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 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-molC-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 (1.8gl(-1)) was obtained, at a yield of 8.5% (C-molC-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.
Verhoef
Suzanne
S
TNO Quality of Life, Business Unit Food and Biotechnology Innovations, Julianalaan 67, 2628 BC Delft, The Netherlands; B-Basic, Julianalaan 67, 2628 BC Delft, The Netherlands. suzanne.verhoef@tno.nl
Ruijssenaars
Harald J
HJ
de Bont
Jan A M
JA
Wery
Jan
J
D001483:Base Sequence; D019149:Bioreactors; D001709:Biotechnology; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005285:Fermentation; D007700:Kinetics; D010226:Parabens / Q000378:metabolism*; D010650:Phenylalanine Ammonia-Lyase / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D012997:Solvents; D014443:Tyrosine / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D010226:Parabens; D012997:Solvents; D014443:Tyrosine; D010650:Phenylalanine Ammonia-Lyase; C038193:4-hydroxybenzoic acid