Yang Yaping, Lin Yuheng, Li Lingyun, Linhardt Robert J, Yan Yajun
College of Engineering, University of Georgia, Athens, GA 30602, USA.
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Metab Eng. 2015 May;29:217-226. doi: 10.1016/j.ymben.2015.03.018. Epub 2015 Apr 9.
Malonyl-CoA is the building block for fatty acid biosynthesis and also a precursor to various pharmaceutically and industrially valuable molecules, such as polyketides and biopolymers. However, intracellular malonyl-CoA is usually maintained at low levels, which poses great challenges to efficient microbial production of malonyl-CoA derived molecules. Inactivation of the malonyl-CoA consumption pathway to increase its intracellular availability is not applicable, since it is usually lethal to microorganisms. In this work, we employ synthetic antisense RNAs (asRNAs) to conditionally down-regulate fatty acid biosynthesis and achieve malonyl-CoA enrichment in Escherichia coli. The optimized asRNA constructs with a loop-stem structure exhibit high interference efficiency up to 80%, leading to a 4.5-fold increase in intracellular malonyl-CoA concentration when fabD gene expression is inhibited. Strikingly, this strategy allows the improved production of natural products 4-hydroxycoumarin, resveratrol, and naringenin by 2.53-, 1.70-, and 1.53-fold in E. coli, respectively. In addition, down-regulation of other fab genes including fabH, fabB, and fabF also leads to remarkable increases in 4-hydroxycoumarin production. This study demonstrates a novel strategy to enhance intracellular malonyl-CoA and indicates the effectiveness of asRNA as a powerful tool for use in metabolic engineering.
丙二酰辅酶A是脂肪酸生物合成的原料，也是多种具有药学和工业价值的分子的前体，如聚酮化合物和生物聚合物。然而，细胞内丙二酰辅酶A通常维持在低水平，这对高效微生物生产丙二酰辅酶A衍生分子构成了巨大挑战。使丙二酰辅酶A消耗途径失活以提高其细胞内可用性并不适用，因为这通常对微生物是致命的。在这项工作中，我们利用合成反义RNA（asRNA）有条件地下调脂肪酸生物合成，并在大肠杆菌中实现丙二酰辅酶A的富集。具有环茎结构的优化asRNA构建体表现出高达80%的高干扰效率，当fabD基因表达受到抑制时，导致细胞内丙二酰辅酶A浓度增加4.5倍。引人注目的是，该策略使大肠杆菌中天然产物4-羟基香豆素、白藜芦醇和柚皮素的产量分别提高了2.53倍、1.70倍和1.53倍。此外，下调包括fabH、fabB和fabF在内的其他fab基因也导致4-羟基香豆素产量显著增加。这项研究展示了一种提高细胞内丙二酰辅酶A的新策略，并表明asRNA作为代谢工程中一种强大工具的有效性。
