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

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Affiliations

  • 1. Department of Biotechnology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran.
      Authors
    • Kordesedehi R 1
    • Asadollahi MA 1
    • Biria D 1
    (3 authors)
  • 2. Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran.
      Authors
    • Shahpiri A 2
    (1 author)
  • 3. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
      Authors
    • Nikel PI 3
    (1 author)

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Microbial Biotechnology, 01 Mar 2024, 17(3):e14448
https://doi.org/10.1111/1751-7915.14448 PMID: 38498302 PMCID: PMC10946676

This article is in the Europe PMC Open access subset. Refer to the copyright information in the article for licensing details.
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Abstract 

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.

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Microb Biotechnol. 2024 Mar; 17(3): e14448.
Published online 2024 Mar 18. https://doi.org/10.1111/1751-7915.14448
PMCID: PMC10946676
PMID: 38498302

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

Reihaneh Kordesedehi, 1 Azar Shahpiri,corresponding author 2 Mohammad Ali Asadollahi,corresponding author 1 Davoud Biria, 1 and Pablo Iván Nikel 3
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Abstract

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/P trc 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, H2O2 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.

Abstract

The gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, was expressed in Pseudomonas putida. The cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H2O2 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.

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INTRODUCTION

Pseudomonas putida is a bacterium commonly found in soil and water, and is widely known for its ability to tolerate various chemicals. This feature has made it a popular choice for degrading pollutants in waste streams, such as benzene, toluene, ethylbenzene and xylene (BTEX) (Kour et al., 2022; Shim & Yang, 1999). The high tolerance of certain strains of P. putida is attributed to mechanisms such as modifications in the membrane composition, including a shift in the conformation of membrane unsaturated fatty acids from cis to trans, the presence of efflux pumps that secrete the chaotropic chemicals outside the cell and rapid NADPH supply in response to environmental stresses (Duque et al., 2022; Hallsworth et al., 2003; Nikel et al., 2021; Vallon et al., 2015). Additionally, P. putida has gained attention as a microbial host in industrial biotechnology (Nikel & de Lorenzo, 2018; Timmis, 2002; Volke et al., 2022; Volke, Calero, et al., 2020; Weimer et al., 2020). P. putida ATCC 12633 is a non‐pathogenic member of this genus endowed with a native mandelate pathway. Previous studies have revealed that P. putida can effectively utilize both (R) and (S) enantiomers of mandelate through the mandelate pathway (Scheme 1). Benzoylformate decarboxylase is the third enzyme in this route which catalyses the non‐oxidative conversion of benzoylformate to benzaldehyde. Benzaldehyde is further metabolized through the β‐ketoadipate pathway and tricarboxylic acid (TCA) cycle to yield acetyl‐coenzyme A (CoA) (McLeish et al., 2003).

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The mandelate pathway in Pseudomonas putida ATCC 12633.

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). Also, 2‐HPP is a key component in the pharmaceutical industry for the synthesis of phenylpropane‐1, 2‐diol (Wachtmeister et al., 2016). Benzoylformate decarboxylase catalyses the reaction leading to 2‐HPP through its carboligase activity, typically using α‐ketoacids, such as benzoylformate or benzaldehyde, as donor substrates and acetaldehyde as an acceptor substrate (Cosp et al., 2008; Valinger et al., 2014). The biotransformation of aldehydic substrates can form a new carbon–carbon bond between two asymmetric molecules through carboligation reaction, which is difficult to achieve by chemical methods. 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. Chaotropic solutes such as benzaldehyde and acetaldehyde induce alterations in the permeability of the cell membrane, affecting the transport of substrates and products while diminishing enzymes effectiveness. Consequently, this leads to a decreased biotransformation efficiency and a lower yield of hydroxyketones (Shin & Rogers, 1996; Simmonds & Robinson, 1998). Therefore, cellular stress due to chaotropicity of aldehydes remains a challenge for the biotransformation of benzaldehyde to 2‐HPP.

The genus Deinococcus is known for its extreme tolerance to various stress conditions including radiation, desiccation and other conditions that lead to DNA damage and oxidative stress (Ludanyi et al., 2014). Deinococcus radiodurans R1 is a polyextremophile, red‐pigmented and Gram‐positive bacterium that is highly resistant to different physical and chemical DNA‐damaging stresses (Battista, 2000; Smith et al., 1992), gamma and UV radiation (Anderson, 1956; Battista, 1997) and desiccation (Makarova et al., 2001). D. radiodurans contains a global transcription factor, PprI (inducer of pleiotropic proteins promoting DNA repair, also known as IrrE) (Lu & Hua, 2021). The crystal structure of the D. deserti PprI revealed a distinctive arrangement of three highly conserved domains (Vujičić‐Žagar et al., 2009). It not only activates diverse defence systems, but also greatly expands the range of perturbations in the genome‐wide transcriptional profile of Deinococcus (Lin et al., 2013). Through its three identified domains, it performs a crucial function in directly regulating the expression of other genes such as pprA and recA (Chen et al., 2011; Earl et al., 2002; Hua et al., 2003; Lu et al., 2012; Ohba et al., 2005). Enhanced tolerance against chemicals and various abiotic stress conditions including heat, osmotic pressure, salt and oxidative shock has been reported upon the expression of pprI. For instance, tolerance to organic solvents, such as ethanol and methanol, and Δ1‐dehydrogenation activity of Arthrobacter simplex were improved when PprI was introduced into the strain (Song et al., 2018). Furthermore, an optimal A. simplex strain was developed with improved biotransformation performance to produce prednisone acetate, a steroidal derivative, by the systematic optimization of PprI encoding gene expression levels and using a stronger promoter (Luo et al., 2021). In another study, directed evolution generated PprI mutants that were introduced into Saccharomyces cerevisiae, conferring the enhanced tolerance toward multiple inhibitors present in lignocellulose hydrolysates and thermal stress (Wang et al., 2020). Moreover, the acid stress tolerance of P. putida S16 as a pollutant‐degrading bacterium was boosted by expressing the gene encoding PprI (Zhou et al., 2019). Overexpression of this regulatory gene can also improve the tolerance of Escherichia coli and S. cerevisiae toward ethanol, butanol and osmotic pressure (Chen et al., 2011; Gao et al., 2003; Ma et al., 2011; Pan et al., 2009). Therefore, in the present study, we heterologously expressed the gene encoding PprI from D. radiodurans into P. putida to boost its tolerance against multiple stressors and potentially enhance the 2‐HPP production. The viability and growth of the engineered strain exposed to stressors, including aldehyde substrates, organic solvents, heat, NaCl and H2O2, were tested and compared to those of the control strain. Additionally, biotransformation of benzaldehyde to 2‐HPP using the engineered strain at higher benzaldehyde concentrations was investigated.

