(63b) Cell-Free Metabolic Engineering for Heterologous Pathway Optimization in Pseudomonas Putida KT2440

Authors: 
Rollin, J., National Renewable Energy Laboratory
Johnson, C., National Renewable Energy Laboratory
St. John, P., National Renewable Energy Laboratory
Abraham, P. E., Oak Ridge National Laboratory
Hettich, R., Oak Ridge National Laboratory
Beckham, G. T., National Renewable Energy Laboratory
Cell-free metabolic engineering methods offer unique opportunities to rapidly prototype heterologous pathways of interest. One such pathway, the protocatechuate 4,5-meta cleavage pathway from Sphingobium SYK-6, provides more efficient access to central carbon metabolism and unlocks a unique set of interesting polymer precursor products. Initial efforts to implement this pathway in Pseudomonas putida KT2440, the workhorse organism for biological lignin funneling, have demonstrated effective heterologous pathway activity, but enhancing productivity and eliminating an observed lag phase require enzyme loading optimization, a difficult objective to achieve through in vivo metabolic engineering alone. To address this challenge, we replicated the protocatechuate 4,5-meta cleavage pathway in vitro, monitored the concentrations of pathway intermediates using NMR metabolomics, and parameterized a kinetic model of the system. After validation, this model was used to iteratively optimize enzyme loadings for pathway throughput. The optimal set of enzyme loadings can be translated into genetic sequences using an RBS calculator and integrated into Pseudomonas putida KT2440. These optimal loadings can then be compared with native (Sphingobium SYK-6) and non-optimized (Pseudomonas putida KT2440) protein levels through the use of quantitative proteomics. These results demonstrate a new method for heterologous pathway optimization; longer-term potential for the use of cell-free systems as conversion platforms will also be discussed.