Immanuel Selva Rupa Christinal, Banerjee Deepanwita, Rajankar Mayooreshwar P, Raghunathan Anu
Chemical Engineering Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India.
Chemical Engineering Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India.
Biosystems. 2018 Sep;171:10-19. doi: 10.1016/j.biosystems.2018.06.002. Epub 2018 Jul 3.
Strategies towards optimal violacein biosynthesis, a potential drug molecule, need systems level coordination of enzymatic activities of individual genes in a multigene operon vioABCDE. Constraints-based flux balance analysis of an extended iAF1260 model (iAF1260vio) with a reconstructed violacein module predicted growth and violacein yields in Escherichia coli accurately. Shadow price (SP) analysis identified tryptophan metabolism and NADPH as limiting. Increased tryptophan levels in Δpgi & ΔpheA were validated using in silico gene deletion analysis. Phenotypic phase plane (PhPP) analysis highlighted sensitivity between tryptophan and NADPH for violacein synthesis at molar growth yields. A synthetic VioABCDE operon (SYNO) sequence was designed to maximize Codon Adaptive Index (CAI: 0.9) and tune translation initiation rates (TIR: 2-50 fold higher) in E. coli. All pSYN E. coli transformants produced higher violacein, with a maximum six-fold increase in yields. The rational design E. coli: ΔpheA SYN: gave the highest violacein titers (33.8 mg/l). Such integrated approaches targeting multiple molecular hierarchies in the cell can be extended further to increase violacein yields.
作为一种潜在药物分子的紫菌素生物合成的优化策略，需要多基因操纵子vioABCDE中各个基因的酶活性进行系统水平的协调。基于约束的通量平衡分析扩展的iAF1260模型（iAF1260vio），其重建的紫菌素模块能够准确预测大肠杆菌中的生长和紫菌素产量。影子价格（SP）分析确定色氨酸代谢和NADPH是限制因素。使用计算机基因缺失分析验证了Δpgi和ΔpheA中色氨酸水平的增加。表型相平面（PhPP）分析突出了在摩尔生长产量下色氨酸和NADPH对紫菌素合成的敏感性。设计了一个合成的VioABCDE操纵子（SYNO）序列，以最大化大肠杆菌中的密码子适应指数（CAI：0.9）并调节翻译起始速率（TIR：高2至50倍）。所有pSYN大肠杆菌转化体产生的紫菌素更高，产量最高增加了六倍。合理设计的大肠杆菌：ΔpheA SYN：产生了最高的紫菌素滴度（33.8mg/l）。这种针对细胞中多个分子层次的综合方法可以进一步扩展以提高紫菌素产量。
