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Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae
Author(s) -
Dai Zhubo,
Liu Yi,
Huang Luqi,
Zhang Xueli
Publication year - 2012
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.24547
Subject(s) - farnesyl diphosphate synthase , saccharomyces cerevisiae , metabolic engineering , biochemistry , atp synthase , terpenoid , biology , sulfolobus acidocaldarius , overproduction , yeast , heterologous expression , biosynthesis , chemistry , gene , recombinant dna , archaea
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: 2845–2853. © 2012 Wiley Periodicals, Inc.