EXPERIMENTAL PROCEDURES

Enzymes, reagents, plasmid and microorganism

All chemicals were obtained from Merck (Darmstadt, Germany), Sigma‐Aldrich (USA) and Samchun Chemical Co. (Korea). All reagents were of reagent grade, unless otherwise stated. The expression vector pSEVA234 was used to express pprI (Silva‐Rocha et al., 2013). P. putida ATCC 12633 (PTCC 1694) was purchased from the Persian Type Culture Collection (PTCC), Iran. D. radiodurans R1 ATCC 13939 (IBRC‐M 10806) was obtained from the Iranian Biological Resource Center (IBRC).

Growth conditions

Pseudomonas putida ATCC 12633 was grown in lysogeny broth (LB) medium [5 g L−1 yeast extract, 10 g L−1 tryptone and 10 g L−1 NaCl; 20 g L−1 agar was added to the solid medium when needed]. The mandelate medium, which contained 3 g L−1 ammonium mandelate, 0.2 g L−1 nitrilotriacetic acid, 0.58 g L−1 MgSO4.7H2O, 0.067 g L−1 CaCl2.2H2O, 0.0002 g L−1 (NH4)6 MO7O24.4H2O, 0.002 g L−1 FeSO4.7H2O, 3.4 g L−1 KH2PO4, 6.7 g L−1 Na2HPO4.12H2O and 1 g L−1 yeast extract, was used for the induction of benzoylformate decarboxylase synthesis and mandelate pathway (Hegeman, 1966).

The pprI gene was sourced from D. radiodurans, which was cultivated aerobically on a rotary shaker at 30°C (pH 7.2) using tryptone glucose yeast extract broth (TGYB), containing 5 g L−1 tryptone, 10 g L−1 yeast extract, 1 g L−1 K2HPO4 and 1 g L−1 glucose; 20 g L−1 agar was added when needed. Defined medium broth (DMB) for shaken‐flask cultivation contained 6 g L−1 Na2HPO4, 3 g L−1 KH2PO4, 2 g L−1 NH4Cl, 0.2 g L−1 MgSO4·7H2O, 0.015 g L−1 CaCl2·2H2O and 1 mL trace element solution (1.5 g L−1 FeCl3·6H2O, 0.15 g L−1 H3BO3, 0.03 g L−1 CuSO4·5H2O, 0.18 g L−1 KI, 0.12 g L−1 MnCl2·4H2O, 0.06 g L−1 Na2MoO4·2H2O, 0.023 g L−1 NiCl2·6H2O, 0.12 g L−1 ZnSO4·7H2O, 0.15 g L−1 CoCl2·6H2O and 10 g L−1 EDTA). The medium was supplemented with 5 g L−1 glucose as the sole carbon source (Yu et al., 2016). Bacterial liquid cultures were incubated with shaking at 200 rpm, and kanamycin was added at 50 μg mL−1 to the medium when required.

Construction of a recombinant plasmid for pprI expression in P. putida

Genomic DNA was extracted from actively growing cultures of D. radiodurans following a previously described procedure (Norais et al., 2013). The gene encoding PprI containing 987 bp was amplified by PCR using the forward (5′‐TAT AGA ATT C AG GAG GAA AAA CAT ATG CCC AGT GCC AAC G‐3′) and reverse primers (5′‐ATA TAA GCT TTC ACT GTG CAG CGT CCT GC‐3′). An EcoRI restriction site at the start codon and a HindIII restriction site after the stop codon (underlined in reverse primers) were introduced into the primers. Due to the absence of a ribosomal binding site in the vector pSEVA234, the Shine–Dalgarno motif was placed before the start codon as a ribosome‐binding site (bold in forward primer). The PCR product was then electrophoresed on an agarose gel to confirm that reaction worked. Finally, the DNA fragment encoding PprI was isolated from the agarose gel. The fragment was then ligated into a linear pSEVA vector using T4 ligase (Thermo Fisher Scientific). This new construct was named pSEVA‐PprI. The construct pSEVA‐PprI was introduced into the competent P. putida ATCC 12633 cells using the calcium chloride transformation method. This new strain was named P‐PprI. An empty vector was also transformed into P. putida as a control strain.

