背景:酵母Starmerellabombicola以其高效的槐糖脂生产而闻名,达到(超过)200g/L和2g/(Lh)的滴度和生产率,分别。这种固有的效率导致了槐糖脂的商业化。虽然槐糖脂生物合成途径已经在几年前被阐明,在这项研究中,它被重新审视,真正的关键中间体被揭示。
结果:最近,对过去开发和评估的Starmerellabombicola菌株进行了重新评估,揭示了意想不到的发现。在槐糖脂生物合成基因簇中编码的AT酶是已知的乙酰化槐糖脂的唯一描述的酶,而由SBLE基因编码的SBLE酶被描述为催化(乙酰化)酸性槐糖脂转化为内酯性槐糖脂。描述了两个基因的双重敲除,以导致生成bolaform槐糖脂。然而,用相应的S.bombicola菌株Δsble进行的新实验,ΔatΔsble,和Δat揭示了与当前对SL途径的理解不一致。观察到,Δsble菌株主要产生具有较高乙酰化度的bolaform槐糖脂,而不是酸性槐糖脂。此外,Δat菌株主要产生具有较低乙酰化度的苦参脂和乳酸苦参脂,而ΔatΔsble菌株主要产生具有较低乙酰化度的bolaform槐糖脂。这些结果表明,AT酶不是唯一的酶负责的乙酰化的槐糖脂,而SBLE酶对bolaform糖脂进行分子内酯交换反应,而不是对酸性槐糖脂进行酯化反应。这些发现,加上最近的体外数据,导致我们修改了槐糖脂生物合成途径。
结论:Bolaform槐糖脂而不是酸性槐糖脂是乳酸槐糖脂生物合成途径中的关键中间体。在细胞外S.bombicola野生型培养液中发现了非常少量的Bolaform槐糖脂,因为它们可以非常有效地转化为内酯的槐糖脂。而酸性槐糖脂积累,因为它们不能被转化。此外,槐糖脂的乙酰化并非仅由槐糖脂生物合成基因簇中编码的AT酶进行,而波拉形式的槐糖脂的乙酰化会促进其酯交换。这些发现导致了工业相关的槐糖脂生物合成途径的修订。
BACKGROUND: The yeast Starmerella bombicola is renowned for its highly efficient sophorolipid production, reaching titers and productivities of (over) 200 g/L and 2 g/(L h), respectively. This inherent efficiency has led to the commercialization of sophorolipids. While the sophorolipid biosynthetic pathway has been elucidated a few years ago, in this study, it is revisited and true key intermediates are revealed.
RESULTS: Recently, Starmerella bombicola strains developed and evaluated in the past were reevaluated unveiling unexpected findings. The AT enzyme encoded in the sophorolipid biosynthetic gene cluster is the only described enzyme known to acetylate sophorolipids, while the SBLE enzyme encoded by the SBLE gene is described to catalyze the conversion of (acetylated) acidic sophorolipids into lactonic sophorolipids. A double knockout of both genes was described to result in the generation of bolaform sophorolipids. However, new experiments performed with respective S. bombicola strains Δsble, Δat Δsble, and ∆at revealed inconsistencies with the current understanding of the SL pathway. It was observed that the ∆sble strain produces mainly bolaform sophorolipids with higher acetylation degrees instead of acidic sophorolipids. Furthermore, the ∆at strain produces predominantly bolaform sophorolipids and lactonic sophorolipids with lower acetylation degrees, while the ∆at ∆sble strain predominantly produces bolaform sophorolipids with lower acetylation degrees. These results indicate that the AT enzyme is not the only enzyme responsible for acetylation of sophorolipids, while the SBLE enzyme performs an intramolecular transesterification reaction on bolaform glycolipids instead of an esterification reaction on acidic sophorolipids. These findings, together with recent in vitro data, led us to revise the sophorolipid biosynthetic pathway.
CONCLUSIONS: Bolaform sophorolipids instead of acidic sophorolipids are the key intermediates in the biosynthetic pathway towards lactonic sophorolipids. Bolaform sophorolipids are found in very small amounts in extracellular S. bombicola wild type broths as they are very efficiently converted into lactonic sophorolipids, while acidic sophorolipids build up as they cannot be converted. Furthermore, acetylation of sophorolipids is not exclusively performed by the AT enzyme encoded in the sophorolipid biosynthetic gene cluster and acetylation of bolaform sophorolipids promotes their transesterification. These findings led to the revision of the industrially relevant sophorolipid biosynthetic pathway.