Department of Biosciences, Biotechnology and Environment, University of Bari "Aldo Moro", Campus Universitario, via Orabona 4, Bari, 70125, Italy.
Université Paris-Saclay, INRAE, Micalis Institute, Jouy-en-Josas, 78350, AgroParisTech, France.
Microb Cell Fact. 2023 Jul 13;22(1):128. doi: 10.1186/s12934-023-02137-8.
Mitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries.
In this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica.
The Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.
线粒体载体（MCs）可以深刻影响代谢途径的细胞内通量分布。操纵它们的表达水平，将通量重新导向感兴趣分子的产生，是真核微生物代谢工程的一个有吸引力的目标。非常规酵母解脂耶氏酵母能够利用广泛的底物。作为油脂酵母，它将大部分从那里生成的乙酰辅酶 A 引导到脂质的合成中，这发生在细胞质中。其中，奇数链脂肪酸（OCFAs）是一种很有前途的微生物化合物，在医学、美容、化学和农业等行业有多种应用。
在本研究中，我们鉴定了参与解脂耶氏酵母中的肉碱/乙酰肉碱穿梭的 MC，即 YlCrc1。解脂耶氏酵母 Ylcrc1 敲除菌株无法在乙醇、乙酸和油酸上生长，这表明这种 MC 在将乙酰辅酶 A 从过氧化物酶体和细胞质转运到线粒体中起着至关重要的作用。涉及 YlCRC1 缺失和重组表达来自恶臭假单胞菌的丙酰辅酶 A 转移酶（RePCT）的代谢工程策略，提高了丙酸盐的利用及其转化为 OCFAs。这些遗传修饰和补充葡萄糖和丙酸盐作为唯一碳源的脂生成培养基，导致解脂耶氏酵母中 OCFAs 的积累增加。
涉及 YlCrc1 的解脂耶氏酵母的肉碱/乙酰肉碱穿梭是将过氧化物酶体或细胞质中的乙酰辅酶 A 转运到线粒体的唯一途径。操纵这种载体可以成为涉及细胞质乙酰辅酶 A 的代谢工程方法的一个有前途的目标，正如 YlCRC1 缺失对 OCFAs 合成的影响所证明的那样。
