(65a) Elucidating Metabolic and Regulatory Mechanisms of Microbial Aromatic Utilization for Lignin Valorization

Lignin represents a renewable resource whose bioconversion could displace petroleum-based processes [1, 2]. Research has been conducted to develop a hybrid platform to generate value-added bioproducts from lignin breakdown products (LBPs) obtained by thermo-catalytic depolymerization of waste lignin [1-3]. Traditional model organisms are not well suited for converting LBPs that consist of various toxic aromatic compounds, but non-model organisms have been identified as ideal candidates. One such host is Rhodococcus opacus, which has demonstrated high native tolerance to LBPs and the ability to improve its tolerance and consumption through adaptive evolution. However, the key challenges in such lignin upgrading include our limited understanding of microbial utilization of toxic LBPs at gene levels and limited tools to engineer this organism. To understand and maximize its metabolic potential, we have employed multi-omics approaches, providing a systems-level understanding of the complex metabolism of the wild-type and evolved strains [4-8]. Additionally, we have developed a genetic toolbox for R. opacus engineering [9-12]. Despite recent advances in our understanding of its versatile metabolism and available genetic tools, studies of its gene functions at gene levels are still lagging.

In this presentation, we will discuss our recent efforts to facilitate functional studies of this non-model organism using our genetic toolbox and engineer this promising chassis for lignin valorization. Specifically, we have identified nine putative transcription factor (TF) genes in proximity to gene clusters for degradation of aromatic compounds and confirmed the function of these transcription factors using a gfp reporter gene and multiple deletion analyses. Interestingly, cross-interactions between different aromatic compounds’ consumptions have been observed at the gene regulation levels, implying complex gene regulatory and metabolic networks of this organism. Additionally, we have developed and optimized a gene repression system based on CRISPR interference (CRISPRi) [11], and we will discuss our CRISPRi tool’s utility by demonstrating the inducible accumulation of muconate from aromatics [13]. Altogether, this work represents a considerable achievement to understand and engineer this promising chassis for lignin conversion into value-added chemicals.

[1] Davis, K., Moon, T. S., Tailoring microbes to upgrade lignin. Current opinion in chemical biology 2020, 59, 23-29.

[2] Anthony, W. E., Carr, R. R., DeLorenzo, D. M., Campbell, T. P., et al., Development of Rhodococcus opacus as a chassis for lignin valorization and bioproduction of high-value compounds. Biotechnology for biofuels 2019, 12, 192.

[3] Chatterjee, A., DeLorenzo, D. M., Carr, R., Moon, T. S., Bioconversion of renewable feedstocks by Rhodococcus opacus. Current opinion in biotechnology 2020, 64, 10-16.

[4] Henson, W. R., Campbell, T., DeLorenzo, D. M., Gao, Y., et al., Multi-omic elucidation of aromatic catabolism in adaptively evolved Rhodococcus opacus. Metabolic engineering 2018, 49, 69-83.

[5] Yoneda, A., Henson, W. R., Goldner, N. K., Park, K., et al., Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630. Nucleic Acids Res. 2016, 44, 2240–2254.

[6] Roell, G. W., Carr, R. R., Campbell, T., Shang, Z., et al., A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630. Metabolic engineering 2019, 55, 120-130.

[7] Henson, W. R., Hsu, F.-F., Dantas, G., Moon, T. S., Foston, M., Lipid metabolism of phenol-tolerant Rhodococcus opacus strains for lignin bioconversion. Biotechnology for biofuels 2018, 11, 339.

[8] Hollinshead, W. D., Henson, W. R., Abernathy, M., Moon, T. S., Tang, Y. J., Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel C-metabolite fingerprinting. Biotechnology and bioengineering 2016, 113, 91-100.

[9] DeLorenzo, D. M., Henson, W. R., Moon, T. S., Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630. ACS Synthetic Biology 2017, 6, 1973–1978.

[10] DeLorenzo, D. M., Moon, T. S., Construction of Genetic Logic Gates Based on the T7 RNA Polymerase Expression System in Rhodococcus opacus PD630. ACS Synthetic Biology 2019, 8, 1921-1930.

[11] DeLorenzo, D. M., Rottinghaus, A. G., Henson, W. R., Moon, T. S., Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630. ACS Synthetic Biology 2018, 7, 727-738.

[12] DeLorenzo, D. M., Moon, T. S., Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630. Scientific reports 2018, 8, 6019.

[13] DeLorenzo, D.M., Diao, J., Carr, R., Hu, Y., Moon, T.S., An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus. ACS Synthetic Biology. doi.org/10.1021/acssynbio.0c00591