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Transcriptome analysis of Clostridium beijerinckii adaptation mechanisms in response to ferulic acid
Accepted Manuscript ¨ Title: TranscriptomePlease check Doc ¨ headfor correctness.–> analysis of Clostridium beijerinckii adaptation mechanisms in response to ferulic acid Authors: Jun Liu, Ting Guo, Tao Yang, Jiahui Xu, Chenglun Tang, Dong Liu, Hanjie Ying PII: DOI: Reference:
S1357-2725(17)30043-2 http://dx.doi.org/doi:10.1016/j.biocel.2017.02.009 BC 5084
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The International Journal of Biochemistry & Cell Biology
Received date: Revised date: Accepted date:
21-11-2016 14-2-2017 22-2-2017
Please cite this article as: Liu, Jun., Guo, Ting., Yang, Tao., Xu, Jiahui., Tang, Chenglun., Liu, Dong., & Ying, Hanjie., Transcriptome analysis of Clostridium beijerinckii adaptation mechanisms in response to ferulic acid.International Journal of Biochemistry and Cell Biology http://dx.doi.org/10.1016/j.biocel.2017.02.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Transcriptome analysis of Clostridium beijerinckii adaptation mechanisms in response to ferulic acid
Jun Liua,b,1, Ting Guoc,*, Tao Yang c, Jiahui Xua,b, Chenglun Tanga,b, Dong Liua,b, Hanjie Yinga,b,*
a
State Key Laboratory of Materials-Oriented Chemical Engineering, College of
Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing 211816, China b
National Engineering Technique Research Center for Biotechnology, Nanjing 211816,
China c
College of Food Science and Technology, Central South University of Forestry and
Technology, Changsha, Hunan Province 410004,China
*Corresponding author: Dr. Hanjie Ying (Professor) Tel: +86 25 86990666; Fax: +86 25 86990001; E-mail: [email protected] Dr. Ting Guo (Associate Professor) Tel: +86 20 84178223; Fax: +86 20 84178223; E-mail: [email protected]
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Highlights
MIC of FA against C. beijerinckii 4693::int was 1.5 g/l.
Viability of C. beijerinckii 4693::int was 106.7% in the presence of 0.5g/l FA.
FA altered expression of genes related antibacterial and adaptation mechanisms.
Enzyme ahpC/F was speculated to be related to the ferulic acid tolerance.
Abstract Clostridium beijerinckii 4693::int with high ferulic acid (FA) tolerance was engineered and characterized in our lab. In this study, the minimum inhibition concentrations of FA against C. beijerinckii NCIMB 8052 (wild-type) and 4693::int were 1.0 and 1.5 g/l, respectively; cell viability was 18.5% and 106.7%, respectively, in the presence of 0.5g/l FA. A comparative transcriptome analysis was carried out at two different growth stages to evaluate sensitivity to FA. Genes that were differentially expressed included those related to redox and associated cofactors, riboflavin metabolism, two-component system, glycolysis and butanoate metabolism, and DNA replication as well as those encoding ATP-binding cassette transporters. Cbei_2134 and Cbei_2135 encoding alkyl hydroperoxide reductases are thought to be involved in antibacterial and adaptation mechanisms in C. beijerinckii in the presence of FA.
Key words: Clostridium beijerinckii, ferulic acid tolerance, metabolism pathway, comparative transcriptome analysis
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1. Introduction Low-cost lignocellulosic materials such as wheat straw, bagasse fiber, and corn fiber have been investigated as substrates for butanol production. However, lignocellulose-derived microbial inhibitory compounds (LDMICs) that suppress Clostridia growth and solvent production are generated along with sugars during pretreatment (Wierckx et al., 2011). Phenolic compounds are particularly potent inhibitors (Guo et al, 2013) since they have various functional groups such as aldehydes, ketones, acids and alcohols and side groups such as the methoxy and hydroxyl groups that enhance their toxicity (Adeboye et al., 2014). Ferulic acid (FA) is a common phenolic compound with a benzene ring, methoxy and hydroxyl groups, and a side-chain double bond, that has potent antimicrobial activity (Ezeji et al., 2007; Guo et al., 2010; Lee et al., 2012). We previously described the generation of Clostridium beijerinckii strain M11 with high FA tolerance (0.9 g/l) by atmospheric pressure glow discharge and high-throughput screening (Liu et al., 2016b). We also demonstrated that the Cbei_4693 gene plays an important role in increasing FA tolerance (Liu et al., 2016a). Cbei_4693 encodes a hypothetical protein predicted to be a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase (FMN + NADPH+ H+ = FMNH2 +NADP+, http://www.genome.jp/dbget-bin/www_bget?cbe:Cbei_4693); this led us to hypothesize that
the tolerance of C. beijerinckii to FA is correlated with NADPH level, although the detailed molecular mechanisms remain unclear.
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To clarify the FA metabolic pathway, we evaluated FA toxicity to C. beijerinckii and analyzed the production of FA metabolites by C. beijerinckii. We also carried out a transcriptome analysis to investigate the antibacterial and adaptation mechanisms of C. beijerinckii in response to FA. We identified alkyl hydroperoxide reductase subunit C (ahpC) encoded by Cbei_2134 and alkyl hydroperoxide reductase subunit F (ahpF) encoded by Cbei_2135 as two factors involved in FA tolerance.
2. Materials and methods 2.1 Cells and culture conditions The wild-type strain C. beijerinckii NCIMB 8052, was purchased from American type culture collection (manassas, AV) and the mutant strain C. beijerinckii 4693::int, was generated by disrupting the gene Cbei_4693 in our laboratory (Liu et al., 2016a) and stored at −80°C was used for experiments. After thawing on ice, 200 μl were inoculated in 10 ml YPS medium (3.0 g yeast extract, 5.0 g peptone, 10.0 g soluble starch, 2.0 g ammonium acetate, 2.0 g NaCl, 3.0 g MgSO4·7H2O, 1.0 g KH2PO4, 1.0 g K2HPO4, 0.1 g FeSO4·7H2O in 1 l H2O) (Guo et al. 2011) and incubated anaerobically at 37°C for 12 h. Cells were sub-cultured in fresh YPS medium with a 5% inoculum size for 8 h to reach an optical density at 600 nm (OD600) of approximately 2.0 (secondary seed cells).
2.2 Evaluation of FA toxicity against C. beijerinckii
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2.2.1 Determination of minimum inhibitory concentration (MIC) Erythromycin was used as a positive reference standard for test strains to determine the MIC of FA against C. beijerinckii. A 4-ml volume of P2 medium (30 g/l of glucose; Liu et al., 2015) containing FA or erythromycin (10 μg/ml) was transferred to sterile 24-well plates in an anaerobic chamber overnight at 37°C to remove the oxygen. A 1-ml volume of secondary seed cells was added to the plates; the final FA concentrations were 0, 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, and 2.0 g/l, with each sample prepared in triplicate. After incubation at 37°C for 48 h in the anaerobic chamber, 2 μl (approximately 105 CFU) of the test strains (C. beijerinckii NCIMB 8052 and 4693::int) were spotted on solid YPS medium without FA, and the plates were incubated for 12 h. The lowest concentration of FA yielding no visible growth of bacteria was taken as the MIC.
2.2.2 Field emission scanning electron microscopy (FESEM) FESEM was carried out as previously described, with some modifications (Li et al., 2014). C. beijerinckii 4693::int was inoculated in 50 ml P2 medium (initial OD600 = 0.5) containing FA at concentrations of 0, MIC, 2MIC. After incubation at 37°C for 8 h in an anaerobic chamber, 10 ml of cells were harvested by centrifugation for 10 min at 8000 ×g and washed twice with 10 ml of 0.85% phosphate-buffered saline, then re-suspended in 1 ml of 2.5% glutaraldehyde and maintained at 4°C for 12 h to fix the cells. Following centrifugation for 10 min at 8000 ×g, cells were dehydrated in a
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graded series of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) for 15 min each and vacuum-dried at 50°C. Samples were analyzed by FESEM (Supra-55; Zeiss, Jena, Germany).
