Metabolic engineering of Pseudomonas taiwanensis VLB120 with minimal genomic modifications for high-yield phenol production

Aromatic chemicals are important building blocks for the production of a multitude of everyday commodities. Currently, aromatics production relies almost exclusively on petrochemical processes. To achieve sustainability, alternative synthesis methods need to be developed. Here, we strived for an efficient production of phenol, a model aromatic compound of industrial relevance, from renewable carbon sources using the solvent-tolerant biocatalyst Pseudomonas taiwanensis VLB120. First, multiple catabolic routes for the degradation of aromatics and related compounds were inactivated, thereby obtaining the chassis strain P. taiwanensis VLB120 Delta 5 incapable of growing on 4-hydroxybenzoate (Delta pobA), tyrosine (Delta hpd), and quinate (Muff, Delta quiC1, Delta quiC2). In this context, a novel gene contributing to the quinate catabolism was identified (quiC2). Second, we employed a combination of reverse- and forward engineering to increase metabolic flux towards the product, using leads obtained from the analysis of aromatics producing Pseudomonas putida strains previously generated by mutagenesis. Phenol production was enabled by the heterologous expression of a codon-optimized and chromosomally integrated tyrosine phenol-lyase encoding gene from Pantoea agglomerans AJ2985 (PaTPL2). The genomic modification of endogenous genes encoding TrpE(P29)(0S), AroF-1(P148L), and PheA(T31)(0I), and the deletion of pykA improved phenol production 17-fold, while also minimizing the burden caused by plasmids and auxotrophies. The additional overexpression of known bottleneck enzymes (AroG(fbr), TyrA(fbr)) derived from Escherichia coll. further enhanced phenol titers. The best producing strain P. taiwanensis VLB120 Delta 5-TPL36 reached yields of 15.8% and 18.5% (Cmol/Cmol) phenol from glucose and glycerol, respectively, in a mineral medium without addition of complex nutrients. This is the highest yield ever reported for microbially produced phenol.