Overexpression of endogenous multi‐copper oxidases mcoA and mcoC in Rhodococcus jostii RHA1 enhances lignin bioconversion to 2,4‐pyridine‐dicarboxylic acid

To improve the titre of lignin‐derived pyridine‐dicarboxylic acid (PDCA) products in engineered Rhodococcus jostii RHA1 strains, plasmid‐based overexpression of seven endogenous and exogenous lignin‐degrading genes was tested. Overexpression of endogenous multi‐copper oxidases mcoA, mcoB, and mcoC was found to enhance 2,4‐PDCA production by 2.5‐, 1.4‐, and 3.5‐fold, respectively, while overexpression of dye‐decolorizing peroxidase dypB was found to enhance titre by 1.4‐fold, and overexpression of Streptomyces viridosporus laccase enhanced titre by 1.3‐fold. The genomic context of the R. jostii mcoA gene suggests involvement in 4‐hydroxybenzoate utilization, which was consistent with enhanced whole cell biotransformation of 4‐hydroxybenzoate by R. jostii pTipQC2‐mcoA. These data support the role of multi‐copper oxidases in bacterial lignin degradation, and provide an opportunity to enhance titres of lignin‐derived bioproducts.

renewable aromatic carbon that could potentially be valorized to aromatic chemicals (Zakzeski et al., 2010).Lignin presents a number of practical difficulties for bioconversion, such as difficult bond cleavages, insolubility, and repolymerisation of phenoxy radical intermediates, however, there are several examples of successful chemocatalytic and biocatalytic conversion of polymeric lignin to aromatic monomers (Bugg & Rahmanpour, 2015).In the biocatalytic field, the use of engineered strains of lignin-degrading bacteria such as Rhodococcus jostii RHA1 to generate vanillin (Sainsbury et al., 2013), and Pseudomonas putida KT2440 to generate muconic acid (Vardon et al., 2015), has been a successful strategy, due to the convergent metabolism of diverse lignin degradation metabolites into key intermediates such as protocatechuic acid, thereby removing the need for separation of complex mixtures of depolymerized monomers.
Previous work from our research group has established a biocatalytic route from protocatechuic acid to pyridine-dicarboxylic acid bioproducts in engineered strains of Rhodococcus jostii RHA1, by overexpression of either protocatechuate 4,5-dioxygenase ligAB, or protocatechuate 2,3-dioxygenase praA, followed by cyclisation of the resulting extradiol ring fission products with ammonia (Mycroft et al., 2015), as shown in Figure 1.Titres of 80-125 mg/L from wheat straw lignocellulose feedstock were obtained by plasmid-based expression of ligAB or praA genes in wild-type R. jostii RHA1 (Mycroft et al., 2015).Chromosomal integration of the ligAB genes, removal of the competing β-ketoadipate pathway via deletion of pcaHG genes, and increase in lignin degradation flux by overexpression of Amycolatopsis dyp2, resulted in improved titres of 240-330 mg/L from either wheat straw lignocellulose or soda lignin (Spence et al., 2021).
To date the maximum titres for PDCA production represent conversion yields of 5%-12% from total polymeric lignin in the feedstock, which, although competitive with chemocatalytic lignin conversion methods, would need to be improved to bring the process towards commercial feasibility.Therefore, methods to enhance the titre of PDCA bioproducts are needed.We have previously demonstrated a 1.5-fold enhancement in PDCA titre by overexpression of Amycolatopsis dyp2 (Spence et al., 2021), therefore, in this study we wished to examine whether further enhancements in titre could be achieved by overexpression of endogenous R. jostii lignin-oxidizing genes, or other exogenous lignin-oxidizing genes.
Previous bioinformatic analysis has identified three multi-copper oxidase genes in Rhodococcus jostii RHA1, designated mcoABC (Granja-Travez et al., 2020).Dye-decolorizing peroxidase dypB is known from biochemical work and gene knockout to be active for oxidation of polymeric lignin (Ahmad et al., 2011), and the DypB protein is known to be targeted to an encapsulin nanocompartment (Rahmanpour & Bugg, 2013), whose precise role in metabolism is uncertain.Therefore, each of these genes was overexpressed, also Streptomyces viridosporus small laccase (SLAC, Majumdar et al., 2014) was tested, and the effect on titre of 2,4-PDCA was examined.
