Metabolomics-Driven Identification of the Rate-Limiting Steps in 1-Propanol Production

Frontiers in Microbiology

Naoki Kawase¹  Toshiyuki Ohtake¹  Katsuaki Nitta¹  Claire R. Shen²  James C. Liao³  Sammy Pontrelli⁵  Walter A. Laviña⁶  Sastia P. Putri⁷  Eiichiro Fukusaki⁷ 

[1] Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan;Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan;Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA, United States;Industrial Biotechnology Initiative Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Japan;Institute of Molecular Systems Biology, ETH Zürich, Zurich, Switzerland;Microbiology Division, Institute of Biological Sciences, University of the Philippines Los Baños, Los Baños, Philippines;Osaka University Shimadzu Omics Innovation Research Laboratories, Osaka University, Suita, Japan;

关键词: metabolomics;    rate-limiting steps;    Escherichia coli;    1-propanol;    2-ketobutyrate;    YqhD;   

DOI  :  10.3389/fmicb.2022.871624

来源: DOAJ

【 摘 要 】

The concerted effort for bioproduction of higher alcohols and other commodity chemicals has yielded a consortium of metabolic engineering techniques to identify targets to enhance performance of engineered microbial strains. Here, we demonstrate the use of metabolomics as a tool to systematically identify targets for improved production phenotypes in Escherichia coli. Gas chromatography/mass spectrometry (GC/MS) and ion-pair LC-MS/MS were performed to investigate metabolic perturbations in various 1-propanol producing strains. Two initial strains were compared that differ in the expression of the citramalate and threonine pathways, which hold a synergistic relationship to maximize production yields. While this results in increased productivity, no change in titer was observed when the threonine pathway was overexpressed beyond native levels. Metabolomics revealed accumulation of upstream byproducts, norvaline and 2-aminobutyrate, both of which are derived from 2-ketobutyrate (2KB). Eliminating the competing pathway by gene knockouts or improving flux through overexpression of glycolysis gene effectively increased the intracellular 2KB pool. However, the increase in 2KB intracellular concentration yielded decreased production titers, indicating toxicity caused by 2KB and an insufficient turnover rate of 2KB to 1-propanol. Optimization of alcohol dehydrogenase YqhD activity using an ribosome binding site (RBS) library improved 1-propanol titer (g/L) and yield (g/g of glucose) by 38 and 29% in 72 h compared to the base strain, respectively. This study demonstrates the use of metabolomics as a powerful tool to aid systematic strain improvement for metabolically engineered organisms.

【 授权许可】

Unknown