Jilin province has always ranked first in China for corn yield. The abundant by-products of corn wet milling have great development potential as under-utilized feed resources (Shi et al. 2019). CGM is one of the alternative protein feeds to SBM, but its high fiber content, low crude protein content, and amino acid imbalance limit its application in livestock diets (Zhang et al. 2019b). Our previous study reported that the pretreatment of CGM with sodium bicarbonate effectively solves the problem of low pH caused by corn wet milling, and improves the fermentation rate and nutritional composition of CGM (Chen et al. 2022). For instance, our process significantly improved the number of viable bacteria, enzymes (cellulase, xylanase, β-mannanase, amylase, and protease), peptides, fatty acids, and other active metabolites in CGM. Most remarkably, it increases the hemicellulose degradation rate up to 71.33%. However, whether the lignocellulose-encoding genes of B. velezensis exist in symbiosis and get significantly upregulated in the 48 h sample of FCGM is the key focus of this investigation.
Presently, B. velezensis is mostly studied for biological control and plant growth promotion effects (Wang et al. 2022). Recently, reports on the application of B. velezensis in livestock and poultry are gradually increasing, mainly focusing on the detoxification of mycotoxins in feed (Zearalenone and Aflatoxin B1) (Shu et al. 2018; Wang et al. 2021) and aquatic probiotics (Zhang et al. 2019a). However, its potential application for fermented feeds has not been explored much. Bacillus can enzymatically degrade plant-sourced lignocellulose (Bandounas et al. 2011). In addition, several B. velezensis strains such as GH1-13 (Kim et al. 2017), FZB42 (Chowdhury et al. 2015), LS69 (Liu et al. 2017), 157 (Chen et al. 2018) and LC1 (Li et al. 2020), have cellulose and hemicellulose degradation genes. Here, we performed CAZyme analysis on the twenty-three representative strains of B. velezensis from different sources. Many genes encoding common CAZymes were detected in the genomes of these B. velezensis strains. For instance, cellulolytic enzyme genes such as endoglucanase were common in all the examined B. velezensis strains. The enzymes 6-phospho-β-galactosidase, 6-phospho-β-glucosidase, 6-phospho-α-glucosidase, α-galactosidase, and β-1,3–1,4-glucanase belonging to the GH1, GH4, and GH16 families that participate in cellulose degradation, were also present in all the B. velezensis genomes. Furthermore, β-1,3–1,4-glucanase from the GH16 family, which has been found in other Bacillus species (Teng et al. 2006).
The function of CBMs is to anchor relevant enzymes to the substrate, enhance enzyme activity and increase specificity between the enzyme and substrate (Crouch et al. 2016). CBMs are present in many bacterial and fungal enzymes (Igarashi et al. 2009). CBM2 and CBM16 genes, which participate in cellulose degradation, were present in all the twenty-three B. velezensis strains. CBM2 is a part of many bacterial enzymes and helps enzyme binding to cellulose, xylan, and chitin (Wu et al. 2022). CBM 16 improves enzyme binding to cellulose and glucomannan (Wang and Xu 2019).
