A previous study reported that a novel Aspergillus oryzae strain (CGMCC5992) can synthesize lignin hydrolytic enzymes for lignin degradation from straw. The present work involves the different gene expression of A. oryzae 5992 grown in media using lignin and glucose as carbon sources by suppression subtractive hybridization. Surprisingly, peroxidase was found in up-regulation genes, which is the key enzyme for degrading lignin. This shows that A. oryzae 5992 can secrete peroxidase in the presence of lignin. The functions of up-regulation genes also included gluconeogenesis, repairs, as well as signal and transporter proteins in the cell membrane. In addition, the down-regulation of genes was closely related to the aerobic metabolism of glucose, the fatty acid synthesis of the cell membrane, and the synthesis and utilization of ATP. Therefore, A. oryzae could regulate metabolism using lignin as carbon source, including lignin degradation promotion, glucose metabolism inhibition, and glucose regeneration.
Lignin-induced Expression of Aspergillus oryzae 5992 Genes using Suppression Subtractive Hybridization
Zhicai Zhang,a,b, Hongxue Ding,a Defu Shi,a Lili Xia,a and Maxiaoqi Zhu a
A previous study reported that a novel Aspergillus oryzae strain (CGMCC5992) can synthesize lignin hydrolytic enzymes for lignin degradation from straw. The present work involves the different gene expression of A. oryzae 5992 grown in media using lignin and glucose as carbon sources by suppression subtractive hybridization. Surprisingly, peroxidase was found in up-regulation genes, which is the key enzyme for degrading lignin. This shows thatA. oryzae 5992 can secrete peroxidase in the presence of lignin. The functions of up-regulation genes also included gluconeogenesis, repairs, as well as signal and transporter proteins in the cell membrane. In addition, the down-regulation of genes was closely related to the aerobic metabolism of glucose, the fatty acid synthesis of the cell membrane, and the synthesis and utilization of ATP. Therefore, A. oryzae could regulate metabolism using lignin as carbon source, including lignin degradation promotion, glucose metabolism inhibition, and glucose regeneration.
Keywords: Suppression subtractive hybridization; Aspergillus oryzae; Lignin; Gene; Different expression; Metabolism
Contact information: a: School of Food Science and Biotechnology, Jiangsu University, Zhenjiang 212013, P. R. China; b: Beijing Green Technology and Natural Biotechnology Co. Ltd., Beijing 102300, P. R. China; *Corresponding author: email@example.com
As a complex, non-crystalline, three-dimensional network of aromatic hetero-polymers (Bugg et al. 2011), lignin is the second main constituent of plant biomass after cellulose, accounting for about 20% of plant biomass (Itoh et al. 2003; Wang et al. 2011). In plant tissue, lignin binds the surrounding cellulose to form a natural rugged barrier, limiting the access of cellulase to cellulose (Shi et al. 2008). Therefore, the efficient, rapid, and economically and environmentally friendly destruction of the lignin network is a promising approach for the industrial development and utilization of lignocellulosic substrates, such as bioethanol production and the manufacture of cellulose-based chemicals and materials (Perez et al. 2002).
Different methods have been developed to destroy the lignin of lignocellulosic substrates based on physicochemical technologies, such as microwaving, ionizing radiation, steam explosion, acid or alkali dilution, oxidation, and varying combinations of these (Mosier et al. 2005). However, these typical physical and chemical techniques require not only high amounts of energy (steam or electricity), but also corrosion-resistant and high-pressure reactors, which lead to increased demand for special equipment and cost of pretreatment, as well as secondary pollution (Keller et al. 2003; Brodeur et al. 2011).
