Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose.
Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-dl-phenylalanine and m-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.
Wierckx
Nick J P
NJ
TNO Quality of Life, P.O. Box 342, 7300 AH Apeldoorn, The Netherlands. nick.wierckx@tno.nl
Ballerstedt
Hendrik
H
de Bont
Jan A M
JA
Wery
Jan
J
D001711:Biotransformation; D003001:Cloning, Molecular; D017931:DNA Primers; D005818:Genetic Engineering / Q000379:methods; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D016296:Mutagenesis; D019800:Phenol / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014447:Tyrosine Phenol-Lyase / Q000378:metabolism
D017931:DNA Primers; D019800:Phenol; D014447:Tyrosine Phenol-Lyase; D005947:Glucose