2.2.3 Cytotoxicity assay The antibacterial activity of FA against C. beijerinckii was evaluated with the cytotoxicity assay. The fermentation conditions were the same as for MIC determination except that the P2 medium contained FA at concentrations of 0, 0.5, 0.7, 0.9, 1.0, 1.2, and 1.5 g/l. After incubation for 48 h, 90 μl of sample was transferred to a 96-well plate and 10 μl of Cell Counting Kit-8 (Sigma) were added to each well. Cells without FA served as a control, and fresh P2 medium was used as a blank. Cell viability (%) was calculated as follows: [ODtest − ODblank] / [ODcontrol − ODblank] × 100%. The absorbance was measured at 450 nm using a Multi-Mode Detection Platform (Molecular Devices, Sunnyvale, CA, USA) (Hui et al., 2015).
2.3 Kinetics of FA metabolism P2 medium containing 0.5 g/l FA was used for acetone–butanol–ethanol (ABE) fermentation by C. beijerinckii. The kinetics of FA degradation were investigated by evaluating FA concentration in 5 ml of sample taken from cells grown in 100 ml P2 medium in 250 ml screw-capped bottles without agitation or from a pH control, and the sampling time was 0, 6, 12, and 24 h.
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FA concentration was determined by high-performance liquid chromatography analysis (Agilent 1200 series; Hewlett-Packard, Wilmington, DE, USA) at 280 nm; the mobile phase was 0.3% acetate (70%) and methanol (30%) at a flow rate of 0.8 ml/min, with separation carried out using an Aglient ZORBAX SB-Aq-C18 column (5 μm, 4.6 × 250 mm) at 50°C (Cho et al., 2009). FA metabolites were detected by liquid chromatography mass spectrometry– electrospray ionization (LCMS-ESI) (Timebase U3000-MSQ; Dionex, Sunnyvale, CA, USA) and proton (1H)- and carbon (13C)- nuclear magnetic resonance spectroscopy (NMR) (Nanjing Biopharmaceutical Innovation Platform, Nanjing, China).
2.4 RNA extraction, RNA sequencing (RNA-seq) library construction, and high-throughput sequencing Batch fermentation by C. beijerinckii NCIMB 8052 and 4693::int was carried out in 250-ml screw-capped bottles (two each) containing 100 ml P2 medium (Liu et al., 2015). After inoculation (10% v/v) for 8 h (OD600 = 1.0 ± 0.21), one bottle each of C. beijerinckii NCIMB 8052 and 4693::int was challenged with 0.5 g/l FA as the treatment group, while the other bottle served as the control; 12 h later (i.e., after fermentation for 20 h)—during which time the FA concentration in the medium was reduced by more than half by C. beijerinckii—samples were collected for analysis. Total RNA in each sample was extracted using TRIzol reagent (Takara Bio, Otsu, Japan) according to the manufacture’s protocol. RNA purity and concentration
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were evaluated by 1% agarose gel electrophoresis. Libraries were constructed using the TruSeq RNA Sample Prep kit (Illumina, San Diego, CA, USA). High-throughput sequencing was performed on an Illumina Hiseq 4000 platform by Gene Denovo (Guangzhou, China) (http://www.genedenovo.com).
2.5 Transcriptome assembly and analysis Clean reads were obtained by removing raw reads containing the adaptor or unknown or low-quality sequences; these were then mapped to the C. beijerinckii NCIMB 8052 reference genome using TopHat2 aligner (Kim et al., 2013). Bioconductor edgeR (https://bioconductor.org/) was used for differential expression analysis of RNA-seq expression profiles. Transcripts with greater than 2-fold difference in expression (|log2 ratio| ≥ 1) and false discovery rate ≤ 0.05 were set as thresholds. Differentially expressed genes (DEGs) in FA-challenged relative to control samples were subjected to Gene Ontology (GO) functional analysis (http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (http://www.genome.jp/kegg/).
2.6 Microarray data accession number Microarray data and results have been deposited in the BioSample database, under the following accession numbers: SAMN 05966957-SAMN 05966960.
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3. Results and discussion 3.1 Evaluation of FA toxicity against C. beijerinckii The MICs of FA against C. beijerinckii NCIMB 8052 (MIC8052) and 4693::int (MIC4693::int) were 1.0 and 1.5 g/l, respectively. The OD600 values of the medium containing the two test strains and FA after 48 h were 0.49 ± 0.07 and 0.47 ± 0.01 (as compared to an initial value of 0.50) for MIC8052 and MIC4693::int, respectively. The cytotoxicity assay revealed that the viability of C. beijerinckii 4693::int cells was unaffected by FA concentrations < 0.5g/l, but was reduced to 34.17% at concentrations of 0.7 g/l (18.52% to C. beijerinckii NCIMB 8052 in the presence of 0.5g/l FA) (Fig. 1). Interestingly, the viability of C. beijerinckii 4693::int cells begin keeping the same value, and was lowest, from 1.2 g/l FA in the medium. The concentration of FA that was toxic to C. beijerinckii 4693::int cells (1.2 g/l) was lower than the MIC4693::int (1.5g/l), which was measured during the prolonged lag phase when cell activity was reduced or C. beijerinckii 4693::int was producing spores. To investigate the toxicity of FA to C. beijerinckii in greater detail, we examined changes in cell morphology by FESEM. C. beijerinckii 4693::int cells were treated with FA at concentrations of 0, 1.5, and 3.0 g/l (i.e., 0, MIC, and 2MIC, respectively) for 8 h. Cells exposed to FA had a more wrinkled surface as compared to the smooth surface of untreated cells; moreover, cell damage increased in a concentration-dependent manner (Fig. 2a–c). This demonstrated that FA dramatically increased cell membrane permeability and decreased cell membrane integrity.
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3.2 FA metabolism by C. beijerinckii We investigated the FA metabolic pathway in C. beijerinckii and identified a new metabolic product of FA, hydroFA, in P2 medium by LCMS-ESI and NMR (Fig. S1). FA (0.5 g/l) was completely converted into hydroFA in the absence of 2-methoxy-4-vinylphenol by C. beijerinckii 4693::int after 12 h, but 0.27g/l FA remained in the medium after by C. beijerinckii NCIMB 8052. The degradation rates of FA by C. beijerinckii NCIMB 8052 and 4693::int were 0.02 and 0.04 g/l/h, respectively (Fig. 3). These data illustrate that the degree and rate of FA metabolism is related to the level of NADPH (Liu et al., 2016a). Phenolic acid compounds such as p-coumaric acid, FA, and caffeic acid are metabolized by phenolic acid decarboxylase in Lactobacillus spp. and Saccharomyces cerevisiae, which is not expressed in C. beijerinckii (Filannino et al., 2015; Mukai et al., 2014; Huang et al., 1993). Based on the above-mentioned results, we generated a model of the FA metabolic pathway in C. beijerincklii (Fig. 4). In addition, ABE fermentation was investigated in order to assess the antibacterial activity of hydroFA against C. beijerinckii (Table S1). We found that C. beijerincklii has a natural ability to metabolize FA to a compound with weaker antibacterial activity by reducing the side-chain double bond.
3.3 Comparative transcriptome analysis of wild-type and mutant strains
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To clarify the mechanism of FA toxicity against C. beijerinckii, we carried out a comparative transcriptome analysis by RNA-seq. Of the 5231 reference genes in C. beijerinckii NCIMB8052 (https://www.ncbi.nlm.nih.gov/genome/?term=clostridium+beijerinckii+NCIMB+8052), 4754 (about 90.88%) were examined. There were 2488 genes that were differentially expressed (1383 genes were upregulated and 1105 genes were downregulated) in strain B8052 relative to A8052 (A8052 VS B8052) and 1664 DEGs (691 upregulated and 973 downregulated) in B4693 relative to A4693 (A4693 VS B4693) after treatment with FA, the number of upregulated and downregulated genes in the group of B8052 VS B4693 was 1140, 1485, respectively, and only 411, 228 genes in the group of A8052 VS A4693, respectively (Fig. S2). To assess the functional significance of these transcriptional changes, the DEGs were analyzed in terms of GO and KEGG classifications. Genes involved in redox reactions and associated cofactors, two-component systems, chemotaxis, glycolysis, and butanoate and riboflavin metabolism as well as ATP-binding cassette (ABC) transporters were highly represented. Based on the result of the number of DEGs of wild-type (A8052 VS B8052) was larger than that of the mutant strain(A4693 VS B4693) after treatment with FA, the wild-type strain may be more sensitive to FA challenge than the 4693::int mutant strain in which the Cbei_4693 gene was disrupted.
