Cargando…

Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis

BACKGROUND: Triacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production becau...

Descripción completa

Detalles Bibliográficos
Autores principales: Zhang, Nannan, Mao, Zejing, Luo, Ling, Wan, Xia, Huang, Fenghong, Gong, Yangmin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513132/
https://www.ncbi.nlm.nih.gov/pubmed/28725265
http://dx.doi.org/10.1186/s13068-017-0869-y
_version_ 1783250603752816640
author Zhang, Nannan
Mao, Zejing
Luo, Ling
Wan, Xia
Huang, Fenghong
Gong, Yangmin
author_facet Zhang, Nannan
Mao, Zejing
Luo, Ling
Wan, Xia
Huang, Fenghong
Gong, Yangmin
author_sort Zhang, Nannan
collection PubMed
description BACKGROUND: Triacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels. In bacteria, the final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT). This bifunctional WS/DGAT enzyme is also a key enzyme in biotechnological production of liquid WE via engineering of plants and microorganisms. To date, knowledge about this class of biologically and biotechnologically important enzymes is mainly from biochemical characterization of WS/DGATs from Arabidopsis, jojoba and some bacteria that can synthesize both TAGs and WEs intracellularly, whereas little is known about WS/DGATs from eukaryotic microorganisms. RESULTS: Here, we report the identification and characterization of two bifunctional WS/DGAT enzymes (designated TrWSD4 and TrWSD5) from the marine protist Thraustochytrium roseum. Both TrWSD4 and TrWSD5 comprise a WS-like acyl-CoA acyltransferase domain and the recombinant proteins purified from Escherichia coli Rosetta (DE3) have substantial WS and lower DGAT activity. They exhibit WS activity towards various-chain-length saturated and polyunsaturated acyl-CoAs and fatty alcohols ranging from C(10) to C(18). TrWSD4 displays WS activity with the lowest K (m) value of 0.14 μM and the highest k (cat)/K (m) value of 1.46 × 10(5) M(−1) s(−1) for lauroyl-CoA (C(12:0)) in the presence of 100 μM hexadecanol, while TrWSD5 exhibits WS activity with the lowest K (m) value of 0.96 μM and the highest k (cat)/K (m) value of 9.83 × 10(4) M(−1) s(−1) for decanoyl-CoA (C(10:0)) under the same reaction condition. Both WS/DGAT enzymes have the highest WS activity at 37 and 47 °C, and WS activity was greatly decreased when temperature exceeds 47 °C. TrWSD4 and TrWSD5 are insensitive to ionic strength and reduced WS activity was observed when salt concentration exceeded 800 mM. The potential of T. roseum WS/DGATs to establish novel process for biotechnological production of WEs was demonstrated by heterologous expression in recombinant yeast. Expression of either TrWSD4 or TrWSD5 in Saccharomyces cerevisiae quadruple mutant H1246, which is devoid of storage lipids, resulted in the accumulation of WEs, but not any detectable TAGs, indicating a predominant WS activity in yeast. CONCLUSIONS: This study demonstrates both in vitro WS and DGAT activity of two T. roseum WS/DGATs, which were characterized as unspecific acyltransferases accepting a broad range of acyl-CoAs and fatty alcohols as substrates for WS activity but displaying substrate preference for medium-chain acyl-CoAs. In vivo characterization shows that these two WS/DGATs predominantly function as wax synthase and presents the feasibility for production of WEs by heterologous hosts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-017-0869-y) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-5513132
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-55131322017-07-19 Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis Zhang, Nannan Mao, Zejing Luo, Ling Wan, Xia Huang, Fenghong Gong, Yangmin Biotechnol Biofuels Research BACKGROUND: Triacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels. In bacteria, the final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT). This bifunctional WS/DGAT enzyme is also a key enzyme in biotechnological production of liquid WE via engineering of plants and microorganisms. To date, knowledge about this class of biologically and biotechnologically important enzymes is mainly from biochemical characterization of WS/DGATs from Arabidopsis, jojoba and some bacteria that can synthesize both TAGs and WEs intracellularly, whereas little is known about WS/DGATs from eukaryotic microorganisms. RESULTS: Here, we report the identification and characterization