Elimination of aromatic fusel alcohols as by-products of Saccharomyces cerevisiae strains engineered for phenylpropanoid production by 2-oxo-acid decarboxylase replacement

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Else-Jasmijn Hassing, Joran Buijs, Nikki Blankerts, Marijke A. Luttik, Erik A.de Hulster, Jack T. Pronk, Jean-Marc Daran
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引用次数: 0

Abstract

Engineered strains of the yeast Saccharomyces cerevisiae are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids. Heterologous pathways for production of these compounds use l-phenylalanine and/or l-tyrosine, generated by the yeast shikimate pathway, as aromatic precursors. The Ehrlich pathway converts these precursors to aromatic fusel alcohols and acids, which are undesirable by-products of yeast strains engineered for production of high-value aromatic compounds. Activity of the Ehrlich pathway requires any of four S. cerevisiae 2-oxo-acid decarboxylases (2-OADCs): Aro10 or the pyruvate-decarboxylase isoenzymes Pdc1, Pdc5, and Pdc6. Elimination of pyruvate-decarboxylase activity from S. cerevisiae is not straightforward as it plays a key role in cytosolic acetyl-CoA biosynthesis during growth on glucose. In a search for pyruvate decarboxylases that do not decarboxylate aromatic 2-oxo acids, eleven yeast and bacterial 2-OADC-encoding genes were investigated. Homologs from Kluyveromyces lactis (KlPDC1), Kluyveromyces marxianus (KmPDC1), Yarrowia lipolytica (YlPDC1), Zymomonas mobilis (Zmpdc1) and Gluconacetobacter diazotrophicus (Gdpdc1.2 and Gdpdc1.3) complemented a Pdc strain of S. cerevisiae for growth on glucose. Enzyme-activity assays in cell extracts showed that these genes encoded active pyruvate decarboxylases with different substrate specificities. In these in vitro assays, ZmPdc1, GdPdc1.2 or GdPdc1.3 had no substrate specificity towards phenylpyruvate. Replacing Aro10 and Pdc1,5,6 by these bacterial decarboxylases completely eliminated aromatic fusel-alcohol production in glucose-grown batch cultures of an engineered coumaric acid-producing S. cerevisiae strain. These results outline a strategy to prevent formation of an important class of by-products in ‘chassis’ yeast strains for production of non-native aromatic compounds.

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用2-氧基酸脱羧酶置换法去除酿酒酵母生产苯丙醇的副产物芳香醇醚
酵母工程菌株作为芳香化合物如羟基肉桂酸、二苯乙烯和黄酮类化合物的生产平台被广泛研究。生产这些化合物的异源途径使用由酵母莽草酸途径产生的l-苯丙氨酸和/或l-酪氨酸作为芳香前体。埃利希途径将这些前体转化为芳香醇和酸,这是用于生产高价值芳香化合物的酵母菌株的不良副产物。埃利希途径的活性需要四种酿酒葡萄球菌2-氧酸脱羧酶(2- oadc)中的任何一种:Aro10或丙酮酸脱羧酶同工酶Pdc1、Pdc5和Pdc6。消除酿酒酵母的丙酮酸脱羧酶活性并不是直截了当的,因为它在葡萄糖生长过程中对胞质乙酰辅酶a的生物合成起着关键作用。为了寻找不使芳香2-氧酸脱羧的丙酮酸脱羧酶,研究了11个酵母和细菌2- oadc编码基因。来自乳酸克卢维菌(KlPDC1)、马氏克卢维菌(KmPDC1)、多脂耶氏耶氏菌(YlPDC1)、活动单胞菌(Zmpdc1)和重氮营养菌(Gdpdc1.2和Gdpdc1.3)的同源物补充了酿酒酵母Pdc−菌株对葡萄糖的生长。细胞提取物的酶活性测定表明,这些基因编码的活性丙酮酸脱羧酶具有不同的底物特异性。在这些体外实验中,ZmPdc1、GdPdc1.2或GdPdc1.3对苯丙酮酸没有底物特异性。用这些细菌脱羧酶取代Aro10和Pdc1,5,6,完全消除了葡萄糖培养的香豆酸生产酵母菌株的芳香杂醇生产。这些结果概述了一种策略,以防止在“底盘”酵母菌株中形成一类重要的副产物,用于生产非天然芳香化合物。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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