The Pseudomonas ligninolytic catalytic network reveals the importance of auxiliary enzymes in lignin biocatalysts

Abstract

Lignin degradation by biocatalysts is a key strategy to develop a plant-based sustainable carbon economy and thus alleviate global climate change. This process involves synergy between ligninases and auxiliary enzymes. However, auxiliary enzymes within secretomes, which are composed of thousands of enzymes, remain enigmatic, although several ligninolytic enzymes have been well characterized. Moreover, it is a challenge to understand synergistic lignin degradation via a diverse array of enzymes, especially in bacterial systems. In this study, the coexpression network of the periplasmic proteome uncovers potential accessory enzymes for B-type dye-decolorizing peroxidases (DypBs) in Pseudomonas putida A514. The catalytic network of the DypBs-based multienzyme complex is characterized. DypBs couple with quinone reductases and nitroreductase to participate in quinone redox cycling. They work with superoxide dismutase to induce Fenton reaction for lignin oxidation. A synthetic enzyme cocktail (SEC), recruiting 15 enzymes, was consequently designed with four functions. It overcomes the limitation of lignin repolymerization, exhibiting a capacity comparable to that of the native periplasmic secretome. Importantly, we reveal the synergistic mechanism of a SEC-A514 cell system, which incorporates the advantages of in vitro enzyme catalysis and in vivo microbial catabolism. Chemical analysis shows that this system significantly reduces the molecular weight of lignin, substantially extends the degradation spectra for lignin functional groups, and efficiently metabolizes lignin derivatives. As a result, 25% of lignin is utilized, and its average molecular weight is reduced by 27%. Our study advances the knowledge of bacterial lignin-degrading multienzymes and provides a viable lignin degradation strategy.

Keywords: B-type dye-decolorizing peroxidase; auxiliary enzymes; lignin degradation; quinone redox cycling; synthetic enzyme cocktail.

Fig. 1.

Periplasmic protein expression in P. putida A514. (A) Proteome experimental design for reconstructing the protein coexpression network. The three dots under each condition represent the three biological replicates. All 24 samples were used to reconstruct the network. (B) The submodule of DypB₃₁₅₂. All proteins that possessed links with DypB₃₁₅₂ are shown. Each node represents a protein. (C) Protein expression ratios (log₂R) at early exponential phase for representative proteins directly connected with DypB₃₁₅₂. L: lignin, G: glucose, vs: versus, ALDH: aldehyde dehydrogenase, DHLDH: dihydrolipoamide dehydrogenase, QR: quinone oxidoreductase, Ntr: nitroreductase, GR: glutathione reductase, MO: monooxygenase, SOD: superoxide dismutase, CAT: catalase.

Fig. 2.

Ligninolytic catalytic network of the SEC. (A) Quinone reduction by either QR or Ntr. (B) Hydroquinone oxidation by each DypB. (C–E) Fe³⁺-reducing activity (C), H₂O₂ production (D), and ^•OH production (E) by different enzyme treatments. The production of ^•OH radicals was evaluated by the conversion of 2-deoxyribose into TBARS. (F–H) The lignin degradation (F), lignin molecular weight analysis (G), and ³¹P NMR quantification of the functional groups (H) for kraft lignin treated by the DypBs, NPE, and SEC, respectively. The hydroxyl content of lignin without any treatment (blank) was set as 100% for the comparison. The 0.1 mg DypBs and 1 mg SEC/NPE were used. *P value < 0.05, **P value < 0.01. Data are presented as mean values ± SD of three biological replicates.

Fig. 3.

Synergistic mechanism of the SEC-P. putida A514 system in lignin degradation. (A) Cell growth (line) and lignin degradation (bar) of NPE/SEC-A514 with kraft lignin as the sole carbon source in shake flask cultivations. 1 mg of either NPE or SEC was added when ~10³ CFUs/mL A514 was inoculated in the M9 medium. (B and C) The molecular weight analysis (B) and ³¹P NMR quantification of the functional groups (C) for the kraft lignin treated by SEC, A514, and the SEC-A514 system, respectively. *P value < 0.05, **P value < 0.01, ***P value < 0.001. (D) The real-time relative abundances of the soluble lignin derivates. They were detected by GC-MS. Cluster I: compounds consumed by the SEC-A514 system. Cluster II: compounds that the SEC-A514 system generated and then later consumed. HBA: 4-Hydroxybenzoic acid, VMA: Vanillylmandelic acid, IVA: Isovanillic acid, 3-HBD: 3-Hydroxybenzaldehyde, 4-HBD: 4-Hydroxybenzaldehyde, AC: Acetic acid, HMA: 3-Hydroxy-4-methoxybenzyl alcohol, HBAA: 4-Hydroxybenzeneacetic acid, VA: Vanillyl alcohol, FA: Ferulic acid, MDB: Methyl 2,4-dihydroxybenzoate. B: kraft lignin without any treatment. Data are presented as mean values ± SD of three biological replicates.

Fig. 4.

Lignin degradation by the SEC-P. putida A514 system in bioreactor cultivations. Overall process flow for kraft lignin degradation (A) and APL degradation (B). Cell growth (line) and lignin degradation (bar) of SEC-A514 system with either kraft lignin or APL as the sole carbon source in the 5-L bioreactor. 20 mg SEC was added when ~10⁴ CFUs/mL A514 was inoculated into the M9 medium. A514 (~10⁴ CFUs/mL in bioreactors) in the M9 medium, without any addition, was used as the reference control. Data are mean ± SD of three technical replicates.

Fig. 5.

Schematic diagram of the synergistic lignin degradation mechanism by the SEC-A514 system. (1) Lignin depolymerization. (2) Alleviation of lignin repolymerization. (3) Catabolism of lignin depolymerization products. (4) Oxidative stress response.