Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae
Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae
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通过代谢工程酿酒酵母生产米尔替拉烯
DOI：
10.1002/bit.24547
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发表时间：
2012-11-01
影响因子：
3.8
通讯作者：
Zhang, Xueli
中科院分区：
文献类型：
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作者：
Dai, Zhubo;Liu, Yi;Zhang, Xueli
微生物的代谢工程是生产通常从植物来源提取的有价值的萜类化合物的替代和有吸引力的途径。丹参酮是丹参的活性成分，丹参是一种常用中药，广泛用于治疗多种心血管疾病。作为微生物生产丹参酮，柯巴基二磷酸（CPP）合酶，和正常的CPP贝壳杉烯脱氢酶样基因，其中转换的通用二萜类前体香叶基香叶基二磷酸（GGPP）的miltiradiene（丹参酮合成途径的重要中间体）的一个步骤，被引入到酿酒酵母，导致生产4.2？mg/L米替雷烯。然后研究改善类异戊二烯前体的供应以增加米替雷烯的产量。尽管截短的3-羟基-3-甲基戊二酰辅酶A还原酶（tHMGR）和突变的全局调节因子（upc2.1）基因的过表达确实改善了法呢基二磷酸（FPP）的供应，但米替雷烯的产生并没有增加，而大量的角鲨烯（78？mg/L）。相比之下，miltiradiene生产增加到8.8？通过过量表达FPP合酶（ERG 20）和内源性GGPP合酶（BTS 1）的融合基因以及来自酸热硫化叶菌的异源GGPP合酶（SaGGPS）来改善GGPP的供应。然后用抗生素标记替换附加型质粒中的营养标记，使得工程酵母菌株可以在保持质粒稳定性的情况下使用丰富的培养基以获得更好的细胞生长。过表达ERG 20-BTS 1和SaGGPS基因增加miltiradiene生产从5.4到28.2？毫克/升tHMGR-upc 2.1和ERG 20-BTS 1-SaGGPS基因的组合过表达对米替雷烯的产生有协同作用，使滴度增加到61.8？毫克/升最后，流加发酵进行，和488？mg/L的米替雷烯。在这项工作中改造的酵母菌株提供了一个基础，创造一种替代的方法来生产丹参酮，而不是从植物来源提取。Biotechnol. Bioeng. 2012; 109：28452853。(c)2012 Wiley Periodicals，Inc.
Metabolic engineering of microorganisms is an alternative and attractive route for production of valuable terpenoids that are usually extracted from plant sources. Tanshinones are the bioactive components of Salvia miltiorrhizha Bunge, which is a well-known traditional Chinese medicine widely used for treatment of many cardiovascular diseases. As a step toward microbial production of tanshinones, copalyl diphosphate (CPP) synthase, and normal CPP kaurene synthase-like genes, which convert the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to miltiradiene (an important intermediate of the tanshinones synthetic pathway), was introduced into Saccharomyces cerevisiae, resulting in production of 4.2?mg/L miltiradiene. Improving supplies of isoprenoid precursors was then investigated for increasing miltiradiene production. Although over-expression of a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMGR) and a mutated global regulatory factor (upc2.1) gene did improve supply of farnesyl diphosphate (FPP), production of miltiradiene was not increased while large amounts of squalene (78?mg/L) were accumulated. In contrast, miltiradiene production increased to 8.8?mg/L by improving supply of GGPP through over-expression of a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1) together with a heterologous GGPP synthase from Sulfolobus acidocaldarius (SaGGPS). Auxotrophic markers in the episomal plasmids were then replaced by antibiotic markers, so that engineered yeast strains could use rich medium to obtain better cell growth while keeping plasmid stabilities. Over-expressing ERG20-BTS1 and SaGGPS genes increased miltiradiene production from 5.4 to 28.2?mg/L. Combinatorial over-expression of tHMGR-upc2.1 and ERG20-BTS1-SaGGPS genes had a synergetic effects on miltiradiene production, increasing titer to 61.8?mg/L. Finally, fed-batch fermentation was performed, and 488?mg/L miltiradiene was produced. The yeast strains engineered in this work provide a basis for creating an alternative way for production of tanshinones in place of extraction from plant sources. Biotechnol. Bioeng. 2012; 109: 28452853. (c) 2012 Wiley Periodicals, Inc.