Brambilla Marco, Adamo Giusy Manuela, Frascotti Gianni, Porro Danilo, Branduardi Paola
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2 - 20126 Milan, Italy.
SYSBIO - Centre of Systems Biology, Milano and Roma, Italy.
J Microbiol Biotechnol. 2016 Feb;26(2):326-36. doi: 10.4014/jmb.1508.08002.
Saccharomyces cerevisiae is one of the most employed cell factories for the production of bioproducts. Although monomeric hexose sugars constitute the preferential carbon source, this yeast can grow on a wide variety of nitrogen sources that are catabolized through central nitrogen metabolism (CNM). To evaluate the effects of internal perturbations on nitrogen utilization, we characterized strains deleted or overexpressed in GLT1, encoding for one of the key enzymes of the CNM node, the glutamate synthase. These strains, together with the parental strain as control, have been cultivated in minimal medium formulated with ammonium sulfate, glutamate, or glutamine as nitrogen source. Growth kinetics, together with the determination of protein content, viability, and reactive oxygen species (ROS) accumulation at the single cell level, revealed that GLT1 modulations do not significantly influence the cellular physiology, whereas the nitrogen source does. As important exceptions, GLT1 deletion negatively affected the scavenging activity of glutamate against ROS accumulation, when cells were treated with H2O2, whereas Glt1p overproduction led to lower viability in glutamine medium. Overall, this confirms the robustness of the CNM node against internal perturbations, but, at the same time, highlights its plasticity in respect to the environment. Considering that side-stream protein-rich waste materials are emerging as substrates to be used in an integrated biorefinery, these results underline the importance of preliminarily evaluating the best nitrogen source not only for media formulation, but also for the overall economics of the process.
酿酒酵母是生产生物产品时最常用的细胞工厂之一。尽管单体己糖是优先的碳源，但这种酵母能够利用多种氮源生长，这些氮源通过中心氮代谢（CNM）途径被分解代谢。为了评估内部扰动对氮利用的影响，我们对编码CNM节点关键酶之一谷氨酸合酶的GLT1基因缺失或过表达的菌株进行了表征。这些菌株与作为对照的亲本菌株一起，在以硫酸铵、谷氨酸或谷氨酰胺作为氮源配制的基本培养基中培养。生长动力学以及单细胞水平上蛋白质含量、活力和活性氧（ROS）积累的测定结果表明，GLT1的调控对细胞生理没有显著影响，而氮源则有影响。重要的例外情况是，当用H2O2处理细胞时，GLT1基因缺失对谷氨酸清除ROS积累的活性有负面影响，而Glt1p过量表达导致在谷氨酰胺培养基中的活力降低。总体而言，这证实了CNM节点对内部扰动的稳健性，但同时也突出了其对环境的可塑性。鉴于富含蛋白质的侧流废料正逐渐成为综合生物炼制中使用的底物，这些结果强调了不仅要为培养基配方，而且要为整个工艺的经济性预先评估最佳氮源的重要性。
