Bacterial Transformation of Aromatic Monomers in Softwood Black Liquor

Abstract

The valorization of lignin, a major component of plant-derived biomass, is essential to sustainable biorefining. We identified the major monoaromatic compounds present in black liquor, a lignin-rich stream generated in the kraft pulping process, and investigated their bacterial transformation. Among tested solvents, acetone extracted the greatest amount of monoaromatic compounds from softwood black liquor, with guaiacol, vanillin, and acetovanillone, in an approximately 4:3:2 ratio, constituting ~90% of the total extracted monoaromatic content. 4-Ethanol guaiacol, vanillate, and 4-propanol guaiacol were also present. Bacterial strains that grew on minimal media supplemented with the BL extracts at 1mM total aromatic compounds included Pseudomonas putida KT2442, Sphingobium sp. SYK-6, and Rhodococcus rhodochrous EP4. By contrast, the extracts inhibited the growth of Rhodococcus jostii RHA1 and Rhodococcus opacus PD630, strains extensively studied for lignin valorization. Of the strains that grew on the extracts, only R. rhodochrous GD01 and GD02, isolated for their ability to grow on acetovanillone, depleted the major extracted monoaromatics. Genomic analyses revealed that EP4, GD01, and GD02 share an average nucleotide identity (ANI) of 98% and that GD01 and GD02 harbor a predicted three-component carboxylase not present in EP4. A representative carboxylase gene was upregulated ~100-fold during growth of GD02 on a mixture of the BL monoaromatics, consistent with the involvement of the enzyme in acetovanillone catabolism. More generally, quantitative RT-PCR indicated that GD02 catabolizes the BL compounds in a convergent manner via the β-ketoadipate pathway. Overall, these studies help define the catabolic capabilities of potential biocatalytic strains, describe new isolates able to catabolize the major monoaromatic components of BL, including acetovanillone, and facilitate the design of biocatalysts to valorize under-utilized components of industrial lignin streams.

Bacterial catabolic system of acetovanillone and acetosyringone useful for upgrading aromatic compounds obtained through chemical lignin depolymerization

“…XccA (CT), XccC (BC), and XccB (BCCP) have 55%, 63%, and 45% amino acid sequence identity with SLG_06500, SLG_06510, and SLG_06520, respectively (Table S2). Furthermore, SLG_06500, SLG_06510, and SLG_06520 exhibited 42%, 51%, and 38% amino acid sequence identity, respectively, with apkA (CT), apkB (BC), and apkC (BCCP), the recently predicted AV catabolic enzyme genes in R. rhodochrous GD02 (Table S2) (20). SLG_06490 showed 23% amino acid sequence identity with NagD, a ribonucleotide monophosphatase of E. coli K-12, belonging to the haloacid dehydrogenase (HAD) superfamily (Table S2) (56).…”

“…AV and AS are major lignin-derived aromatic monomers produced in alkaline oxidative depolymerization (13–16) and base-catalyzed depolymerization of lignin (17-19, 62-68). They are also contained in the black liquor produced during the kraft pulping process (20). This study identified acvABCDEF that encodes AV and AS carboxylase components and showed that these genes help produce value-added chemicals from AV.…”

“…Base-catalyzed Indulin AT (a pine kraft lignin) depolymerization resulted in the formation of vanillin, vanillic acid, guaiacol, and AV as major aromatic monomers, similar to the alkaline oxidation of Lignoboost lignin (a softwood kraft technical lignin) (8, 16–19). A black liquor produced in a softwood kraft pulping process, Lignoforce, contained guaiacol, vanillin, and AV as major aromatic monomers (20). Among these major aromatic monomers, microbial catabolic systems of vanillin, vanillic acid, and guaiacol have been characterized (21–29).…”

“…Therefore, chemical production from all of the major aromatic monomers produced by oxidative/base-catalyzed lignin depolymerization has been unachieved. Recently, Eltis and coworkers isolated Rhodococcus rhodochrous GD01 and GD02, which can utilize AV in addition to vanillin, vanillic acid, and guaiacol in the black liquor produced during the Lignoforce kraft pulping of softwood (20). Based on the observation that apkC was induced when GD02 was cultured in black liquor extracts containing AV, apkA - apkB - apkC , presumed to encode biotin-dependent carboxylase, were predicted to be involved in AV catabolism.…”

“…Sphingobium sp. strain SYK-6 can utilize various lignin-derived dimers, such as β- aryl ether, biphenyl, phenylcoumaran, and diarylpropane, as well as monomers, including ferulic acid, vanillin, vanillic acid, and AV (11, 16, 20, 38, 39). In SYK-6 cells, stereoisomers of β-aryl ether model compound, guaiacylglycerol-β-guaiacyl ether, are stereospecifically converted to achiral β-hydroxypropiovanillone (HPV) via Cα- oxidation, ether cleavage, and glutathione removal (Fig.…”