Background: Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunityenhancing activities. However, cytotoxicity remains a challenge for triterpenoid overproduction in microbial cell factories. As lipophilic and relatively small molecular compounds, triterpenes are generally secreted into the extracellular space. The effect of increasing triterpene efflux on the synthesis capacity remains unknown. Results: In this study, we developed a strategy to enhance triterpene efflux through manipulation of lipid components in Y. lipolytica by overexpressing the enzyme Δ9-fatty acid desaturase (OLE1) and disturbing phosphatidic acid phosphatase (PAH1) and diacylglycerol kinase (DGK1). By this strategy combined with two-phase fermentation, the highest lupeol production reported to date was achieved, where the titer in the organic phase reached 381.67 mg/L and the total production was 411.72 mg/L in shake flasks, exhibiting a 33.20-fold improvement over the initial strain. Lipid manipulation led to a twofold increase in the unsaturated fatty acid (UFA) content, up to 61-73%, and an exceptionally elongated cell morphology, which might have been caused by enhanced membrane phospholipid biosynthesis flux. Both phenotypes accelerated the export of toxic products to the extracellular space and ultimately stimulated the capacity for triterpenoid synthesis, which was proven by the 5.11-fold higher ratio of extra/intracellular lupeol concentrations, 2.79-fold higher biomass accumulation and 2.56-fold higher lupeol productivity per unit OD in the modified strains. This strategy was also highly efficient for the biosynthesis of other triterpenes and sesquiterpenes, including α-amyrin, β-amyrin, longifolene, longipinene and longicyclene. Conclusions: In conclusion, we successfully created a high-yield lupeol-producing strain via lipid manipulation. We demonstrated that the enhancement of lupeol efflux and synthesis capacity was induced by the increased UFA content and elongated cell morphology. Our study provides a novel strategy to promote the biosynthesis of valuable but toxic products in microbial cell factories.

BackgroundLupeol exhibits novel physiological and pharmacological activities, such as anti-cancer and immunity enhancement. However, cytotoxicity is still a challenge for triterpenoids overproduction in microbial cell factories. As lipophilic and relatively small-molecular compounds, triterpenes are generally secreted to the extracellular space. The effect of increasing triterpenes efflux on the synthesis capacity remains unknown.ResultsIn this study, we developed a strategy to enhance the triterpenes efflux through manipulation of lipid components in Y. lipolytica by overexpressing the enzyme of Δ9-fatty acid desaturase (OLE1) and disturbing the phosphatidic acid phosphatase (PAH1) and diacylglycerol kinase (DGK1). As a result, we obtained a high-yield lupeol strain with the highest lupeol production of 411.72 mg/L in shake flasks reported to date, reaching a 33.2-fold improvement over the initial strain. The lipid manipulation led to a two-fold increase of unsaturated fatty acids (UFAs) content, up to 61%~73%, and an exceptionally elongated cell morphology, which might be caused by enhanced membrane phospholipids biosynthesis flux. Both of the phenotypes accelerated the export of toxic products to the extracellular space and ultimately stimulated the capacity of triterpenoids synthesis, which were proved by 5.11-fold higher ratio of extra-/intra-cellular lupeol concentrations, 2.7-fold higher biomass accumulation and 2.60-fold higher lupeol productivity per unit OD in modified strains. This strategy was also highly efficient for biosynthesis of other triterpenes and sesquiterpenes, including α-, β-arymin, longifolene, longipinene and longicyclene.ConclusionsTo conclude, we successfully created a high-yield lupeol strain via lipid manipulation. And we demonstrated that the enhancement of lupeol efflux and synthesis capacity was induced by the increased unsaturated fatty acids content and elongated cell morphology. Our study provides a novel strategy to promote the biosynthesis of valuable but toxic products in microbial cell factories.

Risk analysis is an important part of the dam safety risk management. The uncertain factors causing the failure of the gate is very complex, including the gate design and manufacturing, management operation, some man-made factors, etc. The traditional risk analysis method has certain limitation. Bayesian networks can quickly and effectively solve the failure probability of the system and for a direct analysis on the effects of every system component by a two-side inference, so as to locate the system weakness and adopt effective measures. This paper based on Bayesian network, through the example of Bayesian network in the application of dam gate risk analysis.

Background Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunity-enhancing activities. However, cytotoxicity remains a challenge for triterpenoid overproduction in microbial cell factories. As lipophilic and relatively small-molecular compounds, triterpenes are generally secreted into the extracellular space. The effect of increasing triterpene efflux on the synthesis capacity remains unknown.Results In this study, we developed a strategy to enhance triterpene efflux through manipulation of lipid components in Y. lipolytica by overexpressing the enzyme Δ9-fatty acid desaturase (OLE1) and disturbing phosphatidic acid phosphatase (PAH1) and diacylglycerol kinase (DGK1). By this strategy combined with two-phase fermentation, the highest lupeol production reported to date was achieved, where the titer in the organic phase reached 381.67 mg/L and the total production was 411.72 mg/L in shake flasks, exhibiting a 33.20-fold improvement over the initial strain. Lipid manipulation led to a two-fold increase in the unsaturated fatty acid (UFA) content, up to 61%~73%, and an exceptionally elongated cell morphology, which might have been caused by enhanced membrane phospholipid biosynthesis flux. Both phenotypes accelerated the export of toxic products to the extracellular space and ultimately stimulated the capacity for triterpenoid synthesis, which was proven by the 5.11-fold higher ratio of extra/intracellular lupeol concentrations, 2.79-fold higher biomass accumulation and 2.56-fold higher lupeol productivity per unit OD in the modified strains. This strategy was also highly efficient for the biosynthesis of other triterpenes and sesquiterpenes, including α-amyrin, β-amyrin, longifolene, longipinene and longicyclene.Conclusions In conclusion, we successfully created a high-yield lupeol-producing strain via lipid manipulation. We demonstrated that the enhancement of lupeol efflux and synthesis capacity was induced by the increased UFA content and elongated cell morphology. Our study provides a novel strategy to promote the biosynthesis of valuable but toxic products in microbial cell factories.