Conversion and upgrading of S-lignin related syringate by Acinetobacter baylyi ADP1

Background: Lignin holds great potential as an abundant and sustainable source of aromatic compounds, offering a viable alternative to fossil-based resources for producing chemicals and materials. Biological upgrading of lignin-derived aromatics can lead to more comprehensive lignocellulose utilization, thereby enhancing the overall feasibility of production. However, exploring a broader range of potential microbial hosts, pathways, and enzymes is crucial for developing efficient conversion processes. In particular, improving the conversion of S-lignin-related aromatics, such as syringate, remains a key area for future research. Results: In this study, we aimed to investigate the conversion of S-lignin-related syringate in Acinetobacter baylyi ADP1 by exploiting its native vanillate demethylase, VanAB. We discovered that the wild-type strain can efficiently O -demethylate syringate to 3-O-methylgallate (3MGA) and then to gallate, revealing a previously unknown activity of VanAB of A. baylyi ADP1. Conversion dynamics and in vitro characterization showed that VanAB prefers syringate as a substrate over 3MGA. Overexpression of vanAB resulted in simultaneous conversion of syringate and 3MGA, but negatively impacted growth, potentially due to toxic side product formaldehyde and redox imbalance caused by high NADH consumption of the O -demethylation reactions. Native vanAB expression resulted in 3MGA accumulation if syringate was available. We took advantage of this by constructing a strain with heterologous expression of galA , a gallate dioxygenase from Pseudomonas putida KT2440, and demonstrated the conversion of 3MGA into 2-pyrone-4,6,-dicarboxylate (PDC), a precursor for high-quality polyesters. Conclusions: In this study, we discovered a previously unknown activity of syringate conversion in A. baylyi ADP1. By adjusting the expression level of vanAB , syringate can be directed either into gallate or 3MGA, which could be further converted into PDC through the heterologous expression of galA. Our results further highlight the potential and versatility of A. baylyi ADP1 for lignin valorisation.