4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.

<h4>Background</h4>Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated fl...

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Autores principales:	Frederick G Sunstrum, Hannah L Liu, Sharon Jancsik, Lufiani L Madilao, Joerg Bohlmann, Sandra Irmisch
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/eabb92ab86ea4f308b5e3cefb1e74453
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spelling	oai:doaj.org-article:eabb92ab86ea4f308b5e3cefb1e744532021-12-02T20:17:14Z4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.1932-620310.1371/journal.pone.0257478https://doaj.org/article/eabb92ab86ea4f308b5e3cefb1e744532021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0257478https://doaj.org/toc/1932-6203<h4>Background</h4>Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis.<h4>Results</h4>Transcriptome analysis for gene expression patterns related to montbretin biosynthesis identified eight different montbretia AAEs belonging to four different clades. Enzyme characterization identified 4CL activity for two clade IV members, Cc4CL1 and Cc4CL2, converting different hydroxycinnamic acids into the corresponding CoA thioesters. Both enzymes preferred coumaric acid over caffeic acid as a substrate in vitro. While expression of montbretia AAEs did not enhance MbA biosynthesis in N. benthamiana, we demonstrated that both Cc4CLs can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in yeast (Saccharomyces cerevisiae).<h4>Conclusions</h4>Montbretia expresses two functional 4CLs, but neither of them is specific for the formation of caffeoyl-CoA. Based on differential expression analysis and phylogeny Cc4CL1 is most likely involved in MbA biosynthesis, while Cc4CL2 may contribute to lignin biosynthesis. Both Cc4CLs can be used for flavanone production to support metabolic engineering of MbA in yeast.Frederick G SunstrumHannah L LiuSharon JancsikLufiani L MadilaoJoerg BohlmannSandra IrmischPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 10, p e0257478 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Frederick G Sunstrum Hannah L Liu Sharon Jancsik Lufiani L Madilao Joerg Bohlmann Sandra Irmisch 4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.
description	<h4>Background</h4>Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis.<h4>Results</h4>Transcriptome analysis for gene expression patterns related to montbretin biosynthesis identified eight different montbretia AAEs belonging to four different clades. Enzyme characterization identified 4CL activity for two clade IV members, Cc4CL1 and Cc4CL2, converting different hydroxycinnamic acids into the corresponding CoA thioesters. Both enzymes preferred coumaric acid over caffeic acid as a substrate in vitro. While expression of montbretia AAEs did not enhance MbA biosynthesis in N. benthamiana, we demonstrated that both Cc4CLs can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in yeast (Saccharomyces cerevisiae).<h4>Conclusions</h4>Montbretia expresses two functional 4CLs, but neither of them is specific for the formation of caffeoyl-CoA. Based on differential expression analysis and phylogeny Cc4CL1 is most likely involved in MbA biosynthesis, while Cc4CL2 may contribute to lignin biosynthesis. Both Cc4CLs can be used for flavanone production to support metabolic engineering of MbA in yeast.
format	article
author	Frederick G Sunstrum Hannah L Liu Sharon Jancsik Lufiani L Madilao Joerg Bohlmann Sandra Irmisch
author_facet	Frederick G Sunstrum Hannah L Liu Sharon Jancsik Lufiani L Madilao Joerg Bohlmann Sandra Irmisch
author_sort	Frederick G Sunstrum
title	4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.
title_short	4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.
title_full	4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.
title_fullStr	4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.
title_full_unstemmed	4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.
title_sort	4-coumaroyl-coa ligases in the biosynthesis of the anti-diabetic metabolite montbretin a.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/eabb92ab86ea4f308b5e3cefb1e74453
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4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.

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