Characterization of acetovanillone degradation in wild-type and engineered Rhodococcus aromaticivorans RHA1

Area of Science:

• Biotechnology and Industrial Microbiology
• Synthetic Biology
• Biocatalysis and Enzyme Engineering

Background:

• Lignin valorization is key for sustainable chemical production, offering a renewable alternative to petroleum feedstocks.
• Microbial degradation of lignin-derived aromatic compounds (LDACs) is crucial for developing efficient biocatalysts.
• Hydroxyphenylethanones (HPEs) like acetovanillone (AV), 4-hydroxyacetophenone (HAP), and acetosyringone (AS) are significant LDACs with limited reported microbial degradation pathways.

Purpose of the Study:

• To identify and characterize microbial pathways for the degradation of hydroxyphenylethanones (HPEs) in Rhodococcus aromaticivorans RHA1.
• To expand the substrate range of known catabolic enzymes involved in LDAC degradation.
• To compare the efficiency of three homologous HPE catabolic pathways for potential biocatalytic applications.

Main Methods:

• Molecular genetics to identify genes encoding enzymes responsible for AV and HAP degradation in R. aromaticivorans RHA1.
• Enzyme characterization, including kinetic analysis (kcat/KM) of O-demethylase (AgcAB) and hydroxylase (AphAB) activities.
• Heterologous expression of HPE catabolic genes from different strains (R. rhodochrous GD02, A. macra NBRC-14102, S. lignivorans SYK-6) in RHA1 for comparative pathway analysis.

Main Results:

• R. aromaticivorans RHA1 cometabolizes AV and HAP into 3,4-dihydroxyacetophenone (3,4-DHAP) via AgcAB and AphAB enzymes, respectively.
• AV induction of AgcA and AphC suggests a coordinated catabolic response.
• The GD02 pathway demonstrated significantly higher AV conversion rates compared to pathways from A. macra and S. lignivorans, enabling RHA1 growth on HPEs.

Conclusions:

• This study elucidates key enzymes and pathways for HPE degradation, providing insights into aromatic catabolic enzyme substrate preferences.
• Cometabolism plays a vital role in biocatalysis, and engineered microbial cell factories can be developed for lignin valorization.
• The findings facilitate the engineering of microbial systems for the sustainable transformation of lignin into valuable platform chemicals.