BACKGROUND Steviol 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. RESULTS A 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. CONCLUSIONS 5'-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.

中文翻译：

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重新设计和重建甜菊醇生物合成途径，以增强大肠杆菌中甜菊醇的生产。

背景技术甜菊醇糖苷如甜菊糖苷已引起食品和饮料工业的关注。最近，人们努力使用代谢工程技术在微生物中生产这些天然甜味剂。然而，在代谢工程改造的微生物中，甜菊醇的滴度相对较低，因此异源微生物中的甜菊醇生物合成途径需要进行代谢优化。这项研究的目的是通过合成生物学方法重新设计和重建甜菊醇生物合成途径，以便在大肠杆菌中过量生产甜菊醇。结果基因组工程化的大肠杆菌菌株共表达5'非翻译区（UTR）工程化的香叶基香叶基二磷酸合酶，椰油酰基二磷酸合酶和贝壳杉烯合酶，产生623.6±3。分批发酵中的0 mg / L ent-kaurene。5'-UTR工程改造的拟南芥N-末端修饰的Kaurene氧化酶的过表达产生41.4±5 mg / L的ent-kaurenoic酸。通过增加细胞中NADPH / NADP +的比例，可提高对氨基脲的产量（50.7±9.8 mg / L）。源自拟南芥的融合蛋白UtrCYP714A2-AtCPR2的表达（其中trCYP714A2经过5'-UTR工程处理和N末端修饰）在分批发酵中得到38.4±1.7 mg / L甜菊醇。结论5'-UTR工程，融合蛋白方法和氧化还原平衡改善了烧瓶发酵和生物反应器发酵中的甜菊醇效价。