(172e) Terephthalaldehyde As a Platform Chemical Intermediate for Microbial Conversions

Deconstruction of waste polyethylene terephthalate (PET) plastic generates opportunities to create monomers for other classes of materials. For example, we recently designed an enzyme cascade that could produce the useful diamine para-xylylenediamine (pXYL) from terephthalic acid via the dialdehyde terephthalaldehyde (TPAL). However, our cascade featured enzymes whose purification cost, solvent tolerance, and co-factor requirements may pose obstacles to commercialization in the purified enzyme format. Here, we investigated whether we could instead perform transformations of TPAL in growing or resting whole cells. First, we showed that the addition of TPAL to growing cultures of E. coli very efficiently results in formation of 1,4-benzenedimethanol (BDM), a valuable building block for perfumes and dyes. Next, we performed a head-to-head comparison of the solvent tolerance of the ω-transaminase from Chromobacterium violaceum (cvTA) in purified versus resting whole-cell formats at roughly equivalent concentration. Under 40% DMSO, we observed much greater conversion of TPAL to pXYL using resting whole cells transformed to express cvTA than when using purified cvTA. Finally, we investigated whether we could efficiently produce pXYL rather than BDM in growing cells of E. coli by deleting aldehyde reductases. We found that a strain of E. coli previously engineered for reduced aromatic aldehyde reduction (E. coli RARE) was unable to prevent reduction of TPAL. We then performed a TPAL challenge and used RNA-seq to determine additional aldehyde reductase targets and used multiplex automatable genome engineering to create E. coli strains with as many as 10 additional knockouts. Encouragingly, we found that additional knockouts enabled a 1.9-fold higher retention of TPAL and substantially improved pXYL synthesis by 6.8-fold in cells. Overall, our study demonstrates the potential of TPAL as a versatile intermediate in microbial biosynthesis of chemicals that can ultimately be derived from waste PET.