Eugenio Alcalde1, Paul D Fraser2. 1. School Biological Sciences, Royal Holloway, University London, Egham Hill, Egham, Surrey, TW20OEX, UK. 2. School Biological Sciences, Royal Holloway, University London, Egham Hill, Egham, Surrey, TW20OEX, UK. Paul.Fraser@rhul.ac.uk.
Abstract
INTRODUCTION: Xanthophyllomyces dendrorhous is a non-conventional industrial yeast. It has the unique ability among yeasts to produce geranylgeranyl pyrophosphate derived terpenoids such as carotenoids and in particular the high value pigment astaxanthin. OBJECTIVE: In order to fully exploit the industrial potential of Xanthophyllomyces using modern industrial biotechnology approaches the further development of "omic" resources in this organism are required to build on the now sequenced and annotated genome. To contribute to this goal, the present study has developed and implemented an efficient metabolite profiling system comprised of, quenching, extraction and associated GC-MS and UPLC analysis. METHOD: Four quenching methods and five extraction methods compatible with GC-MS and UPLC profiling were tested and validated by analysing steady state metabolite changes of Xanthophyllomyces cultivated at laboratory scale in liquid shake culture at lag, exponential and early and late stationary phases. RESULTS: A customised Automated Mass Spectral Deconvolution and Identification System (AMDIS) library has been created for Xanthophyllomyces, over 400 compounds are present in the library of which 78 are detected and quantified routinely in polar and non-polar derived extracts. A preliminary biochemical network has been constructed. Over a standardised laboratory growth cycle, changes in metabolite levels have been determined to create reference point for future strain improvement approaches and the initial biochemical network construction. Correlation analysis has illustrated that astaxanthin formation correlates positively with different sectors of intermediary metabolism (e.g. the TCA cycle intermediates and amino acid formation), "short" saturated fatty acids and β-carotene, while other metabolites are reduced in response to astaxanthin production. These sectors of intermediary metabolism offer potential future targets for the manipulation resulting in the generation of strains with improved titres of given terpenoids. DISCUSSION: In summary a robust metabolite profiling system for Xanthophyllomyces is in place to further our understanding and potential exploitation of this underutilised industrial yeast.
INTRODUCTION: Xanthophyllomyces dendrorhous is a non-conventional industrial yeast. It has the unique ability among yeasts to produce geranylgeranyl pyrophosphate derived terpenoids such as carotenoids and in particular the high value pigment astaxanthin. OBJECTIVE: In order to fully exploit the industrial potential of Xanthophyllomyces using modern industrial biotechnology approaches the further development of "omic" resources in this organism are required to build on the now sequenced and annotated genome. To contribute to this goal, the present study has developed and implemented an efficient metabolite profiling system comprised of, quenching, extraction and associated GC-MS and UPLC analysis. METHOD: Four quenching methods and five extraction methods compatible with GC-MS and UPLC profiling were tested and validated by analysing steady state metabolite changes of Xanthophyllomyces cultivated at laboratory scale in liquid shake culture at lag, exponential and early and late stationary phases. RESULTS: A customised Automated Mass Spectral Deconvolution and Identification System (AMDIS) library has been created for Xanthophyllomyces, over 400 compounds are present in the library of which 78 are detected and quantified routinely in polar and non-polar derived extracts. A preliminary biochemical network has been constructed. Over a standardised laboratory growth cycle, changes in metabolite levels have been determined to create reference point for future strain improvement approaches and the initial biochemical network construction. Correlation analysis has illustrated that astaxanthin formation correlates positively with different sectors of intermediary metabolism (e.g. the TCA cycle intermediates and amino acid formation), "short" saturated fatty acids and β-carotene, while other metabolites are reduced in response to astaxanthin production. These sectors of intermediary metabolism offer potential future targets for the manipulation resulting in the generation of strains with improved titres of given terpenoids. DISCUSSION: In summary a robust metabolite profiling system for Xanthophyllomyces is in place to further our understanding and potential exploitation of this underutilised industrial yeast.
Authors: John M Halket; Daniel Waterman; Anna M Przyborowska; Raj K P Patel; Paul D Fraser; Peter M Bramley Journal: J Exp Bot Date: 2004-12-23 Impact factor: 6.992
Authors: Mlawule R Mashego; Karl Rumbold; Marjan De Mey; Erick Vandamme; Wim Soetaert; Joseph J Heijnen Journal: Biotechnol Lett Date: 2006-11-08 Impact factor: 2.461
Authors: Alejandra Barbachano-Torres; Lina M Castelblanco-Matiz; Ana C Ramos-Valdivia; Carlos M Cerda-García-Rojas; Luis M Salgado; César M Flores-Ortiz; Teresa Ponce-Noyola Journal: Arch Microbiol Date: 2014-03-28 Impact factor: 2.552
Authors: Jan C Verdoes; Gerhard Sandmann; Hans Visser; Maria Diaz; Minca van Mossel; Albert J J van Ooyen Journal: Appl Environ Microbiol Date: 2003-07 Impact factor: 4.792
Authors: Robert A van den Berg; Huub C J Hoefsloot; Johan A Westerhuis; Age K Smilde; Mariët J van der Werf Journal: BMC Genomics Date: 2006-06-08 Impact factor: 3.969