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 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 () and SLG_07780 (), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. A mutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVA-demethylase oxygenase component gene (), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlled transcriptional regulatory system. A UT26S transformant expressing acquired DDVA uptake capacity, indicating that 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 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 () cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of a -expression plasmid into a 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. 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 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.

Internalization and accumulation of model lignin breakdown products in bacteria and fungi

“…We used these methods to characterize relative intracellular concentrations of four mono-aryls and one di-aryl into four diverse organisms. Prior to this work, only indirect measurements of mono- and di-aryl internalization had been made in microbes [5, 11, 12, 15–20].…”

“…These extracellular lignin biodegradation processes result in a mixture of monomeric and oligomeric aromatic hydrocarbons [8–10]. Low molecular weight lignin breakdown products are then transported into lignolytic bacteria and certain fungi and are subsequently metabolized [5, 11, 12], although evidence for internalization in white rot fungi, including P. chrysosporium , has not been presented to date. It is widely believed that fungi play the primary role in breaking down lignin into low molecular weight species and that bacteria mineralize a large fraction of the low molecular weight material [3, 13, 14].…”

Regulation of vanillate and syringate catabolism by a MarR-type transcriptional regulator DesR in Sphingobium sp. SYK-6

“…For the non-inducing condition, the culture was incubated for a further 2 h without the addition of any substrate. Cells were harvested by centrifugation, washed twice with Wx medium, and resuspended in the same medium to an OD 600 of 5.0. β-galactosidase activity was measured using V630-BIO and expressed as Miller units as previously described 45 .…”

Developing Rhodococcus opacus and Sphingobium sp. coculture systems for valorization of lignin‐derived dimers

“…Additionally, by engineering the native metabolic pathways of Sphingobium sp. SYK‐6, 5,5′‐dehydrodivanillate (DDVA) was converted to 2‐pyrone‐4,6‐dicarboxylate, another kind of functional polymer's precursor (Fujita et al, 2019; Mori et al, 2018). Despite abundant cleavage pathways for lignin‐derived dimers in Sphingobium sp.…”