Exploiting Pseudomonas for the production of methacrylate for the plastics industry
Lead Research Organisation:
University of Strathclyde
Department Name: Inst of Pharmacy and Biomedical Sci
Abstract
The growing world acrylic plastic market is expected to be worth $18bn by 2020. Most of the world's acrylic plastics are produced from unsustainable and polluting fossil fuels. The ability to produce these plastics from renewable feedstocks would be immensely beneficial, with the biosynthetic capabilities of bacteria presenting a viable avenue for production.
Work carried out by Ingenza has led to the successful production of BMA from Alpha-ketoisovaleric acid and n-butanol in Escherichia coli, a common chassis organism. While successful, efforts have only produced BMA in millimolar quantities due to its toxicity to the E. coli chassis organism (in addition to the toxicity of butanol). E. coli was chosen as a chassis organism not due to its suitability for these purposes but principally due to its widespread usage and familiarity. As such it is necessary to explore the use of other chassis organisms and transfer the biosynthetic pathway from E. coli into a more tolerant chassis organism which will be capable of producing BMA in larger industrial quantities (2g/L) from renewable feedstocks.
Pseudomonas putida is a versatile host for the biosynthesis of natural products, due to its solvent tolerance. Solvents generally interfere with bacterial physiology through the disruption of normal membrane function: by partitioning to the cytoplasmic membrane, membrane fluidity is altered and therefore the stability of the bilayer. P. putida modifies its membrane composition through isomerisation of the cis-unsaturated fatty acids of membrane lipids into trans-unsaturated fatty acids (via cis-to-trans isomerase). Headgroup composition can also be employed with similar results. P. putida can also export solvents from the cell preserving the integrity of the cell interior. This is achieved through resistance/nodulation/cell division (RND) efflux pumps which rely on proton motive force to extrude molecules from the cell. Solvent removal from the membrane is a dynamic process, occurring continuously.
Two main questions/aims:
1. Can the desirable traits of Pseudomonas (i.e. solvent tolerance) be transferred into E. coli to improve methacrylate ester tolerance?
2. Can the synthetic pathway be stably maintained in an optimised Pseudomonas host such as Pseudomonas putida e.g. KT2440 or Pseudomonas alkylphenolica.
Milestones:
Adapt and transfer the existing methacrylate ester synthetic pathway into P. putida (months 1-24).
Profile innate fermentative co-product pathways in P. putida (months 12-36).
Optimise fermentation conditions towards the industrial production concept (months 24-48).
Work carried out by Ingenza has led to the successful production of BMA from Alpha-ketoisovaleric acid and n-butanol in Escherichia coli, a common chassis organism. While successful, efforts have only produced BMA in millimolar quantities due to its toxicity to the E. coli chassis organism (in addition to the toxicity of butanol). E. coli was chosen as a chassis organism not due to its suitability for these purposes but principally due to its widespread usage and familiarity. As such it is necessary to explore the use of other chassis organisms and transfer the biosynthetic pathway from E. coli into a more tolerant chassis organism which will be capable of producing BMA in larger industrial quantities (2g/L) from renewable feedstocks.
Pseudomonas putida is a versatile host for the biosynthesis of natural products, due to its solvent tolerance. Solvents generally interfere with bacterial physiology through the disruption of normal membrane function: by partitioning to the cytoplasmic membrane, membrane fluidity is altered and therefore the stability of the bilayer. P. putida modifies its membrane composition through isomerisation of the cis-unsaturated fatty acids of membrane lipids into trans-unsaturated fatty acids (via cis-to-trans isomerase). Headgroup composition can also be employed with similar results. P. putida can also export solvents from the cell preserving the integrity of the cell interior. This is achieved through resistance/nodulation/cell division (RND) efflux pumps which rely on proton motive force to extrude molecules from the cell. Solvent removal from the membrane is a dynamic process, occurring continuously.
Two main questions/aims:
1. Can the desirable traits of Pseudomonas (i.e. solvent tolerance) be transferred into E. coli to improve methacrylate ester tolerance?
2. Can the synthetic pathway be stably maintained in an optimised Pseudomonas host such as Pseudomonas putida e.g. KT2440 or Pseudomonas alkylphenolica.
Milestones:
Adapt and transfer the existing methacrylate ester synthetic pathway into P. putida (months 1-24).
Profile innate fermentative co-product pathways in P. putida (months 12-36).
Optimise fermentation conditions towards the industrial production concept (months 24-48).