Kim Dong Hwan, Hwang Hyun Gyu, Jung Gyoo Yeol
Department of Chemical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, 37673, Gyeongbuk, Korea.
Institute of Environmental and Energy Technology, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, 37673, Gyeongbuk, Korea.
Biotechnol Biofuels Bioprod. 2022 Sep 2;15(1):90. doi: 10.1186/s13068-022-02188-w.
Microbial production of naringenin has received much attention owing to its pharmaceutical applicability and potential as a key molecular scaffold for various flavonoids. In the microbial fermentation, a cheap and abundant feedstock is required to achieve an economically feasible bioprocess. From this perspective, utilizing acetate for naringenin production could be an effective strategy, with the advantages of both low-cost and abundant feedstock. For the efficient production of naringenin using acetate, identification of the appropriate regulatory node of carbon flux in the biosynthesis of naringenin from acetate would be important. While acetyl-CoA is a key precursor for naringenin production, carbon flux between the TCA cycle and anaplerosis is effectively regulated at the isocitrate node through glyoxylate shunt in acetate metabolism. Accordingly, appropriate rerouting of TCA cycle intermediates from anaplerosis into naringenin biosynthesis via acetyl-CoA replenishment would be required.
This study identified the isocitrate and oxaloacetate (OAA) nodes as key regulatory nodes for the naringenin production using acetate. Precise rerouting at the OAA node for enhanced acetyl-CoA was conducted, avoiding extensive loss of OAA by fine-tuning the expression of pckA (encoding phosphoenolpyruvate carboxykinase) with flux redistribution between naringenin biosynthesis and cell growth at the isocitrate node. Consequently, the flux-optimized strain exhibited a significant increase in naringenin production, a 27.2-fold increase (with a 38.3-fold increase of naringenin yield on acetate) over that by the unoptimized strain, producing 97.02 mg/L naringenin with 21.02 mg naringenin/g acetate, which is a competitive result against those in previous studies on conventional substrates, such as glucose.
Collectively, we demonstrated efficient flux rerouting for maximum naringenin production from acetate in E. coli. This study was the first attempt of naringenin production from acetate and suggested the potential of biosynthesis of various flavonoids derived from naringenin using acetate.
由于柚皮素在制药领域的适用性及其作为各种黄酮类化合物关键分子骨架的潜力，其微生物生产受到了广泛关注。在微生物发酵中，需要一种廉价且丰富的原料来实现经济可行的生物过程。从这个角度来看，利用乙酸盐生产柚皮素可能是一种有效的策略，具有低成本和原料丰富的优势。为了利用乙酸盐高效生产柚皮素，确定乙酸盐生物合成柚皮素过程中碳通量的合适调控节点至关重要。虽然乙酰辅酶A是柚皮素生产的关键前体，但在乙酸盐代谢中，通过乙醛酸循环在异柠檬酸节点有效地调节了三羧酸循环（TCA循环）和回补反应之间的碳通量。因此，需要将TCA循环中间体从回补反应通过乙酰辅酶A补充适当重新导向柚皮素生物合成。
本研究确定异柠檬酸和草酰乙酸（OAA）节点是利用乙酸盐生产柚皮素的关键调控节点。通过微调pckA（编码磷酸烯醇丙酮酸羧激酶）的表达，在OAA节点进行精确的重新导向以增强乙酰辅酶A，避免了OAA的大量损失，同时在异柠檬酸节点实现了柚皮素生物合成和细胞生长之间的通量重新分配。结果，通量优化菌株的柚皮素产量显著增加，比未优化菌株提高了27.2倍（乙酸盐上柚皮素产量提高了38.3倍），产生了97.02 mg/L的柚皮素，每克乙酸盐产生21.02 mg柚皮素，这一结果与之前关于传统底物（如葡萄糖）的研究相比具有竞争力。
总体而言，我们证明了在大肠杆菌中通过高效的通量重新导向从乙酸盐中最大限度地生产柚皮素。本研究是首次尝试从乙酸盐生产柚皮素，并表明了利用乙酸盐生物合成源自柚皮素的各种黄酮类化合物的潜力。
