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利用来自黄花蒿的β-香树脂醇合酶在酿酒酵母中工程化生产三萜类化合物。

Engineering triterpene production in Saccharomyces cerevisiae-beta-amyrin synthase from Artemisia annua.

作者信息

Kirby James, Romanini Dante W, Paradise Eric M, Keasling Jay D

机构信息

California Institute for Quantitative Biomedical Research, University of California, Berkeley, CA 94720, USA.

出版信息

FEBS J. 2008 Apr;275(8):1852-9. doi: 10.1111/j.1742-4658.2008.06343.x. Epub 2008 Mar 8.

Abstract

Using a degenerate primer designed from triterpene synthase sequences, we have isolated a new gene from the medicinal plant Artemisia annua. The predicted protein is highly similar to beta-amyrin synthases (EC 5.4.99.-), sharing amino acid sequence identities of up to 86%. Expression of the gene, designated AaBAS, in Saccharomyces cerevisiae, followed by GC/MS analysis, confirmed the encoded enzyme as a beta-amyrin synthase. Through engineering the sterol pathway in S. cerevisiae, we explore strategies for increasing triterpene production, using AaBAS as a test case. By manipulation of two key enzymes in the pathway, 3-hydroxy-3-methylglutaryl-CoA reductase and lanosterol synthase, we have improved beta-amyrin production by 50%, achieving levels of 6 mg.L(-1) culture. As we have observed a 12-fold increase in squalene levels, it appears that this strain has the capacity for even higher beta-amyrin production. Options for further engineering efforts are explored.

摘要

利用从三萜合酶序列设计的简并引物,我们从药用植物青蒿中分离出一个新基因。预测的蛋白质与β-香树脂醇合酶(EC 5.4.99.-)高度相似,氨基酸序列同一性高达86%。将该命名为AaBAS的基因在酿酒酵母中表达,随后进行气相色谱/质谱分析,证实编码的酶为β-香树脂醇合酶。通过改造酿酒酵母中的甾醇途径,我们以AaBAS为例探索了提高三萜产量的策略。通过操纵该途径中的两种关键酶,即3-羟基-3-甲基戊二酰辅酶A还原酶和羊毛甾醇合酶,我们将β-香树脂醇的产量提高了50%,达到了6 mg.L(-1)培养物的水平。由于我们观察到角鲨烯水平增加了12倍,看来该菌株具有更高的β-香树脂醇生产能力。我们还探索了进一步工程改造的方案。

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