Effects of Amino Acids on the Lignocellulose Degradation by Aspergillus Fumigatus Z5
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Miao et al. Biotechnol Biofuels (2019) 12:4 https://doi.org/10.1186/s13068-018-1350-2 Biotechnology for Biofuels RESEARCH Open Access Efects of amino acids on the lignocellulose degradation by Aspergillus fumigatus Z5: insights into performance, transcriptional, and proteomic profles Jiaxi Miao1,2, Mengmeng Wang1,2,3, Lei Ma1,2,3, Tuo Li1,2,3, Qiwei Huang1,2,3, Dongyang Liu1,2,3* and Qirong Shen1,2,3 Abstract Background: As a ubiquitous flamentous fungal, Aspergillus spp. play a critical role in lignocellulose degradation, which was also defned as considerable cell factories for organic acids and industrially relevant enzymes producer. Nevertheless, the production of various extracellular enzymes can be infuenced by diferent factors including nitro- gen source, carbon source, cultivation temperature, and initial pH value. Thus, this study aims to reveal how amino acids afect the decomposition of lignocellulose by Aspergillus fumigatus Z5 through transcriptional and proteomics methods. Results: The activities of several lignocellulosic enzymes secreted by A. fumigatus Z5 adding with cysteine, methio- nine, and ammonium sulfate were determined with the chromatometry method. The peak of endo-glucanase 1 1 1 (7.33 0.03 U mL− ), exo-glucanase (10.50 0.07 U mL− ), β-glucosidase (21.50 0.22 U mL− ), and xylanase ± 1 ± ± (76.43 0.71 U mL− ) were all obtained in the Cys treatment. The secretomes of A. fumigatus Z5 under diferent treat- ments± were also identifed by LC–MS/MS, and 227, 256 and 159 diferent proteins were identifed in the treatments of Cys, Met, and CK (Control, treatment with ammonium sulfate as the sole nitrogen source), respectively. Correlation analysis results of transcriptome and proteome data with fermentation profles showed that most of the cellulose- degrading enzymes including cellulases, hemicellulases and glycoside hydrolases were highly upregulated when cysteine was added to the growth medium. In particular, the enzymes that convert cellulose into cellobiose appear to be upregulated. This study could increase knowledge of lignocellulose bioconversion pathways and fungal genetics. Conclusions: Transcriptome and proteome analyses’ results indicated that cysteine could signifcantly promote the secretion of lignocellulosic enzymes of an efcient lignocellulosic decomposing strain, A. fumigatus Z5. The possible reason for these results is that Z5 preferred to use amino acids such as cysteine to adapt to the external environment through upregulating carbon-related metabolism pathways. Keywords: Lignocellulose, Aspergillus fumigatus Z5, Secretome, Amino acids, Transcriptome, Proteome *Correspondence: [email protected] 3 College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing 210095, China Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Miao et al. Biotechnol Biofuels (2019) 12:4 Page 2 of 19 Background lignocellulose, microbial degradation technology has Various saprotrophic flamentous fungi own a consid- attracted a large amount of attention worldwide because erable capacity of lignocellulose-degrading efciency, of its advantages of having low cost, employing mild which is considered as the most abundant natural mate- reaction conditions, and lack of pollution to the environ- rials, and it is the most abundant resource present in a ment [7]. Due to high extracellular enzymatic activity and variety of plants that humans can easily access and use. a relatively large number of enzymatic species, fungi have Te growing focus on depleting fossil fuels requires a a considerable capacity to degrade cellulose. Meanwhile, shift from nonrenewable carbon sources to renewable the fungi can contribute signifcantly to recycling ligno- biological resources such as lignocellulose. Regardless of cellulosic biomass due to their capacities of secreting a the cause, lignocellulosic materials consist of three main large number of lignocellulolytic enzymes [8]. Terefore, polymers: cellulose (a glucose homopolymer), hemicel- flamentous fungi, including Trichoderma, Aspergillus, lulose, (heteropolymers of pentoses and hexoses), and Penicillium, Acremonium, Myrothecium, Neurospora, and lignin (phenyls, amorphous polymers) [1]. Approximately Chaetomium, have been extensively applied in the cellu- 180 billion tons of cellulose are produced annually by lose industry. A. fumigatus Z5 can efciently decompose plants, making this