Abstract
BackgroundSteviol glycosides such as stevioside have attracted the attention of the food and beverage industry. Recently, efforts were made to produce these natural sweeteners in microorganisms using metabolic engineering. Nonetheless, the steviol titer is relatively low in metabolically engineered microorganisms, and therefore a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. The purpose of this study was to redesign and reconstruct a steviol-biosynthetic pathway via synthetic-biology approaches in order to overproduce steviol in Escherichia coli.ResultsA genome-engineered E. coli strain, which coexpressed 5′ untranslated region (UTR)-engineered geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, and kaurene synthase, produced 623.6 ± 3.0 mg/L ent-kaurene in batch fermentation. Overexpression of 5′-UTR–engineered, N-terminally modified kaurene oxidase of Arabidopsis thaliana yielded 41.4 ± 5 mg/L ent-kaurenoic acid. Enhanced ent-kaurenoic acid production (50.7 ± 9.8 mg/L) was achieved by increasing the cellular NADPH/NADP+ ratio. The expression of a fusion protein, UtrCYP714A2-AtCPR2 derived from A. thaliana, where trCYP714A2 was 5′-UTR–engineered and N-terminally modified, gave 38.4 ± 1.7 mg/L steviol in batch fermentation.Conclusions5′-UTR engineering, the fusion protein approach, and redox balancing improved the steviol titer in flask fermentation and bioreactor fermentation. The expression engineering of steviol-biosynthetic enzymes and the genome engineering described here can serve as the basis for producing terpenoids—including steviol glycosides and carotenoids—in microorganisms.
Highlights
Steviol glycosides such as stevioside have attracted the attention of the food and beverage industry
Some studies [6] have revealed that only a small amount of ent-kaurenoic acid is converted in vivo to steviol (Fig. 1), suggesting that the reaction of hydroxylation of ent-kaurenoic acid needs to be metabolically optimized in the steviol glycoside–biosynthetic pathway
Engineering the ent‐kaurene pathway in E. coli To investigate the effect of a GGPP synthase (GGPPS) expression system on the production of ent-kaurene, the MGI strain expressing genes dxs, dxr, idi, and ispA was chosen as a platform strain for constructing two recombinant ent-kaurene– producing strains (MGI/GGPPS_CDPS_KS and MGIG/ CDPS_KS)
Summary
Steviol glycosides such as stevioside have attracted the attention of the food and beverage industry. The steviol titer is relatively low in metabolically engineered microorganisms, and a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. Given that steviol glycosides contain no calories and taste 200–300-fold sweeter than sucrose [1], these natural sweeteners may help prevent diabetes and obesity [2]. Efforts were made to produce ent-kaurene, ent-kaurenoic acid, steviol, and steviol glycosides in microorganisms by metabolic engineering [5, 6]. The titer of steviol is relatively low in metabolically engineered microorganisms, and a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. The same research group has reported that 1.8 g/L ent-kaurene can be obtained by fed-batch fermentation (in a 5 L bioreactor) under optimized conditions
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