Objectives

Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus.

Results

Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower.

Conclusions

Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

中文翻译：

---

从 GAL 代谢中解耦葡萄糖传感，用于酿酒酵母中的异源乳糖发酵

目标

乳糖直接发酵乙醇系统的开发为乳制品行业生产的大量未充分利用的乳清渗透物提供了市场。对于这个系统，葡萄糖和半乳糖代谢在酿酒酵母中通过删除两个负调节基因GAL80和MIG1并从天然但低效的乳糖发酵酵母马克斯克鲁维酵母中引入必需的乳糖水解酶LAC4和乳糖转运蛋白LAC12来解偶联。

结果

此前，LAC4和LAC12基因整合到MIG1和NTH1基因座以构建菌株 AY-51024M。低乳糖转化率导致我们产生了Δmig1Δgal80来自 AY-5 的二倍体突变株 AY-GM，表现出双生生长和葡萄糖抑制的丧失，随后以显着更高的速率摄取半乳糖并产生比菌株 AY-51024M 更高的乙醇浓度。类似地，在含有 100 g/L 或 150 g/L 乳糖的 5L 生物反应器中，在三个重复的批次处理过程中，奶酪乳清渗透粉溶液 (CWPS) 的乳糖吸收率和乙醇生产率均显着高于 AY -51024M，而整体发酵时间要低得多。

结论

使用 Cre-loxp 系统删除MIG1和GAL80基因以缓解葡萄糖抑制，以及LAC4和LAC12过表达以增加乳糖吸收和转化，为酵母将乳清渗透物副产品发酵成乙醇提供了有效的基础。