IrrE Improves Organic Solvent Tolerance and Δ1-Dehydrogenation

May 10, 2018 - Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, ... of Science and Technology , Tianjin 300457 , People'...
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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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IrrE Improves Organic Solvent Tolerance and Δ1‑Dehydrogenation Productivity of Arthrobacter simplex Bo Song, Qin Zhou, Hai-Jie Xue, Jia-Jia Liu, Yuan-Yuan Zheng, Yan-Bing Shen, Min Wang, and Jian-Mei Luo* Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China S Supporting Information *

ABSTRACT: During steroid bioconversion, organic solvents are widely used for facilitating hydrophobic substrate dissolution in industry. Thus, strains that tolerate organic solvents are highly desirable. IrrE, a global transcriptional factor, was introduced into Arthrobacter simplex with Δ1-dehydrogenation ability. The results evidenced that IrrE did not affect cell biological traits and biotransformation performance under non-stress conditions. However, the recombinant strain achieved a productivity higher than that of the control strain in systems containing more ethanol and substrate, which coincided with cell viability under ethanol stress, the major stress factor during biotransformation. It also demonstrated that IrrE caused genome-wide transcriptional perturbation, and several defense proteins or systems were linked with higher organic solvent tolerance. IrrE simultaneously enhanced cell resistance to various stresses, and its horizontal impacts showed strain and stress dependence. In conclusion, the introduction of exogenous global regulators is an efficient approach to enhance organic solvent tolerance in steroid-transforming strains, resulting in higher productivity. KEYWORDS: Arthrobacter simplex, organic solvent tolerance, Δ1-dehydrogenation, IrrE, regulatory mechanism

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INTRODUCTION Steroids are biological molecules with a carbon skeleton of 4 fused rings (A−D) and a side chain that has up to 10 carbons which fulfill essential physiological functions and widely exist in nature, such as in plants, vertebrates, insects, and lower eukaryotes.1,2 The microbial Δ1-dehydrogenation plays a crucial role in the early steps of steroid degradation by introducing a double bond into the A ring of 3-ketosteroids.3 However, the amount of organic solvents facilitating hydrophobic substrate dissolution is severely limited in the bioconversion system because of the toxicity at high dosage, which adversely limits substrate amount and consequently decreases productivity. Therefore, strains that tolerate organic solvents are highly desirable.4 A variety of efforts have been made to enhance the organic solvent tolerance of microorganisms, but enhancing the tolerance is still a difficult task because the phenotype is, in many cases, a result of multigene regulation.5 Although effective to some extent, simply overexpressing certain functional genes or pathways does not sufficiently achieve the desired phenotype. On the other hand, the long-term adaptive evolution or traditional mutagenesis or genome shuffling approaches have no such limitation but are highly time consuming and labor intensive and the resulting phenotype is hard to transfer into other strains. Recently, the introduction of transcriptional regulators has been promising for organic solvent tolerance improvement since they directly or indirectly manipulate the transcriptional regulatory network.6 IrrE, a global regulator, is responsible for the extraordinary resistance of Deinococcus radiodurans toward ionizing radiation, UV light, desiccation, and a variety of DNA damaging agents.7−9 Recently, it has been shown that the wild-type irrE © XXXX American Chemical Society

can enhance stress tolerances of non-native hosts. These include irradiative, osmotic, heat, salt, and oxidative stresses for Escherichia coli,10−12 ethanol and acid stresses for Zymomonas mobiliz,13 and salt stress for Brassica napus.12,14 The regulator could be laboratory evolved to enhance E. coli tolerances to organic solvent (ethanol and butanol) and acetate stresses15 or major inhibitors in lignocellulosic hydrolysates.16 Additionally, the genome-wide transcriptome and proteome analysis evidenced that IrrE indeed exerted a global regulation role in E. coli and thus led to greater stress tolerances, such as salt and ethanol tolerance.12,17−19 Nonetheless, the heterologous expression of IrrE was mainly performed on Gram-negative bacteria, and its regulatory mechanism study was restricted in a few model microorganisms (such as E. coli). Little is known about the biological impacts of IrrE on Gram-positive bacteria, especially on steroid-transforming strains. Arthrobacter simplex (also termed Pimelobacter simplex), which belongs to the Actinomycetes group, is a Gram-positive bacteria with an overall high G + C content, which had been originally isolated from soil and showed high selectivity and catalytic efficiency20 in Δ1-dehydrogenation. In this paper, the biological influence, regulatory mechanism, and horizontal transfer of IrrE were investigated, where the bioconversion of cortisone acetate (CA) to prednisone acetate (PA) by A. simplex was selected as a reaction model. Our results are encouraging for its further exploitation in enhancing the stress tolerance of steroid-transforming strains. Our results also Received: March 13, 2018 Revised: April 30, 2018 Accepted: May 4, 2018

