggele CaiyinAbstract: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. 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. 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.
Chuanbo Zhang - One of the best experts on this subject based on the ideXlab platform.
-
Fed-Batch Fermentation for Spinosad Production in an Improved Reactor
Transactions of Tianjin University, 2017Co-Authors: Chunzhe Lu, Chuanbo Zhang, Wenyu LuAbstract:As a kind of aerobic bacteria, Saccharopolyspora spinosa exhibits a high demand for oxygen. In the fermentation process, the methods of increasing ventilation and improving agitation speed are usually adopted to achieve higher values of dissolved oxygen. These methods decrease the efficiency of Spinosad biosynthesis. In this study, an improved reactor was designed to solve these problems. The exhaust gas reflux device, impellers, and baffles were improved. Furthermore, we established the kinetic models for the cell growth, substrate consumption and Spinosad generation in batch fermentation process. The simulation results were in good agreement with the experimental data. Spinosad production reached 583.86 mg/L after employing the suitable feeding strategy by fed-batch fermentation in the improved reactor, whereas it was only 157.01 mg/L before optimization. The method described can provide insight to strengthen Spinosad production and can be extended to the culturing process of filamentous aerobic bacteria.
-
suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in saccharopolyspora spinosa
Microbial Cell Factories, 2014Co-Authors: Xiangmei Zhang, Chuanbo Zhang, Fanglong Zhao, Qinggele Caiyin, Dashuai Li, Wenyu LuAbstract: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.
-
Suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in Saccharopolyspora spinosa.
Microbial cell factories, 2014Co-Authors: Xiangmei Zhang, Chuanbo Zhang, Fanglong Zhao, Chaoyou Xue, Jing Yin, Qinggele CaiyinAbstract: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. 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. 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.
-
genome scale metabolic network reconstruction of saccharopolyspora spinosa for Spinosad production improvement
Microbial Cell Factories, 2014Co-Authors: Xiaoyang Wang, Chuanbo Zhang, Meiling Wang, Wenyu LuAbstract:Spinosad is a macrolide antibiotic produced by Saccharopolyspora spinosa with aerobic fermentation. However, the wild strain has a low productivity. In this article, a computational guided engineering approach was adopted in order to improve the yield of Spinosad in S. spinosa. Firstly, a genome-scale metabolic network reconstruction (GSMR) for S.spinosa based on its genome information, literature data and experimental data was extablished. The model was consists of 1,577 reactions, 1,726 metabolites, and 733 enzymes after manually refined. Then, amino acids supplying experiments were performed in order to test the capabilities of the model, and the results showed a high consistency. Subsequently, transhydrogenase (PntAB, EC 1.6.1.2) was chosen as the potential target for Spinosad yield improvement based on the in silico metabolic network models. Furthermore, the target gene was manipulated in the parent strain in order to validate the model predictions. At last, shake flask fermentation was carried out which led to Spinosad production of 75.32 mg/L, 86.5% higher than the parent strain (40.39 mg/L). Results confirmed the model had a high potential in engineering S. spinosa for Spinosad production. It is the first GSMM for S.spinosa, it has significance for a better understanding of the comprehensive metabolism and guiding strain designing of Saccharopolyspora spinosa in the future.
Fanglong Zhao - One of the best experts on this subject based on the ideXlab platform.
-
metabolomics analysis of the effect of dissolved oxygen on Spinosad production by saccharopolyspora spinosa
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2017Co-Authors: Chunzhe Lu, Fanglong Zhao, Feng Li, Wenyu LuAbstract:Spinosad, a universal bio-pesticide, is obtained from the soil actinomycete Saccharopolyspora spinosa. Dissolved oxygen, an important contributing factor in aerobic microbial fermentation, however, is not always available in sufficient amounts. To alleviate oxygen limitation in Spinosad production, three different oxygen vectors, namely oleic acid, toluene, and n-dodecane, were added into early fermentation. Results indicated that n-dodecane was the optimal oxygen vector. Spinosad yield was increased by 44.2% compared to that in the control group in the presence of 0.5% n-dodecane, added after 120 h of incubation. Yields of the test group reached 6.52 mg/g dry cell weight (DCW), while that of the control group was limited to 4.52 mg/g DCW. Metabolomics analysis by gas chromatography coupled to mass spectrometry was performed to demonstrate the metabolism mechanism in the presence and absence of oxygen vector. In total, 78 principal intracellular metabolites in S. spinosa were detected and quantified in the presence and absence of n-dodecane. Levels of some metabolites that were related to the tricarboxylic acid cycle and pentose phosphate pathway varied significantly. Aspartic acid and glucose-1-phosphate levels varied significantly and contributed most in the distinction of the fermentation conditions and phases. The above findings give new insights into the improvement and the metabolomic characteristics of industrial Spinosad production.