Microorganisms have been used to destroy the lignin of lignocellulosic substrates, providing the following advantages: decreased environmental pollution; transformation of waste materials into resources; and realization of the circulatory re-use of resources. Until now, the reported fungi used in lignin degradation can be divided into three categories: white rot fungi, brown rot fungi, and soft rot fungi. White rot fungi have been generally considered to be the relatively stronger fungi for degrading lignin (ten Have and Teunissen 2001; Yelle et al. 2008; Hamed 2013). However, white rot fungi generally offer a poor production of lignin hydrolases since these enzymes are secondary metabolites (Tuor et al. 1995). Although it is cost-effective to pre-treat lignocellulosic substrates using white rot fungi, a large site area is required, which is not suitable for industrial production (Behera et al. 2014). Therefore, it is crucial to discover a strain with the rapid production capacity of lignin hydrolases, which determines whether lignin bio-degradation can be applied to industrial production. Exo-genes of lignin hydrolyases, including manganese peroxidase (MnP) and lignin peroxidase (LiP), have been put into the recipient strains, such as Escherichia coli, Aspergillus niger, and Pichia methanolica using gene cloning technology, and active lignin hydrolyases were successfully obtained (Gu et al. 2000). However, these engineering strains cannot tolerate high concentrations of hydrogen peroxide, another substrate of MnP and LiP, nor can they produce hydrogen peroxide. These two drawbacks directly limit the application of these engineering strains in lignin biodegradation.
Aspergillus oryzae CGMCC5992 has been isolated from the sludge of the Yudai River at the University of Jiangsu and identified according to morphological and molecular biology methods. This strain can produce high amounts of active MnP and LiP (Guo et al. 2014), degrade the lignin of lignocellulosic substrates in a relatively short period, and tolerate high concentrations of hydrogen peroxide (Zhang et al. 2014). Moreover, it is a promising strain for employment in lignin degradation in industrial applications.
Suppression subtractive hybridization (SSH), which combines suppression PCR with subtractive hybridization methods, was developed by Luda Diatchenko in 1966 (Ren et al. 2006). A powerful and efficient hybridization process, SSH can identify two different mRNA populations and isolate genes that are exclusively expressed in one population but not in the other (Diatchenko et al. 1996; Basyuni et al. 2010). This technique has been successfully applied to the identification of various biotic and abiotic stress-responsive genes in recent investigations (Diatchenko et al. 1999; Venkatachalam et al. 2009). In this study, we investigated the genes of A. oryzae CGMCC5992 involved in lignin degradation, explored the molecular mechanism for high-efficiency lignin degradation, and assessed the relationship between gene transcription and the biodegradation of lignin using an SSH-based approach.
Strains and media
A. oryzae CGMCC5992 was isolated in our laboratory and deposited in the China General Microbiological Culture Collection Center (CGMCC). The strain was cultured on potato dextrose agar (PDA) slants at 28 °C for four days, then stored at 4 °C and passaged every seven to nine weeks. The lignin, consisting of 8.7% cellulose, 27.6% hemicelluloses, 48.1% lignin, 7.2% protein or peptone, 4.8% ash, and 3.6% wax, was obtained from an online provider (Yang Hai Chemical Co., Ltd, Ji’nan, Shandong Province, China).
The lignin-degradation medium consisted of 0.5 g/L lignin, 5 g/L peptone, 2 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 0.2 g/L ammonium tartrate, 1 g/L NaCl, 0.1 g/L CoSO4·7H2O, 0.1 g/L CaCl2, 0.1 g/LCuSO4·5H2O, 0.5 g/L MnSO4·H2O, 0.01 g/L H3BO3, 0.1 g/L FeSO4·7H2O, 0.1 g/L ZnSO4·7H2O, 0.001 g/L glycine, and 0.001 g/L vitamin B1.
The control medium consisted of 0.5 g/L glucose, 5 g/L peptone, 2 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 0.2 g/L ammonium tartrate, 1 g/L NaCl, 0.1 g/L CoSO4·7H2O, 0.1 g/L CaCl2, 0.1 g/L CuSO4·5H2O, 0.5 g/L MnSO4·H2O, 0.01 g/L H3BO3, 0.1 g/L FeSO4·7H2O, 0.1 g/L ZnSO4·7H2O, 0.001 g/L glycine, and 0.001 g/L vitamin B1.
Preparation of biomass
A. oryzae spores (1×106) were inoculated into a 250-mL flask containing 100 mL potato dextrose (PD) medium and incubated in a shaking incubator at 30 °C and 125 rpm for two days. A 10% (v/v) inoculum was aseptically added to the 10-mL lignin-degradation medium and control medium, respectively. The fermentation was carried out in a shaking incubator at 30 °C, 125 rpm for seven days. The biomass was obtained through filtration with four layers of gauze, washed three times with 0.01 M phosphate buffer solution (pH 7.4), and stored at −80 °C prior to RNA extraction. The biomass from the lignin-degradation medium and the control medium was labeled as A and B, respectively. In the forward-subtract process, A and B were used as the “test” and “drive,” while they were used as the “drive” and “test” in the reverse-subtract process, respectively.