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DOI: 10.1021/acs.jafc.8b01311 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Transcriptome Analysis of A. simplex Strains by RNA-seq. The overnight cultures of the control strain and IrrE-expressing strain were inoculated in LB medium supplemented with kanamycin (50 μg/ mL) and 6% ethanol, respectively, for an initial OD600 of 0.02. The cells were grown for 46 h at 32 °C on a rotary shaker (160 rpm) and then harvested to isolate the total RNA using Trizol. Each sample had three biological replicates. The library was constructed using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA), and sequencing was conducted on Illumina HiSeq 2000 instrumentation by the commercial service of Genergy Biotechnology Co. Ltd. (Shanghai, China). Trim Galore software was used to dynamically remove the 3′ linker sequence fragments and low quality fragments from sequencing data, and its quality was checked by FastQC 0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The raw data comprised 100 bp paired-end sequences, and the cleaned reads were then mapped to the P. simplex strain VKM Ac-2033D genome (GenBank: CP009896.1) from NCBI which shared the homology up to 99% with A. simplex CPCC 140451 using Bowtie, with one mismatch allowed. The levels of gene expression were quantified using RSEM software.24 The p values obtained were adjusted according to the Benjamini−Hochberg approach for controlling the false discovery rate (FDR). If the FDR was less than 0.05 and the absolute value of the log2 fold change (Log FC) was not less than 1, the gene was considered as significantly expressed. Compared with the reference genome, significantly enriched GO (Gene Ontology, http://geneontology.org/) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were screened with a threshold of corrected p value ≤ 0.05. Quantitative Reverse Transcription-PCR (qRT-PCR). The RNA samples were reverse-transcribed using the Protoscript First Strand cDNA Synthesis Kit (Takara Bio Dalian Co., Ltd., China) following the manufacturer’s instructions. The nucleotide sequences of primers for the selected and reference genes were designed using DNAMAN software and are listed in Table S1. A real-time PCR was performed using a StepOne RealTime PCR System (Applied Biosystems), and the amplification was performed, starting at 30 s at 95 °C, followed by 40 cycles of heating at 95 °C for 10 s, 60 °C for 30 s, and 95 °C for 15 s, and final extension at 60 °C for 1 min. PCR amplification was detected by SYBR Green fluorescence dye (Takara Bio Dalian Co., Ltd., China). A dilution series of genomic DNA was used as the PCR template to construct a standard curve to quantify the expression levels of the tested genes. The 16S rRNA gene was used as the housekeeping gene (internal control) to normalize for differences in total RNA quantity. Relative gene expression levels were calculated by the comparative Ct method (2−ΔΔCt method)25 and then normalized to the control strain levels. PCRs were carried out in triplicate with each of the three different cDNA samples. ROS and Antioxidant Enzyme Activity, Trehalose, Glycerol, and ATP Determination in A. simplex Strains. The cells from different treatment conditions were collected, washed, and resuspended in PBS buffer. They were then further analyzed using the corresponding commercial kits. The detailed procedure is shown in the Supporting Information. Statistical Analyses. All experimental data are presented as the mean ± standard deviation (SD) with at least three replications for each experimental sample. Statistical analyses of the experimental data were performed by one-way analysis of variance (ANOVA), and p < 0.05 was considered as statistically significant.

implied that IrrE might act as a practical regulatory “part” that operates at a level of complexity much higher than those of local regulators or functional genes in the view of synthetic biology, which would be useful for eliciting cellular tolerances that were otherwise difficult to improve by manipulating individual genes or pathways.

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MATERIALS AND METHODS

Microorganisms, Plasmids, Medium, and Culture Conditions. A. simplex CPCC 140451 and Arthrobacter globiformis ATCC 8010 were purchased from the China Pharmaceutical Culture Collection (CPCC) and the American Type Culture Collection (ATCC), respectively. E. coli DH5α and E. coli BL21 were obtained from TaKaRa (Dalian, China). The shuttle vector pART221 was provided by Cristinel Sandu (The Rockefeller University, USA). The IrrE-expressing strain was constructed by transforming recombinant plasmid pART2-irrE. DNA manipulations, nucleotide sequencing, plasmid construction, and transformation, as well as IrrE expression analysis, are described in the Supporting Information. All recombinant strains and the control strains bearing a blank plasmid were cultured in LB medium containing kanamycin (50 μg/mL). For A. simplex and A. globiformis strains, cells were cultured at 32 °C and 160 rpm, whereas E. coli cells were cultured at 37 °C and 200 rpm. Bioconversion of CA to PA by A. simplex Cells and Steroid Analysis. The resting cells were prepared in three consecutive steps for bioconversion of cortisone acetate (CA) to prednisone acetate (PA). (1) Inoculation of strains in 50 mL of LB medium in a 250 mL Erlenmeyer flask and cultivation at 32 °C and 160 rpm for 36−40 h. (2) Next, 1 mL of the cultures from step 1 was used to inoculate 50 mL of LB medium in a 250 mL Erlenmeyer flask. The cells were then cultivated until the OD600 of the culture reached 1.2−1.5, and then 0.05% (w/v) CA used as an inducer was added. (3) After incubation for 16 h at 32 °C and 160 rpm, the cells were harvested, washed, and resuspended in PBS buffer to bring the OD600 to 2.0. Next, 30 mL of the cell suspension was added into a 100 mL Erlenmeyer flask and preincubated at 32 °C and 160 rpm for 15 min to maintain temperature equilibration. Five biotransformation systems were used. (1) System I: 2 g/L CA dry powder was directly added. (2) System II: 2 g/L CA dissolved in 4% (v/v) ethanol was added. (3) System III: 6 g/L CA dissolved in 8% (v/v) ethanol was added. (4) System IV: 6 g/ L CA dissolved in 20 mmol/L HP-β-CD was added. (5) System V: 8 g/L of CA dissolved in 10% (v/v) ethanol was added. Then, bioconversion was conducted at 32 °C and 180 rpm. The growing cells were prepared according to the same procedure as steps 1 and 2 for the resting cells. When the OD600 of the cultures reached 2.0, different concentrations of CA and ethanol were added. Here, apart from system III and system V, system VI containing 8% ethanol and 35 g/L CA and system VII containing 10% ethanol and 35 g/L CA were employed. The other bioconversion conditions were maintained. During the biotransformation process, samples were withdrawn at various time intervals and analyzed by a high performance liquid chromatography (HPLC) method developed in our earlier work.22 The initial PA production rate was calculated from the slope of the initial linear portion of the curve.22 Viable Cell Count and Dehydrogenase Activity Analysis of A. simplex Strains. The resting cells (OD600 approximately 2.0) were added to systems II, III, and V, all without CA. After incubation at 32 °C and 180 rpm for 12 or 30 h, the cultures were withdrawn, serially diluted, plated on LB plates, incubated for 2−3 days at 32 °C, and then counted. The dehydrogenase activity was analyzed by the triphenyl tetrazolium chloride (TTC) method according to our previous procedures.23 The relative dehydrogenase activity was expressed by the ratio of the value in the presence of ethanol divided by the value in the presence of PBS buffer at the same time point.23 Cell Growth and Survival Assay under Various Abiotic Stress Conditions. Cell growth and survival under various abiotic stress conditions were analyzed by the common methods described in literature15,16 with some modifications. The detailed procedure is shown in the Supporting Information.

