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

Abstract

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.

Optimization of microbial cell factories for astaxanthin production: Biosynthesis and regulations, engineering strategies and fermentation optimization strategies

“…The OLE1 gene encodes a Δ9-fatty acid desaturase which is catalyzing the dehydrogenation of the 9-position in saturated fatty acids forming the corresponding unsaturated fatty acid [ 236 ]. The overexpression of this gene has been successfully employed to increase triterpenoids and β-carotene production in yeast by reducing the membrane stress [ 237 , 238 ]. Furthermore, deep knowledge about gene expression and regulation is needed to improve these microbial cell factories, such as epigenetic modifications, non-coding RNAs and post-translational regulations etc., which are still mysterious.…”

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach

“…The optimized fermentation condition attained titers of 100 mg/L α-amyrin and 20 mg/L β-amyrin. At the time of writing, this represents the highest reported mixed amyrin titers in the engineered Y. lipolytica cell factory [ 28 , 29 ]. Importantly, our study validates the case to consider protein design in the metabolic engineering of triterpenoid biosynthesis in the yeast Y. lipolytica .…”

Highly efficient biosynthesis of β-caryophyllene with a new sesquiterpene synthase from tobacco

“…The mixtures were centrifuged at 4000 rpm for 2 min, and the supernatant was removed. The obtained sample was rinsed twice with PB buffer and then analyzed with a TEM [ 44 , 45 ].…”