Bacterial catabolism of acetovanillone, a lignin-derived compound

Abstract

Bacterial catabolic pathways have considerable potential as industrial biocatalysts for the valorization of lignin, a major component of plant-derived biomass. Here, we describe a pathway responsible for the catabolism of acetovanillone, a major component of several industrial lignin streams. Rhodococcus rhodochrous GD02 was previously isolated for growth on acetovanillone. A high-quality genome sequence of GD02 was generated. Transcriptomic analyses revealed a cluster of eight genes up-regulated during growth on acetovanillone and 4-hydroxyacetophenone, as well as a two-gene cluster up-regulated during growth on acetophenone. Bioinformatic analyses predicted that the hydroxyphenylethanone (Hpe) pathway proceeds via phosphorylation and carboxylation, before β-elimination yields vanillate from acetovanillone or 4-hydroxybenzoate from 4-hydroxyacetophenone. Consistent with this prediction, the kinase, HpeHI, phosphorylated acetovanillone and 4-hydroxyacetophenone. Furthermore, HpeCBA, a biotin-dependent enzyme, catalyzed the ATP-dependent carboxylation of 4-phospho-acetovanillone but not acetovanillone. The carboxylase's specificity for 4-phospho-acetophenone (k_cat/K_M = 34 ± 2 mM^-1 s^-1) was approximately an order of magnitude higher than for 4-phospho-acetovanillone. HpeD catalyzed the efficient dephosphorylation of the carboxylated products. GD02 grew on a preparation of pine lignin produced by oxidative catalytic fractionation, depleting all of the acetovanillone, vanillin, and vanillate. Genomic and metagenomic searches indicated that the Hpe pathway occurs in a relatively small number of bacteria. This study facilitates the design of bacterial strains for biocatalytic applications by identifying a pathway for the degradation of acetovanillone.

Keywords: Rhodococcus; acetovanillone; bacterial catabolism; hydroxyacetophenone; lignin.

Fig. 1.

Proposed pathways for degradation of hydroxyphenylethanones (A) and AP (B). In the catabolism of HAP (R = H), HAP is phosphorylated to PAP, then carboxylated to PCAP. In the catabolism of AV (R = OCH₃), AV is phosphorylated to PAV, then carboxylated to PCAV. Pca, protocatechuate; βKA, β-keto adipate; Cat, catechol.

Fig. 2.

Key genes up-regulated during growth on AV, HAP, and AP. Heat map shows log2-fold increase in gene expression during growth on the aromatic substrates versus on citrate. Horizontal lines separate clusters of genes. Genes are fully identified in Table 1 and SI Appendix, Table S1.

Fig. 3.

Phosphorylation of AV and HAP by HpeHI. (A) Extracted ion chromatograms for substrates and products of the HpeHI reaction are labeled, corresponding to both the retention time and m/z of standards. Reaction mixtures of HpeHI with HAP (blue) or AV (red) or no enzyme controls for HAP (dashed, light gray) or AV (dashed, dark gray) are shown. Synthesized standards for the expected products PAP (solid, light gray) and PAV (solid, dark gray) are also shown. (B) Mass spectra of PAP and PAV produced in the reaction (Right) match the expected m/z with <5 ppm error.

Fig. 4.

Transformations catalyzed by HpeCBA and HpeD. (A) AV or PAV were incubated with either HpeCBA or HpeCBA + HpeD as indicated. The product of PAV plus HpeCBA was PCAV, identified by mass spectrometry (SI Appendix, Fig. S9A). The product of PAV plus HpeCBA plus HpeD was 4-hydroxy-3-methoxyphenyl-β-ketopropionate, identified by mass spectrometry (SI Appendix, Fig. S9B). (B) HAP or PAP were incubated with either HpeCBA or HpeCBA plus HpeD, as indicated. The product of PAP plus HpeCBA was PCAP, identified by mass spectrometry (SI Appendix, Fig. S10A). The product of PAV plus HpeCBA plus HpeD was 4-hydroxyphenyl-β-ketopropionate, identified by mass spectrometry (SI Appendix, Fig. S10B). (A and B) Reactions were incubated for 20 min at 30 °C using 250 µM substrate in 20 mM (3-(N-morpholino)propanesulfonic acid (MOPS; I = 0.1 M), pH 7.5, containing 0.5 mM ATP, 4 mM MgCl₂, and 40 mM NaHCO₃. HPLC traces were recorded at 280 nm. Abs, absorbance.

Fig. 5.

Growth of GD02 on lodgepole pine OCF extracts. GD02 was grown at 30 °C in M9+minerals containing OCF extracts at a concentration of 2 mM total aromatic compounds. Depletion of aromatic compounds from culture supernatants was measured using HPLC. Data points represent the average of triplicate experiments and the error bars represent the SD.