Underground isoleucine biosynthesis pathways in E. coli.

Charles Ar Cotton,Alexander Erban,Nicole Paczia,Tobias J Erb,Marian Dempfle,Steffen N Lindner,Luca Schulz,Joachim Kopka,Hai He,Iria Bernhardsgrütter,Alberto De Maria,Stepan Toman,Simon Burgener,Arren Bar-Even,Beau Dronsella

doi:10.7554/elife.54207

Abstract

The promiscuous activities of enzymes provide fertile ground for the evolution of new metabolic pathways. Here, we systematically explore the ability of E. coli to harness underground metabolism to compensate for the deletion of an essential biosynthetic pathway. By deleting all threonine deaminases, we generated a strain in which isoleucine biosynthesis was interrupted at the level of 2-ketobutyrate. Incubation of this strain under aerobic conditions resulted in the emergence of a novel 2-ketobutyrate biosynthesis pathway based upon the promiscuous cleavage of O-succinyl-L-homoserine by cystathionine γ-synthase (MetB). Under anaerobic conditions, pyruvate formate-lyase enabled 2-ketobutyrate biosynthesis from propionyl-CoA and formate. Surprisingly, we found this anaerobic route to provide a substantial fraction of isoleucine in a wild-type strain when propionate is available in the medium. This study demonstrates the selective advantage underground metabolism offers, providing metabolic redundancy and flexibility which allow for the best use of environmental carbon sources.

Highlights

The patchwork model suggests that novel metabolic pathways emerge from the promiscuous activities of enzymes participating in diverse metabolic processes (Lazcano and Miller, 1999; Jensen, 1976; Khersonsky and Tawfik, 2010; Noda-Garcia et al, 2018)
Biosynthesis of isoleucine in E. coli starts with the deamination of threonine to give 2 KB, which is condensed with pyruvate to produce 2-aceto-2-hydroxybutanoate
Strain D5 displayed similar behavior to the DilvA DtdcB strain, that is, while growth without isoleucine was not observed in the first 70 hr, after 70 hr, the replicates started growing. This strongly suggests the emergence of a latent threonine-independent isoleucine biosynthesis pathway

Summary

Introduction

The patchwork model suggests that novel metabolic pathways emerge from the promiscuous activities of enzymes participating in diverse metabolic processes (Lazcano and Miller, 1999; Jensen, 1976; Khersonsky and Tawfik, 2010; Noda-Garcia et al, 2018). Underground metabolism – the network of metabolic conversions which are catalyzed as side reactions of enzymes that have evolved to support other activities (D’Ari and Casadesus, 1998) – provides fertile ground for the evolution of new pathways. A computational analysis suggested that about half of all underground reactions generate metabolites that already exist in the endogenous metabolic network, enabling the emergence of metabolic bypasses for the production of key cellular building blocks (Notebaart et al, 2014). The biosynthesis of isoleucine provides multiple examples of enzyme promiscuity and structural similarity to other pathways, suggesting underground metabolism as a likely origin (Jensen, 1976).

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Conclusion