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

Abstract

Background Valorization of lignin has the potential to significantly improve the economics of lignocellulosic biorefineries. However, its complex structure makes conversion to useful products elusive. One promising approach is depolymerization of lignin and subsequent bioconversion of breakdown products into value-added compounds. Optimizing transport of these depolymerization products into one or more organism(s) for biological conversion is important to maximize carbon utilization and minimize toxicity. Current methods assess internalization of depolymerization products indirectly—for example, growth on, or toxicity of, a substrate. Furthermore, no method has been shown to provide visualization of depolymerization products in individual cells. Results We applied mass spectrometry to provide direct measurements of relative internalized concentrations of several lignin depolymerization compounds and single-cell microscopy methods to visualize cell-to-cell differences in internalized amounts of two lignin depolymerization compounds. We characterized internalization of 4-hydroxybenzoic acid, vanillic acid, p-coumaric acid, syringic acid, and the model dimer guaiacylglycerol-beta-guaiacyl ether (GGE) in the lignolytic organisms Phanerochaete chrysosporium and Enterobacter lignolyticus and in the non-lignolytic but genetically tractable organisms Saccharomyces cerevisiae and Escherichia coli. The results show varying degrees of internalization in all organisms for all the tested compounds, including the model dimer, GGE. Phanerochaete chrysosporium internalizes all compounds in non-lignolytic and lignolytic conditionsmore » at comparable levels, indicating that the transporters for these compounds are not specific to the lignolytic secondary metabolic system. Single-cell microscopy shows that internalization of vanillic acid and 4-hydroxybenzoic acid analogs varies greatly among individual fungal and bacterial cells in a given population. Glucose starvation and chemical inhibition of ATP hydrolysis during internalization significantly reduced the internalized amount of vanillic acid in bacteria. Conclusions Mass spectrometry and single-cell microscopy methods were developed to establish a toolset for providing direct measurement and visualization of relative internal concentrations of mono- and di-aryl compounds in microbes. Utilizing these methods, we observed broad variation in intracellular concentration between organisms and within populations and this may have important consequences for the efficiency and productivity of an industrial process for bioconversion. Subsequent application of this toolset will be useful in identifying and characterizing specific transporters for lignin-derived mono- and di-aryl compounds.« less

Authors:

Barnhart-Dailey, Meghan C.; Ye, Dongmei; Hayes, Dulce C.; Maes, Danae; Simoes, Casey T.; Appelhans, Leah; Carroll-Portillo, Amanda; Kent, Michael S.; Timlin, Jerilyn A.

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Publication Date:

2019-07-03

Research Org.:

Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

Sponsoring Org.:

USDOE National Nuclear Security Administration (NNSA), Office of Defense Nuclear Nonproliferation

OSTI Identifier:

1618765

Alternate Identifier(s):

OSTI ID: 1559560

Report Number(s):

SAND-2019-9902J
Journal ID: ISSN 1754-6834; 175; PII: 1494

Grant/Contract Number:  

190958; AC04-94AL85000; NA0003525

Resource Type:

Published Article

Journal Name:

Biotechnology for Biofuels

Additional Journal Information:

Journal Name: Biotechnology for Biofuels Journal Volume: 12 Journal Issue: 1; Journal ID: ISSN 1754-6834

Publisher:

Springer Science + Business Media

Country of Publication:

Netherlands

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Lignin; bioconversion; transport; single cell analysis; mass spectrometry; monoaryl; diaryl