Department of Chemical and Biomolecular Engineering, Pusan National University, Busan 609-735, Republic of Korea.
J Biotechnol. 2012 Feb 20;157(4):633-40. doi: 10.1016/j.jbiotec.2011.06.008. Epub 2011 Jun 23.
Malonyl-CoA is an intermediary compound that is produced during fatty acid metabolism. Our study aimed to produce the commercially important platform chemical 3-hydroxypropionic acid (3-HP) from its immediate precursor malonyl-CoA by recombinant Escherichia coli strains heterologously expressing the mcr gene of Chloroflexus aurantiacus DSM 635, encoding an NADPH-dependent malonyl-CoA reductase (MCR). The recombinant E. coli overexpressing mcr under the T5 promoter showed MCR activity of 0.015 U mg⁻¹ protein in crude cell extract and produced 0.71 mmol/L of 3-HP in 24h in shake flask cultivation under aerobic conditions with glucose as the sole source of carbon. When acetyl-CoA carboxylase and biotinilase, encoded by the genes accADBCb (ACC) of E. coli K-12 were overexpressed along with MCR, the final 3-HP titer improved by 2-fold, which is 1.6 mM. Additional expression of the gene pntAB, encoding nicotinamide nucleotide transhydrogenase that converts NADH to NADPH, increased 3-HP production to 2.14 mM. The strain was further developed by deleting the sucAB gene, encoding α-ketoglutarate dehydrogenase complex in tricarboxylic acid (TCA) cycle, or blocking lactate and acetate production pathways, and evaluated for the production of 3-HP. We report on the feasibility of producing 3-HP from glucose through the malonyl-CoA pathway.
丙二酰辅酶 A 是脂肪酸代谢过程中产生的一种中间化合物。本研究旨在通过异源表达嗜热栖热菌（Chloroflexus aurantiacus DSM 635）的 mcr 基因，利用重组大肠杆菌生产具有商业重要性的平台化学品 3-羟基丙酸（3-HP），该基因编码一种 NADPH 依赖的丙二酰辅酶 A 还原酶（MCR）。在 T5 启动子的控制下过量表达 mcr 的重组大肠杆菌在粗细胞提取物中的 MCR 活性为 0.015 U mg ⁻¹ 蛋白，在有氧条件下，以葡萄糖为唯一碳源的摇瓶培养 24 小时可产生 0.71mmol/L 的 3-HP。当与 MCR 一起过量表达乙酰辅酶 A 羧化酶和生物素酶，由大肠杆菌 K-12 的基因 accADBCb（ACC）编码时，最终的 3-HP 浓度提高了 2 倍，达到 1.6mmol/L。进一步表达编码烟酰胺核苷酸转氢酶的基因 pntAB，将 NADH 转化为 NADPH，将 3-HP 的产量提高到 2.14mmol/L。通过删除三羧酸（TCA）循环中编码 α-酮戊二酸脱氢酶复合物的 sucAB 基因或阻断乳酸和乙酸的产生途径，对该菌株进行了进一步的开发，并对 3-HP 的生产能力进行了评估。我们报告了通过丙二酰辅酶 A 途径从葡萄糖生产 3-HP 的可行性。