3.4 Expression of genes involved in redox and associated cofactors
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Given that FA detoxification by C. beijerinckii was found to relate to intracellular reducing power, genes expressing redox proteins were analyzed. A previous study analyzing the C. beijerinckii NCIMB 8052 transcriptome reported that short-chain dehydrogenase/reductase (SDR) family proteins are involved in the transformation of FA to furfural alcohol (Zhang et al., 2013). However, genes encoding SDR (Cbei_0869, Cbei_3180, Cbei_0027, Cbei_3633, and Cbei_3527) were more highly expressed in the wild-type strain (A8052 VS B8052) than in C. beijerinckii 4693::int (A4693 VS B4693) after FA challenge (Fig. 5a), indicating that they are unrelated to the detoxification of FA in C. beijerinckii. A GO analysis of DEGs in FA-challenged B8052 VS B4693 showed that two types of redox proteins were upregulated, including those with catalytic (GO: 0003824) and oxidoreductase (GO: 0016491) activities: i.e., alkyl hydroperoxide reductase subunit C (Cbei_2135; ahpC) and alkyl hydroperoxide reductase subunit F (Cbei_2134; ahpF); and ferredoxin-NADP+ reductase (fpr) subunit alpha (Cbei_2182 and Cbei_0661). Genes encoding oxidoreductases—which use peroxide as an acceptor (GO: 0016684)—were upregulated by more than 94- and 95-fold, respectively; whereas the second group of genes was upregulated by more than 7- and 12-fold, respectively, and encoded proteins that use NAD+ or NADP+ as an acceptor and iron-sulfur proteins as donors (GO: 0016730). The remaining oxidoreductases (GO: 0016491) were more highly expressed in the FA-challenged groups (B8052 VS B4693), including hydrogenase (Cbei_1173 and Cbei_0327), riboflavin
biosynthesis
protein
(Cbei_3316,
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Cbei_1224,
and
Cbei_1225),
FAD-dependent oxidoreductase (Cbei_1830, Cbei_4507, Cbei_1910, and Cbei_2543), and
2-hydroxyacid
dehydrogenase
(Cbei_0866,
Cbei_1946,
Cbei_3628,
and
Cbei_4594). The KEGG pathway analysis showed that these genes are mainly related to riboflavin and purine metabolism (Fig. S3), and the expression of these redox enzymes suggested that FA causes damage to nucleotides.. Phenolic compounds induce the accumulation of reactive oxygen species (ROS), which damage DNA, lipids, and proteins and consequently lead to programmed cell death. In addition, organic hydroperoxide or other radical species generated under anaerobic conditions can induce the expression of ahp (Cha et al., 2004), ahpC uses NADPH or NADH as an electron donor to ahpF. ahpC acts as specific alkyl hydroperoxide-scavenging enzyme for protection against the damage by ROS, ahpF is related to thioredoxin reductases possessing an extended additional N-terminal fragment essential to specifically reduce ahpC (Calzi et al.,1997). In addition, Rocha reported that ahpCF play a significant role on resistance to damage from peroxides (Rocha et al., 1999). Besides, the phenolic compounds in hydrolysates could be removed using peroxidase (Cho et al., 2009), and the data of transcriptome analysis (B8052 VS B4693) showed the gene of ahpC and ahpF was upregulated by more than 94- and 95-fold, respectively, then we speculated that the FA metabolism or the FA tolerance is related to the gene Cbei_2134 and Cbei_2135. Besides redox enzymes, cofactors are associated with the intracellular redox reaction. We previously reported that NADPH, a cofactor, is related to FA tolerance in
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C. beijerinckii (Liu at al., 2016a) and may act as an electron donor to the side chain of FA carbon-carbon double bonds. Interestingly, genes encoding components of the electron transport chain complex I (Cbei_4110–Cbei_4112 and Cbei_2987–Cbei_2996) were upregulated in A8052 VS B8052 but were downregulated in A4693 VS B4693, except for Cbei_2986 (Fig. 5b). Complex I is coupled with ATP synthesis via proton pumps or ATPase in the C. beijerinckii cell membrane; genes encoding ATP synthase (Cbei_0412–Cbei_0419) were all downregulated in A8052 VS B8052 and in A4693 VS B4693, suggesting that ATP production was significantly inhibited by FA challenge. It is likely that when FA was added to the medium, H+ ions permeated through the bacterial cell membrane; additionally, the cultures were still in the acidogenic phase and were producing acetate and butyrate, resulting in a decrease in intracellular pH that led to disruption of transmembrane pH potential, thereby inhibiting the synthesis of ATP by F1-F0 ATP synthase (Ibraheem and Ndimba, 2013).
3.5 Expression of two-component system genes The expression of genes associated with the two-component system (membrane transporter genes) was altered in FA-challenged C. beijerinckii NCIMB 8052 or 4693::int (A8052 vs. B8052 and A4693 vs. B4693); specifically, these genes are involved in molecular transducer activity (GO: 0060089)—such as those encoding the protein methyl-accepting chemotaxis sensory transducer (Cbei_0667, Cbei_1390, Cbei_1428, Cbei_1723, Cbei_2186, Cbei_2839, Cbei_3045, and Cbei_4438)—and
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response to chemicals (GO: 0042221) (i.e., chemotaxis proteins: CheA, Cbei_4019, Cbei_4183, Cbei_4307, and Cbei_4829; CheW, Cbei_4018 and Cbei_4304; CheY, Cbei_4305, Cbei_4309, and Cbei_4180) (Fig. 5c). Chemotaxis directs flagellar motion and controls cell movement; indeed, genes encoding flagellar assembly proteins were also altered by FA challenge. In B8052, flagellin domain proteins (Cbei_4274 and Cbei_4289), flagellar biosynthesis proteins (Cbei_4261 and Cbei_4292), and flagellar hook proteins (Cbei_4291, Cbei_4297, and Cbei_4298) were all upregulated relative to the levels in A8052; notably, Cbei_4291 encoding the FliD and Cbei_4292 encoding FliS were upregulated by 5- and 8-fold, respectively, whereas genes encoding the chemotaxis
protein
MotB
(Cbei_4830)
and
the
proton
channel
protein
MotA/TolQ/ExbB (Cbei_1594) were downregulated by 2- and 4-fold, respectively. However, in B4693, the expression of Cbei_4302 and Cbei_4303 (encoding the flagellar motor switch proteins FliN/FliY and FliM, respectively) was slightly increased relative to the levels in A4693, whereas that of genes related to flagellar assembly proteins was mostly unaltered by FA challenge. Additionally, the two-component system includes genes that participate in quorum sensing; those encoding accessory gene regulator protein AgrB (Cbei_0658, Cbei_3171, Cbei_3965, and Cbei_4578) were weakly repressed in C. beijerinckii NCIMB 8052 relative to A8052 and in 4693::int relative to A4693 following FA treatment, but were upregulated by 8- or 9-fold in B4693 as compared to B8052, indicating that disruption of Cbei_4693 enhances quorum sensing in C. beijerinckii.
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3.6 Genes associated with DNA replication and heat shock Phenolic compounds such as FA enhance ROS generation, leading to DNA mutagenesis or unwinding and breakage. In wild-type cells, genes encoding DNA polymerase III (Cbei_1013, Cbei_4951, and Cbei_0820), DNA polymerase I (Cbei_0801), the heat shock protein (Hsp) DnaJ domain protein (Cbei_1414), and Hsp33 (Cbei_1800) were downregulated whereas those encoding the single-strand DNA-binding
protein
ssb
(Cbei_5085),
DNA ligase
LigA/B
(Cbei_0352),
transcription-repair coupling factor mfd (Cbei_0086), and ATP-dependent DNA helicase PcrA (Cbei_0351) were strongly upregulated following FA challenge for 12 h. Unexpectedly, these genes were not or were only weakly induced by 0.5 g/l FA in C. beijerinckii 4693::int (Fig. 5d). The expression of genes encoding Hsps was also significantly altered by FA treatment. Heat shock genes including those in the DnaK and groESL operons were found to be upregulated in response to FA stress and were proposed to contribute to FA tolerance in C. beijerinckii NCIMB 8052 (Lee at al., 2015). We found here that DnaK and groEL operon genes were highly induced in the presence of FA in C. beijerinckii NCIMB 8052 relative to A8052. However, in C. beijerinckii 4693::int, DnaK and groEL genes were downregulated by up to 9- and 3-fold, respectively, relative to A4693 by FA treatment.
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3.7 Other DEGs Genes involved in other cellular functions also showed significant changes in expression after FA challenge for 12 h, including those linked to glycolysis, butanoate and riboflavin metabolism, ABC transporters, and efflux systems (Supplementary material).
