Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran.
Mol Biotechnol. 2023 Sep;65(9):1508-1517. doi: 10.1007/s12033-022-00635-5. Epub 2023 Jan 19.
Cellular pool of malonyl-CoA in Escherichia coli is small, which impedes its utility for overproduction of natural products such as phenylpropanoids, polyketides, and flavonoids. In this study, we report the use of a new metabolic pathway to increase the malonyl-CoA concentration as a limiting metabolite in E. coli. For this purpose, the malonate/sodium symporter from Malonomonas rubra, and malonyl-CoA synthetase (MCS) from Bradyrhizobium japonicum were co-expressed in E. coli. This new pathway allows the cell to actively import malonate from the culture medium and to convert malonate and CoA to malonyl-CoA via an ATP-dependent ligation reaction. HPLC analysis confirmed elevated levels of malonyl-CoA and (2S)-naringenin as a malonyl-CoA-dependent metabolite, in E. coli. A 6.8-fold and more than 3.5-fold increase in (2S)-naringenin production were achieved in the engineered host in comparison with non-engineered E. coli and previously reported passive transport MatBMatC pathway, respectively. This observation suggests that using active transporters of malonate not only improves malonyl-CoA-dependent production but also makes it possible to harness low concentrations of malonate in culture media.
大肠杆菌中丙二酰辅酶 A 的细胞池很小，这阻碍了其用于天然产物（如苯丙素类、聚酮类和类黄酮）的过量生产。在本研究中，我们报告了一种利用新代谢途径来增加丙二酰辅酶 A 浓度作为大肠杆菌中限制代谢物的方法。为此，我们从红平红球菌中表达了丙二酸盐/钠协同转运蛋白，从日本根瘤菌中表达了丙二酰辅酶 A 合成酶（MCS）。该新途径使细胞能够从培养基中主动摄取丙二酸盐，并通过依赖 ATP 的连接反应将丙二酸盐和辅酶 A 转化为丙二酰辅酶 A。HPLC 分析证实大肠杆菌中丙二酰辅酶 A 和（2S）-柚皮素的水平升高，（2S）-柚皮素是丙二酰辅酶 A 的依赖代谢物。与非工程大肠杆菌和之前报道的被动转运 MatBMatC 途径相比，在工程宿主中（2S）-柚皮素的产量分别提高了 6.8 倍和 3.5 倍以上。这一观察结果表明，使用丙二酸盐的主动转运蛋白不仅可以提高丙二酰辅酶 A 依赖性生产，还可以利用培养基中低浓度的丙二酸盐。
