利用合成调控RNA技术提高大肠杆菌异源合成红霉素前体6-脱氧红霉内酯B产量

Enhancing erythromycin precursor 6-dEB production by using synthetic small regulatory RNAs in Escherichia coli

摘要:

红霉素为代表的聚酮类化合物已经成功的在大肠杆菌中实现了异源合成，但其产量仍然较低 (仅 ~10 mg/L)。本研究基于大肠杆菌全基因组代谢模型iAF1260，利用通量平衡分析预测了红霉素母核6-脱氧红霉内酯 (6-Deoxyerythronolide B, 6-dEB) 生物合成的关键靶点，通过合成调控RNA技术 (Synthetic small regulatory RNAs, sRNAs) 对预测的靶点进行验证。结果表明，以弱化lsrC (编码LsrABC转运蛋白) 和ackA (编码乙酸激酶蛋白) 为代表的关键靶点改造可以显著提高6-dEB异源合成，提高幅度可达48.7%。通过弱化靶点的组合，进一步改善了6-dEB的异源合成，产量最终可达22.8 mg/L，比出发菌株产量提高59.9%。本研究发现和确认了6个有效的调控靶点，最终成功地改善了6-dEB在大肠杆菌中的异源合成。研究表明，通量分布比较分析结合sRNAs技术是一种有效的方法提高6-dEB异源合成，也为改善其他代谢产物的异源合成提供了可供借鉴的研究思路。

关键词:大肠杆菌，6-脱氧红霉内酯，异源合成，全基因组代谢模型，sRNA技术

Abstract:

Although heterologous biosynthesis of polyketide erythromycin has been successfully achieved in Escherichia coli, the titer remains at a very low level (~10 mg/L). In this study, based on genome-scale metabolic model of E. coli, in silico method flux distribution comparison analysis was used to discover novel potential targets for heterologous 6-dEB biosynthesis. Synthetic small regulatory RNAs (sRNAs) was used to experimentally test 12 down-regulated targets. The results showed that repression of each of these target genes e.g. lsrC and ackA led to significantly improve heterologous 6-dEB biosynthesis. Using co-repression of lsrC and ackA, 6-dEB titer was improved by 59.9 % in shake-flask with a maximum yield of 22.8 mg/L. This study indicates that combined flux distribution comparison analysis and synthetic small regulatory RNAs is an effective strategy to improve 6-dEB production in E. coli.

Key words:Escherichia coli, 6-deoxyerythronolide B, heterologous biosynthesis, genome-scale metabolic model, synthetic small regulatory RNAs