Center for Microbial Biotechnology, Technical University of Denmark, DK - 2800 Kgs. Lyngby, Denmark.
Microb Cell Fact. 2010 Nov 8;9:84. doi: 10.1186/1475-2859-9-84.
Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis.
Expression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (PDC1 and GDH1) were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the Δpdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in baker's yeast.
Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA) as biological objective function. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories.
香兰素是应用最广泛的香料之一，最初从香草兰（Vanilla planifolia）的干燥豆荚中提取得到。目前，香兰素主要通过化学合成生产。最近，在酿酒酵母（Saccharamyces cerevisiae）中已经实现了从葡萄糖异源生产香草醛的从头合成途径。在这项研究中，我们旨在对这个香草醛细胞工厂进行工程改造，以提高生产力，并开发出一种有吸引力的化学合成替代方法。
在产香兰素的酿酒酵母菌株中表达来自拟南芥的糖基转移酶有助于降低产物毒性。使用一组应用于酵母基因组规模代谢网络的化学计量建模工具，对产生香草醛糖苷的这种工程菌进行了基于计算机的代谢工程策略设计。选择了两个目标（PDC1 和 GDH1）进行实验验证，结果产生了四个工程菌株。在批式培养中，四个突变株中有三个的香草醛 β-D-葡糖苷产率提高了 1.5 倍，而连续培养Δpdc1 突变株的生产力提高了 2 倍。与之前在酿酒酵母中从头合成香草醛的工作相比，该突变株的游离香兰素产量提高了 5 倍。
使用对应于不同生理状态的约束条件被发现极大地影响了基于代谢调整最小化（MOMA）作为生物目标函数的目标预测。根据预测的代谢调整选择目标并进行体内验证，成功地获得了高产菌株。总的来说，我们提出并证明了一种用于改进微生物细胞工厂的计算机设计和目标选择的框架。
