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    【创新进展】国工邓禹团队首次报道了代谢工程大肠杆菌通过己二酸逆降解途径产己二酸

    编辑:文、图:邓禹;审核:刘立明 发布日期:2018-10-17 来源:

    国工邓禹团队首次报道了代谢工程大肠杆菌通过己二酸逆降解途径产己二酸

    己二酸是一种二羧酸,在化学工业,医药和润滑剂制造中有广泛的应用,主要用于生产尼龙6-6纤维和树脂。己二酸目前由石油衍生的原料生产,特别是通过硝酸催化氧化环己酮和环己醇(KA油)的混合物生产。然而,这种化学合成会导致严重的污染和温室气体排放。因此,发现允许可再生和负担得起的己二酸生产的替代方法是很必要的。然而,以前的研究主要集中在直接从不同底物生物中生产己二酸,导致产率和产量低。

    本研究中在大肠杆菌BL21(DE3)中重建了在Thermobifida fusca中鉴定的五步逆己二酸降解途径(RADP)。得到的菌株(Mad136)在摇瓶中产生0.3g/L己二酸,理论产率为11.1%,并且证实了5-羧基-2-戊烯酰-CoA还原酶(Tfu_1647)催化的步骤为RADP的限速步骤。菌株Mad146携带pTrc99A过表达Tfu_1647,摇瓶产量是理论产率的49.5%。通过CRISPR / Cas9进一步消除了主要代谢物竞争碳通量的途径,由于琥珀酰-CoA是己二酸合成的前体,删除了琥珀酸-CoA连接酶基因以促进琥珀酰-CoA的积累。最终的工程菌株Mad123146,其可以在摇瓶中达到理论产率的93.1%,通过补料分批发酵能够产生68.0g/L己二酸。该结果是大肠杆菌中报道的最高己二酸产量。

    本文以“Metabolic engineeringofEscherichia colifor producing adipic acid through the reverse adipate-degradation pathway为题发表于“Metabolic Engineering期刊,国工赵梅为第一作者,邓禹教授和周景文教授为通讯作者。这一研究受到了国家自然科学基金项目(No.31500070, 31600044, 31670095),江苏省自然科学基金(No. BK20150136, BK20150151),中央高校基本科研经费(No. JUSRP51705A, JUSRP51701A)和江苏省杰出教授项目(No. 5926010241150800)支持。

    Metabolic strategies for redirecting carbon flux to adipic acid inEscherichia coli. The reverse adipate-degradation pathway includes five steps inThermobifida fusca: β-ketothiolase (Tfu_0875), 3-hydroxyacyl-CoA dehydrogenase (Tfu_2399), 3-hydroxyadipyl-CoA dehydrogenase (Tfu_0067), 5-Carboxy-2-pentenoyl-

    CoA reductase (Tfu_1647), and adipyl-CoA synthetase (Tfu_2576-7). The ldhA, atoB, and sucD genes were deleted using the CRISPR/Cas9 system. Red font represents genes or enzymes subject to overexpression, and blue font represents those deleted. Solid arrows indicate single steps, and dashed arrows indicate multiple steps. Red arrows represent overexpressed genes or proteins, and blue arrows represent those deleted. ldhA, Llactate dehydrogenase; atoB, acetyl-CoA C-acetyltransferase; sucD, succinyl-CoA synthetase alpha subunit; TCA, tricarboxylic acid cycle; EMP, the glycolysis pathway;AA, adipic acid; AKG, a-ketoglutarate; CIT, citrate; ACO, aconitate; ICI, isocitrate; OAA, oxaloacetate; AcCoA, acetyl CoA; PYR, pyruvate; SucCoA, succinyl-CoA; SUCC, succinic acid; Glc, glucose; Gly, Glycerol; G3P, glycerol 3-phosphate; DHAP, dihydroxyacetone phosphate. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Fed-batch fermentation process associated with strain Mad123146. Substrates consumptions and dissolved oxygen in 5-L bioreactor; (b) Metabolite excretions and cell growth during fed-batch fermentation. The highest titer was achieved in 5-L bioreactor with 1 vvm aeration rate and 400 rpm agitation rate in SOB medium. Error bars represent the s.d. from three independent assays.

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