Tuesday, May 6, 2008 - 10:00 AM
7-04
Production of substituted aromatics from biomass-derived feedstock by engineered solvent-tolerant Pseudomonas putida S12
Pseudomonas putida S12 thrives in minimal medium with a second phase of toluene or 1-octanol. This exceptional solvent tolerance makes strain S12 pre-eminently suited for the production of industrially relevant, but toxic chemicals such as substituted aromatics. We have modified P. putida S12 into a versatile whole-cell biocatalyst with regard to substrate utilization and product formation by different engineering approaches, while employing a systems approach for strain optimization.
In demonstrator cases, tyrosine phenol lyase and phenylalanine ammonia lyase were expressed in P. putida S12 for the production of phenol or t-cinnamate, via L-tyr or L-phe, from glucose and glycerol. The initially modest production characteristics were improved by orders of magnitude by random mutagenesis and high-throughput screening. Transcriptomics analysis of the improved mutants provided useful leads for further targeted optimization and indicated that metabolic fluxes to the aromatic amino acids were significantly increased.
In addition to expanding the range of products and optimizing productivity, we are investigating the efficient utilization of biomass-derived feedstock. Wild-type P. putida S12 readily metabolizes the model renewables glucose and glycerol. However, real-life substrates such as lignocellulose hydrolysates also contain considerable amounts of pentoses that P. putida S12 cannot utilize. By introducing xylose utilization genes from Escherichia coli and subsequent laboratory evolution, a P. putida S12 was obtained that efficiently utilizes xylose as the sole C-source. Utilization of arabinose as well as toxic compounds from lignocellulose hydrolysates is currently investigated for improved efficient production of industrially relevant compounds from cheap biomass-derived feedstock.
In demonstrator cases, tyrosine phenol lyase and phenylalanine ammonia lyase were expressed in P. putida S12 for the production of phenol or t-cinnamate, via L-tyr or L-phe, from glucose and glycerol. The initially modest production characteristics were improved by orders of magnitude by random mutagenesis and high-throughput screening. Transcriptomics analysis of the improved mutants provided useful leads for further targeted optimization and indicated that metabolic fluxes to the aromatic amino acids were significantly increased.
In addition to expanding the range of products and optimizing productivity, we are investigating the efficient utilization of biomass-derived feedstock. Wild-type P. putida S12 readily metabolizes the model renewables glucose and glycerol. However, real-life substrates such as lignocellulose hydrolysates also contain considerable amounts of pentoses that P. putida S12 cannot utilize. By introducing xylose utilization genes from Escherichia coli and subsequent laboratory evolution, a P. putida S12 was obtained that efficiently utilizes xylose as the sole C-source. Utilization of arabinose as well as toxic compounds from lignocellulose hydrolysates is currently investigated for improved efficient production of industrially relevant compounds from cheap biomass-derived feedstock.
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See more of The 30th Symposium on Biotechnology for Fuels and Chemicals (May 4 -- 7, 2008)