Comparative genomic analysis of CAZymes of B. velezensis strains
The assembled genome of B. velezensis LC1, which contains 44 GHs, 38 GTs, 30 CEs, 3 PLs, 6 AAs, and 15 CBMs, was compared to the genomes of other B. velezensis strains. The 20 B. velezensis strains consist of: B. velezensis 10075, B. velezensis 157, B. velezensis FZB42, B. velezensis 83, B. velezensis UCMB5036, B. velezensis LB002, B. velezensis S3-1, B. velezensis At1, B. velezensis BCSo1, B. velezensis JTYP2, B. velezensis BS-37, B. velezensis CN026, B. velezensis B25, B. velezensis DR-08, B. velezensis LPL-K103, B. velezensis GFP-2, B. velezensis T20E-257, B. velezensis GYL4, B. velezensis TB1501, and B. velezensis LS69. Their genomes were comparatively analysed with B. velezensis LC1. At the whole genome level, the B. velezensis LC1 resembles most B. velezensis DR-08 (Fig. 1), while at the CAZyome level, LC1 resembles most B. velezensis S3-1 (Fig. 2).
Genes encoding CAZymes were detected in 21 genomes (Table 1 and Table S1). Sequence analysis results reveal the common and unique features in these lignocellulose degradation genes (Table 1 and Table S1). For example, some cellulolytic enzyme genes such as GH30 and GH5 are common to 21 strains, that also often contain endoglucanases (EC 126.96.36.199). GH1, GH4, and GH16 contribute to cellulose degradation by potentially functioning as 6-phospho-β-galactosidase, 6-phospho-β-glucosidase, and β-1,3-1,4-glucanase, respectively; they frequently appeared in 21 genomes. In addition, common genes capable of hemicellulose degradation were obtained. Arabinan endo-1,5-α-L-arabinosidase, α-N-arabinofuranosidase, and glucuronoxylanase are critical for xylan degradation; they belong to GH43, GH51, and GH30. GH53 hydrolyses (1→4)-β-D-galactosidic linkages in type I arabinogalactans . GH26 functions as β-mannosidase . Some CEs with the potential to deacetylate xylans and xylooligosaccharides, for example, CE3 (acetyl xylan esterase) , and CE 7 (acetylxylan esterase)  were also found frequently. The coexistence of these genes suggests that they play important roles in the enzymatic degradation of cellulose and hemicellulose. We consider these degradation enzymes in B. velezensis to have potential use for biothanol production.
Two PL1 and one PL9 genes are found in 21 genomes and are known to play roles in pectin degradation. The action of pectatelyase on (1→4)-α-D-galacturonan results in the production of oligosaccharides . CE4, which contain polysaccharide deacetylases that function in polysaccharide degradation,  was also detected in the genomes. Moreover, we also identified several non-catalytic modules with binding functions, including CBM3, which binds to cellulose, and CBM6, which binds to amorphous cellulose and β-1,4-xylan .
GH32 contains sucrose-6-phosphate hydrolase and levanase, and are thus expected to function in sucrose hydrolysis . GH13 (α-amylase, α-glucosidase, α-glycosidase, and α-α-phosphorylase) and CBM50 (involved in starch hydrolysis)  were also detected. GH65 (maltose phosphorylase) was identified in several B. velezensis genomes; this sequence is associated with trehalose degradation .
There are few auxiliary activities (AA) genes in all strains. Only the AA10 family was annotated in all strains, while AA4, AA6, and AA7 were only found in strain LC1. AA4 includes vanillyl-alcohol oxidases that exert invertase activity on phenols . In addition, AA7 can biotransform or detoxify lignocellulose . Several Bacillus strains have been considered lignin degraders. For example, Bacillus sp. LD003 affects lignin fractions ; B. pumilus C6 and B. atrophaeus B7 have laccase activity that degrades kraft lignin and the dimer guaiacylglycerol-b-guaiacyl .
Effect of alkali pretreatment on chemical components of bamboo
Lignocellulose-derived ethanol is widely considered a clean liquid fuel . However, raw lignocellulose has a recalcitrant structure that lowers bioconversion efficiency, making pretreatment necessary to make lignocellulose more vulnerable to enzymes or bacteria for the fermentation of ethanol and other products [40,41]. Bamboo lignocellulose pretreated by alkali has a higher enzymatic digestibility than the raw material [42-44]. In the present study, we used sodium hydroxide (NaOH) to pretreat the bamboo powders. Table 2 lists the chemical compositions of pretreated and unpretreated bamboo powder (solid fraction) and shows that dried bamboo contains cellulose, hemicellulose, lignin, ash, neutral detergent fibre, acid detergent fibre, and acid detergent lignin in approximate percentages of 22.13 ± 2.52, 44.94 ± 4.12, 18.71 ± 2.76, 1.94 ± 0.02, 87.72 ± 3.87, 65.59 ± 4.65 and 20.65 ± 1.77, respectively. After alkaline pretreatment, the lignin content decreased to 6.92% ± 0.84%, while the hemicellulose increased to 30.19% ± 2.61%. Cellulose remained stable before and after alkaline pretreatment, which is consistent with previous studies . The results indicate that NaOH pretreament of bamboo efficiently removes lignin without significantly impacting the cellulose.
