Computational Pathway Design for Efficient Bio-Production during Stationary Phase

Enhancing a target compound production is one of important challenges in bio-production using microorganism. Bio-production during the stationary phase, so called “non-growth-associated production”, is expected to lead to a high target yield because the cells do not consume the substrate for growth. In the present study, the mevalonate production using Escherichia colicells during the stationary phase was investigated. Metabolic pathway modification based on the stoichiometry has also been an effective approach for enhancing the target production. A method (solution space design [SSDesign]) that predicts the gene knockout candidates which are suitable for target production under the stationary phase has been developed (Toya et al., Biotech Bioeng, 2015).

2. Materials and Methods

A mevalonate-producing E. coli strain inducing mvaES gene derives from Enterococcus faecaliswas used for the evaluations of bio-process during the stationary phase. The mevalonate-producing strain was aerobically grown on synthetic medium. After the growing, the cells were cultured under the synthetic medium without an essential nutrient such as nitrate, sulfur and magnesium. The network of optimal pathways for non-growth-associated production can be designed from the flux variability (solution space). Elementary mode analysis is a method to decompose the network into its smallest reaction sets, called elementary flux modes (EFMs). The proposed SSDesign determined the area over which EFMs should be removed from the solution space of the parent strain, and explored the gene knockouts that will eliminate these undesirable EFMs.

3. Results and Discussion

The mevalonate yields in the stationary phase increased by 1.5-1.9 times than that in the growth phase. The yield and the specific production rate of mevalonate were highest in magnesium starvation case. The mevalonate is synthesized from acetyl-CoA with NADPH as reductive cofactor in this strain. The metabolic and genetic analysis revealed that the NADPH for mevalonate synthesis was generated in a transhydrogenase reaction.

To evaluate the performance of SSDesign, the method was applied to non-growth-associated succinate production in E. coli. The predicted candidates for succinate production were the deletion mutants ΔpntAB ΔsfcA ΔpykA,F and ΔsfcA ΔmaeB ΔpykA,F Δzwf. According to the solution spaces, these strains allow high growth yield and inevitably produce succinate at zero biomass yield, since their metabolic pathways cannot sustain steady-state without discarding succinate from the cell.