Metabolomics Revealed an Association of Metabolite Changes and Defective Growth in Methylobacterium extorquens AM1 Overexpressing ecm during Growth on Methanol.
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.
School of Life Science, Qingdao Agricultural University, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao, Shandong Province, China.
Jinyu
J
Cui
Department of Microbiology, University of Washington, Seattle, Washington, United States of America.
Nathan M
NM
Good
Kemin Industries, KI Research & Development, Des Moines, Iowa, United States of America.
Bo
B
Hu
School of Life Science, Qingdao Agricultural University, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao, Shandong Province, China.
Jing
J
Yang
Central Laboratory, Qingdao Agricultural University, Qingdao, Shandong Province, China.
Qianwen
Q
Wang
Department of Chemistry, University of Washington, Seattle, Washington, United States of America.
Martin
M
Sadilek
School of Life Science, Qingdao Agricultural University, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao, Shandong Province, China.|Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, China.
Song
S
Yang
D000214:Acyl Coenzyme A / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006038:Glyoxylates / Q000378:metabolism; D055442:Metabolome; D055432:Metabolomics; D000432:Methanol / Q000378:metabolism*; D020601:Methylobacterium extorquens / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*
D000214:Acyl Coenzyme A; D006038:Glyoxylates; C102829:ethylmalonyl-coenzyme A; C031150:glyoxylic acid; D000432:Methanol