Development of a <i>Rhodococcus opacus</i> Cell Factory for Valorizing Lignin to Muconate
Chenggu Cai, Zhaoxian Xu, XU Mei-lin, Mufeng Cai, Mingjie Jin
Abstract
As multiple biochemical pathways exist in nature for lignin synthesis and degradation, lignin valorization through biological pathways has attracted much attention. cis,cis-Muconate, a common metabolic intermediate in lignin biodegradation, is an important platform chemical for the synthesis of a variety of polymers. In this work, Rhodococcus opacus PD630, which is efficient in the assimilation of aromatic compounds, was modified for the first time to accumulate cis,cis-muconate from lignin and lignin-derived aromatics. To achieve efficient genome modification for R. opacus PD630, a markerless gene deletion/insertion system was constructed based on a mutant phenylalanyl-tRNA synthase gene as a counter-selection marker. With the developed gene deletion/insertion system, several R. opacus PD630 mutants were successfully constructed for cis,cis-muconate production from lignin-derived aromatics using catechol and protocatechuate as two key funnel nodes. Results indicated the constructed strains could assimilate at least 15 types of lignin-derived aromatics and real lignin for cis,cis-muconate production. Moreover, both laccase addition and H2SO4 + NaOH combinatorial pretreatment enhanced real lignin valorization to cis,cis-muconate. Ultimately, 1.63 g/L cis,cis-muconate was produced using lignin from corn stover as the substrate by a fed-batch fermentation process. Overall, these results highlight the great potential of using R. opacus as a cell factory for valorizing lignin to cis,cis-muconate and other valuable products.