Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation.
Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation.
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DOI：
10.1099/00221287-144-12-3437
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发表时间：
1998-12
期刊：
影响因子：
1.5
通讯作者：
A. Zeeman;-H.;Luttik;C. Thiele;Johannes;-P.;Van Dijken;Yde Steensmal
中科院分区：
文献类型：
--
作者：
A. Zeeman;-H.;Luttik;C. Thiele;Johannes;-P.;Van Dijken;Yde Steensmal
利用酿酒酵母PDA1基因1.1kb的内部片段筛选，从克鲁维酵母基因组文库中分离到编码线粒体丙酮酸脱氢酶(PDH)复合体E1a亚基的KlPDA1基因。KlPDA1编码的氨基酸序列与酿酒酵母的同源性分别为87%和79%。KIPDA1的破坏导致细胞提取物中PDH活性的完全缺失。突变株在葡萄糖上的最大比生长速率比野生型低3.5倍，而在乙醇上的生长没有受到影响。野生型乳酸克雷伯氏菌CBS 2359表现为Crabtree阴性表型，即在葡萄糖的好氧分批培养中不产生乙醇。相比之下，在同基因Klpda1缺失突变体的有氧培养中，可以产生大量的乙醇和乙醛。在导入完整的KlPDA1基因后，野生型特定生长率恢复，但不像以前在酿酒酵母pda1突变体中发现的那样，通过在亮氨酸存在的情况下进行培养。Klpda1缺失突变体的有氧发酵和生长缓慢表明，在葡萄糖分批培养过程中，PDH旁路的酶(丙酮酸脱羧酶、乙醛脱氢酶和乙酰-CoA合成酶)不能有效地替代PDH复合体。只有在有氧、葡萄糖限制的恒化器培养中，只有在相对较低的生长速率(D=0.10h(-1))下，PDH旁路才能完全取代PDH复合体，从而允许完全的呼吸生长。这导致生物量产量[g生物质(g葡萄糖)-1]低于野生型，因为与通过PDH复合体形成乙酰辅酶A相比，PDH旁路中的ATP消耗更高。
The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.