4. Conclusion Our transcriptome analysis revealed that compared to C. beijerickii NCIMB 8052, C. beijerickii 4693::int with high FA tolerance showed significant alterations in gene expression in the presence of FA, indicating that disrupting the Cbei_4693 gene alters metabolic processes in C. beijerickii NCIMB 8052. Furthermore, Cbei_2134 and Cbei_2135 encoding ahpC and ahpF, respectively, are thought to be involved in C. beijerinckii adaptation mechanisms in response to FA. Our findings indicate that C. beijerickii NCIMB 8052 cells that are metabolically engineered for high tolerance to LDMICs can be applied to the improvement of biofuel production using lignocellulose biomass hydrolysates as substrates.
Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, no. 2013CB733602); National Natural Science Foundation of China (no. 21306032); Priority Academic Program Development of Jiangsu Higher Education
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Institutions; Postdoctoral Science Foundation of Jiangsu Province (no.1302107C); Major Research Plan of the National Natural Science Foundation of China (no. 21390204); Program for Changjiang Scholars and Innovative Research Team in University (no. IRT_14R28); Jiangsu National Synergetic Innovation Center for Advanced Materials; Restructured Institutions Innovation Capacity of Special Funds of the Ministry of Science and Technology of China (no. 2014EG111227).
References 1. Adeboye, P.T., Bettiga, M., Olsson, L., 2014. The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulose hydrolysates. AMB. Express. 4, 46. 2. Calzi, M. L., Poole, L.B., 1997. Requirement for the two AhpF cysteine disulfide centers in catalysis of peroxide reduction by alkyl hydroperoxide reductase. Biochemistry 36, 13357–13364. 3. Cha, M.K., Kim, W.C., Lim, C.J., Kim, K., Kim, I.H., 2004. Escherichia coli periplasmic thiol peroxidase acts as lipid hydroperoxide peroxidase and the principal antioxidative function during anaerobic growth. J. Biol. Chem. 279, 8769-78. 4. Cho, D.H., Lee, Y.J., Um, Y., Sang, B.I. and Kim, Y.H., 2009. Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for
18
butanol production from Clostridium beijerinckii. Appl. Microbiol. Biotechnol. 83, 1035–1043. 5. Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2007.
Butanol poduction from
agricultural residues:impact of degradation products on clostridium beijerinckii growth and butanol fermentation. Biotechnol. Bioeng. 97, 1460-1469. 6. Filannino, P., Bai, Y., Di, Cagno, R., Gobbetti, M., Gänzle, M.G., 2015. Metabolism of phenolic compounds by Lactobacillus spp. during fermentation of cherry juice and broccoli puree, Food. Microbiol. 46, 272-279. 7. Guo, T., He, A.Y., Du, T.F., Zhu, D.W., Liang, D.F., Jiang, M., Wei, P., Ouyang, P.K., 2013. Butanol production from hemicellulosic hydrolysate of corn fiber by a Clostridium beijerinckii mutant with high inhibitor-tolerance. Bioresour. Technol. 135, 379-385. 8. Guo, T., Tang, Y., Xi, Y.L., He, A.Y., Sun, B.J., Wu, H., Liang, D.F., Jiang, M., Ouyang, P.K., 2011. Clostridium beijerinckii mutant obtained by atmospheric pressure glow discharge producing high proportions of butanol and solvent yields. Biotechnol. Lett. 33, 2379-2383. 9. Guo, W., Jia, W., Li, Y., Chen, S., 2010. Performances of lactobacillus brevis for production lactic acid from hydrolysate of lignocellulosics. Appl. Biochem. Biotechnol 161, 124-136. 10. Huang, Z., Dostal, L., Rosazza, J.P., 1993. Microbial transformations of ferulic acid by Saccharomyces cerevisiae and Pseudomonas fluorescens. Appl. Environ.
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Microbiol. 59, 2244-50. 11. Hui, J.H., Li, H., Zheng, X.Y., Ma, H.X., Fan, D.D., Liu, H., Wang, Y.Y., 2015. Control synthesis and self-assembly of calcium apatite at low temperature. Ceramics. International. 41, 6194-6202. 12. Ibraheem, O., Ndimba, B.K., 2013. Molecular adaptation mechanisms employed by ethanologenic bacteria in response to lignocellulose derived inhibitory compounds. Int. J. Biol. Sci. 9, 598-612. 13. Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., Salzberg, S.L., 2013. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome. Bio. 14, R36. 14. Lee, S., Lee, J.H., Mitchell, R.J., 2015. Analysis of Clostridium beijerinckii NCIMB 8052's transcriptional response to ferulic acid and its application to enhance the strain tolerance. Biotechnol. Biofuels. 8:68. 15. Lee, S., Nama, D., Jung, J.Y., Oh, M.K., Sang, B.I., Mitchell, R.J., 2012.Identification of Escherichia coli biomarkers responsive to various lignin-hydrolysate compounds. Bioresour. Technol. 114, 450-456. 16. Li, G.H., Wang, X., Xu, Y.F., Zhang, B.G., Xia, X.D., 2014. Antimicrobial effect and mode of action of chlorogenic acid on staphylococcus aureus. Eur. Food. Res. Technol, 238, 589-596. 17. Liu, J., Guo, T., Shen, X.N., Xu, J.H., Wang, J.Z., Wang, Y.Y., Liu, D., Niu, H.Q., Liang, L., Ying, H.J., 2016a. Engineering Clostridium beijerinckii with the
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Cbei_4693 gene knockout for enhanced ferulic acid tolerance. J. Biotechnol. 229, 53-57. 18. Liu, J., Guo, T., Wang, D., Shen, X.N., Liu, D., Niu, H.Q., Liang, L., Ying, H.J., 2015. Enhanced butanol production by increasing NADH and ATP levels in Clostridium beijerinckii NCIMB 8052 by insertional inactivation of Cbei_4110. Appl. Microbiol. Biotechnol.100, 4985-4996. 19. Liu, J., Guo, T., Wang, D., Xu, J.H., Ying, H.J., 2016b. Butanol production by a Clostridium beijerinckii mutant with high ferulic acid tolerance. Biotechnol. Appl. Biochem. 63, 727-733. 20. Mukai, N., Masaki, K., Fujii, T., Iefuji, H., 2014. Single nucleotide polymorphisms of PAD1 and FDC1 show a positive relationship with ferulic acid decarboxylation ability among industrial yeasts used in alcoholic beverage production, J. Biosci. Bioeng. 118, 50-55. 21. Rocha, E.R., Smith, C.J., 1999. Role of the alkyl hydroperoxide reductase (ahpCF) gene in oxidative stress defense of the obligate Anaerobe bacteroides fragilis. 181, 5701-10. 22. Wierckx, N., Koopman, F., Ruijssenaars, H.J., de Winde, J.H., 2011. Microbial degradation of furanic compounds: biochemistry, genetics and impacts. Appl. Microbiol. Biotechnol. 92, 1095–1105. 23. Zhang, Y., Ezeji, T.C., 2013. Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 to elucidate role of furfural stress during acetone butanol ethanol
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fermentation. Biotechnol Biofuels. 6, 66.
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Figure legends Figure 1. Cytotoxicity of FA to C. beijerinckii 4693::int (a) and NCIMB 8052 (b). OD600 values represent the mean of three experiments.
Figure 2. Scanning electron micrographs of C. beijerinckii 4693::int. (a) Without treatment for 8 h; (b) treated with FA at MIC for 8 h; and (c) treated with FA at 2MIC for 8 h.
Figure 3. Kinetics of FA metabolism in C. beijerinckii 4693::int and NCIMB 8052.
Figure 4. Model of FA metabolism in C. beijerinckii.
Figure 5. Comparative expression patterns of genes encoding reductases and dehydrogenases (a); components of electron transfer chain complex I and associated cofactors (b); two-component system proteins, including those involved in flagellar assembly and chemotaxis (c); and DNA replication components (d). Shown is data for gene expression in C. beijerinckii NCIMB 8052 (or 4693::int) without FA challenge for 8 h [A8052 (A4693)] vs. gene expression in C. beijerinckii NCIMB 8052 (or 4693::int) after 12 h of FA challenge [B8052 (B4693)] [A8052 VS B8052 (A4693 VS B4693)]; gene expression in C. beijerinckii NCIMB 8052 without FA challenge for 8 h vs. gene expression in C.