Identification of secretomes of B. velezensis LC1 grown on medium containing with bamboo powder
To further confirm the lignocellulase system in B. velezensis LC1 cultured in bamboo powder, we analysed the secretomes of B. velezensis LC1 grown on bamboo powder and glucose medium (control). First, the supernatants of a three-day treatment were precipitated with 12% (w/v) trichloroacetic aid (TCA) to obtain proteins. Secretomic protein was detected only in the bamboo powder medium (Fig. 3a). These proteins were analysed by 1D-PAGE (Fig. 3b) and LC–MS/MS. The basic characteristics of the proteins are shown in Fig. 3c. The pI values of most proteins are in the range of 5.0-10.0. A total of 142 proteins were identified; these include a lignocellulolytic enzyme, protease, and other proteins (Table S3). These proteins were then functionally annotated, resulting in the characterisation of 612, 69, 348, and 79 proteins as being enriched in biological processes, cell components, molecular functions, and KEGG pathway functions, respectively (Fig. 3d; Fig. S1; Fig. S2). In addition, degradation-related enzymes including hemicellulases, cellulases, and other GHs were abundant in bamboo powder medium (Table 3).
Hemicellulose is the second most abundant component in lignocellulose; it is hydrolysed into monosaccharides by multiple enzyme systems [46-48]. Arabinogalactan endo-beta-1,4-galactanase (EC 188.8.131.52), which belongs to GH 53, catalyses the hydrolysis of β-1,4-galactosidic bonds in arabinogalactan and galactan side chains . Other GHs exhibit hemicellulose activities. For example, β-xylanases cleave the xylan backbone, while β-xylosidases release the xylose units from xylobiose and xylooligomers. Acetyl xylan esterase is an accessory enzyme that functions synergistically with other enzymes in removing side chain residues from the hemicellulose backbone [50,51]. In the present study, four hemicellulases were identified, consisting of one arabinogalactan endo-β-1,4-galactanase (GH53) (1384349889), one beta-xylanase (GH43) (1384351325), one glucuronoxylanase (GH30) (1384349284), and one acetylxylan esterase (CE1) (1384350660) (Table 3).
Cellulose is hydrolysed synergistically by different enzymes, that is, endoglucanases randomly break internal bonds, then, exoglucanases remove cellobiose, and finally β-glucosidases release glucose from cellobiose [52-55]. β-Glucanases catalyse the degradation of β-glucans and are classified into β-1,3-1,4-glucanases (EC 184.108.40.206), β-1,4-glucanases (EC 220.127.116.11), β-1,3-glucanases (EC 18.104.22.168), and β-1,3(4)-glucanases (EC 22.214.171.124) [56-58]. Microbial β-1,3-1,4-glucanases have been identified as GH 16 and are mainly produced by Bacillus [59-62]. Two different enzymes, an endoglucanase (GH5) (1384349288) and a β-1,3-1,4-glucanase (GH16) (1384351103), which probably participates in cellulose degradation, were identified (Table 3).
AAs play a vital role in lignin degradation . In this study, two AA6s (1384350268 and 1384350569), one AA7 (1384350476), and one AA10 (1384349337) protein were identified. Moreover, other enzymes involved in starch degradation, plant cell wall modifications, and protein degradation (proteases) were detected (Table 3).
Hydrolysis of bamboo powder by B. velezensis LC1 grown on medium with or without bamboo powder
The following B. velezensis strains have attracted attention due to their applications in agriculture and biotechnology: B. velezensis GH1-13 , B. velezensis S3-1 , B. velezensis FZB42 , B. velezensis M75 , B. velezensis LS69 , B. velezensis 9912D , and B. velezensis S499 . B. velezensis LC1, which has shown bamboo lignocellulose degrading ability, was isolated from the intestinal microbiome of Cyrtotrachelus buqueti. The lignocellulose degrading ability of B. velezensis LC1 was demonstrated through bamboo degrading experiments.
Enzymes play critical roles in lignocellulose degradation. For example, lignin peroxidase and manganese peroxidase are important and necessary for the degradation of lignin and laccase, respectively . Endoglucanases, exoglucanases, and β-glucosidases synergistically act to degrade cellulose , while xylanases are essential components of the hemicellulose degrading system . Then we measured cellulase (endoglucanase, β-glucosidase and exoglucanase), hemicellulase (xylanase), and ligninase (laccase, lignin peroxidase and manganese peroxidase) activities, the activities of xylanase, lignin peroxidase, and laccase were significantly higher for bamboo powder than those for glucose (Fig. 4). Endoglucanase, β-glucosidase, and manganese peroxidase activities for bamboo powder were significantly higher than those for glucose after 3 d of treatment. However, exoglucanase activity was higher after 4 d of treatment. These results indicate that higher lignocellulolytic enzyme activities are involved in bamboo lignocellulose degradation.
Then, we measured lignocellulose degradation efficiencies using pretreated bamboo powder. After 6 d of culturing, the degradation products were analysed. The degradation efficiencies of cellulose, hemicellulose, and lignin in bamboo powder were 59.90%, 75.44%, and 23.41%, respectively (Fig. 5a). Several B. velezensis strains have lignocellulose degrading ability [14-18], which supports our finding that B. velezensis LC1 can efficiently degrade bamboo lignocellulose components.
Bamboo used for mushroom cultivation contains abundant cellulose, which is considered suitable for bioethanol production. Much of the research devoted to bamboo lignocellulose bioethanol production have focused on the physical and chemical pretreaments [3,5]. In our study, the ethanol productivity of bamboo was assessed by a continuous reaction of B. velezensis LC1 hydrolysing lignocellulose, Saccharomyces cerevisiae fermenting glucose, and Escherichia coli KO11 fermenting xylose. The bamboo-based ethanol production process is shown in Fig. 5b; its ethanol yield reached 10.44 g/L at 96 h, while the amount of reducing sugar decreased. This indicates that B. velezensis LC1 played an important role in the bioconversion of bamboo lignocellulose into ethanol.
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