Andrejc Darjan, Možir Alenka, Legiša Matic
Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hjadrihova 19, Si-1000, Ljubljana, Slovenia.
Department of Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, Si-1000, Ljubljana, Slovenia.
BMC Biotechnol. 2017 May 8;17(1):41. doi: 10.1186/s12896-017-0362-5.
At first glance, there appears to be a high degree of similarity between the metabolism of yeast (the Crabtree effect) and human cancer cells (the Warburg effect). At the root of both effects is accelerated metabolic flow through glycolysis which leads to overflows of ethanol and lactic acid, respectively. It has been proposed that enhanced glycolytic flow in cancer cells is triggered by the altered kinetic characteristics of the key glycolytic regulatory enzyme 6-phosphofructo-1-kinase (Pfk). Through a posttranslational modification, highly active shorter Pfk-M fragments, which are resistant to feedback inhibition, are formed after the proteolytic cleavage of the C-terminus of the native human Pfk-M. Alternatively, enhanced glycolysis is triggered by optimal growth conditions in the yeast Saccharomyces cerevisiae.
To assess the deregulation of glycolysis in yeast cells, the sfPFKM gene encoding highly active human shorter Pfk-M fragments was introduced into pfk-null S. cerevisiae. No growth of the transformants with the sfPFKM gene was observed on glucose and fructose. Glucose even induced rapid deactivation of Pfk1 activities in such transformants. However, Pfk1 activities of the sfPFKM transformants were detected in maltose medium, but the growth in maltose was possible only after the addition of 10 mM of ethanol to the medium. Ethanol seemed to alleviate the severely unbalanced NADH/NADPH ratio in the sfPFKM cells. However, the transformants carrying modified Pfk-M enzymes grew faster than the transformants with the human native human Pfk-M enzyme in a narrow ecological niche with a low maltose concentration medium that was further improved by additional modifications. Interestingly, periodic extracellular accumulation of phenylacetaldehyde was detected during the growth of the strain with modified Pfk-M but not with the strain encoding the human native enzyme.
Highly active cancer-specific shorter Pfk-M fragments appear to trigger several controlling mechanisms in the primary metabolism of yeast S. cerevisiae cells. These results suggest more complex metabolic regulation is present in S. cerevisiae as free living unicellular eukaryotic organisms in comparison to metazoan human cells. However, increased productivity under broader growth conditions may be achieved if more gene engineering is performed to reduce or omit several controlling mechanisms.
乍一看，酵母的代谢（克奈特效应）与人类癌细胞的代谢（瓦伯格效应）之间似乎存在高度相似性。这两种效应的根源都是糖酵解过程中代谢流加速，分别导致乙醇和乳酸的溢出。有人提出，癌细胞中糖酵解流增强是由关键糖酵解调节酶6-磷酸果糖-1-激酶（Pfk）动力学特性改变引发的。通过翻译后修饰，在天然人Pfk-M的C末端蛋白水解切割后，形成了对反馈抑制有抗性的高活性较短Pfk-M片段。或者，酿酒酵母中的糖酵解增强是由最佳生长条件引发的。
为了评估酵母细胞中糖酵解的失调情况，将编码高活性人较短Pfk-M片段的sfPFKM基因导入缺失pfk的酿酒酵母中。在葡萄糖和果糖上未观察到带有sfPFKM基因的转化体生长。葡萄糖甚至会导致此类转化体中Pfk1活性迅速失活。然而，在麦芽糖培养基中检测到了sfPFKM转化体的Pfk1活性，但只有在培养基中添加10 mM乙醇后，才能在麦芽糖中生长。乙醇似乎缓解了sfPFKM细胞中严重失衡的NADH/NADPH比率。然而，携带修饰后的Pfk-M酶的转化体在低麦芽糖浓度的狭窄生态位中比携带天然人Pfk-M酶的转化体生长得更快，通过进一步修饰可进一步改善。有趣的是，在携带修饰后的Pfk-M的菌株生长过程中检测到苯乙醛的周期性细胞外积累，而在编码天然人酶的菌株中未检测到。
高活性的癌症特异性较短Pfk-M片段似乎触发了酿酒酵母细胞初级代谢中的几种控制机制。这些结果表明，与后生动物人类细胞相比，酿酒酵母作为自由生活的单细胞真核生物存在更复杂的代谢调控。然而，如果进行更多基因工程以减少或省略几种控制机制，可能会在更广泛的生长条件下提高生产力。
