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Title: Systems biology-guided biodesign of consolidated lignin conversion

Abstract

Lignin is the second most abundant biopolymer on the earth, yet its utilization for fungible products is complicated by its recalcitrant nature and remains a major challenge for sustainable lignocellulosic biorefineries. In this study, we used a systems biology approach to reveal the carbon utilization pattern and lignin degradation mechanisms in a unique lignin-utilizing Pseudomonas putida strain (A514). The mechanistic study further guided the design of three functional modules to enable a consolidated lignin bioconversion route. First, P. putida A514 mobilized a dye peroxidase-based enzymatic system for lignin depolymerization. This system could be enhanced by overexpressing a secreted multifunctional dye peroxidase to promote a two-fold enhancement of cell growth on insoluble kraft lignin. Second, A514 employed a variety of peripheral and central catabolism pathways to metabolize aromatic compounds, which can be optimized by overexpressing key enzymes. Third, the β-oxidation of fatty acid was up-regulated, whereas fatty acid synthesis was down-regulated when A514 was grown on lignin and vanillic acid. Therefore, the functional module for polyhydroxyalkanoate (PHA) production was designed to rechannel β-oxidation products. As a result, PHA content reached 73% per cell dry weight (CDW). Further integrating the three functional modules enhanced the production of PHA from kraft lignin andmore » biorefinery waste. Furthermore, this study elucidated lignin conversion mechanisms in bacteria with potential industrial implications and laid out the concept for engineering a consolidated lignin conversion route.« less

Authors:
 [1];  [2];  [3];  [2];  [2];  [4];  [2];  [2];  [4];  [2];  [5];  [2]
  1. Texas A & M Univ., College Station, TX (United States); Zhejiang Univ., Hangzhou (China)
  2. Texas A & M Univ., College Station, TX (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  4. Michigan State Univ., East Lansing, MI (United States)
  5. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1326560
Grant/Contract Number:
AC05-00OR22725
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Green Chemistry
Additional Journal Information:
Journal Name: Green Chemistry; Journal ID: ISSN 1463-9262
Publisher:
Royal Society of Chemistry
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Lin, Lu, Cheng, Yanbing, Pu, Yunqiao, Sun, Su, Li, Xiao, Jin, Mingjie, Pierson, Elizabeth A., Gross, Dennis C., Dale, Bruce E., Dai, Susie Y., Ragauskas, Arthur J., and Yuan, Joshua S. Systems biology-guided biodesign of consolidated lignin conversion. United States: N. p., 2016. Web. doi:10.1039/C6GC01131D.
Lin, Lu, Cheng, Yanbing, Pu, Yunqiao, Sun, Su, Li, Xiao, Jin, Mingjie, Pierson, Elizabeth A., Gross, Dennis C., Dale, Bruce E., Dai, Susie Y., Ragauskas, Arthur J., & Yuan, Joshua S. Systems biology-guided biodesign of consolidated lignin conversion. United States. doi:10.1039/C6GC01131D.
Lin, Lu, Cheng, Yanbing, Pu, Yunqiao, Sun, Su, Li, Xiao, Jin, Mingjie, Pierson, Elizabeth A., Gross, Dennis C., Dale, Bruce E., Dai, Susie Y., Ragauskas, Arthur J., and Yuan, Joshua S. 2016. "Systems biology-guided biodesign of consolidated lignin conversion". United States. doi:10.1039/C6GC01131D. https://www.osti.gov/servlets/purl/1326560.
@article{osti_1326560,
title = {Systems biology-guided biodesign of consolidated lignin conversion},
author = {Lin, Lu and Cheng, Yanbing and Pu, Yunqiao and Sun, Su and Li, Xiao and Jin, Mingjie and Pierson, Elizabeth A. and Gross, Dennis C. and Dale, Bruce E. and Dai, Susie Y. and Ragauskas, Arthur J. and Yuan, Joshua S.},
abstractNote = {Lignin is the second most abundant biopolymer on the earth, yet its utilization for fungible products is complicated by its recalcitrant nature and remains a major challenge for sustainable lignocellulosic biorefineries. In this study, we used a systems biology approach to reveal the carbon utilization pattern and lignin degradation mechanisms in a unique lignin-utilizing Pseudomonas putida strain (A514). The mechanistic study further guided the design of three functional modules to enable a consolidated lignin bioconversion route. First, P. putida A514 mobilized a dye peroxidase-based enzymatic system for lignin depolymerization. This system could be enhanced by overexpressing a secreted multifunctional dye peroxidase to promote a two-fold enhancement of cell growth on insoluble kraft lignin. Second, A514 employed a variety of peripheral and central catabolism pathways to metabolize aromatic compounds, which can be optimized by overexpressing key enzymes. Third, the β-oxidation of fatty acid was up-regulated, whereas fatty acid synthesis was down-regulated when A514 was grown on lignin and vanillic acid. Therefore, the functional module for polyhydroxyalkanoate (PHA) production was designed to rechannel β-oxidation products. As a result, PHA content reached 73% per cell dry weight (CDW). Further integrating the three functional modules enhanced the production of PHA from kraft lignin and biorefinery waste. Furthermore, this study elucidated lignin conversion mechanisms in bacteria with potential industrial implications and laid out the concept for engineering a consolidated lignin conversion route.},
doi = {10.1039/C6GC01131D},
journal = {Green Chemistry},
number = ,
volume = ,
place = {United States},
year = 2016,
month = 7
}