Heterologous expression of pprI

P‐PprI as well as the control strains were grown at 30°C in LB medium in two 250 mL flasks containing 50 mL of LB. Batch cultures were incubated at 30°C with shaking (200 rpm) until the early exponential phase (OD600 ~0.4) was reached. The early exponential phase cells were induced with 0.1 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG). To confirm the heterologous expression of the gene encoding the PprI protein, the cells from 40 mL of culture were harvested by centrifugation 4 h after the addition of IPTG and frozen at −80°C until use. To extract soluble proteins, the frozen pellets were resuspended in 6 mL of pre‐cooled 10 mM Tris–HCl (pH 8.0) disrupted by mild sonication at 4°C and centrifuged at 12,000 × g for 20 min. The soluble proteins recovered in the supernatant phase were analysed using 12% (w/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). For the SDS‐PAGE analysis, 20 μL of the soluble extract was mixed with 6 μL of loading buffer and heated at 95°C for 5 min. After a brief mixing, the mixture was loaded onto a gel.

To extract total protein (soluble and insoluble proteins), the frozen pellets derived from a 40 mL cell culture were resuspended in 6 mL of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8). To 100 μL of the crude lysate, 20 μL of loading buffer containing β‐mercaptoethanol was added directly. The solution was heated for 10 min at 95°C and centrifuged at 4°C, 21,000 × g for 15 min to remove cell debris (Nikel et al., 2008). The gel was prepared using a 12% (w/v) polyacrylamide gel according to Laemmli's protocol in a vertical slab gel apparatus (Bio‐Rad Laboratories), loaded with 20 μL of the supernatant and stained with Coomassie Blue R‐250 (Laemmli, 1970). To assess the solubility of the PprI protein, the intensity of the corresponding bands in the total protein extracted from each strain was compared with that of the soluble fraction.

Assessing the effect of heterologous expression of pprI gene on the cell growth

To examine the effect of the pprI gene expression on growth, the recombinant and control strains were inoculated into an Erlenmeyer flask containing DMB at a ratio of 1:5. IPTG was added when the OD600 of the cells reached 0.4. Growth rate was monitored by measuring the change in optical density at 600 nm every hour.

Tolerance of the engineered strain to different stress conditions

All cell growth and survival studies were performed under various abiotic stress conditions using previously described methods with some modifications (Gallo et al., 2021). Therefore, the following experiments were performed to investigate the effects of PprI introduction on the stress tolerance of the P. putida cells.

First, a single colony of both the recombinant and the control P. putida strains was inoculated into 10 mL of LB liquid medium supplemented with kanamycin (50 μg mL −1). Precultures were incubated for 16 h at 30°C and 200 rpm. Following this, fresh 50 mL LB cultures supplemented with antibiotics were inoculated with the precultures to an initial OD600 of 0.08 and incubated at 30°C and 200 rpm until OD600 reached 0.4. The PprI expression was then induced with 0.1 mM IPTG, and the cultures were incubated at 30°C and 200 rpm for an additional 4 h. The growing cells were then diluted into fresh LB medium containing 50 μg mL−1 kanamycin with an initial OD600 of 0.05 and distributed to 24‐well plates (1 mL per well) supplemented with 0.1 mM IPTG.

The cells were then exposed to different treatments (i) acetaldehyde, 0, 50, 100, 150, 200, 250 mM; (ii) benzaldehyde, 0, 10, 15, 20, 25, 30 mM; (iii) butanol, 0, 0.5, 1, 1.25, 1.5% (v/v); (iv) ethanol, 0, 2, 3, 4, 5, 6% v/v; (V) NaCl, 0, 100, 200, 300, 400, 500, 550, 600, 650, 700, 750 mM; (vi) H2O2, 0, 4, 6, 8, 10, 12, 15 mM; and (vii) heat, 50°C for 0, 10, 20, 30, 40, 50 min. The viability and growth of P‐PprI were tested under the stress conditions and compared with those of the control strain. Growth in each well was determined by measuring the absorbance at OD600 using a microplate reader (BioTek 800 TS) after 16 and 24 h. The minimum inhibitory concentration (MIC) endpoint for each strain was determined as the lowest concentration of each treatment at which bacterial growth was completely inhibited. The experimental setup was prepared independently and measured in triplicates.

In addition, the effect of benzaldehyde treatment on the growth curves of the engineered and control strains was evaluated using a modified protocol described previously by Nikel et al. (2010) and Lieder et al. (2015). In brief, a single colony of the transformed strain was directly inoculated from lysogeny agar (LA) plates into 50 mL of DMB medium containing the appropriate amount of antibiotic. At an OD600 of 0.04, IPTG was added to the Erlenmeyer flasks at a final concentration of 0.1 mM, while the flasks were maintained at 30°C and 200 rpm. When the cells were in the middle of their exponential growth phase and the absorbance reached nearly 1.8, benzaldehyde was added at concentrations of 15 and 20 mM. Finally, cell growth was monitored by turbidity measurements using a biophotometer (Eppendorf) for several days. The cultures were diluted with 9 g L−1 NaCl as necessary.

Spot plating assay

Spot plating assay was performed to enumerate the colonies formed by P‐PprI and the control strain containing an empty vector, following exposure to various stressors.

A single colony of both P‐PprI and control P. putida strains was inoculated into 10 mL of LB liquid medium supplemented with kanamycin (50 μg mL−1) in a 50 mL Falcon tube. Precultures were incubated for 16 h at 30°C and 200 rpm. Subsequently, fresh 50 mL LB cultures supplemented with antibiotics were inoculated with the precultures to achieve an initial OD600 of 0.08 and incubated at 30°C and 200 rpm until OD600 reached 0.4. The PprI expression was then induced with 0.1 mM IPTG, and the cultures were incubated at 30°C and 200 rpm for an additional 4 h.

During the mid‐exponential growth phase, acetaldehyde, benzaldehyde, H2O2, NaCl and ethanol were individually added to each culture at concentrations of 50 mM, 15 mM, 10 mM, 750 mM and 7%, respectively. The cultures were then maintained at the same conditions for additional 24 h.

