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

• Published in: Journal of biotechnology Vol. 132; no. 1; pp. 49 - 56
• Main Authors: Verhoef, Suzanne, Ruijssenaars, Harald J., de Bont, Jan A.M., Wery, Jan
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 15.10.2007; Amsterdam Elsevier; New York, NY
• Subjects: Base Sequence; Biological and medical sciences; Bioreactors; Biotechnology; DNA Primers - genetics; DNA, Bacterial - genetics; Fermentation; Fundamental and applied biological sciences. Psychology; Kinetics; Metabolic engineering; p-Hydroxybenzoate; Parabens - metabolism; Phenylalanine Ammonia-Lyase - metabolism; Plasmids - genetics; Pseudomonas putida; Pseudomonas putida - genetics; Pseudomonas putida - metabolism; Renewable feedstock; Solvent-tolerance; Solvents; Tyrosine - metabolism; Renewable feedstock; Metabolic engineering; Solvent-tolerance; Pseudomonas putida; p-Hydroxybenzoate; Pseudomonadales; Production; Tolerance; Bacteria; Pseudomonadaceae; Solvent
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2007.08.031

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 P. 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 12.9 mM (1.8 g 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, q p 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.