Passive membrane transport of lignin-related compounds

Abstract

Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degradation products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed molecular dynamics simulations for 69 monomeric and dimeric lignin-related phenolics with 3 model membranes to determine the membrane partitioning and permeability coefficients for each compound. The results support an accessible passive permeation mechanism for most compounds, including monolignols, dimeric phenolics, and the flavonoid, tricin. Computed lignin partition coefficients are consistent with concentration enrichment near lipid carbonyl groups, and permeability coefficients are sufficient to keep pace with cellular metabolism. Interactions between methoxy and hydroxy groups are found to reduce membrane partitioning and improve permeability. Only carboxylate-modified or glycosylated lignin phenolics are predicted to require transporters for membrane translocation. Overall, the results suggest that most lignin-related compounds can passively traverse plant and microbial membranes on timescales commensurate with required biological activities, with any potential transport regulation mechanism in lignin synthesis, catabolism, or bioconversion requiring compound functionalization.

Keywords: biological funneling; free energy calculation; lignin biosynthesis; lignin permeability; molecular dynamics.

Fig. 1.

LRCs examined, grouped by their chemistry. On the left are monomeric (single aromatic ring) compounds including monolignols and derivatives encountered via lignin deconstruction processes (H, G, S, and C substitutions from left to right). Other compounds enriched in fragmented lignin streams are also considered, including dimeric LRCs with monomer-dependent R1 and R2 groups, a representative flavonoid (tricin), glucosides (coniferin and syringin), and other aromatics (e.g., cresols). LRCs that can be derived from high-severity processes, such as benzene, are also considered. The red monolignol labeling defines lignin carbon nomenclature.

Fig. 2.

Free energy profiles for diverse G-type LRCs in all 3 membrane compositions (POPC [Left], Z. mays [Middle], and P. putida [Right]), using the molecular center of mass distance from the membrane center as the reaction coordinate. Solid lines indicate the position-dependent free energy. Free energy uncertainties (shaded regions, typically on the order of 0.1 kcal mol⁻¹) are determined from a population of 100 Gibbs samples drawn from the complete dataset. The profiles are superimposed on a simulation snapshot showing membrane lipid heavy atoms (gray, carbons; red, oxygens; blue, nitrogens; and brown, phosphorus atoms), water (white, hydrogens, and light blue, oxygens), and ions (yellow). SI Appendix, Figs. S1–S17 include profiles for all studied LRCs, including diffusivity profiles.

Fig. 3.

(A) Molecular orientation and (B) compound–lipid hydrogen bonding for phenols and vanillate from the REUS calculations in POPC, along with representative snapshots of (C) guaiacol interacting with lipid carbonyls and (D) lipid headgroup phosphates. (A) The mean molecular orientation at a specific distance from the membrane center is computed based on the vector from carbon 1 to carbon 4 (red arrow) and the cosine of the angle between that vector and the membrane normal. The R, R1, and R2 groups vary as shown in Fig. 1. The middle 50% of the distribution is shown using semitransparent shading. (B) Hydrogen bonds between LRCs and lipids depending on penetration depth. Solid lines indicate hydrogen bonds to lipid carbonyl groups, whereas dashed lines quantify hydrogen bonds to lipid headgroups. A hydrogen bond was defined by heavy atoms separated by less than 3.2 Å and a colinear hydrogen within ${30}^{○}$. Comparable figures for other compound classes are presented in SI Appendix, Figs. S20–S36. (C) Small LRCs such as guaiacol (cyan colored carbons) can act as donors in hydrogen bonding interactions (purple dashed lines) with lipid carbonyl oxygens. Methoxy groups can similarly act as acceptors in interactions with water. (D) Depending on the position relative to the membrane center, the hydroxyl group can also act as a donor to phosphate groups in the lipid head. Both C and D use the same color scheme, with nonguaiacol carbons shown in gray, oxygens in red, hydrogens in white, nitrogens in blue, and phosphorus in brown.

Fig. 4.

Comparison of partitioning (Top) and membrane permeability (Bottom; $\log {\mathrm{P}\mathrm{m}}_c$ from SI Appendix, Tables S1–S3) depending on the number of oxygen atoms present in each molecule.