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RESULTS AND DISCUSSION IrrE Expression and Its Biological Effects on A. simplex under a Non-Stress Condition. As shown in Figure S1D, irrE was successfully expressed in A. simplex by Western blot analysis. Under non-stress conditions, IrrE did not affect cell growth behavior or the metabolism ability of glucose. The activity of the KsdD enzyme responsible for the Δ1dehydrogenation reaction in A. simplex was induced by CA and maintained a stable level after 12 h, but there was no remarkable difference between the strains (Figure S2). These B

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Figure 1. PA production curves in different biotransformation systems using the resting cells (A−E) and the growing cells (F−I) as the biocatalysts. (A) System I where 2 g/L CA dry powder without any cosolvent was added. (B) System II where 2 g/L CA solved in 4% (v/v) ethanol was added. (C, F) System III where 6 g/L CA solved in 8% (v/v) ethanol was added. (D) System IV where 6 g/L CA solved in 20 mmol/L HP-β-CD was added. (E, G) System V where 8 g/L CA solved in 10% (v/v) ethanol was added. (H) System VI where 35 g/L CA solved in 8% (v/v) ethanol was added. (I) System VII where 35 g/L CA solved in 10% (v/v) ethanol was added. (1) The control strain. (2) The IrrE-expressing strain. All data for PA production represent the mean values from three independent experiments.

Figure 2. (A) The viable cell count of A. simplex strains after exposure to various reaction systems with or without substrate (CA). (B) The relative dehydrogenase activity of A. simplex strains after exposure to various ethanol concentrations. (1) The control strain. (2) The IrrE-expressing strain.

However, the initial production rate (0.306 g L−1 h−1) in system II was evidently 31.9% higher than that in system I, which could be explained by an increase in the dissolution rate of substrate in the presence of ethanol. In system III, the IrrEexpressing strain not only displayed a much higher initial production rate (0.795 g L−1 h−1) over the control strain (0.347 g L−1 h−1) but also achieved higher PA production (4.85 g/L) at 12 h. Our previous work verified that hydroxypropyl-β-

results revealed that IrrE did not cause biological alteration in unstressed A. simplex. In contrast, IrrE-expressing E. coli displayed a better growth behavior.12,18 Effects of IrrE on Δ1-Dehydrogenation Performance of A. simplex. To lessen the difference in the physiological status between both strains, the resting cells were used as biocatalysts. As shown in Figure 1, both strains displayed a similar PA production process in system I and in system II. C

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Figure 3. (A, B) The cell survival curves of A. simplex strains after exposure to 16% ethanol and 20% methanol. (C, D) The relative OD600 of A. simplex strains after cultivation in LB medium supplemented with ethanol and methanol at different concentrations. (E) The cell viability of A. simplex strains after exposure to various abiotic stresses. (1) The control strain. (2) The IrrE-expressing strain.

cyclodextrin (HP-β-CD) exhibited good biocompatibility with microorganisms when its concentration was less than 30 mmol/ L and widely applied in Δ1-dehydrogenation by A. simplex.26 To explore whether biotransformation performance of both strains was repressed by ethanol, system IV was employed for conversion, where 20 mmol/L HP-β-CD was added as cosolvent instead of ethanol and the CA concentration was correspondingly adjusted to 6 g/L. The PA production curves of both strains followed a similar pattern and achieved the maximum value (approximately 5.0 g/L) at 24 h. Compared with the data from system III, it was clear that the PA yield by the IrrE-expressing strain was still comparable even if the ethanol concentration was elevated to 8%, while the biotransformation performance of the control strain was severely inhibited. In system V, the IrrE-expressing strain reached its highest PA production (6.95 g/L) at 30 h, whereas only traces of PA were detected in the control strain within the entire process of conversion. These observations demonstrated that the IrrE-expressing strain exhibited better biotransformation performance over the control strain in systems containing more ethanol and CA. Effects of IrrE on Cell Viability of A. simplex Strains in Biotransformation Systems. As shown in Figure 2A, after incubation for 12 h in system II, the viable cell count of the IrrE-expressing strain was almost identical with that of the control strain, which had a slight drop but still kept at the same magnitude relative to PBS treatment. It indicated that the complex stresses in system II were too low to produce obvious toxicity to the cells. Upon incubation in systems III and V, a small drop in the viable cell count was observed in the IrrEexpressing strain, but to a much greater extent, a larger drop