An aliquot from each culture was taken and serial dilutions were prepared to obtain dilutions ranging from 10−1 through 10−7 in deionized water. Ten microliter aliquots of each dilution were then spotted onto an LB agar containing 50 μg mL−1 kanamycin. The plates were then allowed to briefly dry, inverted and placed in an incubator at 30°C. After 24 h of incubation, single colonies were counted, and calculations were performed to determine the colony‐forming units (CFU) mL−1 using the following formula:

CFUmL−1=Number of colonies×1/dilution factor×1,000μLmL−1volume plated.

Whole‐cell biotransformations of benzaldehyde and acetaldehyde to 2‐HPP

The engineered (P‐PprI) and control P. putida cells were used as hosts for the whole‐cell bioconversion of benzaldehyde and acetaldehyde to 2‐HPP. Whole‐cell enzyme resting cells were prepared in several consecutive steps for the bioconversion of substrates to 2‐HPP. First, either an engineered or a control strain of P. putida was inoculated into 100 mL Erlenmeyer flasks containing mandelate medium and incubated at 30°C for 16 h. The final concentration of IPTG (0.1 mM) was added when the absorbance of the cells reached an OD600 of 0.4 to induce protein expression. Kanamycin was then added to facilitate plasmid retention, where appropriate.

Subsequently, the seed culture was prepared by inoculation of 10 mL of primary culture containing the P‐PprI and control strains into 100 mL of the same medium in a 500 mL flask. IPTG (0.1 mM) was added during the initial stage of fermentation to induce protein expression. The culture was incubated at 30°C and 200 rpm after induction for an additional 4 h until the OD600 of the culture reached approximately 0.9.

Induced recombinant cells were cultivated by centrifugation (10,000 × g for 10 min at 4°C) and washed twice with 50 mM sodium phosphate buffer (pH 6). These bacterial pellets were used as whole‐benzoylformate decarboxylase biocatalysts to produce 2‐HPP through biotransformation. 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. The effect of initial whole‐cell concentration on the 2‐HPP formation during the biotransformation was also examined. A minimum cell load of 0.033 g WCW mL−1 [0.006 g cell dry weight (CDW) mL−1], corresponding to an OD600 of 20, was selected and the 2‐HPP production was evaluated.

The bioconversion assay was carried out aerobically in a 100 mL Erlenmeyer flask at 30°C with shaking at 200 rpm. During this process, equal portions of the bacterial cultures were withdrawn at specified time intervals and centrifuged at 10,000 × g for 10 min at 4°C to remove the precipitates. Next, 2‐HPP and benzaldehyde in the supernatant were extracted with equal volumes of dichloromethane, followed by vigorous vortexing for 10 min and centrifugation for another 10 min at 20,000 × g at 4°C. The aqueous and organic phases were separated and the lower organic phase containing the desired product was removed using a syringe.

Analysis of 2‐HPP and benzaldehyde

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).

Plasmid stability

Plasmid segregational stability was studied in P. putida as previously described (Volke, Friis, et al., 2020).

RESULTS AND DISCUSSION

Heterologous expression of the gene encoding PprI in P. putida

The nucleotide sequence obtained by PCR from the genomic DNA of D. radiodurans was 100% identical to the open reading frame of gene encoding PprI (WP_010886813) with 987 bp. The translated protein has 328 amino acids, with a theoretical molecular mass (kDa) and pI of 34.7 and 5.3, respectively. The coding sequence of pprI was cloned into pSEVA234 to produce PprI recombinantly in P. putida upon induction with IPTG. A band with the expected molecular weight for PprI was detected in the P‐PprI strain, whereas it was not observed in the control strain (Figure S1). In other studies, the expression of PprI encoding gene with a molecular mass of 36 kDa was confirmed in E. coli strain MGBC‐PprI and Zymomonas mobilis carrying plasmid pBBR‐PprI (Zhang et al., 2010). A robust recombinant strain of Sphingomonas sp., deficient in carotenoid synthesis, was constructed via integration of the irrE gene into the crtB locus, a key gene involved in the synthesis of carotenoids. This strain produced welan gum, a widely used bacterial exopolysaccharide (EPS), in the absence of carotenoid synthesis. The expression of the target protein was identified by SDS‐PAGE, and a band with a molecular mass of approximately 32 kDa was visualized (Liu et al., 2020). The pprI gene expression was also confirmed in P. putida S16 harbouring the recombinant plasmid pME‐GirrE (Zhou et al., 2019). In another study (Song et al., 2018), pprI was successfully expressed in A. simplex with an apparent molecular mass of 37 kDa.

Importantly, under these conditions, the pSEVA234 vector used for expression of pprI was highly stable, and >99.5% of the cells retained the plasmid after 48 h of incubation in LB medium, as assessed by plating the culture and dilutions thereof in the presence or absence of kanamycin (the antibiotic resistance marker in vector pSEVA234 and its derivatives).

The heterologous expression of the pprI gene confers tolerance in response to different stresses

Pseudomonas putida ATCC 12633 suffers from low tolerance to aldehydes. For instance, a benzaldehyde concentration higher than 20 mM and an acetaldehyde concentration above 400 mM seem to denature enzymes and reduce the reaction activity toward 2‐HPP (Simmonds & Robinson, 1998; Wilcocks et al., 1992). Moreover, P. putida ATCC 12633 cannot grow at benzaldehyde concentrations higher than 8 mM in the presence or absence of yeast extract, suggesting that the chaotropic effect of benzaldehyde is not pathway‐ or condition‐specific (Simmonds & Robinson, 1998). Aldehydes are electrophilic compounds that act as chaotropic stressors and can cause damage to macromolecules (Aranda & del Olmo, 2003; Jayakody & Jin, 2021). Their cellular stress and chaotropicity pose a practical limit on the achievable 2‐HPP titers, making them an issue of interest for both academia and industry. Acetaldehyde is a chaotropic solute that can destabilize macromolecular systems, result in single or double breaks in genomic DNA (Jayakody & Jin, 2021; Singh & Khan, 1995) and affect numerous metabolic activities (Aranda & del Olmo, 2003). Chaotropic solutes can also stimulate oxidative stress in cells as a secondary effect and trigger accumulation of reactive oxygen species (ROS) (Hallsworth et al., 2003; Kunjapur & Prather, 2015; Singh & Khan, 1995).

