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C5-technology in Pseudomonas putida S12: Construction, analysis and implementation of D-xylose metabolic pathways

The rising price of oil and impending deficit of fossil resources stimulate the development of alternative processes for the production of chemicals. The production of biochemicals from lignocellulosic biomass is a promising alternative. Lignocellulosic biomass consists of a mixture of sugars that can be converted into valuable products or chemicals by means of bioconversion. It is essential that, in order to establish an economically sound process, the feedstock is utilised as completely as possible. However, due to its heterogeneous nature, lignocellulosic feedstock is often metabolised incompletely. The same problem is encountered with the production of aromatic compounds by the solvent-tolerant micro-organism Pseudomonas putida S12. In order to increase the efficiency of biochemicals production from lignocellulose, strategies were explored to engineer D-xylose metabolic pathways in P. putida S12, to enable the consumption D-xylose, the second most abundant sugar present in lignocellulosic biomass. Two approaches to achieve D-xylose utilization by P. putida S12 were investigated: constructing a phosphorylative pathway using genes from Escherichia coli and establishing an oxidative pathway with genes from Caulobacter crescentus. The resulting strains were both capable of growing on D-xylose as carbon source. Analyses performed on the two strains yielded more insight in the metabolic pathways employed to utilize D-xylose. Also, as a case study for the production of aromatic compounds from D-xylose and a mixture of sugars, a p-hydroxybenzoate producing P. putida S12 strain was engineered with the phosphorylative D-xylose pathway. Chemostat experiments with the resulting strain demonstrated that aromatics production can be significantly improved by feeding mixtures of D-xylose and D-glucose or glycerol. As lignocellulose consists of mixtures of D-xylose and D-glucose, and because glycerol is expected to be a cheap feedstock in the future, these results are promising for the development of a sustainable aromatics production process.