was observed in the control strain. Even after 30 h in system V, the viable cell count of the IrrE-expressing strain was still more than 104 CFU/mL whereas the value of the control strain was dramatically reduced to below 10 CFU/mL. The results were in line with the biotransformation data, where the IrrE-expressing strain possessed good performance while the control strain almost lost its ability in system V (Figure 1G). Additionally, the viable cell count of A. simplex strains after exposure to systems without CA was examined, where the biocatalysts suffered only from a single stress (ethanol). The change patterns in viable cell counts of both strains in the systems without CA were very similar to those in corresponding systems with CA. As shown in Figure 2A, more than 4% ethanol produced stressful impacts on cell viability while the IrrE-expressing strain always displayed an ethanol resistance stronger than that of the control strain within the test range. The viable cell counts of each strain after exposure to complex stresses decreased a little but still kept the same order of magnitude as the respective level under the single (ethanol) stress condition. These results indicated that when its concentration exceeded 4%, ethanol became the major stress factor rather than the substrate (CA) or product (PA). The activity of dehydrogenase reflecting basic metabolic ability was measured at different ethanol concentrations. It is obvious that there are concentration-dependent and time-dependent inhibition effects of ethanol on the relative dehydrogenase activities, but the IrrE-expressing strain presented a higher level over the control strain under the same condition (Figure 2B). These results comprehensively indicated that the IrrE-expressing strain displayed better cell survival and metabolic ability when exposed to systems containing challenging substances, which D

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Figure 4. Quantitative RT-PCR analysis of the relative expression levels of the selected genes in the IrrE-expressing strain after exposure to 6% ethanol for 46 h (compared with the control strain). These selected genes are involved in DNA repair (A), heat shock proteins (B), sigma factors (C), oxidative response (D), trehalose biosynthesis (E), glycerol degradation (F), and energy generation (G). The inserts in (E) and (F) are the schematic representations of the trehalose biosynthesis pathway and the glycerol degradation pathway, respectively, in A. simplex. Genes marked in red and green indicate upregulation and downregulation, respectively. Substances marked in blue represent the effector response to the stress.

consequently led to the superior biotransformation performance. IrrE Protects A. simplex Cells against Various Abiotic Stresses. To confirm the protective role of IrrE on A. simplex cells against organic solvents, cell survival after being shocked

by high stress and cell growth in the presence of moderate stress were comprehensively evaluated. The cell viability after exposure to different stress conditions was investigated in our pre-experiments, and a significant difference between both strains was observed when cells were shocked with 16% (v/v) E

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Figure 5. Intracellular reactive oxygen species (ROS) content (A), the activities of antioxidant enzymes (B), the intracellular trehalose content (C), the intracellular glycerol content (D), and ATP content (E) in A. simplex strains under different shock conditions for 1 h. (i) Growth in LB medium for 46 h. (ii) Growth in LB medium supplemented with 6% ethanol for 46 h. (iii) No stress shock for 1 h. (iv) 16% ethanol shock for 1 h. (v) 20% methanol. (1) The control strain. (2) The IrrE-expressing strain. All data represent the mean values from three independent experiments.

hypothetical proteins. Several common sets of genes involved in general stress response are briefly discussed below. (1) DNA repair. The upregulation of RecA (recombinase A) required for homologous recombination repair and UvrD (helicase II) responsible for nucleotide excision repair (NER) in the IrrEexpressing strain under ethanol stress was further verified by qRT-PCR, achieving 10.1-fold time increase for recA and 10.7fold time increase for uvrD (Figure 4). In D. radiodurans, IrrE was found to stimulate recA expression under ionizing radiation.7−9 Gao claimed that IrrE enhanced the radioresistance of E. coli by upregulating RecA expression.10 (2) Heatshock proteins (HSPs). The transcriptome and qRT-PCR experiments all detected significant upregulation of genes involved in the GroELS system and the DnaK system in the IrrE-expressing strain after ethanol exposure (Table S2 and Figure 4). In accordance with our data, the expression levels of a set of HSPs were induced in IrrE-expressing E. coli after salt shock, including DnaK.12 (3) Efflux pumps. RNA-seq results showed that six genes (KR76_03255, KR76_RS12780, KR76_RS13535, KR76_RS20605, KR76_RS26270, KR76_RS28175) encoding TetR/AcrR family transcriptional regulators and one gene (KR76_RS08350) encoding MarR

ethanol or 20% (v/v) methanol for 1 h or were grown in the systems supplemented with no more than 6% of solvent concentration. As expected, the IrrE-expressing strain showed a much stronger tolerance in the sudden or continuous stress conditions (such as ethanol and methanol) relative to the control strain (Figure 3A−D). Interestingly, IrrE simultaneously protected A. simplex cells against other abiotic stresses, including heat, osmotic pressure, salt, and oxidative shocks (Figure 3E). A similar cross-protection role of IrrE was reported in IrrE-expressing E. coli11,12 and IrrE-expressing Z. mobiliz.13 Effect of IrrE on the Transcriptional Profiles of A. simplex Strains in Response to Ethanol. The combined analysis of RNA-seq and qRT-PCR was performed to compare and verify the transcriptional profile changes between both strains under 6% ethanol for 46 h because few cells could be harvested for the control strain under higher ethanol concentrations. Our results showed that 229 differentially expressed genes (DEGs), with 53 genes being upregulated and 176 genes being downregulated, were observed in the IrrEexpressing strain (Table S2). We noted that about 30% of upregulated and 36% of downregulated genes encoded F

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levels; these processes thus could work together to alleviate organic solvent-induced oxidative stress and consequently confer superior stress tolerance in A. simplex. Additionally, the gene suf B (KR76_RS13950) was increased approximately 15-fold in the IrrE-expressing strain. SufB was involved in the Fe−S cluster assembly system, which is known to protect cells from oxidative damage, and its expression levels were upregulated when E. coli suffered from combined osmotic and heat stress conditions.31 There was additional evidence that compatible solutes (such as trehalose, glycerol, proline, glycine, glutamic acid, and betaine) were involved in protection against organic solvent stress.5 As shown in Figure 5C, under non-stress conditions, trehalose and glycerol contents exhibited no obvious difference between the two strains. However, more trehalose and glycerol accumulated in both strains after exposure to ethanol, indicating that the compatible solutes were stimulated by ethanol. Higher contents of trehalose (10.1 mg/g DCW) and glycerol (0.29 mg/g DCW) were detected in the IrrEexpressing strain under ethanol stress, which was boosted by 42.4% and 38.5% compared to the control strain, respectively. In consonance with RNA-seq data (Table S2), qRT-PCR results revealed that the transcriptional levels of trehalose biosynthesis genes, including otsA (trehalose-6-phosphate synthase), otsB (trehalose-6-phosphate phosphatase), and treS (trehalose synthase (maltose converting)), were upregulated while the transcriptional levels of glycerol degradation genes, including glpA (glycerol-3-phosphate dehydrogenases), glpK (glycerol kinase), glpQ (glycerophosphoryl diester phosphodiesterase), and gpsA (1-acyl-sn-glycerol-3-phosphate acyltransferase), were downregulated in the IrrE-expressing strain under ethanol stress (Figure 4E and F). The change patterns in gene expression fitted well in the compatible solute contents (Figure 5C and D), also implying that regulation of compatible solutes by IrrE indeed took place at the level of synthesis rather than at the level of uptake. In line with our findings, the E. coli strain expressing the wild irrE or tailored irrE markedly accumulated trehalose under osmotic stress conditions11 and glycerol or both compatible solutes under salt stress.12,17,18 Our findings together with previous work supported the idea that more accumulation of trehalose and glycerol was an important protectable mechanism. It was noticeable that the contents of the other compatible solutes (such as glutamate, proline, betaine, and ectoine) did not change obviously after ethanol exposure or could not be identified in A. simplex (data not shown). The engagement of energy-dependent processes, such as HSP-based related protein-refolding processes, and the employment of efflux pumps would suggest that energy generation is a crucial process in dealing with and ameliorating organic solvent stress. ATP is generally used as an index of cell energetics, whose loss is observed after the addition of organic solvents.32−34 As shown in Figure 5E, without stress treatment, the cellular ATP level of the IrrE-expressing strain was almost similar to that of the control strain. Nonetheless, after exposure to 6% ethanol for 46 h, a steep decline in ATP content was observed in both strains (25% for the IrrE-expressing strain and 48% for the control strain). Our results provided an indication that energy consumption was the major cause of the biomass drop in the presence of ethanol described in Figure 2. It was then not surprising that the ATP level (19.0 mmol per cell OD600) in the IrrE-expressing strain was much higher than that of the control strain (13.0 mmol per cell OD600), which implied

family transcriptional regulator were all downregulated to some extent (Table S2). TetR/AcrR family transcriptional regulators were the local repressors of the AcrAB-TolC system,27 and MarR transcriptional regulator was thought to negatively regulate AcrAB expression. 28 Therefore, the depressed expression of these genes would likely lead to AcrAB-TolC upregulation, which, acting as an efflux pump, seemed to have a positive relationship with tolerance to both hexane and cyclohexane in E. coli.28 (4) Sigma factors. IrrE was shown to result in a 3-fold greater expression of RpoS in E. coli after salt shock12 or a 6-fold higher expression of rpoE in Z. mobile under ethanol stress.13 In A. simplex, the genome sequence contains no identifiable rpoS gene. However, the expression levels of rpoE and rpoD, having RpoS-like functions, were decreased by 30% and increased 3.4-fold, respectively, through qRT-PCR analysis (Figure 4). These results indicated that the significant alterations in rpoD and rpoE in the IrrE-expressing strain are possible pathways that regulate RpoD- and RpoE-dependent genes in response to ethanol. (5) Signal transduction. The gene KR76_RS15375 encoded protein CheY, which is a response regulator of two-component signal transduction system (TCSTS). The transcriptome data showed that its transcriptional level was depressed by more than 3.0-fold in the IrrEexpressing strain. On the contrary, acidic pH resulted in a moderate increase of cheY in Helicobacter pyloriunder.29 These up- and downregulated genes made it evident that the contribution of stress tolerance by IrrE was not limited to specific genes or pathways but was related to genome-wide transcriptional perturbation. Effects of IrrE on Key Stress-Responsive Metabolite Contents. It is well-known that organic solvents cause oxidative damage to cells due to intracellular reactive oxygen species (ROS) accumulation, which are highly deleterious for microorganisms by damaging DNA, proteins, and membrane lipids.5 As shown in Figure 5A, similar ROS levels were measured between both strains when grown in the absence of ethanol. After exposure to ethanol, the intracellular ROS contents increased accordingly for both strains, while the ROS level in the IrrE-expressing strain was reduced by about 35.1% compared to that of the control strain. These results indicated that the IrrE-expressing strain suffered from much less oxidative damage, which agreed with the cell viability results (Figures 2 and 3). Similar change patterns were also observed in antioxidant enzyme activities and the expression levels of genes associated with oxidative response. Our finding strongly agrees with the reported IrrE protection role in E. coli and B. napus against salt stress and in E. coli against ethanol stress.7,12,14,18 The series of our results together with previous works suggested that ROS is a stress signal with toxic character and its accumulation stimulated the antioxidant defense system. By qRT-PCR analysis, the gene KR76_RS10090 exhibited the maximum upregulation (36.2-fold), which encoded the mycothiol system anti-sigma-R factor implicated in mycothiol biosynthesis. Mycothiol (MSH) is shown to have functions analogous to those of glutathione (GSH) and thus serves as an antioxidant.30 Considering the relationship between ROS accumulation and DNA damage, we supposed that ethanol directly or indirectly induced many cellular damage events, including ROS accumulation and DNA damage. IrrE might directly sense the cellular stress signals and, thus, boost the scavenging capacity for ROS by activating antioxidant systems consisting of enzymes and antioxidants, as well as reinforcing DNA repair processes by upregulating related gene expression G