Therefore, enhancing tolerance is essential toward a robust 2‐HPP‐producing strain. To achieve this, the present study aimed to heterologously express the gene encoding the prokaryotic regulator PprI of D. radiodurans in a 2‐HPP‐producing P. putida strain. Since pprI encodes a global regulator protein, its expression could lead to the enhanced tolerance toward many stressors. Consequently, the engineered P. putida expressing pprI was exposed to various physicochemical challenges, including benzaldehyde, acetaldehyde, butanol, ethanol, salt, H2O2 and heat treatment, to evaluate its enhanced tolerance.

The heterologous expression of pprI indeed caused an increase in resistance of the P‐PprI strain against acetaldehyde up to 50 mM. In contrast, cells of the control strain were unable to survive at this concentration. The MIC values for acetaldehyde were 100 and 50 mM for P‐PprI and the control strain, respectively, as depicted in Figure 1A.

An external file that holds a picture, illustration, etc. Object name is MBT2-17-e14448-g007.jpg

Growth (expressed as OD600) of the strain carrying PprI (P‐PprI, black) and the control strain containing an empty vector (grey) in LB medium at 30°C and 200 rpm after 24 h. The strains were exposed to different abiotic stresses including (A) acetaldehyde, (B) benzaldehyde, (C) butanol and (D) ethanol. Average values from three biological replicates are shown, with error bars representing standard deviations. Statistical analysis was performed using two‐way ANOVA; significant differences are indicated as *p < 0.05; ***p < 0.001 and ****p < 0.0001.

The chaotropic activity of chaotropic stressors such as benzaldehyde can have many individual stress‐induced effects on the cell (Bhaganna et al., 2010). Benzaldehyde is an aromatic aldehyde that induces cell death by disrupting the integrity of bacterial cell membranes, leading to morphological changes and inhibition of microbial cell growth (Kunjapur et al., 2014; LoPachin & Gavin, 2014; Zaldivar et al., 1999). In this investigation, the recombinant cells expressing pprI were subsequently found to confer significant cross‐tolerance to two of the most potent inhibitors, acetaldehyde and benzaldehyde, that inhibit bacterial cell growth and have irreversible inhibitory effects on the benzoylformate decarboxylase enzyme activity (Iding et al., 2000; Simmonds & Robinson, 1998). Whereas benzaldehyde inhibited the growth of the control strain at 10 mM, the inhibitory effect of benzaldehyde for the P‐PprI strain was only observed at 15 mM. Moreover, the P‐PprI strain displayed higher final OD600 values (cell densities) at all the benzaldehyde concentrations above 10 mM when compared to the control strain (Figure 1B).

Since benzaldehyde is the main stressor substrate used in the production of 2‐HPP, the growth profiles of the P‐PprI strain and P. putida ATCC 12633 in the presence of two inhibitory levels of benzaldehyde, 15 and 20 mM, were also investigated. As shown in Figure 2, all the cells experienced comparable growth rates in the exponential phase. However, the P‐PprI strain exhibited continued growth beyond this phase, whereas the control strain failed to adapt to the stressful conditions and stopped growing. Consequently, the engineered strain demonstrated a significantly higher final OD600, with an approximately 2‐ and 4‐fold increase at 15 mM and 20 mM benzaldehyde concentrations, respectively, in comparison to the control strain.

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Growth curves of strain P‐PprI and the control strains in the presence of 15 and 20 mM benzaldehyde in the medium. Cultures were performed in DMB medium at 30°C and 200 rpm. Data are results of triplicate experiments ± standard deviations.

Improved tolerance and cell survival after exposure to aldehyde substrates is a complicated trait, and the underlying molecular mechanisms remain largely unknown. The DNA repair machinery is likely part of the aldehyde detoxification processes that help to alleviate the growth inhibition caused by aldehydes (Xie et al., 2016). This mechanism also increases the levels of cellular antioxidant metabolites and enzyme activities, which can reduce aldehyde‐mediated membrane damage and apoptosis caused by ROS. In addition, other regulatory mechanisms participate in cellular macromolecule defence pathways, central metabolism and redox shuttles (Jayakody et al., 2018; LoPachin & Gavin, 2014). The expression of many genes related to stress‐responsive proteins, growth‐related proteins, protein kinases, transcriptional regulators, signal transduction and detoxification proteins is probably affected (Lu et al., 2009). Nevertheless, further studies are required to elucidate the molecular mechanisms underlying the pprI expression that contribute to the enhanced aldehyde tolerance in P. putida cells.

Organic solvents can directly or indirectly damage membrane lipids, proteins and genomic DNA (Weber & de Bont, 1996). Chaotropes, such as ethanol and butanol, can cause cellular leakage due to an increase of cell membrane permeability, alteration of Ca2+ homeostasis and oxidative stress (Bhaganna et al., 2010; Cray et al., 2015). Hence, numerous attempts have been made to enhance the tolerance of microorganisms to organic solvents (Schalck et al., 2021; Zhang et al., 2015).

