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

Abstract

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. Graphical abstract

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

“…The cells were then resuspended in 6 mL of resting cell medium containing 200 mM sodium phosphate buffer at pH 7 to reach the intended amount of wet cell weight (WCW) in the reaction medium. The reaction was performed in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM), as previously reported as optimum concentrations (Kordesedehi et al., 2023 ). A higher concentration of benzaldehyde (40 mM) was also used to evaluate whether higher tolerance could improve the 2‐HPP production.…”

“…Analysis of 2‐HPP and benzaldehyde was performed using a gas chromatograph (Agilent 6890) equipped with an FID detector, as described previously (Kordesedehi et al., 2023 ).…”

“…The amount of 2-HPP formation by the recombinant strain was compared with that of the control strain to monitor the effect of pprI overexpression and, consequently, higher aldehyde tolerance on 2-HPP production. We tracked the changes in 2-HPP production by the recombinant cells compared to the control strain using optimized substrate concentrations determined in our recent study (Kordesedehi et al, 2023). The production of 2-HPP in the engineered strain was enhanced from 1.2 g L −1 (control strain) to 1.6 g L −1 for the P-PprI strain when using 20 mM benzaldehyde and 600 mM acetaldehyde with 0.033 g WCW mL −1 after an incubation of 3 h. This corresponds to a yield of 0.74 g 2-HPP g −1 benzaldehyde for the recombinant strain, suggesting a 35% improvement in the yield compared to the control strain.…”

“…Benzoylformate decarboxylase can also catalyse carboligation side reactions, making it a valuable tool in the production of ( S )‐2‐hydroxypropiophenone (2‐HPP) (Knoll et al., 2006 ; Kordesedehi et al., 2023 ). 2‐HPP is a valuable platform chemical that is used as a synthon in organic chemistry and as a precursor of antifungal agents (Fang et al., 2000 ; Gala et al., 1996 ).…”

“…While P. putida strains are generally thought to withstand solvent stressors, the growth of these strains can be severely impaired by benzaldehyde and other aldehydes (Simmonds & Robinson, 1998 ). Since it is well known that the resting (non‐growing) cells are suitable for producing chemicals with growth‐inhibitory or chaotropic substrates (Cray et al., 2015 ; Dvořák et al., 2020 ; Sun et al., 2018 ), among other benefits, it was previously envisioned that bioconversion with benzoylformate decarboxylase in a resting cell process could be particularly useful (De María et al., 2008 ; Kordesedehi et al., 2023 ; Wilcocks et al., 1992 ). High concentration of aldehyde substrates has an inhibitory effect on both the pure enzyme and the whole‐cell catalyst.…”