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Figure 6. A proposed model for the regulation of IrrE on organic solvent tolerance of A. simplex.

responsive metabolite contents and antioxidant enzyme activities were observed when both strains encountered other stressful conditions, such as 16% ethanol or 20% methanol shock for 1 h (Figure 3), but the change ratio of the IrrEexpressing strain to that of the control strain greatly depended on the type of and levels of stress that the cells suffered from. As illustrated by the network diagram (Figure 6), IrrE, functioning as a global transcriptional factor, not only may switch on diverse defense systems, including DNA repair, HSPs, efflux pumps, ROS detoxification, compatible solute accumulation, and energy generation, but also may modulate native global transcriptional factors and signal transduction, producing more defense proteins and other defense pathways directly or indirectly. ROS detoxification plays an irreplaceable role in alleviating oxidative damage imposed by organic solvents. The metabolism of energy is particularly important to provide energy to HSP-based related protein-refolding processes and efflux pump employment. Compatible solute accumulation assists the reduction of damage induced by organic solvents. Although the initial causes of cellular stresses (organic solvents) and the end consequences (cell growth and survival), as well as the enhanced gene expression levels and stress-responsive metabolites in different pathways, are clear, the interrelationships among different functional pathways are yet unclear and await further studies. PA Productivity of the IrrE-Expressing Strain in a Practical Conversion Process. Similar to the bioconversion results from the resting cells, the growing cells of the IrrEexpressing strain exhibited PA production superior to that of the control strain. It was noteworthy that the growing cells were more favorable for PA production than the resting cells. In system VI, the maximum PA production by the IrrE-expressing strain was 14.0 g/L versus only 9.0 g/L for the control strain (Figure 1H). In system VII, the IrrE-expressing strain exhibited a higher productivity and reached a maximum PA of 21.1 g/L at 48 h, which was 51% higher than the PA productivity in system VI, since more ethanol promoted more CA dissolution.

that more energy was elicited in the form of ATP by IrrE to run all adaptive mechanisms and consequently contributed to better growth characterization in the presence of ethanol (Figure 3). Several differentially expressed genes associated with energy production were selected from the RNA-seq data and further verified by qRT-PCR. In the tricarboxylic acid (TCA) cycle, the gene sucB showed the largest increase, achieving an almost 28fold increase in the IrrE-expressing strain. Dihydrolipoamide succinyltransferase (SucB) is a subunit of the 2-oxoglutarate dehydrogenase complex, which is a key enzyme of the TCA cycle.35 In the glycolysis (EMP) pathway, the upregulation of gapA encoding 3-phosphoglyceraldehyde dehydrogenase reached an 6.4-fold increase. Its product catalyzes the reaction from glyceraldehyde-3-phosphate (G3P) to 1,3-diphosphoglycerate (1,3-BPG) by using NAD+ as a coenzyme and, thus, plays a crucial role in catabolic and anabolic carbohydrate metabolism.36 In the oxidative pentose phosphate (PP) pathway, zwf encoding glucose-6-phosphate dehydrogenase (otherwise referred to as G6PDH) and gnd encoding 6phosphogluconate dehydrogenase in the IrrE-expressing strain had about a 3.7-fold and 4.1-fold upregulation, respectively, after ethanol treatment compared to that of the control strain. Their products were responsible for producing most of the cytoplasmic NADPH by catalyzing the two irreversible reactions, glucose-6-phosphate → 6-phospho-gluconate → ribulose-5-phosphate.37 In agreement with our finding, IrrE upregulated the expression levels of genes involved in energy generation processes, including EMP, TCA cycle, and PP pathway, which was linked with greater tolerance in E. coli under salt or ethanol stress.12,17,18 Taken together with the boosted expression levels of genes encoding HSPs and genes associated with efflux pumps, we supposed that solvent-induced damage of cellular macromolecules and the need to repair or resynthesize such molecules would increase energetic needs in response to solvent toxicity. The activated IrrE indeed played a key role in keeping sufficient cellular energetic levels to deal with ethanol stress. Similar alteration patterns in the key stressH

