DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5′-Dehydrodivanillate Uptake by Sphingobium sp. Strain SYK-6

Abstract

The microbial conversion of lignin-derived aromatics is a promising strategy for the industrial utilization of this large biomass resource. However, efficient application requires an elucidation of the relevant transport and catabolic pathways. In <i>Sphingobium</i> sp. strain SYK-6, most of the enzyme genes involved in 5,5'-dehydrodivanillate (DDVA) catabolism have been characterized, but the transporter has not yet been identified. Here, we identified SLG_07710 (<i>ddvK</i>) and SLG_07780 (<i>ddvR</i>), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. A <i>ddvK</i> mutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVA <i>O</i>-demethylase oxygenase component gene (<i>ligXa</i>), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlled <i>ligXa</i> transcriptional regulatory system. A <i>Sphingobium japonicum</i> UT26S transformant expressing <i>ddvK</i> acquired DDVA uptake capacity, indicating that <i>ddvK</i> encodes the DDVA transporter. DdvK, probably requiring the proton motive force, was suggested to be a novel MFS transporter on the basis of the amino acid sequence similarity. Subsequently, we evaluated the effects of <i>ddvK</i> overexpression on the production of the DDVA metabolite 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers. A SYK-6 mutant of the PDC hydrolase gene (<i>ligI</i>) cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of a <i>ddvK</i>-expression plasmid into a <i>ligI</i> mutant increased the growth rate in DDVA and the amounts of DDVA converted and PDC produced after 48 h by 1.35- and 1.34-fold, respectively. These results indicate that enhanced transporter gene expression can improve metabolite production from lignin derivatives.<b>IMPORTANCE</b> The bioengineering of bacteria to selectively transport and metabolize natural substrates into specific metabolites is a valuable strategy for industrial-scale chemical production. The uptake of many substrates into cells requires specific transport systems, and so the identification and characterization of transporter genes are essential for industrial applications. A number of bacterial major facilitator superfamily transporters of aromatic acids have been identified and characterized, but many transporters of lignin-derived aromatic acids remain unidentified. The efficient conversion of lignin, an abundant but unutilized aromatic biomass resource, to value-added metabolites using microbial catabolism requires the characterization of transporters for lignin-derived aromatics. In this study, we identified the transporter gene responsible for the uptake of 5,5'-dehydrodivanillate, a lignin-derived biphenyl compound, in <i>Sphingobium</i> sp. strain SYK-6. In addition to characterizing its function, we applied this transporter gene to the production of a value-added metabolite from 5,5'-dehydrodivanillate.