Cellulose is synergistically hydrolyzed by different enzymes, for instance, endoglucanases randomly cut the amorphous region of the cellulose polysaccharide chain to generate cellobiose(Guo et al. 2018). However, in the lack of exoculcellulase, B. velezensis may not directly hydrolyze cellobiose, which limits the cellulose degradation ability of B. velezensis. All the twenty-three B. velezensis strains lack the enzymes for complete cellulose degradation, and therefore it is necessary to add appropriate exogenous enzymes for collaborative treatment. In addition, common genes from the GH11, GH43, GH51, and GH30 families participate in hemicellulose degradation including endo-β-1,4-xylanase, arabinoxylan arabinofuranohydrolase, arabinan endo-1,5-α-L-arabinosidase, 1,4-β-xylosidase, α-N-arabinofuranosidase, and glucuronoxylanase and are the key factors for xylan degradation. Hemicellulose, the second most abundant lignocellulose component, can be hydrolyzed to monosaccharides by a variety of enzymatic systems (Tang et al. 2021). The catalytic site of endo-1,4-β-xylanase from the GH11 family is the β-1,4-xylosidic linkages of xylan, producing short xylooligosaccharides after xylan cleavage (Monica and Kapoor 2021). In the field of pulp bleaching, xylanases from the GH11 family are favored over xylanases from the GH10 family. The GH11 xylanases are smaller in size (~ 20 kDa), lack cellulase activity, and easily penetrate the cellulose fiber network without damaging the fiber (Bai et al. 2015). The function of β-xylosidase and β-mannosidase is to release xylose units from xylobiose and xylooligosaccharides (Barker et al. 2010), and the hydrolysis of the terminal mannose of mannan polysaccharides (Malgas et al. 2015). Some CEs (acetylxylan esterase) have the potential to deacetylate xylan and degrade xylose oligosaccharides, such as CE3, which were also found in our study. CE3 can enhance the solubilization of xylan (Zhang et al. 2011). In addition, several CE4 polysaccharide deacetylases, which participate in the degradation of plant polysaccharides, were also found in B. velezensis strains. In addition to the peptidoglycan N-deacetylates involved in chitin degradation, CE4 are a highly specific class of acetylxylan esterases, but they cannot degrade acetyl galactoglucomannan or acetylated manno-compounds (Biely 2012). Therefore, the hemicellulose-encoding genes that exist in B. velezensis may have potential applications in the food, feed, paper, and biofuel industries. Also, the PL1 (two) and PL9 (one) genes were present in all the twenty-three B. velezensis genomes, which participate in pectin degradation. The end of oligosaccharides is mainly subjected to elimination cleavage of (1 → 4)-α-D-galacturonan by a pectin lyase (See-Too et al. 2017). Levanase belonging to the GH32 family participates in sucrose hydrolysis (Bezzate et al. 1994). α-amylase and α-glucosidase from the GH13 family participate in starch hydrolysis (Graebin et al. 2016). There were also some auxiliary CAZymes (such as AA4, AA6, AA7, and AA10) in the twenty-three B. velezensis strains. Vanillyl-alcohol oxidases (VAO), a member of the AA4 family, can transform various phenolic compounds with side chains located in the aromatic ring counter-position (Xu et al. 2021). In addition, AA7 enzymes associated with biotransformation or detoxification of lignocellulosic compounds were also detected (Levasseur et al. 2013). AA10 proteins, which mainly act on chitin or cellulose, are a class of copper-dependent lytic polysaccharide monooxygenases (LPMOs) (Forsberg et al. 2014). Several Bacillus strains possess ligninase activity. For instance, B. pumilus C6 and B. atrophaeus B7 have laccase activity and degrade kraft lignin and dimer guaiacylglycerol-b-guaiacyl (Huang et al. 2013). Bacillus sp. LD003 is mainly adsorbed on the lignin component decolorizing Azure B, methylene blue, toluidine blue O and other dyes (Bandounas et al. 2011). Therefore, the presence of these lignocellulase genes in B. velezensis genomes suggests that the bacteria can help hemicellulose degradation, including some amount of cellulose, starch, and pectin.
Combined with the results in Table 1 and Fig. 1, in total, 108 CAZymes were found in the twenty-three B. velezensis strains, while the other homologous family genes showed some differences. Geographic origin and habitat can influence the function of CAZymes from lignocellulosic degrading bacteria. For example, B. velezensis LC1 is an endophytic bacterium isolated from the gut of Cyrtotrachelus buqueti that can degrade bamboo lignocellulose (Li et al. 2020). B.velezensis CL-4, examined in this study, is an intestinal bacterium isolated from chicken cecal contents and has non-starch polysaccharide (NSP) degradation activity. In the poultry cecum, most contents are undigested starch and NSP that are fermented by microorganisms or residual digestive enzymes to complete the digestion process (Chen et al. 2022). Some B. velezensis strains, such as GH1-13 (Kim et al. 2017), FZB42 (Chowdhury et al. 2015) and LS69 (Liu et al. 2017), are symbiotic rhizobacteria that can hydrolyze a series of polysaccharides, proteins and other compounds, which help their colonization in the rhizosphere. In general, the presence of such homologous family genes implies the same function and degradation mode, but the bacteria habitat may affect the specificity during the evolution of B. velezensis.