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Figure 7. Survival ratio (A) and the relative OD600 (B) of A. globiformis strains and E. coli strains after treatment with different conditions. (1) The control strain. (2) The IrrE-expressing strain. All data represent the mean values from three independent experiments.

between the assays used, the strain employed, and the stress conditions chosen. On the basis of the above transcriptome and qRT-PCR data, a series of native and single functional genes, showing upregulated expression levels in the IrrE-expressing strain upon ethanol stress, were selected and respectively overexpressed in A. simplex. These genes were involved in DNA repair (uvrD and recA), HSPs (groEL and dnaK), oxidative response (sod and katG), and trehalose synthesis (otsA, otsB, and treS). These overexpressing strains showed a tolerance to organic solvents higher than that of the control strain, but the enhancement effects greatly differed among different genes (unpublished results). These findings further confirmed the involvement of DNA damage repair, heat shock protein, oxidative response, and trehalose accumulation accounting for greater organic solvent tolerance mediated by IrrE. Nevertheless, these overexpressing strains presented an organic solvent tolerance lower than that of the IrrE-expressing strain obtained in this study. Why does the phenomenon appear? The gene manipulation by overexpression of single functional genes precludes the simultaneous exploration of multiple gene modifications and thus often has a limitation in reaching a global phenotype optimum controlled by complex interactions of multiple genes and gene products. As revealed by our transcriptome results, the number of genes regulated by IrrE under ethanol stress in A. simplex exceeded 200, which was much more than the number of genes altered by mutated sigma factors (around 100) in the application of gTME in E. coli for enhancement of ethanol tolerance.38 Thus, IrrE extended the perturbation range of the transcriptional profiles, which might lead to larger improvements of strain tolerances. Further

Surprisingly, there was no detectable PA in the control strain during the entire process (Figure 1). Protection Role of IrrE on Different Types of Microorganisms against Various Abiotic Stresses. A. globisformis and E. coli were selected as the additional hosts because the former that belongs to Gram-positive bacteria is also a typical strain with Δ1-dehydrogenation ability and the latter that belongs to Gram-negative bacteria is the most common model microorganism. After horizontal gene transfer, the IrrE-expressing strain exhibited a resistance much stronger than that of the control strain when challenged with the same stress (Figure 7), which indicated that IrrE has great potential as a practical regulatory “part” in the view of synthetic biology. Interestingly, IrrE-mediated tolerance showed strain and stress dependence. For example, the positive impacts of IrrE on the cell resistance to organic solvents and oxidative stress could be well transferred among the three bacteria, while the enhanced survival after acid and alkaline shock was observed only in IrrEexpressing E. coli and the better survival after heat shock only in IrrE-expressing A. simplex. It was interesting to note that IrrE did not function in Saccharomyces cerevisiae (data not shown), which was due to the fact that prokaryotic regulators were likely inactive in the eukaryotic hosts, and vice versa. These paradoxical findings are possibly due to the idea that, although IrrE often activated a wide range of pathways, some pathways IrrE activated might not be specific for the targeted stress tolerance phenotype since the cell adaptation response to stress was very complex and greatly differed from the type and level of stress as well as microorganisms present.5 We also noted that the data from E. coli strains had some discrepancies with previous studies,11,12,15 which were explained by variations I

