Enhanced chaotrope tolerance and (S)-2-hydroxypropiophenone production by recombinant Pseudomonas putida engineered with Pprl from Deinococcus radiodurans.

Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other aldehydes into valuable α-hydroxyketones, such as (S)-2-hydroxypropiophenone. However, poor tolerance of this strain toward chaotropic aldehydes hampers efficient biotransformation processes. To circumvent this problem, we expressed the gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, in P. putida. Fine-tuned gene expression was achieved using an expression plasmid under the control of the LacIQ /Ptrc system, and the cross-protective role of PprI was assessed against multiple stress treatments. Moreover, the stress-tolerant P. putida strain was tested for 2-hydroxypropiophenone production using whole resting cells in the presence of relevant aldehyde substrates. P. putida cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H2 O2 and thermal stress, thereby reflecting the multistress protection profile conferred by PprI. Additionally, the engineered cells converted aldehydes to 2-hydroxypropiophenone more efficiently than the parental P. putida strain. 2-Hydroxypropiophenone concentration reached 1.6 g L-1 upon a 3-h incubation under optimized conditions, at a cell concentration of 0.033 g wet cell weight mL-1 in the presence of 20 mM benzaldehyde and 600 mM acetaldehyde. Product yield and productivity were 0.74 g 2-HPP g-1 benzaldehyde and 0.089 g 2-HPP g cell dry weight-1 h-1 , respectively, 35% higher than the control experiments. Taken together, these results demonstrate that introducing PprI from D. radiodurans enhances chaotrope tolerance and 2-HPP production in P. putida ATCC 12633.

Optimized enantioselective (S)-2-hydroxypropiophenone synthesis by free- and encapsulated-resting cells of Pseudomonas putida.

BACKGROUND: Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction. RESULTS: The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL- 1. 2-HPP titer, yield and productivity using the free cells were 1.2 g L- 1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g- 1 DCW h- 1, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)-polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product. CONCLUSIONS: Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones.