Lignin catabolic pathways reveal unique characteristics of dye‐decolorizing peroxidases in Pseudomonas putida

Abstract

Summary Lignin is one of the largest carbon reservoirs in the environment, playing an important role in the global carbon cycle. However, lignin degradation in bacteria, especially non‐model organisms, has not been well characterized either enzymatically or genetically. Here, a lignin‐degrading bacterial strain, Pseudomonas putida A514, was used as the research model. Genomic and proteomic analyses suggested that two B subfamily dye‐decolorizing peroxidases (DypBs) were prominent in lignin depolymerization, while the classic O2‐dependent ring cleavage strategy was utilized in central pathways to catabolize lignin‐derived aromatic compounds that were funnelled by peripheral pathways. These enzymes, together with a range of transporters, sequential and expression‐dose dependent regulation and stress response systems coordinated for lignin metabolism. Catalytic assays indicated these DypBs show unique Mn2+ independent lignin depolymerization activity, while Mn2+ oxidation activity is absent. Furthermore, a high synergy between DypB enzymes and A514 cells was observed to promote cell growth (5 × 1012 cfus/ml) and lignin degradation (27%). This suggested DypBs are competitive lignin biocatalysts and pinpointed limited extracellular secretion capacity as the rate‐limiting factor in bacterial lignin degradation. DypB production was, therefore, optimized in recombinant strains and a 14,141‐fold increase in DypB activity (56,565 U/l) was achieved, providing novel insights for lignin bioconversion.

Development of a CRISPR/Cas9n-based tool for metabolic engineering of Pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion

“…Through this approach, we genetically investigated ferulic acid-to-PHA bioconversion in detail and further constructed the engineered P. putida KTc9n20 strain to successfully expand ferulic acid valorization, in addition to its well-known ferulic acid-to-vanillin biotransformation. Together with a microbial lignin depolymerization system under development 5 , exploration of PHA regulation mechanism under aromatic compounds, and further improvements in culture conditions in the near future, could provide insights into microbial carbon sink as well as improving lignin-consolidated bioprocessing schemes.…”

“…Cells were lysed in the SDS loading buffer, and then supernatant cell lysates were subjected to 12% gel SDS-PAGE separation. In addition, Cas9n protein was purified by Ni-NTA column (Takara) and separated by SDS-PAGE as previously described 5 .…”

“…erulic acid is a ubiquitous phenolic component of lignocellulose, which is linked to hemicellulose and lignin via ester linkages, and is released to the environment by fungal and bacterial esterase enzymes [1][2][3] . Moreover, ferulic acid is a intermediate metabolite during lignin biodegradation 4,5 . Considering that lignocellulose is an abundant carbon compound on the earth 6 , investigation of ferulic acid catabolism not only provides insights into the global carbon cycle 7 , but also contributes to the development in biocatalytic conversion of ferulic acid to valuable bioproducts 8,9 .…”

Biochemical features of dye‐decolorizing peroxidases: Current impact on lignin degradation

“…Substrates tested include a variety of aromatic compounds classified as dyes [50,[53][54][55][56], beta-carotene [57], and aromatic sulfides [48]; some of which are poorly metabolized by other heme peroxidases. Numerous studies have shown that DyPs play a key role in the degradation of lignin [54,55,[58][59][60][61][62][63][64][65]. Their catalytic properties make them very interesting because the bacterial enzymes can be easily engineered, heterologously expressed in E. coli and purified [65] avoiding the issues of using a eukaryotic hosts.…”

A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage‐derived IncY plasmid

“…The plasmid encodes a DyP-decolorizing peroxidase unit involved in detoxification. DyP decolorizing peroxidases of fungi and bacteria have an unknown physiological function, but wide biotechnological applicability as they degrade lignin and oxidize a variety of aromatic compounds, dyes, and other small molecules (Lin et al, 2019;Singh et al, 2013). A 32% identical DyP homolog is encoded on the Fec10 and E. coli K-12 chromosome.…”