School of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Korea.
Biotechnol Bioeng. 2011 Mar;108(3):621-31. doi: 10.1002/bit.22988. Epub 2010 Nov 12.
Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses.
尽管酿酒酵母能够将半乳糖发酵成乙醇，但从半乳糖中生产乙醇的得率和生产率明显低于从葡萄糖中生产的。采用反向代谢工程方法提高酿酒酵母对半乳糖的乙醇产量和生产率。将全基因组扰动文库导入酿酒酵母中，然后使用三种不同的富集方法筛选快速发酵半乳糖的转化体。对分离转化体中遗传扰动的表征揭示了三个靶基因，其过表达可增强对半乳糖的利用。一个确证靶基因（编码磷酸甘露糖异构酶的 SEC53）和两个新靶基因（编码小核 RNA 的 SNR84 和编码转录通用抑制剂的 TUP1 截断形式）被鉴定为可能改善半乳糖发酵的过表达靶基因。SEC53 的过表达可能具有与过表达编码磷酸葡萄糖变位酶的 PGM2 相似的机制。虽然其机制尚不清楚，但 SNR84 的过表达改善了半乳糖的生长和乙醇生产。过表达截断的 TUP1（tTUP1）基因对半乳糖发酵的改善最为显著，与对照菌株相比，半乳糖消耗速率和乙醇生产率分别提高了 250%。此外，过表达 tTUP1 显著缩短了从葡萄糖切换到半乳糖时出现的滞后期。基于这些结果，我们提出了一个假设，即没有 C 端抑制域的突变 Tup1 可能通过部分减轻葡萄糖抑制作用，更早、更高地表达 GAL 基因。tTUP 的过表达确实增加了 GAL 基因（GAL1、GAL4 和 GAL80）的 mRNA 水平。本研究结果表明，通过过表达鉴定的靶标（SNR84 和 tTUP1）改变全局调控网络与过表达半乳糖同化途径中的限速代谢基因（PGM2）一样有效，可提高酿酒酵母对半乳糖的高效发酵。此外，由于半乳糖是海洋植物生物质（如红海藻）、奶酪乳清和糖蜜中的丰富糖之一，这些结果在生物燃料领域将具有工业应用价值。