Our results did not show a significant increase in cell viability in the presence of butanol of the engineered strain when compared to the control strain. Both the recombinant and the control strains exhibited an MIC value of 1% (v/v) butanol, which impeded any growth for both strains (Data S1). Nevertheless, after 24 h, the cells containing the exogenous gene showed superior growth at 1% (v/v) butanol and reached an absorbance level of 0.355, whereas no further growth was detected for the control strain (Figure 1C). PprI simultaneously enhanced cell resistance to various stresses in a stress‐ and strain‐dependent fashion, with the butanol resistance phenotype resulting from multigene regulation (Song et al., 2018). This paradoxical finding possibly arises from the fact that some pathways activated by PprI might not be specific to the butanol stress tolerance phenotype (Cuenca et al., 2016; Ramos et al., 2015). An artificially evolved exogenous global regulator may be a suitable strategy to confer cross‐tolerance to butanol in Pseudomonas strains. Laboratory‐evolved PprI mutants have been shown to improve E. coli tolerance to ethanol and butanol stress (Chen et al., 2011).

Ethanol is an important chaotropic compound that inhibits β‐galactosidase activity and reduces water activity (Bell et al., 2013; Bhaganna et al., 2010; Hallsworth, 1998; Hallsworth & Nomura, 1999). The P‐PprI strain also showed the improved tolerance to ethanol. Whereas the growth of control cells was completely restrained at 5% (v/v) ethanol after 16 h, the recombinant strain tolerated 4 and 5% (v/v) ethanol and proliferated well (Data S1). Additionally, the OD600 of P‐PprI reached a maximum of 0.65 after 24 h in the presence of 6% (v/v) ethanol, whereas the control strain displayed significantly impaired growth (Figure 1D). Helalat et al. (2019) reported a similar cross‐protection role of the pprI gene in increasing the tolerance of different pprI‐expressing cells of S. cerevisiae to a range of compounds, including ethanol, salt and butanol. The authors also confirmed an increase in the yield of biofuel production.

The saline and osmotic stressor, NaCl, modifies the cell's osmotic balance. Various concentrations of NaCl were applied to evaluate the osmotic status of the cells and assess their resistance to salts. The cell growth rate was monitored for 16 h (Data S1) and 24 h (Figure 3A). The P‐PprI strain showed a significantly higher tolerance to NaCl and higher cell growth than the control strain. The control strain exhibited slightly improved growth at salt concentrations of up to 400 mM, whereas the P‐PprI strain demonstrated remarkable growth compared to the control strain when exposed to the salt concentrations between 500 and 750 mM. The P‐PprI strain was able to tolerate up to 750 mM NaCl and grew well while the control strain displayed impaired growth. Tolerance to osmotic stressor is regulated by several unidentified genes and molecular mechanisms. Following exposure to hyperosmotic conditions, the expression of protein kinases, such as the Ndk‐encoding gene, is likely to be upregulated in the P. putida cells expressing pprI (Lu et al., 1995; Pan et al., 2009). Accordingly, the transcription levels of trehalose biosynthetic genes are elevated in the pprI‐expressing microorganisms and trehalose accumulation can serve as a crucial osmoprotectant disaccharide under osmotic stress conditions (Ma et al., 2011). On the other hand, ATP level is upregulated in the cells with activated PprI in the presence of salt or ethanol stress as an index of cell energy and expression of genes involved in energy generation processes (Chen et al., 2012; Pan et al., 2009; Zhao et al., 2015). The P. putida cells synthesize and accumulate low‐molecular‐weight hydrophilic compounds such as glycerol and trehalose. These compounds can protect their macromolecular machinery from the adverse effects of hydrophilic chaotropes like ethanol and benzyl alcohol, as well as hydrophobic substances such as benzene and toluene. Additionally, they help mitigate the inhibitory impact of kosmotropic stressors, such as NaCl, under challenging conditions (Bhaganna et al., 2010, 2016). The content of these intracellular compatible solutes, that is, trehalose and glycerol, is further stimulated by the pprI gene in the recombinant strains following exposure to ethanol and salt osmotic conditions (Chen et al., 2012; Ma et al., 2011; Pan et al., 2009; Song et al., 2018; Zhao et al., 2015). Therefore, it appears that the expression of the pprI gene in different cells intensifies organism's tolerance to ethanol and salt, based on the findings of the current study and previous studies (Ma et al., 2011; Pan et al., 2009; Zaldivar et al., 1999; Zhang et al., 2010; Zhao et al., 2015).

An external file that holds a picture, illustration, etc. Object name is MBT2-17-e14448-g003.jpg

Growth (expressed as OD600) of the strain carrying PprI (P‐PprI, black) and the control strain containing an empty vector (grey) in LB medium at 30°C and 200 rpm after 24 h. The strains were exposed to different abiotic stresses including (A) sodium chloride (NaCl), (B) oxidative hydrogen peroxide (H2O2) and (C) heat (50°C). Average values from three biological replicates are shown, with error bars representing standard deviations. Statistical analysis was performed using two‐way ANOVA; significant differences are indicated as *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001.

Some cellular stressors such as oxidative damage and extreme temperature can reduce or prevent cell functions, impair or damage cell membranes and macromolecules, and may be lethal (Noel et al., 2023). Hydrogen peroxide (H2O2) is an ROS that can damage the cell membrane and the crucial biomolecules, including DNA and proteins (Misra & Fridovich, 1976). Overexpression of pprI can significantly and specifically induce pprA and recA expression in Deinococcus spp. (Hua et al., 2003). PprA, a pleiotropic protein‐promoting DNA repair, has been reported to stimulate a catalase activity in E. coli, leading to the enhanced levels of antioxidant metabolites in cells and increased tolerance to hydrogen peroxide (Kota & Misra, 2006). Moreover, PprI has a significant effect on the oxidative stress response of Deinococcus by improving the RecA encoding gene expression and promoting the activity of KatG, an enzyme with a catalase‐peroxidase function (Hua et al., 2003).

