Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae.
Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae.
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DOI：
10.1186/1754-6834-5-65
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发表时间：
2012-09-06
影响因子：
6.3
通讯作者：
Boles E
中科院分区：
文献类型：
--
作者：
Brat D;Weber C;Lorenzen W;Bode HB;Boles E
支链醇异丁醇表现出作为替代生物燃料的上级物理化学性质。酵母酿酒酵母在发酵过程中自然产生少量的异丁醇作为副产物，这是由缬氨酸的催化作用引起的。作为S.酿酒酵母广泛用于工业应用，并且可以容易地通过基因工程进行修饰，这种微生物是用于发酵生产更高量的异丁醇的有希望的宿主。通过将缬氨酸生物合成酶Ilv 2、Ilv 5和Ilv 3从线粒体基质重新定位到细胞质中，可以提高异丁醇的生产。为了防止这三种酶输入到酵母线粒体中，构建了N-末端缩短的Ilv 2、Ilv 5和Ilv 3版本，它们缺乏线粒体靶向序列。SDS-PAGE和免疫荧光分析证实了截短酶的表达和重新定位。生长试验或酶测定证实了酶活性。异丁醇的生产只增加在没有缬氨酸和线粒体缬氨酸合成途径的同时阻断。在使截短的缬氨酸生物合成基因的密码子使用适应于高度表达的糖酵解基因的密码子使用之后，异丁醇生产甚至可以得到更大的增强。最后，选择合适的酮异戊酸脱羧酶Aro 10和醇脱氢酶Adh 2并过表达。最高异丁醇滴度为0.63 g/L，产率接近15 mg/g葡萄糖。通过对线粒体缬氨酸合成酶的重新定位和优化以及Aro 10脱羧酶和Adh 2乙醇脱氢酶的过表达，在酵母中成功建立了胞质异丁醇生产途径。通过阻断与线粒体缬氨酸途径的竞争和通过从发酵培养基中省略缬氨酸来产生驱动力。丙酮酸脱羧酶基因的额外缺失和辅因子不平衡的工程化应导致甚至更高的异丁醇产量。
The branched chain alcohol isobutanol exhibits superior physicochemical properties as an alternative biofuel. The yeast Saccharomyces cerevisiae naturally produces low amounts of isobutanol as a by-product during fermentations, resulting from the catabolism of valine. As S. cerevisiae is widely used in industrial applications and can easily be modified by genetic engineering, this microorganism is a promising host for the fermentative production of higher amounts of isobutanol. Isobutanol production could be improved by re-locating the valine biosynthesis enzymes Ilv2, Ilv5 and Ilv3 from the mitochondrial matrix into the cytosol. To prevent the import of the three enzymes into yeast mitochondria, N-terminally shortened Ilv2, Ilv5 and Ilv3 versions were constructed lacking their mitochondrial targeting sequences. SDS-PAGE and immunofluorescence analyses confirmed expression and re-localization of the truncated enzymes. Growth tests or enzyme assays confirmed enzymatic activities. Isobutanol production was only increased in the absence of valine and the simultaneous blockage of the mitochondrial valine synthesis pathway. Isobutanol production could be even more enhanced after adapting the codon usage of the truncated valine biosynthesis genes to the codon usage of highly expressed glycolytic genes. Finally, a suitable ketoisovalerate decarboxylase, Aro10, and alcohol dehydrogenase, Adh2, were selected and overexpressed. The highest isobutanol titer was 0.63 g/L at a yield of nearly 15 mg per g glucose. A cytosolic isobutanol production pathway was successfully established in yeast by re-localization and optimization of mitochondrial valine synthesis enzymes together with overexpression of Aro10 decarboxylase and Adh2 alcohol dehydrogenase. Driving forces were generated by blocking competition with the mitochondrial valine pathway and by omitting valine from the fermentation medium. Additional deletion of pyruvate decarboxylase genes and engineering of co-factor imbalances should lead to even higher isobutanol production.
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影响因子：
2.8
作者：
Hamacher, T;Becker, J;Boles, E
通讯作者：
Boles, E
影响因子：
4.8
作者：
Dickinson, JR;Eshantha, L;Hewlins, MJE
通讯作者：
Hewlins, MJE
影响因子：
2.1
作者：
de la Plaza, M;de Palencia, PF;Requena, T
通讯作者：
Requena, T
影响因子：
3.2
作者：
Boer, Viktor M.;Tai, Siew Leng;Daran, Jean-Marc
通讯作者：
Daran, Jean-Marc
影响因子：
4.4
作者：
Becker, J;Boles, E
通讯作者：
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