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Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus

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 li...

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Autores principales: Awad, Dania, Brueck, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992551/
https://www.ncbi.nlm.nih.gov/pubmed/31797019
http://dx.doi.org/10.1007/s00216-019-02254-7
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author Awad, Dania
Brueck, Thomas
author_facet Awad, Dania
Brueck, Thomas
author_sort Awad, Dania
collection PubMed
description 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. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00216-019-02254-7) contains supplementary material, which is available to authorized users.
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spelling pubmed-69925512020-02-11 Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus Awad, Dania Brueck, Thomas Anal Bioanal Chem Research Paper 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. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00216-019-02254-7) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2019-12-04 2020 /pmc/articles/PMC6992551/ /pubmed/31797019 http://dx.doi.org/10.1007/s00216-019-02254-7 Text en © The Author(s) 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Research Paper
Awad, Dania
Brueck, Thomas
Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus
title Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus
title_full Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus
title_fullStr Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus
title_full_unstemmed Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus
title_short Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus
title_sort optimization of protein isolation by proteomic qualification from cutaneotrichosporon oleaginosus
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992551/
https://www.ncbi.nlm.nih.gov/pubmed/31797019
http://dx.doi.org/10.1007/s00216-019-02254-7
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