Therefore, the P‐PprI and control strains were examined for their responses to H2O2 stress (Figure 3B). The P‐PprI strain tolerated up to 12 mM H2O2 and grew well, whereas the control strain could only survive at H2O2 levels below 8 mM. With 8 mM of the oxidative stress agent, the OD600 for the P‐PprI strain was 1.03 after 24 h, while the control strain only grew up to 0.14. The results after16 h of exposure to H2O2 display a similar trend, as shown in the Data S1 (Figure S3F). Similarly, Gao et al. (2003) showed that the heterologous expression of pprI confers tolerance in E. coli to H2O2.

We further studied the effect of heterologous pprI expression on P‐PprI tolerance to thermal stress. Therefore, the P‐PprI and control strains were incubated at 50°C for different time intervals, and the temperature was then shifted to 30°C for 16 h to examine heat stress survival. The control strain was unable to grow under optimum culture conditions after being exposed to 50°C for 50 min, whereas the P‐PprI strain exhibited the ability to grow in lysogeny broth (LB) medium after 16 h despite that the same thermal stress was applied (Figure 3C). Moreover, the P‐PprI strain was able to grow in LB medium after 16 h even when they had been exposed to 50°C for as long as 50 min, while the control strain was not able to grow under such conditions (Figure 3C). The minimum inhibitory time for the control strain was 50 min at 50°C, after which it failed to grow even under optimum culture conditions.

Previous studies have shown that the expression level of sufB increased in a pprI‐expressing strain of E. coli in response to cellular damage. This gene can subsequently alleviate the adverse effects of osmotic pressure and heat stress conditions in microorganisms (Gunasekera et al., 2008; Song et al., 2018). Furthermore, stress‐inducible ATP‐dependent protease can be induced as a key protein at high temperatures by the pprI‐expressing microorganisms (Han & Lee, 2006). Therefore, PprI potentially can protect P. putida from a thermal stress.

These results suggest that the gene encoding PprI regulates a cluster of genes with functions related to different metabolic and signalling pathways, ultimately enhancing defence against diverse abiotic stresses, such as oxidative, osmotic and thermal shocks (Pan et al., 2009).

Spot plating assay of cells exposed to stressors

A spot plating assay was performed to further explore improved tolerance of the recombinant strain toward stressors. To this end, the survival of the P‐PprI and control strains was assessed following stressors exposure using spot plating technique. In general, the results revealed that P‐PprI strain grew better and forms more colonies on LB agar medium supplemented with kanamycin compared to the control strain after 24 h of exposure to different stressors (Table 1).

TABLE 1

Spot plating assay for evaluating the tolerance of control and recombinant strains against different stressors.

StressorConcentrationSurvival (CFU mL−1) of Pseudomonas putida Fold‐change in survival
P‐PprIATCC 12633 (control)
Benzaldehyde15 mM3 × 109 2 × 109 1.5
Acetaldehyde50 mM1 × 104 0N.A.
Ethanol7% (v/v)4 × 107 6 × 106 6
H2O2 10 mM2 × 104 1 × 104 2
NaCl750 mM5 × 1012 2 × 1012 2.5

Evaluating the cell survival in the presence of the aldehyde showed that the strain P‐PprI formed 50% more colonies compared to the control strain when exposed to 15 mM benzaldehyde. Moreover, the control strain did not form any colonies on the medium containing 50 mM acetaldehyde, which supports the results obtained in liquid cultures. Further, our spot plating assay suggested enhanced tolerance of the P‐PprI strain upon exposure to 7% (v/v) ethanol as the number of colonies for the recombinant strain increased by 5.7‐fold compared to the control strain. The enhanced phenotype was also verified under oxidative stress conditions, since the total number of the colonies on a plate containing 10 mM H2O2 was 2‐times higher for strain P‐PprI that for the control strain. Finally, regarding the inhibitory effects of NaCl, we also conducted a spot platting test following treatment of P‐PprI and control strains subjected to 750 mM NaCl. Quantified colonies for both experimental setups support the observed results with in liquid cultures, confirming higher salt tolerance for the recombinant strain as a result of expressing the gene encoding PprI from D. radiodurans.

2‐HPP production by control and engineered resting cells

The development of biocatalytic strategies is of particular interest for the low‐cost and efficient enantiopure synthesis of fine chemicals like α‐hydroxyketones. The most important function of the induced P. putida cells cultivated in a mandelate medium is the bioconversion of aldehyde substrates into the corresponding α‐hydroxyketones such as 2‐HPP. As such, 2‐HPP production was investigated using a biocatalytic strategy that fulfils green chemistry principles using both the recombinant and the control whole cells, starting from an inexpensive aldehyde substrate. Resting cells can be employed for catalysing a carboligation reaction leading to 2‐HPP because their physiological status and metabolic activity are easy to control (de Carvalho, 2017). To this end, P. putida cells were collected after growing in the ammonium mandelate medium and resuspended in sodium phosphate buffer. Thus, an efficient resting cell‐based bioprocess was developed to alleviate growth‐coupled limitations. 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. Increasing the biomass load did not further improve 2‐HPP production.

When the benzaldehyde concentration was increased to 40 mM, 2‐HPP titers of 1.55 and 1.1 g L−1 were obtained for the P‐PprI and control strains, respectively. While the P‐PprI strain produced 50% more 2‐HPP from 40 mM benzaldehyde, the 2‐HPP titers for both strains were still lower than when 20 mM benzaldehyde was used. Notably, an increase in the biomass load correlated with biotransformation efficiency, leading to a 2‐ to 3‐fold increase in the 2‐HPP production in certain cases (Figure 4). For instance, for a biomass load of 0.099 and 0.0165 g WCW mL−1 of recombinant cells, the 2‐HPP titers were 2.9 and 1.1 g L−1, respectively. Under these conditions, >90% retained the PprI‐expressing plasmid.

