(332b) Advanced Biological and Chemical Design for Lignin Bioconversion

Yuan, J., Texas A&M University
Xie, S., Texas A&M University
Zhao, C., Texas A&M University
Pu, Y., Oak Ridge National Laboratory
Lin, F., Texas A&M University
Sun, S., Texas A&M University
Dai, S., Texas A&M University
Ragauskas, A. J., School of Chemistry and Biochemistry, Georgia Institute of Technology

The utilization of lignin for fungible fuels and chemicals represents one of the most imminent challenges in modern biorefinery. However, bioconversion of lignin is highly challenging due to its recalcitrant nature as a phenolic heteropolymer. We first addressed the challenges by revealing the chemical and biological mechanisms for synergistic lignin degradation by a bacterial and enzymatic system, which significantly improved lignin consumption, cell growth and lipid yield. The R. opacus cell growth increased exponentially in response to the level of laccase treatment, indicating the synergy between laccase and cells in lignin degradation. Other treatments like iron and hydrogen peroxide showed limited impact on cell growth. Chemical analysis of lignin under various treatments further confirmed the synergy between laccase and cells at chemical level. 31P NMR suggested that laccase, R. opacus cell and Fenton reaction reagents promoted the degradation of different types of lignin functional groups, elucidating the chemical basis for the synergistic effects. 31P NMR further revealed that laccase treatment had the most significant impact for degrading the abundant chemical groups. The results were further confirmed by the molecular weight analysis, the HSQC analysis and lignin quantification by Prussian Blue assay. The cell-laccase fermentation led to a 17-fold increase of lipid production. Overall, the study indicated that laccase and R. opacus can synergize to degrade lignin efficiently, likely through rapid utilization of monomers generated by laccase to promote reaction toward depolymerization. Based on the study, consolidated lignin conversion was engineered using laccase and R. opacus. An efficient ligninase secretion system has been established through proteomics-guided optimization of promoter/rbs, secretion peptide, and others. The advanced biodesign has achieved over 70U/mL of laccase production and nearly 1000 fold increase of cell growth. The integration of biological and chemical design has led to the lipid titer of over 1g/L on insoluble Kraft lignin. The study has paved a path for more efficient conversion of lignin and waste material from biorefinery.