RNA isolation and cDNA synthesis
Total RNA was extracted using a Fungi RNA kit (R6618, Sigma, USA) following the manufacturer’s instructions. The mRNA was extracted and purified using PolyATtract® mRNA isolation system II (25200, Promega, USA) according to the manufacturer’s instructions. The cDNA synthesis was carried out using 1 μg mRNA with the PCR-selectTM cDNA subtraction kit (Clotech, USA). Differential screening was performed using a PCR-select differential screening kit (BD Biosciences, USA) according to the manufacturer’s instructions. Spots exhibiting at least a five-fold higher intensity with forward-subtracted probes compared with the reverse-subtracted probes were scored as positives.
DNA sequencing of different genes
The cDNA of positive spots was sequenced using an ABI 3730 automatic DNA sequencer (USA). A total of 300 positive clones were randomly sequenced at the GenScript Corporation (Nanjing, China). Sequence alignment and homology searches were performed using “TIGR” and “NCBI BLAST” databases available on the J. Craig Venter Institute website (blast.jcvi.org) (Chan et al. 2006; Johnson et al. 2008). E-value scores of less than 10−5 were considered significant, indicating homology between the obtained sequences and database sequences.
Validation of Differential Expression of Genes by Reverse Transcription PCR (RT-PCR)
The differentially expressed genes selected randomly from the ESTs were further validated by RT-PCR using gene-specific primer pairs designed by DNAMAN. The cDNAs of A. oryzae biomass growing in two different media were amplified. The quality and specificity of amplified products were confirmed by visualization on a 1% agarose gel.
RESULTS AND DISCUSSION
Purity Analysis of Total RNA and mRNA
UV absorption of RNA from biomasses A and B revealed a uniform peak at 257 nm without any shoulder peaks. Their A260/280 ratios were 1.9 and 2.01, respectively, suggesting little contamination by polysaccharides and proteins (Leite et al. 2012). Electrophoresis of the extracted RNA exhibited distinct 28S, 18S, and 5s rRNA bands (Fig. 1a), indicating high purity, integrity, and no obvious degradation of total RNA (Ping et al. 2010). The high-quality mRNA strip was obtained from total RNA through electrophoresis on 1.0% agarose gel, and the diffusion band (Fig. 1b) suggested that the size of mRNA ranged from several hundred base pair (bp) to over several thousand bp (kb) and met the requirement of the SSH experiment.
Fig. 1. (a) Agarose gel electrophoresis of total RNA from Aspergillus oryzae from the lignin-degradation medium and the control medium. (b) mRNA isolated from total RNA. A: The lignin-degradation medium; B: the control medium.
The Construction of cDNA Subtractive Library
To identify the genes associated with lignin degradation, SSH cDNA libraries (both forward and reverse) were constructed from high-quality mRNA. A total of 6,000 cDNA clones derived from the SSH-cDNA libraries, including 3,200 from the forward library and 2,800 from the reverse one, were successfully amplified.
A total of 87 positive clones with the strongest signals were sequenced, including 55 EST sequences from the forward library and 32 EST sequences from the reversed library. These sequence data had been validated.
Functional Classification of Different Genes
Based on sequence similarities to known genes in A. oryzae, 89 different genes in both carbon sources (glucose and lignin) were categorized into several groups according to their functions as follows: (1) hydrolysis; (2) energy metabolism; (3) transporters; (4) synthesis; (5) transcription; (6) protein synthesis; (7) signal transduction; (8) protein destination and storage; (9) division; (10) cell metabolism regulation and control; (11) cell structure; (12) unknown proteins; (13) intracellular traffic; and (14) no significant similarity to the NCBI BLAST search database.
