Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, DK-2800 Kgs, Lyngby, Denmark.
Biotechnol Biofuels. 2011 Jul 28;4:21. doi: 10.1186/1754-6834-4-21.
Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials.
The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD) complex medium under aerobic conditions, respectively.
Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial improvement in isobutanol production for the reference strain, but not for the ILV2 ILV3 ILV5 overexpression strain. This result implies that other constraints besides the enzyme activities for the supply of 2-ketoisovalerate may become bottlenecks for isobutanol production after ILV2, ILV3, and ILV5 have been overexpressed, which most probably includes the valine inhibition to Ilv2.
由于异丁醇具有较高的能量密度和较低的吸湿性，因此它可以成为比乙醇更好的生物燃料。此外，异丁醇的支链结构使其辛烷值高于同分异构体正丁醇。由于酿酒酵母对醇类具有相对耐受性、在工业发酵中具有稳健性，并且有可能将异丁醇生产与木质纤维素材料的发酵相结合，因此选择酿酒酵母作为生产宿主。
通过在厌氧发酵葡萄糖的矿物培养基中，同时过表达支链氨基酸代谢中的生物合成基因 ILV2、ILV3 和 ILV5，将异丁醇的产率从 0.16mg/g 葡萄糖提高到 0.97mg/g 葡萄糖。通过过表达编码细胞质支链氨基酸转氨酶的 BAT2，异丁醇的产率进一步提高了一倍。在过表达 ILV2、ILV3 和 ILV5 的菌株中过表达编码 Ilv2 调节亚基的 ILV6，将异丁醇的产率降低了三倍。在摇瓶中的有氧培养中，过表达 ILV2、ILV3 和 ILV5 的菌株和对照菌株在矿物培养基中的异丁醇产量分别为 3.86mg/g 和 0.28mg/g 葡萄糖。在有氧条件下的酵母提取物/蛋白胨/葡萄糖（YPD）复杂培养基中，它们分别增加到 4.12mg/g 和 2.4mg/g 葡萄糖。
在缬氨酸代谢中过表达基因 ILV2、ILV3、ILV5 和 BAT2 导致酿酒酵母中异丁醇产量增加。在过表达 ILV2、ILV3 和 ILV5 的菌株中过表达 ILV6 产生了负面影响，可能是通过增加 Ilv2 对缬氨酸抑制的敏感性，从而削弱了过表达 ILV2、ILV3 和 ILV5 对异丁醇生产的积极影响。过表达 ILV2、ILV3 和 ILV5 的菌株和对照菌株的有氧培养表明，在培养物中提供氨基酸可大大提高对照菌株的异丁醇产量，但对过表达 ILV2、ILV3 和 ILV5 的菌株没有影响。这一结果表明，在过表达 ILV2、ILV3 和 ILV5 之后，除了提供 2-酮异戊酸的酶活性之外，其他限制因素可能成为异丁醇生产的瓶颈，这很可能包括对 Ilv2 的缬氨酸抑制。
