Improving Dipicolinic Acid Production By Bacillus Subtilis during Stationary Phase Based on 13c-Metabolic Flux Analysis

Bacillus subtilis is an attractive microorganism for bio-production because the genome can be easily edited to give the desirable abilities. It was revealed that the genome reduction provides useful properties for enhancing and maintaining the recombinant proteins productivity during a stationary phase (Morimoto et al., 2008). Dipicolinic acid (DPA), major component of B. subtilis spore, is a useful chemical as one of the precursors for bio-plastic. We constructed the DPA producing B. subtilis strains from wild-type 168 and its genome-reduced strain MGB874 by changing the promoter of spoVFAB genes encoding DPA synthase for the expression. The DPA was mainly produced during stationary phase in both strains, and the yield and the production rate were improved by the genome reduction. In the present study, we investigated the metabolic flows on the central carbon metabolism during the stationary phase, and performed the metabolic engineering for further enhancing the DPA productivity.

The DPA producing B. subtilis MGB874 strain was aerobically cultured on synthetic media which containing glucose and glutamate as carbon sources. After the cell growth stopped, the ¹³C-labeled glucose was added in the culture, and the ¹³C enrichments of intracellular metabolites were measured by gas chromatography–mass spectrometry and capillary electrophoresis time–of–flight mass spectrometry. The flux distribution was optimized to minimize the difference between the experimentally measured ¹³C-enrichments of metabolites and those calculated from the flux distribution. The estimated flux distribution revealed that the uptake glucose was mostly excreted as acetoin and the large part of DPA was derived from glutamate. We conducted the rational metabolic design based on the ¹³C-metabolic flux distribution, and successfully improved the productivity of DPA during the stationary phase.