We performed transcriptomic analysis to compare the DEGs involved in lignocellulosic degradation that were significantly upregulated between the FCGM-0 h and FCGM-48 h samples. As shown in Table 2, GH13 is the main gene family involved in starch hydrolysis. GH43, GH51, and CE4 gene families are involved in hemicellulose hydrolysis. GH1, GH4, and PL1 gene families are involved in cellulose and pectin degradation. Notably, many highly expressed DEGs in FCGM-48 h were related to xylan degradation. The expression of xylan 1,4-β-xylosidase was found to be upregulated 5.29-folds. This enzyme attacks the β (→4 glycosidic bond on D-xylan, removing the D-xylose residue at the non-reducing end (Jamaldheen et al. 2019). The enzymes involved in xylan side chain removal, (two genes encoding α-N-arabinofuranosidase) were upregulated by 2.03 times and 3.36 times, respectively. These enzymes hydrolyze the terminal non-reducing α-L-arabinofuranoside residues and produce α-L-arabinosides (Lagaert et al. 2014). Acetoxylan esterase, an accessory enzyme, works with other enzymes to remove the backbone side-chain residues of xylan (Zhang et al. 2011). Three genes encoding acetoxylan esterases were highly expressed in this study. Genes encoding accessory enzymes involved in arabinan polymers breakdown (two genes encoding arabinan endo-1,5-α-L-arabinosidase) were upregulated by 2.15 times and 4.58 times, respectively. These enzymes improve the endo-hydrolysis of (1 → 5)-α-arabinofuranosidic linkages in (1 → 5) arabinans (Sunna and Antranikian 1997). Hence, the transcriptome analysis indicated a higher hemicellulose degradation rate in FCGM-48 h samples.
The DEGs involved in cellulose degradation mainly including maltose-6'-phosphate glucosidase and 6-phospho-β-galactosidase. The expression level of maltose-6'-phosphate glucosidase increased by 6.61 times. This enzyme hydrolyses a variety of 6-phospho-α-D-glucosides (Thompson et al. 1995). α-glucosidase and α-amylase genes, involved in starch degradation, were also upregulated by 2.13 times and 1.94 times. α-amylase participates in endo-hydrolysis of (1 → 4)-α-D-glucosidic linkages in starch, glycogen, and related polysaccharides and oligosaccharides (Janeček et al. 2014). The main role of α-glucosidase is to hydrolyze terminal non-reducing (1 → 4) -linked α-D-glucose residues and release D-glucose (Graebin et al. 2016). Pectin degradation is mainly carried out by the PL1 family of enzymes, which break down the pectin component in CGM.
The functions of GT enzymes are mainly associated with cell structure, storage, and signaling. They can biosynthesize an infinite number of oligosaccharides, polysaccharides, and glycoconjugates by transferring sugar residues from activated sugar donors to specific acceptor molecules forming glycosidic bonds (Coutinho et al. 2003). In this study, glycosyl transferases from the GT1, GT2, GT4, and GT28 families were significantly upregulated, which directly affected the biosynthesis of oligosaccharides, polysaccharides, cell wall chitin, and peptidoglycan required for bacterial metabolism (Klutts et al. 2006). Such observations of up-regulation were also found in some fungi such as Aspergillus fumigatus and Aspergillus tamarii (Miao et al. 2015). In addition, bacterial and fungal-secreted enzymes can be affected by the culture formats (Wang et al. 2010a). For A. niger, pretreated sugarcane bagasse improved the secretion of endoglucanase and xylanase in solid-state fermentation, while the submerged fermentation favored the production of β-glucosidase (Midorikawa et al. 2018). Therefore, culture format may also affect the B. velezensis secretion of lignocellulase during the fermentation of CGM. However, the enzyme production of B. velezensis during submerged fermentation of CGM still needs to be verified by subsequent experiments.
In conclusion, we found that B. velezensis CL-4 fermentation promoted arabinoxylan degradation in CGM, followed by the partial degradation of cellulose, pectin, and starch. Furthermore, transcriptome and CAZyme analysis revealed the common lignocellulase in the twenty-three strains of B. velezensis, which can be utilized for the degradation of cellulose and hemicellulose components of biomass. The different characteristics of B. velezensis secreted lignocellulase may be related to their habitats. Importantly, exogenous cellulase can be combined with B. velezensis to further improve the degradation of cellulose components in CGM.