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(2) Xiong, L. B.; Sun, W. J.; Liu, Y. J.; Wang, F. Q.; Wei, D. Z. Enhancement of 9 alpha-hydroxy-4-androstene-3,17-dione production from soybean phytosterols by deficiency of a regulated intramembrane proteolysis metalloprotease in Mycobacterium neoaurum. J. Agric. Food Chem. 2017, 65, 10520−10525. (3) Donova, M. V.; Egorova, O. V. Microbial steroid transformations: current state and prospects. Appl. Microbiol. Biotechnol. 2012, 94, 1423−1447. (4) Heipieper, H. J.; Neumann, G.; Cornelissen, S.; Meinhardt, F. Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems. Appl. Microbiol. Biotechnol. 2007, 74, 961−973. (5) Torres, S.; Pandey, A.; Castro, G. R. Organic solvent adaptation of Gram positive bacteria: applications and biotechnological potentials. Biotechnol. Adv. 2011, 29, 442−452. (6) Lin, Z. L.; Zhang, Y.; Wang, J. Q. Engineering of transcriptional regulators enhances microbial stress tolerance. Biotechnol. Adv. 2013, 31, 986−991. (7) Earl, A. M.; Mohundro, M. M.; Mian, I. S.; Battista, J. R. The IrrE protein of Deinococcus radiodurans R1 is a novel regulator of recA expression. J. Bacteriol. 2002, 184, 6216−6224. (8) Hua, Y.; Narumi, I.; Gao, G.; Tian, B.; Satoh, K.; Kitayama, S.; Shen, B. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans. Biochem. Biophys. Res. Commun. 2003, 306, 354−360. (9) Lu, H. M.; Gao, G. J.; Xu, G. Z.; Fan, L.; Yin, L. F.; Shen, B. H.; Hua, Y. J. Deinococcus radiodurans PprI switches on DNA damage response and cellular survival networks after radiation damage. Mol. Cell. Proteomics 2009, 8, 481−494. (10) Gao, G.; Tian, B.; Liu, L.; Sheng, D.; Shen, B.; Hua, Y. DNA Repair 2003, 2, 1419−1427. (11) Ma, R.; Zhang, Y.; Hong, H.; Lu, W.; Lin, M.; Chen, M.; Zhang, W. Improved osmotic tolerance and ethanol production of ethanologenic Escherichia coli by IrrE, a global regulator of radiationresistance of Deinococcus radiodurans. Curr. Microbiol. 2011, 62, 659− 664. (12) Pan, J.; Wang, J.; Zhou, Z.; Yan, Y.; Zhang, W.; Lu, W.; Ping, S.; Dai, Q.; Yuan, M.; Feng, B.; Hou, X.; Zhang, Y.; Ruiqiang, M.; Liu, T.; Feng, L.; Wang, L.; Chen, M.; Lin, M. IrrE, a global regulator of extreme radiation resistance in Deinococcus radiodurans, enhances salt tolerance in Escherichia coli and Brassica napus. PLoS One 2009, 4, e4422. (13) Zhang, Y.; Ma, R. Q.; Zhao, Z. L.; Zhou, Z. F.; Lu, W.; Zhang, W.; Chen, M. irrE, an exogenous gene from Deinococcus radiodurans, improves the growth of and ethanol production by a Zymomonas mobilis strain under ethanol and acid stresses. J. Microbiol. Biotechnol. 2010, 20, 1156−1162. (14) Wang, J.; Guo, C.; Dai, Q. L.; Feng, B.; Zuo, K. J.; Lin, M. Salt tolerance conferred by expression of a global regulator IrrE from Deinococcus radiodurans in oilseed rape. Mol. Breed. 2016, 36, 88. (15) Chen, T. J.; Wang, J. Q.; Yang, R.; Li, J. C.; Lin, M.; Lin, Z. L. Laboratory-evolved mutants of an exogenous global regulator, IrrE from Deinococcus radiodurans, enhance stress tolerances of Escherichia coli. PLoS One 2011, 6, e16228. (16) Wang, J. Q.; Zhang, Y.; Chen, Y. L.; Lin, M.; Lin, Z. L. Global regulator engineering significantly improved Escherichia coli tolerances toward inhibitors of lignocellulosic hydrolysates. Biotechnol. Bioeng. 2012, 109, 3133−3142. (17) Chen, T. J.; Wang, J. Q.; Zeng, L. L.; Li, R. Z.; Li, J. C.; Chen, Y. L.; Lin, Z. L. Significant rewiring of the transcriptome and proteome of an Escherichia coli strain harboring a tailored exogenous global regulator IrrE. PLoS One 2012, 7, e37126. (18) Zhao, P.; Zhou, Z. F.; Zhang, W.; Lin, M.; Chen, M.; Wei, G. H. Global transcriptional analysis of Escherichia coli expressing IrrE, a regulator from Deinococcus radiodurans, in response to NaCl shock. Mol. BioSyst. 2015, 11, 1165−1171. (19) Zhou, Z. F.; Zhang, W.; Chen, M.; Pan, J.; Lu, W.; Ping, S. Z.; Yan, Y. L.; Hou, X. G.; Yuan, M. L.; Zhan, Y. H.; Lin, M. Genomewide transcriptome and proteome analysis of Escherichia coli expressing

studies are required to identify its direct target genes and interacting partners. Taken together, the introduction of an exogenous global regulator, IrrE, could efficiently enhance A. simplex cell tolerance to organic solvents and various abiotic stresses, which consequently could endow steroid-transforming strains with better productivity in systems containing more ethanol and substrate. The comprehensive analysis of transcriptome and key stress-responsive metabolites evidenced that a global perturbation was generated by IrrE and that multiple defense proteins or systems were involved in the improved organic solvent tolerance, including antioxidant system, compatible solute biosynthesis and degradation (such as trehalose and glycerol), energy generation, and general stress responses (such as DNA repair, HSPs, and native global transcriptional factors). In addition, the protective function of IrrE was transferred among various microorganisms and showed obvious strain and solvent specificity. Our finding could provide a new strategy and application example for the construction of industrial steroid-transforming strains with high efficiency.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b01311. Primers used for real-time quantitative PCR in this study (Table S1); the construction workflow of recombinant plasmid and the verification of the recombinant strain (Figure S1); the effects of IrrE on the cell under the nonstress condition (Figure S2); the Δ1-dehydrogenation of cortisone acetate (CA) to prednisone acetate (PA) by A. simplex (Figure S3); HPLC chromatographs of CA to PA bioconversion by the resting cells of A. simplex strains (Figure S4); representation of differentially expressed genes in selected gene ontology (GO) categories in IrrEexpressing A. simplex after exposure to 6% ethanol for 46 h (Figure S5) (PDF) Summary of the differentially expressed genes in the IrrEexpressing strain relative to the control strain (Table S2) (XLSX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (86)-022-60601256. Fax: (86)-022-60602298. Address: 89 PO Box, No. 29, St. No. 13 Tianjin Economic-Technological Development Area (TEDA), Tianjin 300457, P. R. China. ORCID

Jian-Mei Luo: 0000-0003-2012-4417 Funding

This work was financially supported by the National Natural Science Foundation of China (21306138 and 21646017) and the College Student’s Laboratory Innovation Fund Project of Tianjin University of Science and Technology (1704A302). Notes

The authors declare no competing financial interest.

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