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beijerinckii 4693::int without FA challenge for 8 h (A8052 VS A4693); and gene expression in C. beijerinckii NCIMB 8052 after 12 h of FA challenge vs. gene expression in C. beijerinckii 4693::int after 12 h of FA challenge (B8052 VS B4693).
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S1357-2725(17)30043-2 http://dx.doi.org/doi:10.1016/j.biocel.2017.02.009 BC 5084
To appear in:
The International Journal of Biochemistry & Cell Biology
Received date: Revised date: Accepted date:
21-11-2016 14-2-2017 22-2-2017
Please cite this article as: Liu, Jun., Guo, Ting., Yang, Tao., Xu, Jiahui., Tang, Chenglun., Liu, Dong., & Ying, Hanjie., Transcriptome analysis of Clostridium beijerinckii adaptation mechanisms in response to ferulic acid.International Journal of Biochemistry and Cell Biology http://dx.doi.org/10.1016/j.biocel.2017.02.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Transcriptome analysis of Clostridium beijerinckii adaptation mechanisms in response to ferulic acid
Jun Liua,b,1, Ting Guoc,*, Tao Yang c, Jiahui Xua,b, Chenglun Tanga,b, Dong Liua,b, Hanjie Yinga,b,*
a
State Key Laboratory of Materials-Oriented Chemical Engineering, College of
Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing 211816, China b
National Engineering Technique Research Center for Biotechnology, Nanjing 211816,
China c
College of Food Science and Technology, Central South University of Forestry and
Technology, Changsha, Hunan Province 410004,China
*Corresponding author: Dr. Hanjie Ying (Professor) Tel: +86 25 86990666; Fax: +86 25 86990001; E-mail: [email protected] Dr. Ting Guo (Associate Professor) Tel: +86 20 84178223; Fax: +86 20 84178223; E-mail: [email protected]
1
Highlights
MIC of FA against C. beijerinckii 4693::int was 1.5 g/l.
Viability of C. beijerinckii 4693::int was 106.7% in the presence of 0.5g/l FA.
FA altered expression of genes related antibacterial and adaptation mechanisms.
Enzyme ahpC/F was speculated to be related to the ferulic acid tolerance.
Abstract Clostridium beijerinckii 4693::int with high ferulic acid (FA) tolerance was engineered and characterized in our lab. In this study, the minimum inhibition concentrations of FA against C. beijerinckii NCIMB 8052 (wild-type) and 4693::int were 1.0 and 1.5 g/l, respectively; cell viability was 18.5% and 106.7%, respectively, in the presence of 0.5g/l FA. A comparative transcriptome analysis was carried out at two different growth stages to evaluate sensitivity to FA. Genes that were differentially expressed included those related to redox and associated cofactors, riboflavin metabolism, two-component system, glycolysis and butanoate metabolism, and DNA replication as well as those encoding ATP-binding cassette transporters. Cbei_2134 and Cbei_2135 encoding alkyl hydroperoxide reductases are thought to be involved in antibacterial and adaptation mechanisms in C. beijerinckii in the presence of FA.
Key words: Clostridium beijerinckii, ferulic acid tolerance, metabolism pathway, comparative transcriptome analysis
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1. Introduction Low-cost lignocellulosic materials such as wheat straw, bagasse fiber, and corn fiber have been investigated as substrates for butanol production. However, lignocellulose-derived microbial inhibitory compounds (LDMICs) that suppress Clostridia growth and solvent production are generated along with sugars during pretreatment (Wierckx et al., 2011). Phenolic compounds are particularly potent inhibitors (Guo et al, 2013) since they have various functional groups such as aldehydes, ketones, acids and alcohols and side groups such as the methoxy and hydroxyl groups that enhance their toxicity (Adeboye et al., 2014). Ferulic acid (FA) is a common phenolic compound with a benzene ring, methoxy and hydroxyl groups, and a side-chain double bond, that has potent antimicrobial activity (Ezeji et al., 2007; Guo et al., 2010; Lee et al., 2012). We previously described the generation of Clostridium beijerinckii strain M11 with high FA tolerance (0.9 g/l) by atmospheric pressure glow discharge and high-throughput screening (Liu et al., 2016b). We also demonstrated that the Cbei_4693 gene plays an important role in increasing FA tolerance (Liu et al., 2016a). Cbei_4693 encodes a hypothetical protein predicted to be a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase (FMN + NADPH+ H+ = FMNH2 +NADP+, http://www.genome.jp/dbget-bin/www_bget?cbe:Cbei_4693); this led us to hypothesize that
the tolerance of C. beijerinckii to FA is correlated with NADPH level, although the detailed molecular mechanisms remain unclear.
3
To clarify the FA metabolic pathway, we evaluated FA toxicity to C. beijerinckii and analyzed the production of FA metabolites by C. beijerinckii. We also carried out a transcriptome analysis to investigate the antibacterial and adaptation mechanisms of C. beijerinckii in response to FA. We identified alkyl hydroperoxide reductase subunit C (ahpC) encoded by Cbei_2134 and alkyl hydroperoxide reductase subunit F (ahpF) encoded by Cbei_2135 as two factors involved in FA tolerance.
2. Materials and methods 2.1 Cells and culture conditions The wild-type strain C. beijerinckii NCIMB 8052, was purchased from American type culture collection (manassas, AV) and the mutant strain C. beijerinckii 4693::int, was generated by disrupting the gene Cbei_4693 in our laboratory (Liu et al., 2016a) and stored at −80°C was used for experiments. After thawing on ice, 200 μl were inoculated in 10 ml YPS medium (3.0 g yeast extract, 5.0 g peptone, 10.0 g soluble starch, 2.0 g ammonium acetate, 2.0 g NaCl, 3.0 g MgSO4·7H2O, 1.0 g KH2PO4, 1.0 g K2HPO4, 0.1 g FeSO4·7H2O in 1 l H2O) (Guo et al. 2011) and incubated anaerobically at 37°C for 12 h. Cells were sub-cultured in fresh YPS medium with a 5% inoculum size for 8 h to reach an optical density at 600 nm (OD600) of approximately 2.0 (secondary seed cells).
2.2 Evaluation of FA toxicity against C. beijerinckii
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2.2.1 Determination of minimum inhibitory concentration (MIC) Erythromycin was used as a positive reference standard for test strains to determine the MIC of FA against C. beijerinckii. A 4-ml volume of P2 medium (30 g/l of glucose; Liu et al., 2015) containing FA or erythromycin (10 μg/ml) was transferred to sterile 24-well plates in an anaerobic chamber overnight at 37°C to remove the oxygen. A 1-ml volume of secondary seed cells was added to the plates; the final FA concentrations were 0, 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, and 2.0 g/l, with each sample prepared in triplicate. After incubation at 37°C for 48 h in the anaerobic chamber, 2 μl (approximately 105 CFU) of the test strains (C. beijerinckii NCIMB 8052 and 4693::int) were spotted on solid YPS medium without FA, and the plates were incubated for 12 h. The lowest concentration of FA yielding no visible growth of bacteria was taken as the MIC.
2.2.2 Field emission scanning electron microscopy (FESEM) FESEM was carried out as previously described, with some modifications (Li et al., 2014). C. beijerinckii 4693::int was inoculated in 50 ml P2 medium (initial OD600 = 0.5) containing FA at concentrations of 0, MIC, 2MIC. After incubation at 37°C for 8 h in an anaerobic chamber, 10 ml of cells were harvested by centrifugation for 10 min at 8000 ×g and washed twice with 10 ml of 0.85% phosphate-buffered saline, then re-suspended in 1 ml of 2.5% glutaraldehyde and maintained at 4°C for 12 h to fix the cells. Following centrifugation for 10 min at 8000 ×g, cells were dehydrated in a
5
graded series of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) for 15 min each and vacuum-dried at 50°C. Samples were analyzed by FESEM (Supra-55; Zeiss, Jena, Germany).
2.2.3 Cytotoxicity assay The antibacterial activity of FA against C. beijerinckii was evaluated with the cytotoxicity assay. The fermentation conditions were the same as for MIC determination except that the P2 medium contained FA at concentrations of 0, 0.5, 0.7, 0.9, 1.0, 1.2, and 1.5 g/l. After incubation for 48 h, 90 μl of sample was transferred to a 96-well plate and 10 μl of Cell Counting Kit-8 (Sigma) were added to each well. Cells without FA served as a control, and fresh P2 medium was used as a blank. Cell viability (%) was calculated as follows: [ODtest − ODblank] / [ODcontrol − ODblank] × 100%. The absorbance was measured at 450 nm using a Multi-Mode Detection Platform (Molecular Devices, Sunnyvale, CA, USA) (Hui et al., 2015).
