Metabolomics Revealed an Association of Metabolite Changes and Defective Growth in Methylobacterium extorquens AM1 Overexpressing ecm during Growth on Methanol.

Jinyu Cui,Song Yang,Bo Hu,Jing Yang,Nathan M Good,Martin Sadilek,Qianwen Wang,Ivan A Berg

doi:10.1371/journal.pone.0154043

Abstract

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multi-carbon compounds. The ethylmalonyl-CoA (EMC) pathway is one of the central assimilatory pathways in M. extorquens during growth on C1 and C2 substrates. Previous studies had shown that ethylmalonyl-CoA mutase functioned as a control point during the transition from growth on succinate to growth on ethylamine. In this study we overexpressed ecm, phaA, mcmAB and found that upregulating ecm by expressing it from the strong constitutive mxaF promoter caused a 27% decrease in growth rate on methanol compared to the strain with an empty vector. Targeted metabolomics demonstrated that most of the central intermediates in the ecm over-expressing strain did not change significantly compared to the control strain; However, poly-β-hydroxybutyrate (PHB) was 4.5-fold lower and 3-hydroxybutyryl-CoA was 1.6-fold higher. Moreover, glyoxylate, a toxic and highly regulated essential intermediate, was determined to be 2.6-fold higher when ecm was overexpressed. These results demonstrated that overexpressing ecm can manipulate carbon flux through the EMC pathway and divert it from the carbon and energy storage product PHB, leading to an accumulation of glyoxylate. Furthermore, untargeted metabolomics discovered two unusual metabolites, alanine (Ala)–meso-diaminopimelic acid (mDAP) and Ala–mDAP–Ala, each over 45-fold higher in the ecm over-expressing strain. These two peptides were also found to be highly produced in a dose-dependent manner when glyoxylate was added to the control strain. Overall, this work has explained a direct association of ecm overexpression with glyoxylate accumulation up to a toxic level, which inhibits cell growth on methanol. This research provides useful insight for manipulating the EMC pathway for efficiently producing high-value chemicals in M. extorquens.

Highlights

Methanol is emerging as an important renewable feedstock for the chemical industry with a global production of 53 million tons in 2011 and an expected annual production increase of 10–20% [1]
The specific activity for ethylmalonyl-CoA mutase (Ecm) in cell-free extracts was 4-fold higher in the NG115 (44±4 nmol.mg-1.min-1) strain compared to the control strain (11±1 nmol.mg-1.min-1)
Glyoxylate, an integral intermediate connecting the EMC pathway and the serine cycle, was found to be 2.6-fold higher in the NG115 strain compared to the NG107 strain

Summary

Introduction

Methanol is emerging as an important renewable feedstock for the chemical industry with a global production of 53 million tons in 2011 and an expected annual production increase of 10–20% [1]. Biological conversion of methanol by methylotrophic bacteria provides a potentially important route for the production of industrial chemicals. M. extorquens, a facultative methylotrophic α-proteobacterium capable of using single carbon compounds and multiple carbon compounds as carbon and energy sources, has served as the best-characterized model organism for studying one-carbon metabolism [2,3]. Success converting methanol into platform chemicals has been limited due to the regulatory complexity and metabolic redundancy of methylotrophic metabolism. In M. extorquens assimilation during methylotrophic metabolism involves three interlocked cycles: the serine cycle, the ethylmalonyl-CoA (EMC) pathway and the PHB cycle [7,8]. The main function of the EMC pathway is to regenerate glyoxylate for reincorporation into the serine cycle during the C1 assimilation [9,10,11,12,13]

Methods

Results

Conclusion