Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route

Abstract

Background3-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.ResultsHere 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.ConclusionsWe 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 materialThe online version of this article (10.1186/s12934-017-0798-2) contains supplementary material, which is available to authorized users.

Adaptive laboratory evolution of native methanol assimilation in Saccharomyces cerevisiae

“…Recent attempts have been made to produce valuable metabolites using methylotrophs. For example, Methylobacterium extorquens AM1 has been engineered to produce 3-hydroxypropionic acid from methanol 8 . The methylotrophic yeast Pichia pastoris (renamed Komagataella phaffii 9 ) is currently used industrially for production of recombinant proteins 10 .…”

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor

“…For constructing the sequential reactions in M. extorquens AM1, DNA fragments of thiM and MTH_47 and the fragments of adhE2 , thiM and MTH_47 were assembled into the Bam HI– Sac I restriction sites of pCM80 plasmid under the mxaF promoter to obtain pCB1 plasmid and pCB3 plasmid. Cell extracts of recombinants of M. extorquens AM1 were generated as described previously with slight modifications [15, 34]. Briefly, 50 mL of cells at the late exponential phase (OD 600 = 1.2 ± 0.1) were harvested and resuspended in 7 mL of buffer (50 mM Tris–HCl, 10 mM MgCl 2 , 20 mM KCl, pH 8.0).…”

Chemical Production from Methanol Using Natural and Synthetic Methylotrophs

“…By overexpression of malonyl‐CoA reductase (Mcr) from Chloroflexus aurantiacus in M. extorquens AM1, the engineered strain produced 0.07 g L −1 of 3HP. [ 22 ] It is worthy to note that there may be an oxidative route for 3HP degradation present in M. extorquens AM1. The putative 3HP degradation pathway is through assimilation into propionyl‐CoA, which enters ethylmalonyl‐CoA pathway (represented by the gray line in Figure 2).…”