Hormone-sensitive lipase couples intergenerational sterol metabolism to reproductive success

Triacylglycerol (TG) and steryl ester (SE) lipid storage is a universal strategy to maintain organismal energy and membrane homeostasis. Cycles of building and mobilizing storage fat are fundamental in (re)distributing lipid substrates between tissues or to progress ontogenetic transitions. In this study, we show that Hormone-sensitive lipase (Hsl) specifically controls SE mobilization to initiate intergenerational sterol transfer in Drosophila melanogaster. Tissue-autonomous Hsl functions in the maternal fat body and germline coordinately prevent adult SE overstorage and maximize sterol allocation to embryos. While Hsl-deficiency is largely dispensable for normal development on sterol-rich diets, animals depend on adipocyte Hsl for optimal fecundity when dietary sterol becomes limiting. Notably, accumulation of SE but not of TG is a characteristic of Hsl-deficient cells across phyla including murine white adipocytes. In summary, we identified Hsl as an ancestral regulator of SE degradation, which improves intergenerational sterol transfer and reproductive success in flies.

Steryl Ester Formation and Accumulation in Steroid-Degrading Bacteria

ABSTRACT Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here, we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis, as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol, and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when it was grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole-cell systems in the biotechnological production of SEs for use in functional foods and cosmetics. IMPORTANCE Oleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen-limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

Steryl ester formation and accumulation in steroid-degrading bacteria

ABSTRACTSteryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl- and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole cell systems in the biotechnological production of SEs for use in functional foods and cosmetics.IMPORTANCEOleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

High-level soluble expression and enzymatic characterization of Burkholderia sp. lipase with steryl ester hydrolysis activity in E. coli

Background Burkholderia cepacia lipase is an important industrial biocatalyst for biodiesel production and chiral pharmaceutical synthesis. Heterologous soluble expression of lipase lipA gene from B. cepacia in Escherichia coli highly depends on co-expression of its cognate foldase gene, lipB. However, the interaction between recombinant lipase LipA and chaperonin LipB is rather complicated and confusing. In this research, various systems of lipA/lipB co-expression combinations are investigated to obtain high-level soluble expression of lipA, respectively. Results The best co-expression combination system for lipA and lipB is E. coli Origami 2 (DE3)/pETDuet-lipB(MCS1)/lipA(MCS2). The soluble expression level of lipA is 100.4 U/OD600 towards 4-nitrophenyl laurate hydrolysis. The recombinant LipA can be rapidly isolated from cell-free supernatant of recombinant E. coli lysate using HisTrap HP affinity chromatography column, and the lipA/LipB complex is obtained. Enzymatic characterization analysis shows that the purified LipA is a mesothermal and alkaline enzyme. LipA displays preference for medium-chain-length acyl groups (C10-C12) and sn-1,3 regioselectivity. Besides triacylglycerol hydrolase activity (EC. 3.1.1.3), LipA also displays steryl ester hydrolase activity (EC. 3.1.1.13). The specific activity of LipA towards 4-nitrophenyl decanoate and cholesterol linoleate are 638.9 U/mg and 1111.5 mU/mg, respectively. Conclusions Host strain E. coli Origami 2 (DE3), lipB locus at MCS1 on the dual expression cassette plasmid pETDuet, and low-temperature induction contribute to the soluble expression of lipA. Recombinant LipA displays both triacylglycerol hydrolase activity and steryl ester hydrolase activity.

Pemisahan squalene dan fatty acid sterol esters dari soybean oil deodorizer distillate

Depending on conditions in the refining process, soybean oil deodorizer distillate (SODD) contains 42-51% free fatty acids (FFA) and 16-25% triacylglycerol (TAG). Bioactive compounds such as tocopherols, free phytosterols, fatty acid steryl esters (FASE) and squalene also make up a significant portion of SODD. The efficient removal of FFA and TAG is a crucial step in the isolation and purification of bioactive compounds from SODD. In this work, a modified soxhlet extraction technique was developed and its optimal operation conditions were determined for the efficient removal of FFA and TAG from SODD. Starting with SODD that contains 3.53% FASE and 1.99% squalene, it was possible to obtain a fraction enriched with FASE (22.0%, recovery 91.3%) and squalene (8.63%, recovery 100%) by this modified soxhlet extraction under the following operation conditions: SODD/(silica gel) = 1/3 (w/w), extraction temperature = -6°C, number of extraction/h = 8.7. FFA remained in this FASE and squalene enriched fraction is 38.7% and there was no detectable TAG. The percentage removal of FFA and TAG for this FASE and squalene enriched fraction are 84.29% and 100%, respectively. The advantages of modified soxhlet extraction over open-column chromatography are that less solvent usage, larger sample size per batch and shorter operation time.Keywords: Fatty acid steryl ester; free fatty acid; modified soxhlet extraction; soybean oil deodorizer distillate; squalene; triacylglycerol AbstrakDalam proses pemurnian minyak kedelai, produk samping dari proses penghilangan bau (soybean oil deodorizer distillate, SODD) mengandung 42-52% free fatty acids (FFA) dan 18-28% triacylglycerols (TAG). Komponen bioaktif seperti tocopherols, free phytosterols, fatty acid steryl esters (FASE) dan squalene juga mempunyai kontribusi yang besar dalam komposisi SODD. Efisiensi penghilangan FFA dan TAG adalah langkah yang sangat penting dalam pemisahan dan pemurnian komponen bioaktif dari SODD. Dalam penelitian ini, modifikasi soxhlet ekstraksi telah ditemukan dan optimisasi kondisi operasi ditentukan berdasarkan efisiensi penghilangan FFA dan TAG dari SODD. Fraksi lemak non polar (nonpolar lipid fraction, NPLF) dengan kandungan FASE (22.0%, recovery 91.3%) dan squalene (8.63%, recovery 100%) telah diperoleh dengan menggunakan modifikasi soxhlet ektraksi dengan kondisi operasi: SODD/(silica gel) = 1/3 (w/w), dan suhu ekstraksi = -6°C. FFA dan TAG yang tersisa di NPLF secara berurutan adalah 38.7% dan 0%. Persentase penghilangan FFA dan TAG di NPLF secara berurutan adalah 84.29% and 100%. Keuntungan menggunakan modifikasi soxhlet ekstraksi dibandingkan dengan silica gel column chromatography adalah sedikitnya pelarut yang digunakan, besarnya sampel per unit batch yang digunakan, dan pendeknya waktu operasi.Kata kunci: Free fatty acid, fatty acid steryl ester, modifikasi soxhlet ekstraksi, soybean oil deodorizer distillate, squalene, triacylglycerols