González-Siso María Isabel, Touriño Alba, Vizoso Ángel, Pereira-Rodríguez Ángel, Rodríguez-Belmonte Esther, Becerra Manuel, Cerdán María Esperanza
Grupo de Investigación EXPRELA, Departamento de Bioloxía Celular e Molecular, Facultade de Ciencias, Universidade da Coruña, Campus de A Coruña, 15071-, A Coruña, Spain.
Microb Biotechnol. 2015 Mar;8(2):319-30. doi: 10.1111/1751-7915.12160. Epub 2014 Sep 3.
In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (Δklndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the Δklndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K. lactis mitochondria. Permeabilized cells of the Δklndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The Δklndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The Δklndi1 mutation shifts the K. lactis metabolism from respiratory to fermentative: the Δklndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non-fermentable carbon sources such as lactate. The biotechnological benefit of the Δklndi1 mutant for bioethanol production from waste cheese whey lactose was proved.
在本文中，我们通过构建和表征编码线粒体替代性内部脱氢酶的单基因中的无效突变体（Δklndi1），报道了呼吸型酵母乳酸克鲁维酵母的代谢工程。Δklndi1突变体的分离线粒体显示外源NADH的氧化速率未受影响，但基质NADH无氧化；这证实了KlNdi1p是乳酸克鲁维酵母线粒体中唯一的内部NADH脱氢酶。Δklndi1突变体的透化细胞未显示基质NADH的氧化，这表明将NADH从线粒体基质转移到细胞质以被外部脱氢酶氧化的穿梭系统不起作用。在缺乏营养的情况下，Δklndi1突变会缩短时序寿命。谷胱甘肽还原酶耗竭会增加KlNDI1的表达。Δklndi1突变使乳酸克鲁维酵母的代谢从呼吸型转变为发酵型：Δklndi1菌株的呼吸速率降低，葡萄糖产生乙醇的量增加，而它在诸如乳酸等不可发酵碳源中无法生长。证明了Δklndi1突变体在利用废奶酪乳清乳糖生产生物乙醇方面的生物技术优势。
