In situ phenol removal from fed-batch fermentations of solvent tolerant Pseudomonas putida S12 by pertraction

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.

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Bioproduction of p-Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium Pseudomonas putida S12 in a Two-Phase Water-Decanol Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.02186-08 ◽

2008 ◽

Vol 75 (4) ◽

pp. 931-936 ◽

Cited By ~ 87

Author(s):

Suzanne Verhoef ◽

Nick Wierckx ◽

R. G. Maaike Westerhof ◽

Johannes H. de Winde ◽

Harald J. Ruijssenaars

Keyword(s):

Pseudomonas Putida ◽

Value Added ◽

Batch Cultivation ◽

Volumetric Productivity ◽

Acid Decarboxylase ◽

Efficient Production ◽

Second Phase ◽

Fed Batch ◽

Solvent Tolerant ◽

Fed Batch Cultivation

ABSTRACT Two solvent-tolerant Pseudomonas putida S12 strains, originally designed for phenol and p-coumarate production, were engineered for efficient production of p-hydroxystyrene from glucose. This was established by introduction of the genes pal and pdc encoding l-phenylalanine/l-tyrosine ammonia lyase and p-coumaric acid decarboxylase, respectively. These enzymes allow the conversion of the central metabolite l-tyrosine into p-hydroxystyrene, via p-coumarate. Degradation of the p-coumarate intermediate was prevented by inactivating the fcs gene encoding feruloyl-coenzyme A synthetase. The best-performing strain was selected and cultivated in the fed-batch mode, resulting in the formation of 4.5 mM p-hydroxystyrene at a yield of 6.7% (C-mol of p-hydroxystyrene per C-mol of glucose) and a maximum volumetric productivity of 0.4 mM h−1. At this concentration, growth and production were completely halted due to the toxicity of p-hydroxystyrene. Product toxicity was overcome by the application of a second phase of 1-decanol to extract p-hydroxystyrene during fed-batch cultivation. This resulted in a twofold increase of the maximum volumetric productivity (0.75 mM h−1) and a final total p-hydroxystyrene concentration of 21 mM, which is a fourfold improvement compared to the single-phase fed-batch cultivation. The final concentration of p-hydroxystyrene in the water phase was 1.2 mM, while a concentration of 147 mM (17.6 g liter−1) was obtained in the 1-decanol phase. Thus, a P. putida S12 strain producing the low-value compound phenol was successfully altered for the production of the toxic value-added compound p-hydroxystyrene.

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Conjugal TOL Transfer from Pseudomonas putida to Pseudomonas aeruginosa: Effects of Restriction Proficiency, Toxicant Exposure, Cell Density Ratios, and Conjugation Detection Method on Observed Transfer Efficiencies

Applied and Environmental Microbiology ◽

10.1128/aem.71.1.51-57.2005 ◽

2005 ◽

Vol 71 (1) ◽

pp. 51-57 ◽

Cited By ~ 34

Author(s):

Catalina Arango Pinedo ◽

Barth F. Smets

Keyword(s):

Pseudomonas Aeruginosa ◽

Pseudomonas Putida ◽

Strong Dependence ◽

Plasmid Transfer ◽

Mass Action ◽

Tol Plasmid ◽

Toxicant Exposure ◽

Transfer Frequency ◽

Cell Densities

ABSTRACT The effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the TOL plasmid between Pseudomonas spp. was investigated by examinations of filter matings. A Pseudomonas putida KT2442-derived strain carrying a gfp-tagged variant of the TOL plasmid was used as a donor, and both restriction-deficient (PAO1162N) and -proficient (PAO2002N) Pseudomonas aeruginosa strains were used as recipients. The in situ enumeration of conjugation events allowed us to obtain frequency estimates that were unbiased by transconjugant growth or plasmid retransfer. We observed a strong dependence of the plasmid transfer frequency on the initial donor-to-recipient ratio of surface matings, which invalidated the use of mass action-based plasmid transfer kinetic estimators. Careful control of the initial parental cell densities permitted evaluations of the true effects of restriction proficiency and toxicant exposure on TOL transfer. At standard donor-to-recipient ratios (10−3 for PAO1162N and 2 Ã¯Â¿Â½ 101 for PAO2002N) and total cell densities (105 cells/mm2 for PAO1162N and 106 cells/mm2 for PAO2002N), plasmid transfer frequencies without toxicant exposure were approximately 10−7 (events/mm2)−1 for PAO1162N and 10−11 (events/mm2)−1 for PAO2002N based on in situ observations of conjugation events. The enumeration of transconjugants via selective plating yielded transfer frequencies that were up to 1 order of magnitude lower. Premating exposure to sodium dodecyl sulfate (1 to 10 mM) significantly increased the transfer frequency for the restriction-proficient strain PAO2002N (P < 0.05) but not for the restriction-deficient strain PAO1162N. On the other hand, premating exposure to ethanol, toluene, or phenol had no positive effect on the plasmid transfer frequency. Clearly, restriction proficiency provides a strong barrier to interspecific transfer of the TOL plasmid, and this barrier was only marginally attenuated by recipient exposure to toxicants within the ranges examined.

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Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonas putida KT2440

Applied Microbiology and Biotechnology ◽

10.1007/s00253-005-0191-7 ◽

2006 ◽

Vol 71 (4) ◽

pp. 423-431 ◽

Cited By ~ 52

Author(s):

Zhiyong Sun ◽

Juliana A. Ramsay ◽

Martin Guay ◽

Bruce A. Ramsay

Keyword(s):

Pseudomonas Putida ◽

Cell Density ◽

High Cell Density ◽

Batch Cultivation ◽

Feeding Strategies ◽

High Cell ◽

Fed Batch ◽

Pseudomonas Putida Kt2440 ◽

Fed Batch Cultivation

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Effect of Organic Solvents on the Yield of Solvent-Tolerant Pseudomonas putida S12

Applied and Environmental Microbiology ◽

10.1128/aem.65.6.2631-2635.1999 ◽

1999 ◽

Vol 65 (6) ◽

pp. 2631-2635 ◽

Cited By ~ 70

Author(s):

Sonja Isken ◽

Antoine Derks ◽

Petra F. G. Wolffs ◽

Jan A. M. de Bont

Keyword(s):

Growth Rate ◽

Pseudomonas Putida ◽

Organic Solvents ◽

Critical Concentration ◽

Bacterial Membrane ◽

Maximal Growth ◽

Wild Type ◽

Active Efflux ◽

Maximal Growth Rate ◽

Solvent Tolerant

ABSTRACT Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h−1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.

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