调节 TCA 循环促进大肠杆菌利用乙酸合成 (R)-3-羟基丁酸的研究

Adjusting the TCA Cycle for Promoting the Production of (R)-3-Hydroxybutyric Acid from Acetate inEscherichia coli

摘要

乙酸是常见的非糖类碳源之一，具有成本低、来源广等优点。TCA(Tricarboxylic Acid) 循环是工程大肠杆菌以乙酸为碳源合成 (R)-3-羟基丁酸（R-3HB）的主要竞争途径，合理调控 TCA 循环能够提高R-3HB 的产量和得率。本研究构建了一种能够通过相对荧光强度反映R-3HB 浓度的高效筛选方法，并使用携带随机不同 crRNA 的 CRISPRi 系统，对 TCA 循环进行不同强度的抑制，筛选出 5 株R-3HB 的潜在高产菌株，在摇瓶水平进行发酵验证。其中，SX11 菌株在摇瓶条件下能够生产 2.12 g/L 的R-3HB，相较于对照组提高 31.7%；且该菌株在 64 h 时的得率为 0.41 g/g，相较于对照组提高 115.8%。基于 SX11 菌株中的 crRNA 序列构建了对应的 CRISPRi 质粒进行回补验证，大肠杆菌 BW25113(pTrcAB7, pBADP2, pSC101-gltA) 的R-3HB 产量和得率分别为 2.04 g/L和 0.37 g/g，相对原始菌株 (pTrcAB7, pBADP2) 分别提高 13.3% 和 94.7%。该结果证明了含有gltAcrRNA2^*的 CRISPRi 系统能够有效调控 TCA 循环，促进菌株利用乙酸合成R-3HB。本文为提高以乙酰-CoA 为前体的产物产量提供了新思路。

Abstract

Acetate is a common non-carbohydrate carbon source with the advantages of low cost and abundant supply. When engineeredEscherichia coli is used to synthesize (R)-3-hydroxybutyric acid (R-3HB) with acetate as the sole carbon source, the TCA cycle acts as the major competitive metabolic pathway. Appropriately regulating the TCA cycle is therefore an effective strategy to improve the titer and yield ofR-3HB. In this study, an efficient screening method was established, which could reflect the relative abundance ofR-3HB via fluorescence intensity. A CRISPRi library containing random crRNA sequences was further applied to inhibit the TCA cycle at varying intensities. Combining the efficient screening method with the CRISPRi system, five candidate strains with potential highR-3HB production capacity were obtained and subsequently verified by shake-flask fermentation. Among them, strain SX11 produced 2.12 g/LR-3HB, representing a 31.7% increase compared with the control strain. At 64 h of fermentation, the yield ofR-3HB in SX11 reached 0.41 g/g, which was 115.8% higher than that of the control group. Based on the crRNA sequence identified from strain SX11, a corresponding CRISPRi plasmid was constructed for validation. The recombinant strain BW25113(pTrcAB7, pBADP2, pSC101-gltA) achieved anR-3HB titer of 2.04 g/L and a yield of 0.37 g/g, which were 13.3% and 94.7% higher than those of the control, respectively. These results confirm that the CRISPRi system carryinggltAcrRNA2^* can effectively modulate the TCA cycle, thereby enhancingR-3HB titer and yield from acetate. This work provides a novel strategy for improving the production of acetyl-CoA-derived biochemicals.

关键词

大肠杆菌 / 乙酸 / (R)-3-羟基丁酸 / 高效筛选 / CRISPRi

Key words

Escherichia coli / acetate / (R)-3-hydroxybutyric acid / efficient screening / CRISPRi

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基金资助

国家重点研发计划(2021YFC2103500)