2.3 Kinetics of FA metabolism P2 medium containing 0.5 g/l FA was used for acetone–butanol–ethanol (ABE) fermentation by C. beijerinckii. The kinetics of FA degradation were investigated by evaluating FA concentration in 5 ml of sample taken from cells grown in 100 ml P2 medium in 250 ml screw-capped bottles without agitation or from a pH control, and the sampling time was 0, 6, 12, and 24 h.
6
FA concentration was determined by high-performance liquid chromatography analysis (Agilent 1200 series; Hewlett-Packard, Wilmington, DE, USA) at 280 nm; the mobile phase was 0.3% acetate (70%) and methanol (30%) at a flow rate of 0.8 ml/min, with separation carried out using an Aglient ZORBAX SB-Aq-C18 column (5 μm, 4.6 × 250 mm) at 50°C (Cho et al., 2009). FA metabolites were detected by liquid chromatography mass spectrometry– electrospray ionization (LCMS-ESI) (Timebase U3000-MSQ; Dionex, Sunnyvale, CA, USA) and proton (1H)- and carbon (13C)- nuclear magnetic resonance spectroscopy (NMR) (Nanjing Biopharmaceutical Innovation Platform, Nanjing, China).
2.4 RNA extraction, RNA sequencing (RNA-seq) library construction, and high-throughput sequencing Batch fermentation by C. beijerinckii NCIMB 8052 and 4693::int was carried out in 250-ml screw-capped bottles (two each) containing 100 ml P2 medium (Liu et al., 2015). After inoculation (10% v/v) for 8 h (OD600 = 1.0 ± 0.21), one bottle each of C. beijerinckii NCIMB 8052 and 4693::int was challenged with 0.5 g/l FA as the treatment group, while the other bottle served as the control; 12 h later (i.e., after fermentation for 20 h)—during which time the FA concentration in the medium was reduced by more than half by C. beijerinckii—samples were collected for analysis. Total RNA in each sample was extracted using TRIzol reagent (Takara Bio, Otsu, Japan) according to the manufacture’s protocol. RNA purity and concentration
7
were evaluated by 1% agarose gel electrophoresis. Libraries were constructed using the TruSeq RNA Sample Prep kit (Illumina, San Diego, CA, USA). High-throughput sequencing was performed on an Illumina Hiseq 4000 platform by Gene Denovo (Guangzhou, China) (http://www.genedenovo.com).
2.5 Transcriptome assembly and analysis Clean reads were obtained by removing raw reads containing the adaptor or unknown or low-quality sequences; these were then mapped to the C. beijerinckii NCIMB 8052 reference genome using TopHat2 aligner (Kim et al., 2013). Bioconductor edgeR (https://bioconductor.org/) was used for differential expression analysis of RNA-seq expression profiles. Transcripts with greater than 2-fold difference in expression (|log2 ratio| ≥ 1) and false discovery rate ≤ 0.05 were set as thresholds. Differentially expressed genes (DEGs) in FA-challenged relative to control samples were subjected to Gene Ontology (GO) functional analysis (http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (http://www.genome.jp/kegg/).
2.6 Microarray data accession number Microarray data and results have been deposited in the BioSample database, under the following accession numbers: SAMN 05966957-SAMN 05966960.
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3. Results and discussion 3.1 Evaluation of FA toxicity against C. beijerinckii The MICs of FA against C. beijerinckii NCIMB 8052 (MIC8052) and 4693::int (MIC4693::int) were 1.0 and 1.5 g/l, respectively. The OD600 values of the medium containing the two test strains and FA after 48 h were 0.49 ± 0.07 and 0.47 ± 0.01 (as compared to an initial value of 0.50) for MIC8052 and MIC4693::int, respectively. The cytotoxicity assay revealed that the viability of C. beijerinckii 4693::int cells was unaffected by FA concentrations < 0.5g/l, but was reduced to 34.17% at concentrations of 0.7 g/l (18.52% to C. beijerinckii NCIMB 8052 in the presence of 0.5g/l FA) (Fig. 1). Interestingly, the viability of C. beijerinckii 4693::int cells begin keeping the same value, and was lowest, from 1.2 g/l FA in the medium. The concentration of FA that was toxic to C. beijerinckii 4693::int cells (1.2 g/l) was lower than the MIC4693::int (1.5g/l), which was measured during the prolonged lag phase when cell activity was reduced or C. beijerinckii 4693::int was producing spores. To investigate the toxicity of FA to C. beijerinckii in greater detail, we examined changes in cell morphology by FESEM. C. beijerinckii 4693::int cells were treated with FA at concentrations of 0, 1.5, and 3.0 g/l (i.e., 0, MIC, and 2MIC, respectively) for 8 h. Cells exposed to FA had a more wrinkled surface as compared to the smooth surface of untreated cells; moreover, cell damage increased in a concentration-dependent manner (Fig. 2a–c). This demonstrated that FA dramatically increased cell membrane permeability and decreased cell membrane integrity.
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3.2 FA metabolism by C. beijerinckii We investigated the FA metabolic pathway in C. beijerinckii and identified a new metabolic product of FA, hydroFA, in P2 medium by LCMS-ESI and NMR (Fig. S1). FA (0.5 g/l) was completely converted into hydroFA in the absence of 2-methoxy-4-vinylphenol by C. beijerinckii 4693::int after 12 h, but 0.27g/l FA remained in the medium after by C. beijerinckii NCIMB 8052. The degradation rates of FA by C. beijerinckii NCIMB 8052 and 4693::int were 0.02 and 0.04 g/l/h, respectively (Fig. 3). These data illustrate that the degree and rate of FA metabolism is related to the level of NADPH (Liu et al., 2016a). Phenolic acid compounds such as p-coumaric acid, FA, and caffeic acid are metabolized by phenolic acid decarboxylase in Lactobacillus spp. and Saccharomyces cerevisiae, which is not expressed in C. beijerinckii (Filannino et al., 2015; Mukai et al., 2014; Huang et al., 1993). Based on the above-mentioned results, we generated a model of the FA metabolic pathway in C. beijerincklii (Fig. 4). In addition, ABE fermentation was investigated in order to assess the antibacterial activity of hydroFA against C. beijerinckii (Table S1). We found that C. beijerincklii has a natural ability to metabolize FA to a compound with weaker antibacterial activity by reducing the side-chain double bond.
3.3 Comparative transcriptome analysis of wild-type and mutant strains
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To clarify the mechanism of FA toxicity against C. beijerinckii, we carried out a comparative transcriptome analysis by RNA-seq. Of the 5231 reference genes in C. beijerinckii NCIMB8052 (https://www.ncbi.nlm.nih.gov/genome/?term=clostridium+beijerinckii+NCIMB+8052), 4754 (about 90.88%) were examined. There were 2488 genes that were differentially expressed (1383 genes were upregulated and 1105 genes were downregulated) in strain B8052 relative to A8052 (A8052 VS B8052) and 1664 DEGs (691 upregulated and 973 downregulated) in B4693 relative to A4693 (A4693 VS B4693) after treatment with FA, the number of upregulated and downregulated genes in the group of B8052 VS B4693 was 1140, 1485, respectively, and only 411, 228 genes in the group of A8052 VS A4693, respectively (Fig. S2). To assess the functional significance of these transcriptional changes, the DEGs were analyzed in terms of GO and KEGG classifications. Genes involved in redox reactions and associated cofactors, two-component systems, chemotaxis, glycolysis, and butanoate and riboflavin metabolism as well as ATP-binding cassette (ABC) transporters were highly represented. Based on the result of the number of DEGs of wild-type (A8052 VS B8052) was larger than that of the mutant strain(A4693 VS B4693) after treatment with FA, the wild-type strain may be more sensitive to FA challenge than the 4693::int mutant strain in which the Cbei_4693 gene was disrupted.
