Improved Quantitative Approach for Monitorization of Gangliosides Structural Diversity in Fungal Cell Factories by LC-MS/MS

Separations

article

Javier-Fernando Montero-Bullón¹  Javier Martín-González¹  Gloria Muñoz-Fernández¹  Alberto Jiménez¹  José Luis Revuelta¹ 

[1] Metabolic Engineering Group, Department of Microbiology and Genetics, University of Salamanca

关键词: LC;    MS/MS;    mass spectrometry;    liquid chromatography;    glycosphingolipids;    ceramides;    gangliosides;    GM3;    GD3;   

DOI  :  10.3390/separations9120432

学科分类：社会科学、人文和艺术（综合）

来源: mdpi

【 摘 要 】

Gangliosides are glycolipids occurring in higher animals, with a sphingoid core in the form of ceramide, bound to a glycan moiety including several units of sialic acid. Gangliosides are involved in important (patho)-physiological processes as components of cell membranes in humans, which has led to intensive study and interest in production strategies. Their structural variability depends on the combination of a sphingoid base, a fatty acyl chain, and an attached oligosaccharide. The combinatorial diversity differs and grows exponentially in synthetic biology approaches, e.g., use of microbial cell factories. A specific analytical platform accounting for this complexity is not available to date. However, quantification of the intermediates of the whole biosynthetic route is needed to boost projects on biotechnological ganglioside production. In this study, a fast high-throughput quantitative LC-MS/MS methodology was developed to cover analysis of gangliosides, with a wider structural perspective adapted to fungal organisms. This work was achieved using metabolically engineered strains that further allowed to test detection in biological complex matrixes. Ganglioside backbones—hitherto uncharacterized—with the five most common fungal sphingoid bases and both simple and hydroxylated fatty acids were subjected to characterization. The addition of glycans to the polar head was also successfully monitored with up to 4 units—corresponding to GD3 which bears two sialic acid units and furthermore represents the common precursor for the whole ganglio-series. This platform represents an improved methodology to study the biochemical diversity associated to gangliosides for natural and metabolically engineered biosynthetic pathways.

【 授权许可】

CC BY