期刊
METABOLIC ENGINEERING
卷 67, 期 -, 页码 41-52出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2021.05.007
关键词
Acyl carrier protein; p-Coumaric acid; Directed evolution; Dynamic regulation network; Malonyl-CoA; (2S)-Naringenin
资金
- National Key Research and Development Program of China [2019YFA0904800]
- National Science Fund for Excellent Young Scholars [21822806]
- National Natural Science Foundation of China [31900066, 21877053, 31770097]
- Air Force Office of Scientific Research [FA9550-14-1-0089]
- National First-class Discipline Program of Light Industry Technology and Engineering [LITE2018-24]
- Fundamental Research Funds for the Central Universities [JUSRP12056, JUSRP51705A]
The study developed a growth coupled NCOMB DRN network for real-time control of intracellular supply of malonyl-CoA through dynamic metabolic engineering. The network showed a significant improvement in (2S)-naringenin production and cell growth, demonstrating its high efficiency for improving pathway production.
Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH, acpS, acpT, acs, and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2S)-naringenin production (523.7 +/- 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production.
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