3.4 Expression of genes involved in redox and associated cofactors
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Given that FA detoxification by C. beijerinckii was found to relate to intracellular reducing power, genes expressing redox proteins were analyzed. A previous study analyzing the C. beijerinckii NCIMB 8052 transcriptome reported that short-chain dehydrogenase/reductase (SDR) family proteins are involved in the transformation of FA to furfural alcohol (Zhang et al., 2013). However, genes encoding SDR (Cbei_0869, Cbei_3180, Cbei_0027, Cbei_3633, and Cbei_3527) were more highly expressed in the wild-type strain (A8052 VS B8052) than in C. beijerinckii 4693::int (A4693 VS B4693) after FA challenge (Fig. 5a), indicating that they are unrelated to the detoxification of FA in C. beijerinckii. A GO analysis of DEGs in FA-challenged B8052 VS B4693 showed that two types of redox proteins were upregulated, including those with catalytic (GO: 0003824) and oxidoreductase (GO: 0016491) activities: i.e., alkyl hydroperoxide reductase subunit C (Cbei_2135; ahpC) and alkyl hydroperoxide reductase subunit F (Cbei_2134; ahpF); and ferredoxin-NADP+ reductase (fpr) subunit alpha (Cbei_2182 and Cbei_0661). Genes encoding oxidoreductases—which use peroxide as an acceptor (GO: 0016684)—were upregulated by more than 94- and 95-fold, respectively; whereas the second group of genes was upregulated by more than 7- and 12-fold, respectively, and encoded proteins that use NAD+ or NADP+ as an acceptor and iron-sulfur proteins as donors (GO: 0016730). The remaining oxidoreductases (GO: 0016491) were more highly expressed in the FA-challenged groups (B8052 VS B4693), including hydrogenase (Cbei_1173 and Cbei_0327), riboflavin
biosynthesis
protein
(Cbei_3316,
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Cbei_1224,
and
Cbei_1225),
FAD-dependent oxidoreductase (Cbei_1830, Cbei_4507, Cbei_1910, and Cbei_2543), and
2-hydroxyacid
dehydrogenase
(Cbei_0866,
Cbei_1946,
Cbei_3628,
and
Cbei_4594). The KEGG pathway analysis showed that these genes are mainly related to riboflavin and purine metabolism (Fig. S3), and the expression of these redox enzymes suggested that FA causes damage to nucleotides.. Phenolic compounds induce the accumulation of reactive oxygen species (ROS), which damage DNA, lipids, and proteins and consequently lead to programmed cell death. In addition, organic hydroperoxide or other radical species generated under anaerobic conditions can induce the expression of ahp (Cha et al., 2004), ahpC uses NADPH or NADH as an electron donor to ahpF. ahpC acts as specific alkyl hydroperoxide-scavenging enzyme for protection against the damage by ROS, ahpF is related to thioredoxin reductases possessing an extended additional N-terminal fragment essential to specifically reduce ahpC (Calzi et al.,1997). In addition, Rocha reported that ahpCF play a significant role on resistance to damage from peroxides (Rocha et al., 1999). Besides, the phenolic compounds in hydrolysates could be removed using peroxidase (Cho et al., 2009), and the data of transcriptome analysis (B8052 VS B4693) showed the gene of ahpC and ahpF was upregulated by more than 94- and 95-fold, respectively, then we speculated that the FA metabolism or the FA tolerance is related to the gene Cbei_2134 and Cbei_2135. Besides redox enzymes, cofactors are associated with the intracellular redox reaction. We previously reported that NADPH, a cofactor, is related to FA tolerance in
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C. beijerinckii (Liu at al., 2016a) and may act as an electron donor to the side chain of FA carbon-carbon double bonds. Interestingly, genes encoding components of the electron transport chain complex I (Cbei_4110–Cbei_4112 and Cbei_2987–Cbei_2996) were upregulated in A8052 VS B8052 but were downregulated in A4693 VS B4693, except for Cbei_2986 (Fig. 5b). Complex I is coupled with ATP synthesis via proton pumps or ATPase in the C. beijerinckii cell membrane; genes encoding ATP synthase (Cbei_0412–Cbei_0419) were all downregulated in A8052 VS B8052 and in A4693 VS B4693, suggesting that ATP production was significantly inhibited by FA challenge. It is likely that when FA was added to the medium, H+ ions permeated through the bacterial cell membrane; additionally, the cultures were still in the acidogenic phase and were producing acetate and butyrate, resulting in a decrease in intracellular pH that led to disruption of transmembrane pH potential, thereby inhibiting the synthesis of ATP by F1-F0 ATP synthase (Ibraheem and Ndimba, 2013).
3.5 Expression of two-component system genes The expression of genes associated with the two-component system (membrane transporter genes) was altered in FA-challenged C. beijerinckii NCIMB 8052 or 4693::int (A8052 vs. B8052 and A4693 vs. B4693); specifically, these genes are involved in molecular transducer activity (GO: 0060089)—such as those encoding the protein methyl-accepting chemotaxis sensory transducer (Cbei_0667, Cbei_1390, Cbei_1428, Cbei_1723, Cbei_2186, Cbei_2839, Cbei_3045, and Cbei_4438)—and
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response to chemicals (GO: 0042221) (i.e., chemotaxis proteins: CheA, Cbei_4019, Cbei_4183, Cbei_4307, and Cbei_4829; CheW, Cbei_4018 and Cbei_4304; CheY, Cbei_4305, Cbei_4309, and Cbei_4180) (Fig. 5c). Chemotaxis directs flagellar motion and controls cell movement; indeed, genes encoding flagellar assembly proteins were also altered by FA challenge. In B8052, flagellin domain proteins (Cbei_4274 and Cbei_4289), flagellar biosynthesis proteins (Cbei_4261 and Cbei_4292), and flagellar hook proteins (Cbei_4291, Cbei_4297, and Cbei_4298) were all upregulated relative to the levels in A8052; notably, Cbei_4291 encoding the FliD and Cbei_4292 encoding FliS were upregulated by 5- and 8-fold, respectively, whereas genes encoding the chemotaxis
protein
MotB
(Cbei_4830)
and
the
proton
channel
protein
MotA/TolQ/ExbB (Cbei_1594) were downregulated by 2- and 4-fold, respectively. However, in B4693, the expression of Cbei_4302 and Cbei_4303 (encoding the flagellar motor switch proteins FliN/FliY and FliM, respectively) was slightly increased relative to the levels in A4693, whereas that of genes related to flagellar assembly proteins was mostly unaltered by FA challenge. Additionally, the two-component system includes genes that participate in quorum sensing; those encoding accessory gene regulator protein AgrB (Cbei_0658, Cbei_3171, Cbei_3965, and Cbei_4578) were weakly repressed in C. beijerinckii NCIMB 8052 relative to A8052 and in 4693::int relative to A4693 following FA treatment, but were upregulated by 8- or 9-fold in B4693 as compared to B8052, indicating that disruption of Cbei_4693 enhances quorum sensing in C. beijerinckii.
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3.6 Genes associated with DNA replication and heat shock Phenolic compounds such as FA enhance ROS generation, leading to DNA mutagenesis or unwinding and breakage. In wild-type cells, genes encoding DNA polymerase III (Cbei_1013, Cbei_4951, and Cbei_0820), DNA polymerase I (Cbei_0801), the heat shock protein (Hsp) DnaJ domain protein (Cbei_1414), and Hsp33 (Cbei_1800) were downregulated whereas those encoding the single-strand DNA-binding
protein
ssb
(Cbei_5085),
DNA ligase
LigA/B
(Cbei_0352),
transcription-repair coupling factor mfd (Cbei_0086), and ATP-dependent DNA helicase PcrA (Cbei_0351) were strongly upregulated following FA challenge for 12 h. Unexpectedly, these genes were not or were only weakly induced by 0.5 g/l FA in C. beijerinckii 4693::int (Fig. 5d). The expression of genes encoding Hsps was also significantly altered by FA treatment. Heat shock genes including those in the DnaK and groESL operons were found to be upregulated in response to FA stress and were proposed to contribute to FA tolerance in C. beijerinckii NCIMB 8052 (Lee at al., 2015). We found here that DnaK and groEL operon genes were highly induced in the presence of FA in C. beijerinckii NCIMB 8052 relative to A8052. However, in C. beijerinckii 4693::int, DnaK and groEL genes were downregulated by up to 9- and 3-fold, respectively, relative to A4693 by FA treatment.
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3.7 Other DEGs Genes involved in other cellular functions also showed significant changes in expression after FA challenge for 12 h, including those linked to glycolysis, butanoate and riboflavin metabolism, ABC transporters, and efflux systems (Supplementary material).
