MODEL BASED OPTIMIZATION OF THE TERPENOIDS BIOSYNTHESIS IN E. coli

Terpenoids are a family of compounds with high industrial interest and the development of biotechnological production methods is essential to achieve more sustainable alternatives to traditional extraction and synthesis methods. The modification and engineering of the catalytic activity (kcat) have been shown to be a feasible strategy in the biotechnological realm. Accordingly, we introduce a novel optimization strategy based in the modification of the kcat of the enzymes and applied it to the maximization of the terpenoids synthesis in E. coli. This approach is fairly general and can be applied alone or in conjunction with classic optimization strategies such as the modification of enzymatic specific activities. For this purpose we first build up a reliable dynamic mathematical model of the alternative mevalonate pathway synthesis leading terpenoids biosynthesis in E. coli through the methyl-D-erythritol 4-phosphate (MEP) pathway. This model includes the 2-C-methyl-D-erythritol 2, 4-diphosphate (MEC) pumps that mediate MEC extracellular extrusion. Although the physiological significance of the MEC extrusion is still discussed and their biological function is not clear, we find that this process is a must to guarantee bacteria homeostasis and cell viability. We have identified the enzyme IspA as a bottleneck of the terpenoids biosynthesis, which is dual substrate for the two bisubstrate final reactions. Here are presented different ways to overcome this enzymatic flux restriction by modification of the IspA kcat or by introduction of new enzymes with parallel function. Our results show that the MEP pathway optimized solutions for kcat can yield a maximum of 17.68 fold increment in the terpenoids biosynthetic flux when the kcat are modified. This maximal solution involves the modification of 8 kcat and the corresponding Km's and Kd's. Remarkably, this increase doesn't imply a change in the total enzyme concentration of the cell. This is favorable output since enzyme overproduction can compromise cell functionality. We also apply the overexpression of the enzyme activity approach and then we have compared and combined both strategies including scenarios as the deletion or import of the genes expressing enzymes not naturally present in E. coli. A combined strategy of enzymes concentrations and kcat modifications with changes up ti six enzyme levels allows a flux increment of 21.22 fold the basal value. It involves the incorporation of two IspA with similar GPP and IPP bisubstrate functions than the native one but low affinity for the DMAPP as substrate.