An external file that holds a picture, illustration, etc. Object name is MBT2-17-e14448-g006.jpg

Effect of cell density on the 2‐HPP production by strain P‐PprI (black) and the control strain containing an empty vector (grey) after 3 h of incubation. Reactions were performed in 100‐mL Erlenmeyer flasks in the presence of 40 mM benzaldehyde and 600 mM acetaldehyde at 30°C and 200 rpm. Average values from three biological replicates are shown, with error bars representing standard deviations. Statistical analysis was performed using two‐way ANOVA; significant differences are indicated as **p < 0.01; ***p < 0.001 and ****p < 0.0001.

The recombinant P. putida consumed most of the benzaldehyde (about 92%) when OD600 reached 60, and a significant amount of 2‐HPP (2.9 g L−1) was excreted after this time. At cell concentrations exceeding 0.099 g WCW mL−1, benzaldehyde was nearly fully utilized, leaving less than 0.3 g L−1 of the unconsumed benzaldehyde remaining (Figure 5).

An external file that holds a picture, illustration, etc. Object name is MBT2-17-e14448-g004.jpg

Effect of biomass density on remaining benzaldehyde concentrations using strain P‐PprI (Black) and the control strain containing an empty vector (grey) after 3 h of incubation. Reactions were performed in 100‐mL Erlenmeyer flasks with 40 mM benzaldehyde and 600 mM acetaldehyde at 30°C and 200 rpm. Average values from three biological replicates are shown, with error bars representing standard deviations. Statistical analysis was performed using two‐way ANOVA; significant differences are indicated as *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001.

However, a further increase in the biomass did not result in a higher production. This was primarily due to the high biomass load, leading to an increased cell aggregation, which might restrain the substrate uptake and a respiratory function of the cells (Lu et al., 2009). Overall, the recombinant strain outperformed the control strain in terms of the 2‐HPP production, particularly in a system with a higher benzaldehyde concentration. This was accompanied by an improved cell viability at the high benzaldehyde concentrations, which is a major stress factor during biotransformation.

The results indicated that a multistress‐tolerant strain can more efficiently produce 2‐HPP relative to the control strain and should be regarded as a promising strategy when designing recombinant Pseudomonas biocatalysts to valorize aromatic and aliphatic substrates producing α‐hydroxyketones.

PprI has been implicated to improve the enzymatic activity of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) as well as the ethanol production in ethanologenic E. coli and Z. mobilis strains (Ma et al., 2011; Zhang et al., 2010). Gómez‐Álvarez et al. (2022) demonstrated an enhanced bioconversion of lignin‐derived aromatics into the building block pyridine 2,4‐dicarboxylic acid by resting cells of engineered P. putida KT2440 overexpressing a transporter (pcaK) and a monooxygenase (pobA).

CONCLUSION

In recent years, there has been a growing interest in exploiting P. putida as a powerful and engineerable host for industrial biotechnology and biocatalytic applications. P. putida is ubiquitous in soil and water, and is therefore exposed to multiple sources of stress. Such exposure to physicochemical challenges has caused evolution of several defence mechanisms over time. However, this bacterium is still sensitive to the high concentrations of some chemicals, such as aldehydes and alcohols. Expression of a gene encoding the global regulator PprI from D. radiodurans in this study conferred tolerance to several chemical and environmental stresses. In particular, the engineered cells tolerated higher concentrations of H2O2 than the control cells. Also, the engineered strain was more tolerant to benzaldehyde and accumulated the highest 2‐HPP titers in a biotransformation process involving acetaldehyde and benzaldehyde as stressor chemicals. Therefore, the engineered strain may be useful for other processes in which cells are exposed to the high concentrations of stressor compounds. However, the mechanisms behind the improved tolerance resulting from the pprI expression are not fully understood, and further research is needed to reveal the molecular mechanisms underlying the phenotypes reported herein.

AUTHOR CONTRIBUTIONS

Reihaneh Kordesedehi: Conceptualization; data curation; formal analysis; investigation; methodology; visualization; writing – original draft. Azar Shahpiri: Data curation; formal analysis; project administration; resources; supervision; validation; visualization; writing – review and editing. Mohammad Ali Asadollahi: Data curation; formal analysis; funding acquisition; project administration; resources; supervision; validation; visualization; writing – review and editing. Davoud Biria: Conceptualization; data curation; validation; writing – review and editing. Pablo Iván Nikel: Data curation; formal analysis; funding acquisition; resources; validation; visualization; writing – review and editing.

FUNDING INFORMATION

Financial support received from Iran National Science Foundation (INSF) through the contract number 4002334 is appreciated. Grants from the Novo Nordisk Foundation [NNF20CC0035580 and TARGET (NNF21OC0067996)] to P.I.N. are gratefully acknowledged.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ETHICS STATEMENT

None declared.

Supporting information

Data S1

Notes

Kordesedehi, R. , Shahpiri, A. , Asadollahi, M.A. , Biria, D. & Nikel, P.I. (2024) Enhanced chaotrope tolerance and (S)‐2‐hydroxypropiophenone production by recombinant Pseudomonas putida engineered with Pprl from Deinococcus radiodurans . Microbial Biotechnology, 17, e14448. Available from: 10.1111/1751-7915.14448 [CrossRef] [Google Scholar]

Contributor Information

Azar Shahpiri, ri.ca.tui@iriphahs.a.

Mohammad Ali Asadollahi, ri.ca.iu.tsa@ihallodasa.am.

DATA AVAILABILITY STATEMENT

The datasets generated during and/or analysed during the current study are included in this published article. They are also available from the corresponding author on a reasonable request.

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