Bioproduction of p-hydroxybenzoate from renewable feedstock by solvent-tolerant Pseudomonas putida S12

Pseudomonas putida strain S12palB1 was constructed that produces p-hydroxybenzoate from renewable carbon sources via the central metabolite L-tyrosine. P. putida S12palB1 was based on the platform strain R putida S12TPL3, which has an optimised carbon flux towards L-tyrosine. Phenylalanine ammonia lyase (Pal) was introduced for the conversion Of L-tyrosine into p-coumarate, which is further converted into p-hydroxybenzoate by endogenous enzymes. p-Hydroxybenzoate hydroxylase (PobA) was inactivated to prevent the degradation of p-hydroxybenzoate. These modifications resulted in stable accumulation of p-hydroxybenzoate at a yield of 11% (C-mol C-mol(-1)) on glucose or on glycerol in shake flask cultures. In a glycerol-limited fed-batch fermentation, a final p-hydroxybenzoate concentration of 12.9mM (1.8g l(-1)) was obtained, at a yield of 8.5% (C-mol C-mol(-1)). A 2-fold increase of the specific p-hydroxybenzoate production rate (q(p)) was observed when L-tyrosine was supplied to a steady-state C-limited chemostat culture of P. putida S12palB1. This implied that L-tyrosine availability was the bottleneck for p-hydroxybenzoate production under these conditions. When p-coumarate was added instead, qp increased by a factor 4.7, indicating that Pal activity is the limiting factor when sufficient L-tyrosine is available. Thus, two major leads for further improvement of the p-hydroxybenzoate production by P. putida S12palB1 were identified. (C) 2007 Elsevier B.V. All rights reserved.