Indigo is widely used in textile industries for denim garments dyeing and is mainly produced by chemical synthesis which, however, raises environmental sustainability issues. Bio-indigo may be produced by fermentation of metabolically engineering bacteria, but current methods are economically incompetent due to low titer and the need for an inducer. To address these problems, we first characterized several synthetic promoters in E. coli and demonstrated the feasibility of inducer-free indigo production from tryptophan using the inducer-free promoter. We next coupled the tryptophan-to-indigo and glucose-to-tryptophan pathways to generate a de novo glucose-to-indigo pathway. By rational design and combinatorial screening, we identified the optimal promoter-gene combinations, which underscored the importance of promoter choice and expression levels of pathway genes. We thus created a new E. coli strain that exploited an indole pathway to enhance the indigo titer to 123 mg/L. We further assessed a panel of heterologous tryptophan synthase homologs and identified a plant indole lyase (TaIGL), which along with modified pathway design, improved the indigo titer to 235 mg/L while reducing the tryptophan byproduct accumulation. The optimal E. coli strain expressed 8 genes essential for rewiring carbon flux from glucose to indole and then to indigo: mFMO, ppsA, tktA, trpD, trpC, TaIGL and feedback-resistant aroG and trpE. Fed-batch fermentation in a 3-L bioreactor with glucose feeding further increased the indigo titer (≈965 mg/L) and total quantity (≈2183 mg) at 72 h. This new synthetic glucose-to-indigo pathway enables high-titer indigo production without the need of inducer and holds promise for bio-indigo production.

中文翻译：

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大肠杆菌中的自诱导合成途径增强了葡萄糖的可持续靛蓝生产


靛蓝广泛用于纺织工业的牛仔服装染色，主要通过化学合成生产，但引发了环境可持续性问题。生物靛蓝可能通过代谢工程细菌的发酵产生，但由于滴度低且需要诱导剂，目前的方法在经济上是不可行的。为了解决这些问题，我们首先表征了大肠杆菌中的几种合成启动子，并证明了使用无诱导剂启动子从色氨酸生产无诱导剂靛蓝的可行性。接下来，我们将色氨酸到靛蓝和葡萄糖到色氨酸通路耦合，以产生从头葡萄糖到靛蓝通路。通过理性设计和组合筛选，我们确定了最佳的启动子-基因组合，这强调了启动子选择和通路基因表达水平的重要性。因此，我们创造了一种新的大肠杆菌菌株，它利用吲哚途径将靛蓝滴度提高到 123 mg/L。我们进一步评估了一组异源色氨酸合酶同源物，并确定了一种植物吲哚裂解酶 （TaIGL），它与改进的途径设计一起，将靛蓝滴度提高到 235 mg/L，同时减少了色氨酸副产物积累。最佳大肠杆菌菌株表达了 8 个基因，这些基因对于将碳通量从葡萄糖重新连接到吲哚，然后重新连接到靛蓝至关重要：mFMO、ppsA、tktA、trpD、trpC、TaIGL 和抗反馈的 aroG 和 trpE。在 3 L 生物反应器中补料分批发酵，葡萄糖补料在 72 h 时进一步提高了靛蓝滴度 （≈965 mg/L） 和总量 （≈2183 mg）。这种新的合成葡萄糖-靛蓝途径无需诱导剂即可实现高滴度靛蓝生产，并为生物靛蓝生产带来希望。