Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus

Dania Awad,Thomas Brueck

doi:10.1007/s00216-019-02254-7

Abstract

In the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus, due to its broad feedstock catabolism and high lipid yield. However, this yeast has a recalcitrant cell wall and high cell lipid content, which complicates efficient and unbiased protein extraction for downstream proteomic analysis. Optimization efforts of protein sample preparation from C. oleaginosus in the present study encompasses the comparison of 8 lysis methods, 13 extraction buffers, and 17 purification methods with respect to protein abundance, proteome coverage, applicability, and physiochemical properties (pI, MW, hydrophobicity in addition to COG, and GO analysis). The optimized protocol presented in this work entails a one-step extraction method utilizing an optimal lysis method (liquid homogenization), which is augmented with a superior extraction buffer (50 mM Tris, 8/2 M Urea/Thiourea, and 1% C7BzO), followed by either of 2 advantageous purification methods (hexane/ethanol or TCA/acetone), depending on subsequent applications and target studies. This work presents a significant step forward towards implementation of efficient C. oleaginosus proteome mining for the identification of potential targets for genetic optimization of this yeast to improve lipogenesis and production of specialty lipids.Graphical abstract

Highlights

Climate change drives the development of sustainable bioprocesses in the chemical and pharmaceutical industry
As it is difficult to predict which protocol could result in optimal proteome coverage of the non-model oleaginous yeast C. oleaginosus, we provide a comprehensive study that qualifies protein preparation methods and their downstream applicability based on qualitative and quantitative methods of proteins and lipids
This study addresses the three most challenging aspects of protein sample preparation by examining 7 methods of disintegration methods, 13 extraction buffers for protein solubilization, and 17 methods of purification/ delipidation for optimal protein sample preparation from the oleaginous yeast C. oleaginosus

Summary

Introduction

Climate change drives the development of sustainable bioprocesses in the chemical and pharmaceutical industry. The latter yeast species, C. oleaginosus is metabolically capable of converting a wide range of carbohydrates (glucose, galactose, cellobiose, xylose, sucrose, and lactose) and complex biomass-derived residual substrates (whey, glycerol, volatile fatty acids, ethanol, and N-acetylglucosamine) into lipids (60% w/w)[1, 6, 7]. The fatty acid profile of C. oleaginosus mimics that of plant-oils with 16–33% C16:0 and 43–57% C18:1 and can be used to generate biodiesel and oleochemical specialty products [8]. Development of an industrially relevant strain requires a comprehensive understanding of the complex genomic, proteomic, and metabolic system networks that determine and control microbial oleaginicity [3]. Finding novel genes and pathways committed to oleaginicity should facilitate strain engineering for improved lipid titers, robustness, and technoeconomics of the microbial production of fatty acid derivatives [3].

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Results

Conclusion