Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant
Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant
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DOI：
10.1128/aem.00186-08
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发表时间：
2008-03-01
影响因子：
4.4
通讯作者：
Domingues, Lucilia
中科院分区：
文献类型：
--
作者：
Guimaraes, Pedro M. R.;Francois, Jean;Domingues, Lucilia
构建发酵乳糖的酿酒酵母菌株具有重要的生物技术意义，特别是在奶酪乳清发酵方面。此前已构建了表达乳酸克鲁维酵母LAC12(乳糖渗透酶)和LAC4(P-半乳糖苷酶)基因的絮凝型嗜乳糖酿酒酵母重组菌株，但在乳糖发酵中效率较低。因此，该菌株经历了进化工程过程(在乳糖介质中连续转移和稀释)，从而产生了进化重组菌株，其消耗乳糖的速度是原始重组菌株的两倍，产生的乙醇比原始重组菌株多30%。我们在进化株中发现了两个针对LAC结构的分子事件：LAC4和LAC12之间基因间隔区(启动子)1,593-bp的缺失，以及与原始重组体相比，质粒拷贝数减少了约10倍。结果表明，在原重组菌株中，完整的启动子不能介导乳糖对LAC4和LAC12转录的诱导，而在进化菌株中，缺失证实了这两个基因的转录诱导。我们认为，异源LAC基因在进化重组体中的表达调控是通过两个基因拷贝数的减少和启动子结构变化导致的对LAC4和LAC12转录诱导水平的不同之间的相互作用完成的。然而，我们的结果并不排除其他可能的突变，这些突变可能有助于乳糖发酵表型的改善。这项研究说明了简单的进化工程方法在菌株改良中的有效性。该进化菌株高效发酵三倍浓缩奶酪乳清，为乳糖发酵提供了一种有吸引力的替代方案。
The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (P-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media.