(602r) Bacteria Engineered for Bio-Styrene Production

Styrene is an important commodity chemical with versatile commercial applications, predominantly as a monomer building block for the production of many useful polymers and co-polymers. Today, all styrene is produced via chemocatalytic routes from petroleum-derived benzene or ethylbenzene according to energy intensive processes.  Through de novo metabolic pathway engineering, we have pathway engineered a functional pathway to instead produce styrene from renewable feedstocks. By over-expressing both PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae in a phenylalanine over-producing Escherichia coli host we have engineered the first styrene-producing microbe.  Initial shake flask cultures produced ~300 mg/L styrene, approaching the determined toxicity threshold.  Subsequent works have since sought to overcome several of critical limitations of this preliminary work.  Here we present our progress in this regard on several fronts.  First, we have increased and balanced pathway flux through the systematic tuning of the expression of PAL2, the previous bottleneck enzyme. This was achieved through the systematic tuning of each of gene copy number, promoter strength, and RBS strength.  Next, we have sought to overcome the toxicity issue by the ‘bottom up’ engineering of an aromatic tolerant Pseudomonas sp. to produce styrene from glucose and other renewable substrates.  At the same time, the ability to circumvent styrene toxicity through its in situ recovery has been explored with the use of solvent extraction, gas stripping, and membrane pervaporation. At last we have sought to improve the sustainability and economics of bio-styrene production by engineering photosynthetic cyanobacteria to produce styrene from CO₂ and light inputs alone.