TY - BOOK

T1 - Identification and heterologous expression of biosynthetic genes for glucosinolate production

AU - Wang, Cuiwei

N1 - Må ikke offentliggøres

PY - 2019

Y1 - 2019

N2 - Glucosinolates (GLS) are amino acid-derived defense compounds characteristic of the Brassicales order. They have received increased attention since they have been shown to and several types of cancer. These health beneficial effects have made GLSs attractive as dietary supplements. However, the concentration of GLSs is often low and highly regulated in plants natively producing GLSs. This has primed a desire to produce GLSs in microorganisms to provide a rich source and enable intake of a well-defined dose. In this thesis, I focus on the chain-elongated phenylalanine-derived 2-phenylethyl GLS, phenylalanine-derived benzyl GLS and chain-elongated methionine-derived 4- methylsulfinylbutyl GLS, which have been reported to have health beneficial effects. I identified the enzymes in the 2-phenylethyl GLS pathway and successfully produced 2- phenylethyl GLS in Nicotiana benthamiana. I deciphered that the enzymes BCAT4 and MAM1 in the chain elongation from Barbarea vulgaris are critical in the profile of chainelongated products. For the first time, I characterized the role of members of CYP79C family, CYP79C1 and CYP79C2 in GLS biosynthesis. Even though the genes are not expressed in Arabidopsis heterologous expression shows that they are involved in the production of 2- methylpropyl GLS, 1-methylpropyl GLS, benzyl GLS and indolyl-3-methyl GLS. Additionally, I engineered the pathway of benzyl GLS in yeast and optimized the production by modulating sulfur metabolism, increasing the activity of sulfotransferase SOT16 and enhancing the flux at the entry point to the pathway. Finally, I attempted to produce the precursor of 4-methylsulfinylbutyl GLS, dihomo-methionine, in yeast by expressing the chain elongation pathway of Arabidopsis. The one-cycle product homo-methionine but notdihomo-methionine was produced, which may be attributed to the absence of the large subunit of isopropylmalate isomerase as identified by targeted proteomics.

AB - Glucosinolates (GLS) are amino acid-derived defense compounds characteristic of the Brassicales order. They have received increased attention since they have been shown to and several types of cancer. These health beneficial effects have made GLSs attractive as dietary supplements. However, the concentration of GLSs is often low and highly regulated in plants natively producing GLSs. This has primed a desire to produce GLSs in microorganisms to provide a rich source and enable intake of a well-defined dose. In this thesis, I focus on the chain-elongated phenylalanine-derived 2-phenylethyl GLS, phenylalanine-derived benzyl GLS and chain-elongated methionine-derived 4- methylsulfinylbutyl GLS, which have been reported to have health beneficial effects. I identified the enzymes in the 2-phenylethyl GLS pathway and successfully produced 2- phenylethyl GLS in Nicotiana benthamiana. I deciphered that the enzymes BCAT4 and MAM1 in the chain elongation from Barbarea vulgaris are critical in the profile of chainelongated products. For the first time, I characterized the role of members of CYP79C family, CYP79C1 and CYP79C2 in GLS biosynthesis. Even though the genes are not expressed in Arabidopsis heterologous expression shows that they are involved in the production of 2- methylpropyl GLS, 1-methylpropyl GLS, benzyl GLS and indolyl-3-methyl GLS. Additionally, I engineered the pathway of benzyl GLS in yeast and optimized the production by modulating sulfur metabolism, increasing the activity of sulfotransferase SOT16 and enhancing the flux at the entry point to the pathway. Finally, I attempted to produce the precursor of 4-methylsulfinylbutyl GLS, dihomo-methionine, in yeast by expressing the chain elongation pathway of Arabidopsis. The one-cycle product homo-methionine but notdihomo-methionine was produced, which may be attributed to the absence of the large subunit of isopropylmalate isomerase as identified by targeted proteomics.

M3 - Ph.D. thesis

BT - Identification and heterologous expression of biosynthetic genes for glucosinolate production

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -