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 conditions 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. Electronic supplementary material The ...

Development of a Biosensor for Detection of Benzoic Acid Derivatives in Saccharomyces cerevisiae

“…After 65 h of cultivation, the strain ySCC185-UbiC produced 1.32 mM of pHBA while the control strain ySCC185-30 did not produce any detectable amounts of it ( Figure 6A). The production yield achieved in our strain is half of that reported by Averesch et al (2017). This difference might be attributed to the modifications included in our parental strain (ySCC185-F) or differences in the media composition/culture conditions.…”

“…Thus, all tested metabolites have different pK a s and their diffusion through the plasma membrane of yeast might be slightly different. Moreover, it is known that the rates of import of benzoic acid derivatives (p-coumaric acid and pHBA) in S. cerevisiae are different (Barnhart-Dailey et al, 2019). However, as pHBA, 2HBA, pABA, 2NBA, benzoic acid, and aspirin all activate sBAD in vivo under the conditions tested, we assume that they are transported in the cell in order to activate the intracellular sBAD and trigger the corresponding fluorescence signal.…”

“…We next addressed whether sBAD would support real-time monitoring of in vivo pHBA metabolite production. To do so, we engineered the ySCC185-F strain to overproduce pHBA as previously described (Averesch et al, 2017). We first deleted the TRP3 gene and then substituted the wild-type version of ARO7 with the ARO4K229L gene.…”

“…We first deleted the TRP3 gene and then substituted the wild-type version of ARO7 with the ARO4K229L gene. This strain named ySCC185-F-A can produce higher yields of chorismate compared to the ySCC185-F strain (Averesch et al, 2017). ySCC185-F-A was then further modified to express the ubiC (chorismate lyase) gene from E. coli under the constitutive promoter TDH3, leading to the strain ySCC185-UbiC.…”

“…Biotechnology offers an alternative for pHBA production, based on the shikimate pathway (Lee and Wendisch, 2017). Recently, researchers have engineered yeast to overproduce pHBA, increasing the flux to chorismate and expressing, in the modified strain, the chorismate lyase gene (UbiC) from E. coli (Averesch et al, 2017).…”