Abstract
The yeast strain (Saccharomyces cerevisiae) MTCC 3157 was selected for combinatorial biosynthesis of plant sesquiterpene amorpha-4,11-diene. Our main objective was to overproduce amorpha 4-11-diene, which is a key precursor molecule of artemisinin (antimalarial drug) produced naturally in plant Artemisia annua through mevalonate pathway. Farnesyl diphosphate (FPP) is a common intermediate metabolite of a variety of compounds in the mevalonate pathway of yeast and leads to the production of ergosterols, dolichol and ubiquinone, and so forth. In our studies, FPP converted to amorphadiene (AD) by expressing heterologous amorphadiene synthase (ADS) in yeast. First, ERG9 (squalane synthase) promoter of yeast was replaced with repressible methionine (MET3) promoter by using bipartite gene fusion method. Further to overcome the loss of the intermediate FPP through competitive pathways in yeast, fusion protein technology was adopted and farnesyldiphosphate synthase (FPPS) of yeast has been coupled with amorphadiene synthase (ADS) of plant origin (Artemisia annua L.) where amorphadiene production was improved by 2-fold (11.2 mg/L) and 4-fold (25.02 mg/L) in yeast strains YCF-002 and YCF-005 compared with control strain YCF-AD (5.5 mg/L), respectively.
Highlights
Microbial fermentation ensures production of industrially important metabolites in large quantities
The minimum concentration of methionine for expression of ERG9 was determined by growing the ERG9 repressed yeast cells in shake flasks supplemented with varying quantities of methionine (Figure 2)
When yeast cells grown in the absence of methionine, cells propagated exponentially up to 10 h and followed by a slight drop in the growth and immediately after 12 h cells followed exponential phase up to 20 h
Summary
Microbial fermentation ensures production of industrially important metabolites in large quantities. Apart from the proteins, nature offers diverse classes of complex metabolites (isoprenoids) that are utilized in the food, cosmetic, and pharmaceutical industries and so forth [1]. Many of these complex metabolites are produced naturally in low quantities in plants that are difficult or expensive to cultivate. Systems, and synthetic biology principles and methods allowed easy transfer of heterologous pathways from natural plant producer to a suitable microbial host such as yeast and E. coli [2,3,4]. E. coli was the most studied host for metabolic engineering of isoprenoid by modulating
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