重构异戊二烯焦磷酸代谢以促进萜类化合物的合成

徐显皓, 吕雪芹, 崔世修, 刘延峰, 夏洪志, 李江华, 堵国成, 李兆丰, Rodrigo Ledesma-Amaro, 陈坚, 刘龙

工程（英文） ›› 2023, Vol. 28 ›› Issue (9) : 166-178.

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工程（英文） ›› 2023, Vol. 28 ›› Issue (9) : 166-178. DOI: 10.1016/j.eng.2023.03.019
研究论文
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重构异戊二烯焦磷酸代谢以促进萜类化合物的合成

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Remodeling Isoprene Pyrophosphate Metabolism for Promoting Terpenoids Bioproduction

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摘要

萜类化合物是最大的一类天然产物。它们以异戊二烯焦磷酸(IPP)作为基本单元构建而成。使用细胞工厂合成萜类化合物已经引起了极大关注。而迄今为止，IPP的供应不足仍然是高效合成萜类化合物的主要挑战。在本项研究中，我们发现在枯草芽孢杆菌（Bacillus subtilis）中，中心代谢和IPP供应之间的代谢通量分配不平衡阻碍了IPP的积累。因此，我们构建了一系列响应丙酮酸和/或丙二酰辅酶A的双输入多输出（two-input-multi-output, TIMO）基因回路对IPP的代谢途径进行了改造，使IPP的积累增加了将近4倍。随后，设计了一种IPP代谢网络重构策略以提高三种萜类化合物的产量，包括维生素K2 (MK-7, 4.1倍)、番茄红素(9倍)和β-胡萝卜素（0.9倍）。在50-L的生物反应器中，MK-7的产量达到1549.6 mg∙L−1，是迄今为止报道的最高产量。本文提出了一种基于TIMO基因回路的IPP代谢网络重构框架，可用于复杂代谢网络的协同精细调控，从而实现萜类化合物的高效合成。

Abstract

Terpenoids are the largest family of natural products. They are made from the building block isoprene pyrophosphate (IPP), and their bioproduction using engineered cell factories has received a great deal of attention. To date, the insufficient metabolic supply of IPP remains a great challenge for the efficient synthesis of terpenoids. In this work, we discover that the imbalanced metabolic flux distribution between the central metabolism and the IPP supply hinders IPP accumulation in Bacillus subtilis (B. subtilis). Therefore, we remodel the IPP metabolism using a series of genetically encoded two-input-multi-output (TIMO) circuits that are responsive to pyruvate or/and malonyl-CoA, resulting in an IPP pool that is significantly increased by up to four-fold. As a proof-of-concept validation, we design an IPP metabolism remodeling strategy to improve the production of three valuable terpenoids, including menaquinone-7 (MK-7, 4.1-fold), lycopene (9-fold), and β-carotene (0.9-fold). In particular, the titer of MK-7 in a 50-L bioreactor reached 1549.6 mg∙L−1, representing the highest titer reported so far. Thus, we propose a TIMO genetic circuits-assisted IPP metabolism remodeling framework that can be generally used for the synergistic fine-tuning of complicated metabolic modules to achieve the efficient bioproduction of terpenoids.

关键词

萜类 / 基因回路 / 枯草芽孢杆菌 / 异戊二烯焦磷酸

Keywords

Terpenoids / Genetic circuits / Bacillus subtilis / Isoprene pyrophosphate

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徐显皓, 吕雪芹, 崔世修. 重构异戊二烯焦磷酸代谢以促进萜类化合物的合成. Engineering. 2023, 28(9): 166-178 https://doi.org/10.1016/j.eng.2023.03.019

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