polysaccharide a substantial organic various agricultural enzymes with the help of cellobiohy- carbon pool on earth [2]. It is one of the most widely dis- drolases which belong to the glycoside hydrolase (GH) tributed and most abundant substances on earth and one families and carbohydrate-active enzymes (CAZy) of the cheapest renewable resources. Plant cellulose is including GH1, GH3, GH5, GH9, GH12, GH44, GH45, mainly degraded by various microorganisms into organic and lytic polysaccharide monooxygenases (LPMO) [9, carbon sources and then transformed into the most sub- 10]. In addition, A. fumigatus Z5 genomes encode many stantial material fows in the biosphere. Terefore, the other CAZy such as polysaccharide lyases (PLs) and car- importance of cellulose as a renewable energy source has bohydrate esterases (CEs), for the degradation hemicel- become the subject of research and commerce. Neverthe- luloses and pectin [10]. less, the critical step in the use of cellulose is its hydroly- Many studies on the microbial degradation of ligno- sis into monomeric sugars and its eventual conversion to cellulose have mainly focused on microbial resources, valuable chemicals and energy [3]. enzyme properties and synthetic regulation, and enzyme Lignocellulolytic enzymes are a series of enzymes genetic engineering [11, 12]. However, selection of the related to lignocellulose degradation, including pecti- specifc nutritional factors that infuence the biodegra- nases, cellulases, hemicellulases, manganese peroxidase dation ability of lignocellulosic fungi and its concrete (MnP), lignin peroxidase (LiP), and laccase (Lac), [4]. As mechanisms is still rarely reported at present. Nitrogen the major components of lignocellulolytic enzymes, cel- sources are indispensable during the secretion process of lulase consists of at least three types of enzymes: endo- various extracellular enzymes by A. fumigatus Z5, espe- glucanases (EC 3.2.1.4) which act randomly on insoluble cially for specifc kinds such as amino acids and peptides. and soluble cellulose chains; exo-glucanases (cellobiohy- As one of the important nutritional factors, amino acids, drolases EC 3.2.1.91), which respond to liberate cellobi- and their analogs are known to stimulate the enzyme ose from the reducing and nonreducing ends of cellulose production of various fungi, such as α-amylase and xyla- chains; and β-glucosidases (EC 3.2.1.21), which liberate nases [13, 14]; unfortunately, this biological mechanism is glucose from cellobiose [5]. Each component contains not yet clear. Here, amino acids were added into culture multiple isoenzymes, such as the Trichoderma reesei medium containing rice straw powder, and the efect of cellulase system, including at least fve endonucleases amino acids on the cellulose production of A. fumiga- (EGI–EGV), two exonucleases (CBHI, CBHII), and two tus Z5 was explored to reveal the intrinsic mechanism β-glucosidases (BGI, BGII). Enzymes degrading the through the combination of transcriptome and proteome hemicelluloses (called hemicellulases) are well character- analysis methods, which can reveal the specifc lifestyle ized, and are classifed according to their substrate spe- of each fungal species and the strategy that each species cifcities, such as xylanase, lichenase, and laminarinase. utilizes for lignocellulose conversion [15–17]. Transcrip- Pectinase is an enzyme that can break down pectin. Te tomics can help reveal a synergistic response of a fun- degradation of lignocellulose requires the synergistic gal strain to the external environment and nutritional action of all these enzymes mentioned above, especially changes, and proteomics is a useful tool to discover pro- cellulases and hemicellulases. fle and identify various proteins in response to special Most of the hydrolytic enzymes are secreted by various environment. microbes, including bacteria, actinomycetes, and fla- Te objective of this study is to reveal how amino acids mentous fungi, which have been screened from various (cysteine and methionine) afect lignocellulose biodeg- habitats [1, 6]. Among the diferent methods of utilizing radation by the efcient lignocellulose-degrading strain Miao et al. Biotechnol Biofuels (2019) 12:4 Page 3 of 19 A. fumigatus Z5. Moreover, the RNA-seq transcriptome factors to evaluate the biodegradation of rice straw by profles and the 4-plex 2D HPLC–MS/MS quantita- A. fumigatus Z5, and the endo-glucanases, exo-glu- tive proteomic profle were applied to analyze the genes canases, β-glucosidases,