101. Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route
- Author
-
Min Zhang, Li-Ping Zhu, Yi-Ming Yang, Song Yang, Yuan-Ming Zhou, Guang-Yuan Wang, Jing Yang, Bo Hu, and Wen-Jing Chen
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae ,lcsh:QR1-502 ,Bioengineering ,3-Hydroxypropionic acid ,Reductase ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,lcsh:Microbiology ,Mass Spectrometry ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Methylobacterium extorquens ,Coenzyme A Ligases ,medicine ,Metabolomics ,Reassimilation ,Lactic Acid ,Promoter Regions, Genetic ,Escherichia coli ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,Dicarboxylic Acid Transporters ,Carbon Isotopes ,biology ,Strain (chemistry) ,Research ,Methanol ,biology.organism_classification ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,Reductive route ,Batch Cell Culture Techniques ,Isotope Labeling ,13C-labeling ,Acyl Coenzyme A ,Genetic Engineering ,Biotechnology - Abstract
Background 3-Hydroxypropionic acid (3-HP) is an important platform chemical, serving as a precursor for a wide range of industrial applications such as the production of acrylic acid and 1,3-propanediol. Although Escherichia coli or Saccharomyces cerevisiae are the primary industrial microbes for the production of 3-HP, alternative engineered hosts have the potential to generate 3-HP from other carbon feedstocks. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating 3-HP from one-carbon feedstock methanol. Results Here we constructed a malonyl-CoA pathway by heterologously overexpressing the mcr gene to convert methanol into 3-HP in M. extorquens AM1. The engineered strains demonstrated 3-HP production with initial titer of 6.8 mg/l in shake flask cultivation, which was further improved to 69.8 mg/l by increasing the strength of promoter and mcr gene copy number. In vivo metabolic analysis showed a significant decrease of the acetyl-CoA pool size in the strain with the highest 3-HP titer, suggesting the supply of acetyl-CoA is a potential bottleneck for further improvement. Notably, 3-HP was rapidly degraded after the transition from exponential phase to stationary phase. Metabolomics analysis showed the accumulation of intracellular 3-hydroxypropionyl-CoA at stationary phase with the addition of 3-HP into the cultured medium, indicating 3-HP was first converted to its CoA derivatives. In vitro enzymatic assay and β-alanine pathway dependent 13C-labeling further demonstrated that a reductive route sequentially converted 3-HP-CoA to acrylyl-CoA and propionyl-CoA, with the latter being reassimilated into the ethylmalonyl-CoA pathway. The deletion of the gene META1_4251 encoding a putative acrylyl-CoA reductase led to reduced degradation rate of 3-HP in late stationary phase. Conclusions We demonstrated the feasibility of constructing the malonyl-CoA pathway in M. extorquens AM1 to generate 3-HP. Furthermore, we showed that a reductive route coupled with the ethylmalonyl-CoA pathway was the major channel responsible for degradation of the 3-HP during the growth transition. Engineered M. extorquens AM1 represents a good platform for 3-HP production from methanol. Electronic supplementary material The online version of this article (10.1186/s12934-017-0798-2) contains supplementary material, which is available to authorized users.
- Published
- 2017