Using design of experiments to guide genetic optimization of engineered metabolic pathways.

Design of experiments (DoE) is a term used to describe the application of statistical approaches to interrogate the impact of many variables on the performance of a multivariate system. It is commonly used for process optimization in fields such as chemical engineering and material science. Recent advances in the ability to quantitatively control the expression of genes in biological systems open up the possibility to apply DoE for genetic optimization. In this review targeted to genetic and metabolic engineers, we introduce several approaches in DoE at a high level and describe instances wherein these were applied to interrogate or optimize engineered genetic systems. We discuss the challenges of applying DoE and propose strategies to mitigate these challenges. ONE-SENTENCE SUMMARY: This is a review of literature related to applying Design of Experiments for genetic optimization.

A novel pathway for initial biotransformation of dinitroaniline herbicide butralin from a newly isolated bacterium Sphingopyxis sp. strain HMH.

Butralin (N-sec- Butyl-4-tert-butyl-2,6-dinitroaniline) is a highly persistent dinitroaniline herbicide frequently detected in the environment. In this study, butralin-degrading soil bacterium, Sphingopyxis sp. strain HMH was isolated from agricultural soil samples. Based on whole genome sequence analysis of the strain HMH, the gene encoding a nitroreductase NfnB was identified and expressed in Escherichia coli (E. coli), and protein was purified to homogeneity. NfnB is a flavin-nitroreductase, found to be a functional tetramer, composed of subunit molecular mass of 25 kDa. The metabolites from butralin degradation by strain HMH and purified NfnB were identified using ultra performance liquid chromatography high resolution mass spectrometry (UPLC-HRMS), and a novel mechanism of butralin degradation was proposed. NfnB selectively nitro-reduced butralin into N- (sec-Butyl)-4-(tert-butyl)-6-nitrobenzene- 1,2-diamine, followed by formation of 5-(tert-Butyl)-3 -nitrobenzene-1,2-diamine and butanone by N- dealkylation through possible hydroxylation reaction onto the carbon linked amine of the N-(sec-Butyl) moiety. In our study, we could not detect the hydroxylated product 2-(2-Amino-4-tert-butyl-6-nitro- phenylamino)-butan-2-ol) (carbinolamine), instead its Schiff base product (E)-2-(Butan-2-yildeneamino)-5- (tert-butyl)-3-nitroaniline was detected. The release of butanone was further confirmed by derivatization with 2,4- dinitrophenylhydrazine (DNPH) followed by MS analysis. In conclusion, this study explores a novel multi-functional flavin- nitroreductase family enzyme NfnB, catalyzing unique and sequential nitroreduction and N-dealkylation through oxidative hydroxylation of dinitroaniline herbicide butralin.