Biofuel production by biodegradable lignocellulose has far-reaching prospects [13]. Research on the degradation system of lignocellulose-degrading microorganisms has great significance to its practical application and development. However, few microorganisms, only white rot fungi, have been reported that can degrade lignin and cellulose at the same time [18]. In the actual degradation of biomass materials, the degradation of lignin, cellulose and hemicellulose often occurs simultaneously and is interrelated. In our research, we found that DF3-3 has good degradation performance on both lignin and cellulose. More experiments and new approaches are needed in future work to better develop and apply DF3-3.
The enzymology for bacterial lignin degradation has been well-studied in recent years, and some bacterial specific enzymes for lignin degradation have been reported. Like Cα-dehydrogenase (LigD) [48], glutathione-dependent β-etherase enzymes (LigE, F, G) have been identified from Sphingomonas paucimobilis SYK-6 [49], a demethylase enzyme (LigX) from Pseudomonas paucimobilis [50], and DyP-type peroxidases from Rhodococcus jostii RHA1 [51]. Recent reports have shown multicopper oxidases that demonstrate laccase activities [52, 53]. In our study, we found that DF3-3 had Lac, Mnp and Lip activities. Genome research identified three multicopper oxidase coding genes (gene 5348, gene 4491, gene 2609), and gene 5348 has high similarity (96.64%) to the laccase structural protein gene of Streptomyces griseorubens (GGQ64023.1) [54], showing that DF3-3 has the ability to encode laccase at the genetic level. No gene encoding manganese peroxidase has been detected, but a catalase/peroxidase gene (gene3898) was found in DF3-3, which was 99.87% similar to KatG from Streptomyces sp. Akac8 [8]. The fur gene (gene3899) encoding a transcription regulator appears downstream, and these were reported as possible manganese peroxidase-encoding genes in Streptomyces reticuli [43, 44], suggesting that DF3-3 can exhibit manganese peroxidase activity. In addition, a gene (gene 6937) encoding a dye decolouring peroxidase was observed. The peroxidase encoded by it has a broad spectrum of substrates, which is also commonly reported in the depolymerization of lignin in some bacteria [22, 45, 46].
The hydrogen peroxide-producing enzyme system mainly participates in lignin degradation in the capacity of auxiliary enzymes [47]. These enzymes include glyoxal oxidase, aryl alcohol oxidase, quinone reductase and related dehydrogenases [48]. They produce hydrogen peroxide to support degradation by other peroxidases. The alcohol dehydrogenase (gene 2768), alcohol dioxygenase (gene 3045), and aldehyde dehydrogenase (aldH) annotated in the DF3-3 genome are thought to be involved in the process of lignin degradation. In addition, catalase removes the hydrogen peroxide produced by these reactions quickly enough to prevent oxidative damage to [4Fe-4S]-clusters in proteins and protect the body from toxification [24, 49]. The catalase-encoding gene (katE) identified in DF3-3 is thought to be involved in lignin degradation.
According to the GC-MS results, 2,4-di-tert-butylphenol and 2,2'-methyl bis(4-methyl-6-tert-butyl phenol) were involved in the metabolic processes of DF3-3. Recent studies have also shown that there is a 2,4-di-tert-butylphenol metabolic pathway for microbial degradation of lignin [55]. Considering the resorcinol pathway and its correlative gene, it is speculated that there may be a similar pathway in DF3-3. It may retain the structure of the tert-butyl side face and be metabolized by the meta-cleavage pathway. The formation of 2,2'-methyl bis(4-methyl-6-tert-butyl phenol) may come from the same metabolic intermediate as 2,4-di-tert-butylphenol. However, there are few studies on microbial degradation of this kind of structure at present. As an environmental pollutant, 4-tert-butylphenol can be degraded by several reported bacteria [56, 57]. However, to better understand this purification process, its specific metabolic process and some of the enzymes involved require further study.
The lignin degradation pathway of actinomycetes was first studied through research on the culture medium and metabolites [31, 58]. As the understanding of molecular biology increased, people began to seek more direct evidence. Masai et al. [23] first established a relatively complete pathway of lignin degradation and metabolism by means of enzymology and genomics. In Sphingomonas paucimobilis SYK-6, β-aryl ether cleavage catalysis [59], the biphenyl ring cleavage pathway [60], the ferulate catabolic pathway [61], the O-demethylation systems of vanillate and syringate [62], the protocatechuate 4,5-cleavage pathway [63], and multiple 3-O-methylagallate catabolic pathways [64] were described. Eleven lignin metabolic pathways were found in the genome of Cupriavidus necator, among which the β-ketoadipate pathway also included four branches: catechol, chlorocatechol, methylcatechol and protocatechuate ortho ring-cleavage [42]. In our study, evidence of a lignin degradation pathway was found in the metabolites and genetics. The products detected by GC-MS are also related to five lignin metabolic pathways. However, some unusual products detected may point to new branches. We speculate that 4-hydroxyphenylpyruvate (14) and 3-phenylpyruvic acid (12) may be transformed from phenylalanine and enter the homologous pathway and β-ketoadipate pathway, respectively, for further metabolism. A large number of genes related to the β-ketoadipate pathway have been detected in DF3-3, but there are still some genes involved in the reaction process that have not been compared, and for some genes, the reaction process in which they specifically participate has not been further elucidate. Perhaps there are related reactions in DF3-3 which are different from those in other bacteria. The heterologous expression of these gene fragments is helpful to the establishment and production of efficient engineered bacteria with biological enzymes.
Recent studies have shown that bacterial degradation of lignin has complex growth condition-specific regulation [65]. Because of the diversity of structure of lignan compounds, different reactions may occur in the degradation process of different substrate. Based on genome data alone, we predicted two other pathways the phenylacetate-CoA pathway and the 2,3-dihydroxyphenylpropionic acid pathway, but did not find the related metabolites by GC-MS. This absence might be related to the substrates. To further verify the degradation process, transcriptome analysis, proteomic analysis and other biological methods are needed to study the enzymes and the genes involved in their degradation pathways to understand the biological function of DF3-3 in the degradation of lignin.