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Investigating the potential of Rhodococcus to transform lignin and plastic streams Roccor, Raphael


Members of the genus Rhodococcus have considerable potential as biocatalyst to transform aromatic by-product and waste streams into sustainable chemicals. Such streams include lignin of woody biomass and polyethylene terephthalate (PET), a plastic. Both are abundant polymers that are difficult to valorize due to their recalcitrance. Herein, I investigated the potential of Rhodococcus jostii RHA1 to transform lignin and PET. First, I tested if RHA1’s lignin degrading ability can be enhanced by secreting bacterial lignin-modifying enzymes via the twin arginine translocation (Tat) system. I studied the growth of RHA1 on lignin in combination with small laccase (sLac) secretion and analyzed the sLac reaction products. The results indicate that the Tat system can be exploited to efficiently secrete lignin-modifying enzymes but that this strategy may not yield enhanced growth on lignin as toxic 2,6-dimethoxybenzoquione was produced. Second, I investigated the metabolism of RHA1 on a lignin-enriched stream of corn stover. A time-resolved transcriptomic study showed that RHA1 co-expressed various aromatic and organic acid pathways during exponential growth. I tested the requirement of relevant pathways by growth experiments with mutant strains. When entering the stationary phase, RHA1 highly up-regulated the propane, the alkylphenol, and the alkylguaiacol pathways suggesting that these could serve an unknown role other than the catabolism of their associated compounds. Third, I examined the PET-transform potential of RHA1 by secreting the PET-hydrolyzing enzymes, PETase and MHETase, using the Sec secretion system. While translocation of active enzyme was limited, expression of the Sec secretion signal MHETase construct allowed RHA1 to grow on two PET breakdown products: mono-(2-hydroxyethyl) terephthalate and bis(2-hydroxyethyl) terephthalate. Furthermore, I analyzed the metabolism of RHA1 on ethylene glycol, another PET breakdown product, using transcriptomics and described new gene candidates for the degradation of short chain alcohols and their dependence on the cofactor mycofactocin. Taken together, my results provide insights into utilizing RHA1 as a lignin and PET transforming biocatalyst and reveal remaining barriers. The data will inform engineering strategies to develop efficient RHA1 strains that yield high-value oleochemicals from biomass and plastic waste streams.

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