Auto-inducible synthetic pathway in E. coli enhanced sustainable indigo production from glucose

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
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Abstract

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.

大肠杆菌中的自动诱导合成途径增强了葡萄糖靛蓝的可持续生产。
靛蓝被广泛应用于纺织业的牛仔服染色,主要通过化学合成法生产,但这会引发环境可持续性问题。生物靛蓝可通过代谢工程细菌发酵生产,但由于滴度低且需要诱导剂,目前的方法在经济上并不可行。为了解决这些问题,我们首先对大肠杆菌中的几种合成启动子进行了鉴定,并利用无诱导剂启动子证明了利用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的途径和葡萄糖到色氨酸的途径耦合起来,生成了一条全新的葡萄糖到靛蓝的途径。通过合理设计和组合筛选,我们确定了最佳的启动子-基因组合,这凸显了启动子选择和途径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,利用吲哚途径将靛蓝滴度提高到 123 毫克/升。我们进一步评估了一组异源色氨酸合成酶同源物,并确定了一种植物吲哚裂解酶(TaIGL),该酶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的积累。最佳大肠杆菌菌株表达了 8 个从葡萄糖到吲哚再到靛蓝的碳通量重新连接所必需的基因:mFMO、ppsA、tktA、trpD、trpC、TaIGL 以及反馈抗性 aroG 和 trpE。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,有望用于生物靛蓝生产。
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来源期刊
Metabolic engineering
Metabolic engineering 工程技术-生物工程与应用微生物
CiteScore
15.60
自引率
6.00%
发文量
140
审稿时长
44 days
期刊介绍: Metabolic Engineering (MBE) is a journal that focuses on publishing original research papers on the directed modulation of metabolic pathways for metabolite overproduction or the enhancement of cellular properties. It welcomes papers that describe the engineering of native pathways and the synthesis of heterologous pathways to convert microorganisms into microbial cell factories. The journal covers experimental, computational, and modeling approaches for understanding metabolic pathways and manipulating them through genetic, media, or environmental means. Effective exploration of metabolic pathways necessitates the use of molecular biology and biochemistry methods, as well as engineering techniques for modeling and data analysis. MBE serves as a platform for interdisciplinary research in fields such as biochemistry, molecular biology, applied microbiology, cellular physiology, cellular nutrition in health and disease, and biochemical engineering. The journal publishes various types of papers, including original research papers and review papers. It is indexed and abstracted in databases such as Scopus, Embase, EMBiology, Current Contents - Life Sciences and Clinical Medicine, Science Citation Index, PubMed/Medline, CAS and Biotechnology Citation Index.
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