Table 1. Characterization of Differentially Expressed Genes Randomly Selected from the Forward Library
Table 2. Characterization of Differentially Expressed Genes Randomly Selected from the Reversed Library
The data showed that the up-regulated genes were from the forward library, while the down-regulated genes were from the reverse library. Among the up-regulated genes, the largest functional gene group corresponded to the category of hydrolytic enzymes, including glutathione peroxidase, endo-1,3(4)-β-glucanase, acetate-CoA ligase, cytochrome P450 alkane hydroxylase, alkaline protease, ubiquitin, polyubiquitin UbiD/Ubi4, endo-mannanase, glycosyl hydrolase, 1,3-beta-glucanosyl-transferase, thaumatin-like protein, alkaline serine protease, acid phosphatase, glutaminase, and metalloproteinase. Glutathione peroxidase is a type of oxidoreductase produced by microorganisms or plants that is capable of making phenol and amine compounds to be oxidized. It has been reported that peroxidase can break down lignin (Beckett et al. 2015). We obtained sequences from A. oryzae in the medium using lignin as carbon source. The gene sequences of glutathione peroxidase were obtained from NCBI database.
The gene sequences of A. oryzae from 11 to 86 and 91 to 103 were identical to the sequences of glutathione peroxidase from 569 to 648. The matched sequences are shown in bold red in Figs. 3 and 4.
Fig. 3. The gene sequences of A. oryzae
Fig. 4. The gene sequences of glutathione peroxidase
Based on this high homology, one can infer that A. oryzae generated peroxidase to degrade lignin in the medium using lignin as a carbon source. In some literature, glutathione peroxidase could catalyze the reaction of converse H2O2 into ·OH in the presence of GSH to degrade organic substance (Navrot et al. 2006; Passaia and Margis-Pinheiro 2015).
It has been generally accepted that alkaline proteinase and metalloproteinase can catalyze the hydrolysis reaction of protein (Birkedal-Hansen et al. 1993; Anwar and Saleemuddin 1998), and ubiquitin can hydrolyze protein (Baarends et al. 1999). Glutaminase can hydrolyze glutamine to glutamic acid and participate in transdeamination, by which the amino acids from protein hydrolysis are changed into fatty acids (Koibuchi et al. 2000). Based on these ideas, the up-regulation of these four genes indicates the increase of protein hydrolysis using lignin as a carbon source. As two key components in plant cell walls, mannans and various 1,4-β-linked glucan polymers play an important structural role in plants (Whitney et al. 1998). The key endo-β-mannanase (EC 220.127.116.11) can randomly hydrolyze mannan by breaking the internal β-1,4-D-mannopyranosyl bond in the mannan backbone (Yuan et al. 2007), and endo-1,3(4)-β-glucanase can degrade crystalline cellulose (Nicol et al. 1998). The up-regulation of these two enzymes in the presence of lignin reveals that the semi-cellulose and cellulose hydrolysis by the strain was enhanced in response to lignin.
The expression of cytochrome P450 alkane hydroxylase was increased in the presence of lignin. As a heme-thiolate enzyme, it can collectively catalyze a range of relatively specific monooxygenase reactions and transform diverse lipophilic compounds into more polar metabolites (Yadav and Loper 1999). Cytochrome P450 alkane hydroxylase is an inducible enzyme and is not synthesized in the absence of alkane. The up-regulation of this enzyme indicates that alkane existed in the medium. However, there was no alkane in the medium, nor was there the catabolism of protein, cellulose, or semi-cellulose. The only source of alkane was the hydrolysate of the lignin caused by lignin hydrolytic enzymes (Yan et al. 2008). Therefore, the presence of cytochrome P450 alkane hydroxylase may indirectly prove the possibility that the strain synthesized lignin hydrolytic enzymes to degrade lignin. These digestive enzymes were synthesized in the cytoplasm and secreted outside the cells in vesicle form.
The second largest gene group corresponded to functions relating to transporters, including synaptobrevin, SNARE protein, v-SNARE protein, C2H2 transcription factors, and phthalate transporters. Synaptobrevin, a highly conserved membrane protein originally described as a component of brain synaptic vesicles, may play a general role in membrane trafficking and in the secretion of digestive enzymes (Chin et al. 1993). SNARE protein mediates the specificity of vesicle trafficking by defining membranes compatible for docking and fusion (Bock and Scheller 1999). v-SNARE drives Ca2+-triggered membrane fusion at a millisecond-timescale (Kesavan et al. 2007). These proteins may involve the transfer of hydrolytic enzymes from intracellular to extracellular status.
