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Biomass and its derivatives have broad applications in the fields of bio-catalysis, energy storage, environmental remediation. The structure and components of biomass, which are vital parameters affecting corresponding performances of derived products, need to be fully understood for further regulating the biomass and its derivatives. Herein, tobacco is taken as an example of biomass to introduce the typical characterization techniques in unraveling the structural information, chemical components, and properties of biomass and its derivatives. Firstly, the structural information, chemical components and application for biomass are summarized. Then the characterization techniques together with the resultant structural information and chemical components are introduced. Finally, to promote a wide and deep study in this field, the perspectives and challenges concerning structure and composition charaterization in biomass and its derivatives are put forward.

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Tobacco, a widely cultivated crop, has been extensively utilized by humans for an extended period. However, the tobacco industry generates a significant amount of organic waste, and the effective utilization of this tobacco waste has been limited. Currently, most tobacco waste is either recycled as reconstituted tobacco sheets or disposed of in landfills. However, tobacco possesses far more potential value than just these applications. This article provides an overview of the diverse uses of tobacco waste in agriculture, medicine, chemical engineering, and energy sectors. In the realm of agriculture, tobacco waste finds primary application as fertilizers and pesticides. In medical applications, the bioactive compounds present in tobacco are fully harnessed, resulting in the production of phenols, solanesol, polysaccharides, proteins, and even alkaloids. These bioactive compounds exhibit beneficial effects on human health. Additionally, the applications of tobacco waste in chemical engineering and energy sectors are centered around the utilization of lignocellulosic compounds and certain fuels. Chemical platform compounds derived from tobacco waste, as well as selected fuel sources, play a significant role in these areas. The rational utilization of tobacco waste represents a promising prospect, particularly in the present era when sustainable development is widely advocated. Moreover, this approach holds significant importance for enhancing energy utilization.

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Industrial tobacco waste was mainly treated via a reconstituted tobacco process using the paper-making method, which involves aqueous concentrated tobacco waste extract (cTWE) fermentation (aging). The fermentation was done to improve the quality of reconstituted tobacco. However, cTWE is a multi-stress environment that is characterized by low pH (about 4), as well as high sugar (above 150 g/L) and nicotine (above 15 g/L) content. In this study, a specific selection strategy was used to successfully isolate multi-stress-resistant bacterial or fungal strains, that exhibited positive effects on cTWE fermentation, thereby improving the quality of final products. A potential strain Zygosaccharomyces parabailii MC-5K3 was used for the bioaugmentation of cTWE fermentation and it significantly influenced the microbial diversity of the fermented cTWE. Zygosaccharomyces was observed to be the only dominant fungal genus instead of some pathogenic bacterial genera, with an abundance of over 95% after four days, and still more than 80% after a week. Meanwhile, metabolomics profiling showed significant concentration decrease with regard to some flavor-improving relative metabolites, such as 3-hydroxybenzoic acid (log2FC = - 5.25) and sorbitol (log2FC = - 5.54). This finding is extrapolated to be the key influence factor on the quality of the fermented cTWE. The correlation analysis also showed that the alterations in microbial diversity in the fermented cTWE led to some important differential metabolite changes, which finally improved various properties of tobacco products.

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Nicotine-degrading Pseudomonas sp. JY-Q is a preferred strain utilized in reconstituted tobacco process for tobacco waste treatment. However, its efficiency of nicotine metabolism still requires to be improved via genomic technology such as promoter engineering based on genomic information. Concerning upstream module of nicotine metabolic pathway, we found that two homologous genes of nicotine dehydrogenase (nicA2 and nox) coexisted in strain JY-Q. However, the transcriptional amount of nox was 20-fold higher than that of nicA2. Thus, the nicA2 expression required improvement. Combinatorial displacement was accomplished for two predicted endogenous promoters, named as PnicA2 and Pnox for nicA2 and nox, respectively. The mutant with Pnox as the promoters for both nicA2 and nox exhibited the best nicotine metabolic capacity which increased by 66% compared to the wild type. These results suggested that endogenous promoter replacement is also feasible for function improvement of metabolic modules and strain enhancement of biodegradation capacity to meet real environment demand.

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Nicotine and nicotinic acid (NA) are both considered to be representatives of N-heterocyclic aromatic compounds, and their degradation pathways have been revealed in Pseudomonas species. However, the cooccurrence of these two pathways has only been observed in Pseudomonas sp. strain JY-Q. The nicotine pyrrolidine catabolism pathway of strain JY-Q consists of the functional modules Nic1, Spm, and Nic2. The module enzyme, 3-succinoylpyridine monooxygenase (Spm), catalyzes transformation of 3-succinoyl-pyridine (SP) to 6-hydroxy-3-succinoyl-pyridine (HSP). There exist two homologous but not identical Spm enzymes (namely, Spm1 and Spm2) in JY-Q. However, when spm1 and spm2 were both in-frame deleted, the mutant still grew well in basic salt medium (BSM) supplemented with nicotine as the sole carbon/nitrogen nutrition, suggesting that there exists an alternative pathway responsible for SP catabolism in JY-Q. NicAB, an enzyme accounting for NA hydroxylation, contains reorganized domains similar to those of Spm. When the JY-Q_nicAB gene (nicAB in strain JY-Q) was introduced into another Pseudomonas strain, one that is unable to degrade NA, the resultant recombinant strain exhibited the ability to transform SP to HSP, but without the ability to metabolize NA. Here, we conclude that NicAB in strain JY-Q exhibits an additional role in SP transformation. The other genes in the NA cluster, NicXDFE (Nic2 homolog), then also exhibit a role in subsequent HSP metabolism for energy yield. This finding also suggests that the cooccurrence of nicotine and NA degradation genes in strain JY-Q represents an advantage for JY-Q, making it more effective and flexible for the degradation of nicotine.IMPORTANCE 3-Succinoyl-pyridine (SP) and 6-hydroxy-3-succinoyl-pyridine (HSP) are both valuable chemical precursors to produce insecticides and hypotensive agents. SP and HSP could be renewable through the nicotine microbial degradation pathway, in which 3-succinoylpyridine monooxygenases (Spm) account for transforming SP into HSP in Pseudomonas sp. strain JY-Q. However, when two homologous Spm genes (spm1 and spm2) were knocked out, the mutant retained the ability to degrade nicotine. Thus, in addition to Spm, JY-Q should have an alternative pathway for SP conversion. In this research, we showed that JY-Q_NicAB was responsible for this alternative SP conversion. Both of the primary functions for nicotinic acid dehydrogenation and the additional function for SP metabolism were detected in a recombinant strain harboring JY-Q_NicAB. As a result, both nicotinic acid and nicotine degradation pathways in JY-Q contribute to its remarkable nicotine tolerance and nicotine degradation availability. These findings also provide one more metabolic engineering strategy for accumulation for value-added intermediates.

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Nicotine contamination in tobacco waste effluent (TWE) from tobacco industry is a serious threat to public health and environment. Microbial degradation is an impending approach to remove nicotine and transform it into some other high value chemicals. Pseudomonas sp. JY-Q exhibits high efficiency of degradation, which can degrade 5 g/L of nicotine within 24 h. In strain JY-Q, we found the co-occurrence of two homologous key enzymes NicA2 and Nox, which catalyze nicotine to N-methylmyosmine, and then to pseudooxylnicotine via simultaneous hydrolysis. In this study, recombinant NicA2 and Nox were expressed in E. coli BL21(DE3) and purified. In vitro, the activity of recombinant NicA2 and Nox was accelerated by adding co-factor NAD+, suggesting that they worked as dehydrogenases. The optimal reaction conditions, substrate affinity, catabolism efficiency, pH-stability and thermal-stability were determined. Nox showed lower efficiency, but at a higher stability level than NicA2. Nox exhibited wider pH range and higher temperature as optimal conditions for the enzymatic reaction. In addition, The Nox showed higher thermo-stability and acid-stability than that of NicA2. The study on enzymatic reaction kinetics showed that Nox had a lower Km and higher substrate affinity than NicA2. These results suggest that Nox plays more significant role than NicA2 in nicotine degradation in TWE, which usually is processed at low pH (4-5) and high temperature (above 40 °C). Genetic engineering is required to enhance the affinity and suitability of NicA2 for an increased additive effect on homologous NicA2 and Nox in strain JY-Q.

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Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. strain JY-Q still has difficulties degrading high concentrations of nicotine. In this study, we investigated the effect of two homologous transcriptional regulators and endogenous ectopic strong promoters on the efficiency of nicotine degradation. Comparative genomics analysis showed that two homologous transcriptional regulators, namely, NicR2A and NicR2Bs (NicR2B1 plus NicR2B2), can repress nicotine degradation gene expression. When both nicR2A and nicR2Bs were deleted, the resulting mutant JY-Q ΔnicR2A ΔnicR2B1 ΔnicR2B2 (QΔABs) exhibits a 17% higher nicotine degradation efficiency than wild-type JY-Q. Transcriptome sequencing (RNA-seq) analysis showed that the transcription levels (fragments per kilobase per million [FPKM] value) of six genes were higher than those of the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, PRS28250 , PRS09985 , and PRS24685 , were identified. Their promoter activities were evaluated by comparing their expression levels using reverse transcriptase quantitative PCR (RT-qPCR). We found that the transcription levels of RS28250, RS09985, and RS24685 were respectively 16.8, 2.6, and 1.6 times higher than that of hspB2, encoding 6-hydroxy-3-succinylpyridine hydroxylase, which is involved in nicotine degradation. Thus, two strong endogenous promoters, namely, PRS28250 and PRS09985 , were selected to replace the original promoters of nic2 gene clusters. The effect of the endogenous ectopic promoter was also related to the position of target gene clusters. When the promoter PRS28250 replaced the promoter of hspB2, the resultant mutant QΔABs-ΔPhspB2 ::PRS28250 exhibited nicotine-degrading efficiency 69% higher than that of JY-Q. This research suggests a feasible strategy to enhance strains' capacity for nicotine degradation by removal of repressing regulatory proteins and replacing the target promoter with strong endogenous ectopic promoters.IMPORTANCE This study evaluated the differential effects of homologous NicR2A and NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. strain JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild-type (WT) strain JY-Q by removing repressing regulatory proteins NicR2A and NicR2Bs and replacing the target promoter with strong endogenous ectopic promoters. The resulting mutants exhibited high tolerance and degradation of nicotine. These findings should be beneficial for improving the pollutant-degrading capacity of natural strains through genomic modification.

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