Abstract
BackgroundLupeol 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.ResultsIn 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.ConclusionsIn 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.
Highlights
Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunityenhancing activities
To decrease the intractable cytotoxicity, extensive efforts aimed at creating oleaginous subcellular organelles by altering lipid-droplet composition and size potentially improved the terpene partition coefficient in oil droplets and the storage space so that lipophilic terpenes can accumulate in these compartments [9,10,11,12,13,14,15]
The unsaturated fatty acid (UFA) content in the control strain (LU-9) was only 35%, but the UFA proportions in all the high-production strains increased up to 61–73%, approximately twofold higher than that in the control strain. These findings suggested that overexpressing OLE1 and knocking out PAH1-DGK1 mainly enhanced the proportion of UFAs instead of total fatty acid accumulation
Summary
Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunityenhancing activities. Lupeol, a typical triterpenoid, caused severe damage to cell viability even at a relatively low concentration of 60 mg/L [3, 8]. Researchers successfully increased lycopene accumulation by creating supersized lipid droplets through manipulation of triacylglycerol (TAG) metabolism in Saccharomyces cerevisiae, which resulted in the highest yield (73.3 mg/g cdw and 2.37 g/L lycopene) reported in S. cerevisiae to date [10]. Gao et al [12] successfully applied lipid droplets as a storage sink to enhance β-carotene production in Y. lipolytica, resulting in a β-carotene yield of up to 4 g/L in fed-batch fermentation, the highest level attained so far
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