of two bifunctional WS/DGAT enzymes (designated TrWSD4 and TrWSD5) from the marine protist Thraustochytrium roseum. Both TrWSD4 and TrWSD5 comprise a WS-like acyl-CoA acyltransferase domain and the recombinant proteins purified from Escherichia coli Rosetta (DE3) have substantial WS and lower DGAT activity. They exhibit WS activity towards various-chain-length saturated and polyunsaturated acyl-CoAs and fatty alcohols ranging from C(10) to C(18). TrWSD4 displays WS activity with the lowest K (m) value of 0.14 μM and the highest k (cat)/K (m) value of 1.46 × 10(5) M(−1) s(−1) for lauroyl-CoA (C(12:0)) in the presence of 100 μM hexadecanol, while TrWSD5 exhibits WS activity with the lowest K (m) value of 0.96 μM and the highest k (cat)/K (m) value of 9.83 × 10(4) M(−1) s(−1) for decanoyl-CoA (C(10:0)) under the same reaction condition. Both WS/DGAT enzymes have the highest WS activity at 37 and 47 °C, and WS activity was greatly decreased when temperature exceeds 47 °C. TrWSD4 and TrWSD5 are insensitive to ionic strength and reduced WS activity was observed when salt concentration exceeded 800 mM. The potential of T. roseum WS/DGATs to establish novel process for biotechnological production of WEs was demonstrated by heterologous expression in recombinant yeast. Expression of either TrWSD4 or TrWSD5 in Saccharomyces cerevisiae quadruple mutant H1246, which is devoid of storage lipids, resulted in the accumulation of WEs, but not any detectable TAGs, indicating a predominant WS activity in yeast. CONCLUSIONS: This study demonstrates both in vitro WS and DGAT activity of two T. roseum WS/DGATs, which were characterized as unspecific acyltransferases accepting a broad range of acyl-CoAs and fatty alcohols as substrates for WS activity but displaying substrate preference for medium-chain acyl-CoAs. In vivo characterization shows that these two WS/DGATs predominantly function as wax synthase and presents the feasibility for production of WEs by heterologous hosts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-017-0869-y) contains supplementary material, which is available to authorized users. BioMed Central 2017-07-15 /pmc/articles/PMC5513132/ /pubmed/28725265 http://dx.doi.org/10.1186/s13068-017-0869-y Text en © The Author(s) 2017 Open AccessThis 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. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Zhang, Nannan
Mao, Zejing
Luo, Ling
Wan, Xia
Huang, Fenghong
Gong, Yangmin
Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis
title Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis
title_full Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis
title_fullStr Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis
title_full_unstemmed Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis
title_short Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis
title_sort two bifunctional enzymes from the marine protist thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-coa:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513132/
https://www.ncbi.nlm.nih.gov/pubmed/28725265
http://dx.doi.org/10.1186/s13068-017-0869-y
work_keys_str_mv AT zhangnannan twobifunctionalenzymesfromthemarineprotistthraustochytriumroseumbiochemicalcharacterizationofwaxestersynthaseacylcoadiacylglycerolacyltransferaseactivitycatalyzingwaxesterandtriacylglycerolsynthesis
AT maozejing twobifunctionalenzymesfromthemarineprotistthraustochytriumroseumbiochemicalcharacterizationofwaxestersynthaseacylcoadiacylglycerolacyltransferaseactivitycatalyzingwaxesterandtriacylglycerolsynthesis
AT luoling twobifunctionalenzymesfromthemarineprotistthraustochytriumroseumbiochemicalcharacterizationofwaxestersynthaseacylcoadiacylglycerolacyltransferaseactivitycatalyzingwaxesterandtriacylglycerolsynthesis
AT wanxia twobifunctionalenzymesfromthemarineprotistthraustochytriumroseumbiochemicalcharacterizationofwaxestersynthaseacylcoadiacylglycerolacyltransferaseactivitycatalyzingwaxesterandtriacylglycerolsynthesis
AT huangfenghong twobifunctionalenzymesfromthemarineprotistthraustochytriumroseumbiochemicalcharacterizationofwaxestersynthaseacylcoadiacylglycerolacyltransferaseactivitycatalyzingwaxesterandtriacylglycerolsynthesis
AT gongyangmin twobifunctionalenzymesfromthemarineprotistthraustochytriumroseumbiochemicalcharacterizationofwaxestersynthaseacylcoadiacylglycerolacyltransferaseactivitycatalyzingwaxesterandtriacylglycerolsynthesis