Periodic Reporting for period 1 - PELIG (Characterisation of a Novel Pathway for Lignin Fragment Degradation in Rhodococcus jostii)
Reporting period: 2016-10-06 to 2018-10-05
In nature, microorganisms including bacteria could degrade lignin and consume it as carbon source for growth, which has attracted considerable attention from researchers exploring the relevant metabolic pathways and intercepting that process to accumulate highly valuable products. Theoretically aromatic catabolic pathways can be engineered to funnel the products to different biological intermediates, thus opening up a new field of metabolic engineering for lignin utilisation. Hence, the discovery of unknown lignin metabolic pathways and gene products in bacteria is of importance. The overall objective of this study is to explore and manipulate a novel lignin metabolic pathway catalysed by benzoylformate decarboxylase in Rhodococcus jostii for lignin-based renewable chemicals production via a synthetic biology approach.
Results showed that a 1 L fermentation yielded about 9 mg purified recombinant R.jBFD and 7.6 mg P.fBFD with specific decarboxylase activities of 28 U/mg and 63 U/mg respectively. Then we tested the abilities of both the enzymes to catalyse the lignin model compounds aryl C3 molecules containing COCH(OH)CH2(OH) and analogues However, the HPLC traces of enzymatic reactions under the optimal conditions (pH=7, 25 ℃) showed that even after 12 h there were no measurable conversion of these substrates, which suggested that BFD would not accept the tested compounds as substrates under the current conditions. Meanwhile, it was found that both the BFD were able to catalyse the C-C bond formation between vanillin and acetaldehyde due to the formation of 2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one, but they were unable to cleavage the C-C bond when using the product as substrate. The alignment between these two proteins showed that they shared about 37% sequence identity and 20% sequence similarity, which indicated that they possessed the similar structure and function. However, the decarboxylase activity and carboligase activity of P.fBFD is much stronger than R.jBFD under the same conditions. Comparison of the X-ray structures of both enzymes identified one residue in each (Ser73 in RHA1 and Ala73 in Pf-5) that were likely to be involved in determining the activity. Site-directed mutagenesis was conducted to interchange the residues in both R.jBFD and P.fBFD. LC/MS results revealed that BFD variant containing the A73S mutation was found to lose most of its activity, while BFDS73A mutant showed an enhanced activity. These findings demonstrated that steric restraint might be predominantly responsible for the differences in activity. Based on the result of this project, another lignin model compounds-3-methoxy-4-hydroxy-benzoylformate and its analogues-will be tested in future work.
Meanwhile the researcher tried to design a deletion system to remove genes encoding BFD in both RHA1 and pf-5. However, although construction of the mutagenic plasmid for ΔR.jbfd and ΔP.fbfd genetic knockout strains were successful, the researchers haven’t obtain any proof of plasmid integration into the chromosomal DNA.
The results from the Action have been presented in 3 high-level international conferences (2 poster presentation, 1 flash oral presentation), and will be written up for publication in scientific journals in the next 6 months. The biochemical characterisation of BFD catalysed pathway studied in this project may boost future projects considering the break-down of lignin.