Metabolic Detoxification of 2-Oxobutyrate by Remodeling Escherichia coli Acetate Bypass.

Yu Fang,Xiaoyuan Wang,Jianli Wang,Jie Song,Hailing Zhang,Shuyan Zhang,Lianghong Yin,Zhen Wang,Xiaoqing Hu

doi:10.3390/metabo11010030

Abstract

2-Oxobutyrate (2-OBA), as a toxic metabolic intermediate, generally arrests the cell growth of most microorganisms and blocks the biosynthesis of target metabolites. In this study, we demonstrated that using the acetate bypass to replace the pyruvate dehydrogenase complex (PDHc) in Escherichia coli could recharge the intracellular acetyl-CoA pool to alleviate the metabolic toxicity of 2-OBA. Furthermore, based on the crystal structure of pyruvate oxidase (PoxB), two candidate residues in the substrate-binding pocket of PoxB were predicted by computational simulation. Site-directed saturation mutagenesis was performed to attenuate 2-OBA-binding affinity, and one of the variants, PoxBF112W, exhibited a 20-fold activity ratio of pyruvate/2-OBA in substrate selectivity. PoxBF112W was employed to remodel the acetate bypass in E. coli, resulting in l-threonine (a precursor of 2-OBA) biosynthesis with minimal inhibition from 2-OBA. After metabolic detoxification of 2-OBA, the supplies of intracellular acetyl-CoA and NADPH (nicotinamide adenine dinucleotide phosphate) used for l-threonine biosynthesis were restored. Therefore, 2-OBA is the substitute for pyruvate to engage in enzymatic reactions and disturbs pyruvate metabolism. Our study makes a straightforward explanation of the 2-OBA toxicity mechanism and gives an effective approach for its metabolic detoxification.

Highlights

Microbial cells in the processes of growth and production frequently suffer from intracellular toxic metabolites and adverse environmental factors, such as glyoxal [1], methylglyoxal [2], coniferyl aldehyde [3], furfural [4,5] and so on. 2-Oxobutyrate (2OBA), known as 2-ketobutyric acid or α-ketobutyrate, belongs to the class of alphaketo acids known as short-chain keto acids next to pyruvate [6]
The sufficient supply of the precursor intermediate oxaloacetate for L-threonineoverproducing strains is vital to L-threonine maximization, whereas the inadequacy of reduced cofactors (NADPH and ATP) and α-ketoglutarate from the TCA cycle is the limitation of L-threonine biosynthesis in engineered E. coli strains [35,36]
The intracellular NADH is converted into NADPH for L-threonine synthesis through pyridine nucleotide transhydrogenase

Summary

Introduction

Microbial cells in the processes of growth and production frequently suffer from intracellular toxic metabolites and adverse environmental factors, such as glyoxal [1], methylglyoxal [2], coniferyl aldehyde [3], furfural [4,5] and so on. 2-Oxobutyrate (2OBA), known as 2-ketobutyric acid or α-ketobutyrate, belongs to the class of alphaketo acids known as short-chain keto acids next to pyruvate [6]. In the biosynthesis process of 2-OBA derivatives, such as L-isoleucine [9,10], L-2-aminobutyric acid [11], 1propanol [12] and 1-butanol [13,14], the expression level of threonine deaminase catalyzing the breakdown of L-threonine to generate 2-OBA tends to be limited to avoid the toxic accumulation of 2-OBA, which imposes a restriction on the improvement of its downstream flux and target product productivity This type of biological toxicity retards the growth of the fermentation strains, and blocks the biosynthesis of target products. Most 2-OBA products on the market are chemically synthesized from the condensation products of diethyloxalate and ethylpropionate or the condensation products of acetaldehyde [15]

Methods

Results

Conclusion