Abstract
Identification of efficient key enzymes in biosynthesis pathway and optimization of the fitness between functional modules and chassis are important for improving the production of target compounds. In this study, the taxadiene biosynthesis pathway was firstly constructed in yeast by transforming ts gene and overexpressing erg20 and thmgr. Then, the catalytic capabilities of six different geranylgeranyl diphosphate synthases (GGPPS), the key enzyme in mevalonic acid (MVA) pathway catalyzing famesyl diphosphate (FPP) to geranylgeranyl diphosphate (GGPP), were predicted using enzyme-substrate docking strategy. GGPPSs from Taxus baccata x Taxus cuspidate (GGPPSbc), Erwinia herbicola (GGPPSeh), and S. cerevisiae (GGPPSsc) which ranked 1st, 4th and 6th in docking with FPP were selected for construction. The experimental results were consistent with the computer prediction that the engineered yeast with GGPPSbc exhibited the highest production. In addition, two chassis YSG50 and W303-1A were chosen, and the titer of taxadiene reached 72.8 mg/L in chassis YSG50 with GGPPSbc. Metabolomic study revealed that the contents of tricarboxylic acid cycle (TCA) intermediates and their precursor amino acids in chassis YSG50 was lower than those in W303-1A, indicating less carbon flux was divided into TCA cycle. Furthermore, the levels of TCA intermediates in the taxadiene producing yeasts were lower than those in chassis YSG50. Thus, it may result in more carbon flux in MVA pathway in chassis YSG50, which suggested that YSG50 was more suitable for engineering the taxadiene producing yeast. These results indicated that computer-aided protein modeling directed isoenzyme selection strategy and metabolomic study could guide the rational design of terpenes biosynthetic cells.
Highlights
In the past few years, producing natural products by synthetic biology strategies has attracted more and more attention [1,2,3]
A large number of studies have shown that inducing the functional modules into Saccharomyces cerevisiae or Escherichia coli chassis is a feasible way for terpenes production [5,6,7,8,9,10,11,12,13,14,15,16,17,18]
The ts was controlled by a strong constructive promoter tdh3, and the plasmid was transformed into W303-1A, obtaining strain SyBE_001188
Summary
In the past few years, producing natural products by synthetic biology strategies has attracted more and more attention [1,2,3]. Production of taxadiene has reached a yield of 1020 mg/L in E. coli after fermentation optimization [18]. E. coli platform may not be suitable for downstream processing and enzyme modifying though it is capable for the initial biosynthetic steps for terpene hydrocarbons [19]. S. cerevisiae is more suitable for further construction for synthesis steps and industrial production [15,20,21]. Production of taxadiene in yeast has not been able to meet the expectation. It has only a yield of 8.7 mg/L according to our knowledge [13]. Improvement of the terpene production is of great value for synthetic biology research
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