-
suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in saccharopolyspora spinosa
Microbial Cell Factories, 2014Co-Authors: Xiangmei Zhang, Chuanbo Zhang, Fanglong Zhao, Qinggele Caiyin, Dashuai Li, Wenyu LuAbstract: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.
-
Suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in Saccharopolyspora spinosa.
Microbial cell factories, 2014Co-Authors: Xiangmei Zhang, Chuanbo Zhang, Fanglong Zhao, Chaoyou Xue, Jing Yin, Qinggele CaiyinAbstract: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. 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. 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.
-
a comparative metabolomics analysis of saccharopolyspora spinosa wt wh124 and lu104 revealed metabolic mechanisms correlated with increases in Spinosad yield
Bioscience Biotechnology and Biochemistry, 2013Co-Authors: Fanglong Zhao, Meiling Wang, Xiaoyang Wang, L U WenyuAbstract:Metabolomics analysis of three Saccharopolyspora spinosa strains (wild type strain WT, ultraviolet mutant strain WH124, and metabolic engineering strain LU104) with different Spinosad producing levels was performed by liquid chromatograph coupled to mass spectrometry (LC-MS). The metabolite profiles were subjected to hierarchal clustering analysis (HCA) and principal component analysis (PCA). The results of HCA on a heat map revealed that the large numbers of primary metabolism detected were more abundant in WH124 and less abundant in LU104 during the early fermentation stage as compared to the WT strain. PCA separated the three strains clearly and suggested nine metabolites that contributed predominantly to the separation. These biomarkers were associated with central carbon metabolism (succinic acid, α-ketoglutarate, acetyl-CoA, and ATP), amino acid metabolism (glutamate, glutamine, and valine), and secondary metabolism (pseudoaglycone), etc. These findings provide insight into the metabolomic character...
-
stepwise increase of Spinosad production in saccharopolyspora spinosa by metabolic engineering
Biochemical Engineering Journal, 2013Co-Authors: Yuejiao Duan, Fanglong Zhao, Wenyu LuAbstract:Abstract Rational metabolic and cellular engineering approaches are useful in improving strain performance. In the last years, several studies not only clarified the biosynthetic pathway of Spinosad, but also provided useful information of metabolic restrictions in the spinosyn biosynthetic pathway. However, these studies overlooked the problem that the spinosyn biosynthetic pathway was unbalanced: the expression of six genes in the spinosyn biosynthetic pathway was insufficient; Saccharopolyspora spinosa accumulated useless compounds because of insufficient expression of SpnK . So a rational strain improvement strategy was developed to tune the unbalanced spinosyn biosynthetic pathway. First, we overexpressed spnK to increase the amount of the flux from rhamnosylated aglycone to pseudoaglycones (PSA). Then six genes ( spnP , spnO , spnN , spnQ , spnR , and spnS ) involved in forosamine biosynthesis and spnK were co-expressed in S. spinosa LU102 to convert the accumulated PSA to Spinosad. The yield of Spinosad in S. spinosa LU102 was 214 mg/L, which was 2.6-fold higher than that in the wild-type S. spinosa (82 mg/L). Finally, Spinosad production in the tuned S. spinosa LU104 was further increased to 405 mg/L, which was a 5.0-fold enhancement compared with the wild-type S. spinosa , by duplicating spnP , spnO , spnN , spnQ , spnR , spnS , spnK , gtt , gdh and kre genes.
