ABSTRACT

Blastobotrys raffinosifermentans is an ascomycetous yeast with biotechnological applications, recently shown to be an oleaginous yeast accumulating lipids under nitrogen limitation. Diacylglycerol acyltransferases (DGATs) act in the lipid storage pathway, in the last step of triacylglycerol biosynthesis. Two DGAT families are widespread in eukaryotes. We first checked that B. raffinosifermentans strain LS3 possessed both types of DGAT, and we then overexpressed the native DGAT-encoding genes, DGA1 and DGA2, separately or together. DGA2 (from the DGAT1 family) overexpression was sufficient to increase lipid content significantly in LS3, to up to 26.5% of dry cell weight (DCW), 1.6 times the lipid content of the parental strain (16.90% of DCW) in glucose medium under nitrogen limitation. By contrast, DGA1 (of the DGAT2 type) overexpression led to a large increase (up to 140-fold) in the amount of the corresponding transcript, but had no effect on overall lipid content relative to the parental strain. Analysis of the expression of the native genes over time in the parental strain revealed that DGA2 transcript levels quadrupled between 8 and 24 h in the N-limited lipogenic medium, whereas DGA1 transcript levels remained stable. This survey highlights the predominant role of the DGAT1 family in lipid accumulation and demonstrates the suitability of B. raffinosifermentans for engineering for lipid production.

中文翻译：

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天然酰基转移酶编码基因 DGA1 和 DGA2 在过表达时对 Blastobotrys raffinosifermentans 脂质积累的影响不同

摘要

Blastobotrys raffinosifermentans是一种具有生物技术应用的子囊菌酵母，最近被证明是一种在氮限制下积累脂质的产油酵母。二酰基甘油酰基转移酶 (DGAT) 在三酰基甘油生物合成的最后一步中的脂质储存途径中起作用。两个 DGAT 家族在真核生物中广泛存在。我们首先检查B. raffinosifermentans菌株 LS3 是否具有两种类型的 DGAT，然后我们分别或一起过表达天然 DGAT 编码基因DGA1和DGA2。DGA2（来自 DGAT1 家族）过表达足以显着增加 LS3 中的脂质含量，高达细胞干重 (DCW) 的 26.5%，是亲本菌株在氮气下葡萄糖培养基中脂质含量（DCW 的 16.90%）的 1.6 倍局限性。相比之下，DGA1（DGAT2 型）过表达导致相应转录物数量大幅增加（高达 140 倍），但相对于亲本菌株对总体脂质含量没有影响。对亲本菌株中天然基因随时间的表达分析表明，在 N 限制的脂肪生成培养基中，DGA2转录水平在 8 到 24 小时之间翻了两番，而DGA1转录水平保持稳定。该调查突出了 DGAT1 家族在脂质积累中的主要作用，并证明了B. raffinosifermentans对脂质生产工程的适用性。