期刊
METABOLIC ENGINEERING
卷 67, 期 -, 页码 417-427出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2021.08.003
关键词
Flavonoids; Riboswitch; Multi-level expression optimization; Synthetic biology
资金
- Ministry of Science and ICT [NRF-2018M3D3A1A01055754, NRF-2016K1A1A2912829]
- Korea Institute of Energy Technology Evaluation and Planning
- Ministry of Trade, Industry, and Energy (MOTIE) of the Republic of Korea [20194030202330]
- Korea Evaluation Institute of Industrial Technology (KEIT) [20194030202330] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
Recombinant microbes provide a promising alternative for producing naringenin, a key molecular scaffold for flavonoids. In this study, a systematic strategy for multi-level optimization of biosynthetic pathways was introduced, resulting in a significant increase in naringenin production. Through the construction of a combinatorial library covering both transcription and translation stages, improved strains were identified, leading to a 3-fold increase in naringenin production compared to traditional methods.
Recombinant microbes have emerged as promising alternatives to natural sources of naringenin-a key molecular scaffold for flavonoids. In recombinant strains, expression levels of the pathway genes should be optimized at both transcription and the translation stages to precisely allocate cellular resources and maximize metabolite production. However, the optimization of the expression levels of naringenin generally relies on evaluating a small number of variants from libraries constructed by varying transcription efficiency only. In this study, we introduce a systematic strategy for the multi-level optimization of biosynthetic pathways. We constructed a multi-level combinatorial library covering both transcription and translation stages using synthetic T7 promoter variants and computationally designed 5 '-untranslated regions. Furthermore, we identified improved strains through high-throughput screening based on a synthetic naringenin riboswitch. The most-optimized strain obtained using this approach exhibited a 3-fold increase in naringenin production, compared with the parental strain in which only the transcription efficiency was modulated. Furthermore, in a fed-batch bioreactor, the optimized strain produced 260.2 mg/L naringenin, which is the highest concentration reported to date using glycerol and p-coumaric acid as substrates. Collectively, this work provides an efficient strategy for the expression optimization of the biosynthetic pathways.
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