4. Conclusion Our transcriptome analysis revealed that compared to C. beijerickii NCIMB 8052, C. beijerickii 4693::int with high FA tolerance showed significant alterations in gene expression in the presence of FA, indicating that disrupting the Cbei_4693 gene alters metabolic processes in C. beijerickii NCIMB 8052. Furthermore, Cbei_2134 and Cbei_2135 encoding ahpC and ahpF, respectively, are thought to be involved in C. beijerinckii adaptation mechanisms in response to FA. Our findings indicate that C. beijerickii NCIMB 8052 cells that are metabolically engineered for high tolerance to LDMICs can be applied to the improvement of biofuel production using lignocellulose biomass hydrolysates as substrates.
Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, no. 2013CB733602); National Natural Science Foundation of China (no. 21306032); Priority Academic Program Development of Jiangsu Higher Education
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Institutions; Postdoctoral Science Foundation of Jiangsu Province (no.1302107C); Major Research Plan of the National Natural Science Foundation of China (no. 21390204); Program for Changjiang Scholars and Innovative Research Team in University (no. IRT_14R28); Jiangsu National Synergetic Innovation Center for Advanced Materials; Restructured Institutions Innovation Capacity of Special Funds of the Ministry of Science and Technology of China (no. 2014EG111227).
References 1. Adeboye, P.T., Bettiga, M., Olsson, L., 2014. The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulose hydrolysates. AMB. Express. 4, 46. 2. Calzi, M. L., Poole, L.B., 1997. Requirement for the two AhpF cysteine disulfide centers in catalysis of peroxide reduction by alkyl hydroperoxide reductase. Biochemistry 36, 13357–13364. 3. Cha, M.K., Kim, W.C., Lim, C.J., Kim, K., Kim, I.H., 2004. Escherichia coli periplasmic thiol peroxidase acts as lipid hydroperoxide peroxidase and the principal antioxidative function during anaerobic growth. J. Biol. Chem. 279, 8769-78. 4. Cho, D.H., Lee, Y.J., Um, Y., Sang, B.I. and Kim, Y.H., 2009. Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for
18
butanol production from Clostridium beijerinckii. Appl. Microbiol. Biotechnol. 83, 1035–1043. 5. Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2007.
Butanol poduction from
agricultural residues:impact of degradation products on clostridium beijerinckii growth and butanol fermentation. Biotechnol. Bioeng. 97, 1460-1469. 6. Filannino, P., Bai, Y., Di, Cagno, R., Gobbetti, M., Gänzle, M.G., 2015. Metabolism of phenolic compounds by Lactobacillus spp. during fermentation of cherry juice and broccoli puree, Food. Microbiol. 46, 272-279. 7. Guo, T., He, A.Y., Du, T.F., Zhu, D.W., Liang, D.F., Jiang, M., Wei, P., Ouyang, P.K., 2013. Butanol production from hemicellulosic hydrolysate of corn fiber by a Clostridium beijerinckii mutant with high inhibitor-tolerance. Bioresour. Technol. 135, 379-385. 8. Guo, T., Tang, Y., Xi, Y.L., He, A.Y., Sun, B.J., Wu, H., Liang, D.F., Jiang, M., Ouyang, P.K., 2011. Clostridium beijerinckii mutant obtained by atmospheric pressure glow discharge producing high proportions of butanol and solvent yields. Biotechnol. Lett. 33, 2379-2383. 9. Guo, W., Jia, W., Li, Y., Chen, S., 2010. Performances of lactobacillus brevis for production lactic acid from hydrolysate of lignocellulosics. Appl. Biochem. Biotechnol 161, 124-136. 10. Huang, Z., Dostal, L., Rosazza, J.P., 1993. Microbial transformations of ferulic acid by Saccharomyces cerevisiae and Pseudomonas fluorescens. Appl. Environ.
19
Microbiol. 59, 2244-50. 11. Hui, J.H., Li, H., Zheng, X.Y., Ma, H.X., Fan, D.D., Liu, H., Wang, Y.Y., 2015. Control synthesis and self-assembly of calcium apatite at low temperature. Ceramics. International. 41, 6194-6202. 12. Ibraheem, O., Ndimba, B.K., 2013. Molecular adaptation mechanisms employed by ethanologenic bacteria in response to lignocellulose derived inhibitory compounds. Int. J. Biol. Sci. 9, 598-612. 13. Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., Salzberg, S.L., 2013. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome. Bio. 14, R36. 14. Lee, S., Lee, J.H., Mitchell, R.J., 2015. Analysis of Clostridium beijerinckii NCIMB 8052's transcriptional response to ferulic acid and its application to enhance the strain tolerance. Biotechnol. Biofuels. 8:68. 15. Lee, S., Nama, D., Jung, J.Y., Oh, M.K., Sang, B.I., Mitchell, R.J., 2012.Identification of Escherichia coli biomarkers responsive to various lignin-hydrolysate compounds. Bioresour. Technol. 114, 450-456. 16. Li, G.H., Wang, X., Xu, Y.F., Zhang, B.G., Xia, X.D., 2014. Antimicrobial effect and mode of action of chlorogenic acid on staphylococcus aureus. Eur. Food. Res. Technol, 238, 589-596. 17. Liu, J., Guo, T., Shen, X.N., Xu, J.H., Wang, J.Z., Wang, Y.Y., Liu, D., Niu, H.Q., Liang, L., Ying, H.J., 2016a. Engineering Clostridium beijerinckii with the
20
Cbei_4693 gene knockout for enhanced ferulic acid tolerance. J. Biotechnol. 229, 53-57. 18. Liu, J., Guo, T., Wang, D., Shen, X.N., Liu, D., Niu, H.Q., Liang, L., Ying, H.J., 2015. Enhanced butanol production by increasing NADH and ATP levels in Clostridium beijerinckii NCIMB 8052 by insertional inactivation of Cbei_4110. Appl. Microbiol. Biotechnol.100, 4985-4996. 19. Liu, J., Guo, T., Wang, D., Xu, J.H., Ying, H.J., 2016b. Butanol production by a Clostridium beijerinckii mutant with high ferulic acid tolerance. Biotechnol. Appl. Biochem. 63, 727-733. 20. Mukai, N., Masaki, K., Fujii, T., Iefuji, H., 2014. Single nucleotide polymorphisms of PAD1 and FDC1 show a positive relationship with ferulic acid decarboxylation ability among industrial yeasts used in alcoholic beverage production, J. Biosci. Bioeng. 118, 50-55. 21. Rocha, E.R., Smith, C.J., 1999. Role of the alkyl hydroperoxide reductase (ahpCF) gene in oxidative stress defense of the obligate Anaerobe bacteroides fragilis. 181, 5701-10. 22. Wierckx, N., Koopman, F., Ruijssenaars, H.J., de Winde, J.H., 2011. Microbial degradation of furanic compounds: biochemistry, genetics and impacts. Appl. Microbiol. Biotechnol. 92, 1095–1105. 23. Zhang, Y., Ezeji, T.C., 2013. Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 to elucidate role of furfural stress during acetone butanol ethanol
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fermentation. Biotechnol Biofuels. 6, 66.
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Figure legends Figure 1. Cytotoxicity of FA to C. beijerinckii 4693::int (a) and NCIMB 8052 (b). OD600 values represent the mean of three experiments.
Figure 2. Scanning electron micrographs of C. beijerinckii 4693::int. (a) Without treatment for 8 h; (b) treated with FA at MIC for 8 h; and (c) treated with FA at 2MIC for 8 h.
Figure 3. Kinetics of FA metabolism in C. beijerinckii 4693::int and NCIMB 8052.
Figure 4. Model of FA metabolism in C. beijerinckii.
Figure 5. Comparative expression patterns of genes encoding reductases and dehydrogenases (a); components of electron transfer chain complex I and associated cofactors (b); two-component system proteins, including those involved in flagellar assembly and chemotaxis (c); and DNA replication components (d). Shown is data for gene expression in C. beijerinckii NCIMB 8052 (or 4693::int) without FA challenge for 8 h [A8052 (A4693)] vs. gene expression in C. beijerinckii NCIMB 8052 (or 4693::int) after 12 h of FA challenge [B8052 (B4693)] [A8052 VS B8052 (A4693 VS B4693)]; gene expression in C. beijerinckii NCIMB 8052 without FA challenge for 8 h vs. gene expression in C.
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beijerinckii 4693::int without FA challenge for 8 h (A8052 VS A4693); and gene expression in C. beijerinckii NCIMB 8052 after 12 h of FA challenge vs. gene expression in C. beijerinckii 4693::int after 12 h of FA challenge (B8052 VS B4693).
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