Each gene was separately cloned into expression vector pTipQC2, used previously for gene expression in R. jostii RHA1 (Mycroft et al., 2015).Each recombinant vector was then transformed into R. jostii pcaHG::ligAB(Ptpc5) in which the chromosomal pcaHG genes had been replaced with the Sphingobium SYK-6 ligAB genes, under the control of a constitutive Ptpc5 promoter, as published previously (Spence et al., 2021).Each recombinant construct was then grown on minimal M9 media containing 1% Green Value Protobind soda lignin for 7 days at 30°C, and supernatant analysed by C 18 reverse phase HPLC for production of 2,4-PDCA.
The genomic context of the R. jostii mcoA and mcoC genes was examined.As shown in Figure 3a, the mcoA gene (RHA1_ro02377) is colocated with phenol degradation genes such as phenol hydroxylase genes RHA1_ro02379 and RHA1_ro02380, and the catABC genes encoding the catechol intradiol cleavage pathway, and is located immediately adjacent to RHA1_ro02376 encoding a 4-hydroxybenzoyl CoA thioesterase.In contrast, the mcoC gene (RHA1_ro01580) is not colocated with any aromatic degradation genes (see Figure 3b).Phylogenetic analysis (see Figure 3c F I G U R E 1 Catabolic pathway for generation of 2,4-PDCA from lignin, showing insertion of ligAB genes (Mycroft et al., 2015), and deletion of pcaHG genes (Spence et al., 2021).PDCA, pyridine-dicarboxylic acid.
4-hydroxybenzaldehyde or vanillic acid by R. jostii pTipQC2-mcoA was increased by 40%-80%, compared with R. jostii pTipQC2 over 24 h, but no increase in rate of consumption of 4-hydroxyphenylacetic acid was observed (see Table 1, data in Figure S3).These data are consistent with a role of McoA in oxidation of 4-hydroxybenzoate or related low molecular weight phenols.
The results show that overexpression of lignin-oxidizing genes in R. jostii RHA1 can improve the titre of aromatic bioproducts, which is a useful strategy for maximizing yields of lignin bioproducts.The overall yield of 2,4-PDCA from Protobind lignin is 3.7% for R. jostii overexpressing mcoA and 5.1% for R. jostii overexpressing mcoC, compared 2.4% reported by Spence et al (Spence et al., 2021).It is interesting that overexpression of multi-copper oxidase genes mcoA and mcoC is more effective than dye-decolorizing peroxidase dypB, which is consistent with a role for multi-copper oxidases in lignin depolymerization, a point debated in the literature (Granja-Travez et al., 2020;Munk et al., 2015).
Both R. jostii mcoA and mcoC genes have TAT signal sequences, implying that they are exported to the cell surface, where the initial phase of lignin oxidation takes place, although mcoA appears to be involved in small molecule phenolic oxidation.Co-expression of R. jostii dypB and encapsulin genes gave no enhancement in PDCA titre, implying that targetting of DypB to the encapsulin nanocompartment does not enhance polymeric lignin breakdown in whole cells (Rahmanpour & Bugg, 2013).Overexpression of fungal laccases in Polyporus brumalis (Ryu et al., 2013) and Phanerochaete chrysosporium (Linares et al., 2018) has also been reported to enhance rates of lignocellulose breakdown.

| MATERIALS AND METHODS
Lignin biotransformation: Cultures of each recombinant strain were grown on M9 minimal media containing 1% (wt/vol) Green Value Protobind soda lignin, containing 50 µg/mL chloramphenicol, and were induced by addition of 5 µg/mL thiostrepton every 48 h, at 30°C in an orbital shaker (180 rpm), for 7 days.Samples of culture supernatant were analysed by C 18 reverse phase chromatography as described previously (Spence et al., 2021).Characterization of Green Value Protobind lignin has been reported previously (Constant et al., 2016).