The third gene group consisted of cell structure proteins, including integral membrane proteins, plasma membrane proteolipids, and glycolipid-anchored surface proteins. The up-regulation of these genes indicates that lignin as a carbon source altered the content of different components in the cell membrane. The β-1,3-glucanosyl transferases internally split the β-1,3-glucan molecule and transferred the newly-generated reducing end to the non-reducing end of another β-1,3-glucan molecule, resulting in the elongation of the β-1,3-glucan side chains (Caracuel et al. 2005). The existence of UDP-sugar pyrophospharylase implies that the biomass of A. oryzae synthesized the glycogen as energy storage from other materials (Schnurr et al. 2006). Therefore, the up-regulation of these two enzymes shows that A. oryzae increased gluconeogenesis when it degraded lignin. The up-regulation of the heat shock protein shows that the A. oryzae mycelia enhanced the function to protect the cell itself and to repair the damage caused by various stimuli (Whitley et al. 1999). Here, the heat shock protein mainly protected cells from damage from H2O2,which was synthesized by the mycelia itself, and it also acted as another substrate of lignin by enzymatic hydrolysis. The other genes corresponded to cell division and some transcription. In conclusion, the up-regulation of these genes indicates that the hydrolytic effect, gluconeogenesis, and the repairing function were significantly enhanced and their protein contents in the cell membrane were also increased.
Protein kinases contain a highly conserved catalytic region and a less conserved regulatory domain, and they are usually modular enzymes that play a role in nearly every aspect of cell biology (Lakshminarayan et al. 2008; Shen et al. 2014). C2H2 zinc finger protein binds to speciﬁc DNA sequences via its zinc ﬁngers, and it modulates gene expression during diverse biological processes such as cell growth, differentiation, tumorigenesis, embryogenesis, and apoptosis (Jiang et al. 2007). It is believed that GTP-binding proteins are the main regulators of vesicular transport in eukaryotic cells, by which glucose is transferred from the outer membrane to the site where glucose is decomposed (Rothman 1994). These proteins are all concerned with glucose metabolism; therefore their down-regulation suggests that glucose metabolism was limited. Another vital functional gene group corresponded to the category of metabolism, including acyl-CoA desaturase, stearic acid desaturase, phosphatidate cytidylyltransferase, malate synthase, aconitate hydratase, ATP synthase F1-sector subunit beta, and plasma membrane ATPase 2. Acyl-CoA desaturase and stearic acid desaturase are the key enzymes of fatty acid catabolism (Hodson and Fielding 2013). Phosphatidate cytidylyltransferase catalyzes the synthesis reaction of triglyceride, which is mainly in the cell membrane (Longmuir and Johnston 1980). The down-regulation of these genes further revealed the changes in cell membrane components. Aconitate hydratase and malate synthase are the rate-limiting enzymes of the tricarboxylic acid cycle and glyoxylate cycles, respectively (Umemura et al. 1997). Their down-regulation indicated that the aerobic metabolism, ATP synthesis, and utilization were inhibited, which was demonstrated by the down-regulation of ATP synthase F1-sector subunit beta and plasma membrane ATPase 2. Taken together, the down-regulation of genes demonstrates that glucose catabolism, lipid synthesis metabolism, and energy metabolism were all suppressed, and the components of the cell membrane were also changed.
- The present study is the first report of the different gene expressions of A. oryzae grown in the medium containing lignin and glucose, respectively.
- The discovery of peroxidase from up-regulation genes in the medium using lignin as a carbon source explained that A. oryzae could degrade lignin in the straw. This finding provided evidence of the micromechanism interacting between the microorganism and lignin.
- From the number of over-expressed unigenes, it was also highly convincing that A. oryzae could hydrolyze protein, cellulose, and hemicelluloses in the straw.
- Surprisingly, the strain possessed the capability for gluconeogenesis and changed the components of the cell membrane in the presence of lignin, including the increase of protein components and the decrease of lipids.
- Lignin, as a carbon source, suppressed the aerobic metabolism of glucose, and it also reduced the synthesis and utilization of energy. The ability of mycelia’s resistance to various stimuli and self-repair was significant.
This work was supported by grants from the Department of Sequencing, Shanghai, and the National Natural Science Foundation of China (No. 31101269), the Natural Science Foundation of Jiangsu Province (No. BK2011154), the Nantong Social Development Fund (No. S2011003), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Article submitted: March 11, 2015; Peer review completed: May 15, 2015; Revised version received and accepted: Aug. 26, 2015; Published: August 31, 2015.