Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose

Abstract

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.

FIG. 1.

Phenol production by different P. putida S12TPL strains in shake flask cultures in MMG with salicylate. ▪, S12TPL; □, S12TPL1; ▴, S12TPL2; ▵, S12TPL3. The data points are the averages of duplicate experiments. The maximum variation between duplicates was less than 10%.

FIG. 2.

Phenol production during typical fed-batch cultivation of P. putida S12TPL3. Glucose was used as the sole carbon source. The arrow indicates the time that feeding was started. ▴, cell concentration; ▪, phenol concentration; □: ammonium concentration.

FIG. 3.

Phenol production during typical biphasic fed-batch cultivation of P. putida S12TPL3 under nitrogen-limiting conditions. The solid arrow indicates the time that feeding was started, and the open arrow indicates when octanol addition was started. □, phenol concentration in the octanol phase; ▪, total phenol concentration, calculated as described in Materials and Methods; ▴, percentage of CO₂ (vol/vol) in the effluent gas.