Shiqi Luo,Wei Song,Guipeng Hu,Jian Wen,Xiaomin Li,Cong Gao,Jia Liu,Wanqing Wei,Liming Liu
Graphical Here, we have constructed a dual-enzyme ( Ec TnaA, Ma FMO) cascade pathway for the biosynthesis of indigo from L-tryptophan and enhanced the enzymatic activity of Ma FMO through mechanism-guided rational design. Subsequently, by optimizing promoters and increasing copy numbers to regulate the expression levels of Ec TnaA and Ma FMO D197E , under optimal conditions, E. coli IND-13 achieved the highest yield to date using glucose as the substrate. Indigo is widely used in dyes, medicines and semiconductors materials due to its excellent dyeing efficiency, antibacterial, antiviral, anticancer, anti-corrosion, and thermostability properties. Here, a biosynthetic pathway for indigo was designed, integrating two enzymes ( Ec TnaA, Ma FMO) into a higher L-tryptophan-producing the strain Escherichia coli TRP. However, the lower catalytic activity of Ma FMO was a bottleneck for increasing indigo titers. To overcome this limitation, the enzyme activity of Ma FMO was enhanced through mechanism-guided rational design. The optimal mutant obtained in this study, Ma FMO D197E , whose k cat / K m was 1.34 times that of the wild type, and its specific activity was 2.36 times that of the wild type. In addition, the expression levels of Ec TnaA and Ma FMO D197E were regulated by optimizing the promoters and increasing the copy number to generate the strain E. coli IND-13. Finally, in the optimal fermentation conditions (220 rpm, 0.05 % Tween-80), the strain E. coli IND-13 achieved the indigo titer of 568.52 mg/L in a 5-L bioreactor, with the yield and productivity of 2.62 mg/g and 12.96 mg/L/h (the highest to date), respectively. The results presented here can lay a foundation for further construction of cell factories for indigo and its derivatives with industrial application potential.
The structure–property relationship and adsorption mechanism of a novel collector for cassiterite, 2-carboxyethylphenylphosphinic acid (CEPPA), were comprehensively investigated and compared with those of styrene phosphonic acid (SPA) by density functional theory (DFT) calculations and flotation tests. The net Mulliken charge and frontier molecular orbital (FMO) theory study results indicated that CEPPA exhibited stronger electron-donating ability and higher chemical reactivity than SPA. The flotation test results demonstrated that CEPPA had stronger collecting ability for cassiterite in a much wider effective pH range than SPA. The adsorption simulation results confirmed that the CEPPA molecule was adsorbed on cassiterite (110) surface by two hydrogen bonds. CEPPA monoanion and dianion were adsorbed on cassiterite (110) surface by a series of O-Sn bonds, and the interactions between the O atoms in CEPPA phosphinic group with the Sn atoms on cassiterite (110) surface were stronger than those between the O atoms in CEPPA carboxyl group with the Sn atoms on cassiterite (110) surface. The formation of the O-Sn bonds was mainly contributed from the 2p orbital of O atoms and 5s and 5p orbitals of Sn atoms. The various adsorption configurations of CEPPA on cassiterite accounted for the better collecting ability of CEPPA for cassiterite than that of SPA. The quantitative analysis results were in good agreement with the qualitative experimental test results in a previous paper.
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