Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast <i>Yarrowia lipolytica</i> through Metabolic Engineering and Process Optimization

et al. 2020

Biotechnol Biofuels

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.

Section: Resultsmentioning

confidence: 99%

Section: Engineering Of Mva and Lipid Metabolism To Improve Lupeol Prmentioning

confidence: 99%

High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components

et al. 2020

Biotechnol Biofuels

“…As such, we manipulated eight structural and regulatory genes related to lipid metabolism in LU-9 ( Fig. 2A), including β-oxidation disruption (knockout of PXA1, MFE1, PEX10 or POT1) [31][32][33], and lipid synthesis regulation (overexpression of the limiting step gene ACC1, OLE1…”

Section: Engineering Of Mva and Lipid Metabolism To Improve Lupeol Prmentioning

confidence: 99%

High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components

et al. 2020

Preprint

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.

“…As such, we manipulated eight structural and regulatory genes related to lipid metabolism in LU-9 ( Fig. 2A), including β-oxidation disruption (by knocking out PXA1, MFE1, PEX10 or POT1) [38][39][40] and lipid synthesis regulation (by overexpressing the limiting step genes ACC1, OLE1 and its activator MGA2 G643R and disturbing the regulatory and structural genes RPD3, SNF1, LRO1, PAH1 and DGK1) [41,42] ( Fig. 2A.…”

Section: Engineering Of Mva and Lipid Metabolism To Improve Lupeol Prmentioning

confidence: 99%

High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components

et al. 2020

Preprint

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.