Understand and optimize natural and synthetic metabolic networks of microorganisms

Background & ObjectivesIn 4-10 years from now, it is likely that the next generation synthetic methylotrophs obtainedfrom this project will be further developed for production of chemicals (e.g. bulk chemicals,fuels) from methanol, after which commercial production may be feasible. The choice of themolecules to be produced is the first step for developing such platform strains. In addition tothe value added, the choice of the molecule has to take into account the nature of the carbonsubstrate and its assimilation pathway. In the synthetic methylotrophic E. coli expressing MUT2pathway, each mole of methanol converted through Mdh generates one mole of NADH thusone can think that its consumption will favor NADH dependent chemicals production. This hasbeen recently demonstrated in a synthetic methylotrophic E. coli expressing the MUT1pathway where the conversion of extra NADH into NADPH resulted in twofold improvement ofNADPH dependent lysine production [47]. Interestingly it was also observed that coupling theNAD dependent conversion of methanol to a NADH sink eliminated any NADH accumulationand led to over twofold more methanol oxidation [48]. Thus choosing a molecule whichproduction is NADH-dependent has the potential to boost both the assimilation of methanoland it conversion into this molecule. This can be further improved if the molecule is directlyplugged to the MUT2 pathway at the DHA level. Both the 3-hydroxypropionic acid (3-HP) and1,3 propanediol (1,3-PDO) gather all these features. These are important platform chemicalsthat can be converted into commercially valuable compounds (i.e. polymers, lubricants..) [49,50]. They can be produced from glycerol which is one step after DHA using a NAD(H)-consuming process. However, the main problems hampering the current commercialproduction of 1,3-PDO or 3-HP from glycerol using recombinant microorganisms areassociated with the toxicity of 3-HPA and inefficient NAD(H) regeneration. Therefore, I proposeto solve those problem by testing 2 metabolic pathways allowing the co-production 1,3-PDOor 3-HP from methanol in processes as shown in figure 3. Both pathways allow balanced redoxdemands and prevent 3-HPA toxicity by encapsulating the pathway 2 into a BMC. In theory,the production of redox equivalents by the glycerol oxidative branch of the pathway (i.e. 3-HP)being balanced by the reductive branch (i.e. 1,3-PDO), this should led to the conversion of the3HPA into equimolar mixture of 3HP and 1,3PDO. In addition, it has been previously shownthat the products can be easily separated from each other [51].