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  • Background: Higher ratios of syringyl-to-guaiacyl (S/G) lignin components of Populus were shown to improve sugar release by enzymatic hydrolysis using commercial blends. Cellulolytic microbes are often robust biomass hydrolyzers and may offer cost advantages; however, it is unknown whether their activity can also be significantly influenced by the ratio of different monolignol types in Populus biomass. Hydrolysis and fermentation of autoclaved, but otherwise not pretreated Populus trichocarpa by Clostridium thermocellum ATCC 27405 was compared using feedstocks that had similar carbohydrate and total lignin contents but differed in S/G ratios. Results: Populus with an S/G ratio of 2.1 was converted moremore » rapidly and to a greater extent compared to similar biomass that had a ratio of 1.2. For either microbes or commercial enzymes, an approximate 50% relative difference in total solids solubilization was measured for both biomasses, which suggests that the differences and limitations in the microbial breakdown of lignocellulose may be largely from the enzymatic hydrolytic process. Unexpectedly, the reduction in glucan content per gram solid in the residual microbially processed biomass was similar (17–18%) irrespective of S/G ratio, pointing to a similar mechanism of solubilization that proceeded at different rates. Fermentation metabolome testing did not reveal the release of known biomass-derived alcohol and aldehyde inhibitors that could explain observed differences in microbial hydrolytic activity. Biomass-derived p-hydroxybenzoic acid was up to ninefold higher in low S/G ratio biomass fermentations, but was not found to be inhibitory in subsequent test fermentations. Cellulose crystallinity and degree of polymerization did not vary between Populus lines and had minor changes after fermentation. However, lignin molecular weights and cellulose accessibility determined by Simons’ staining were positively correlated to the S/G content. Conclusions: Higher S/G ratios in Populus biomass lead to longer and more linear lignin chains and greater access to surface cellulosic content by microbe-bound enzymatic complexes. Substrate access limitation is suggested as a primary bottleneck in solubilization of minimally processed Populus, which has important implications for microbial deconstruction of lignocellulose biomass. Our findings will allow others to examine different Populus lines and to test if similar observations are possible for other plant species.« less
  • Background: Higher ratios of syringyl-to-guaiacyl (S/G) lignin components of Populus were shown to improve sugar release by enzymatic hydrolysis using commercial blends. Cellulolytic microbes are often robust biomass hydrolyzers and may offer cost advantages, however, it is unknown whether their activity can also be significantly influenced by the ratio of different monolignol types in Populus biomass. Hydrolysis and fermentation of autoclaved but otherwise not pretreated Populus trichocarpa by Clostridium thermocellum ATCC 27405 was compared using feedstocks that had similar carbohydrate and total lignin contents but differed in S/G ratios. Results: Populus with an S/G ratio of 2.1 was converted moremore » rapidly and to a greater extent compared to similar biomass that had a ratio of 1.2. For either microbes or commercial enzymes, an approximate 50% relative difference in total solids solubilization was measured for both biomasses, which suggests that the differences and limitations in the microbial breakdown of lignocellulose may be largely from the enzymatic hydrolytic process. Unexpectedly, the reduction in glucan content per gram solid in the residual microbially processed biomass was similar (17 18 %) irrespective of S/G ratio, pointing to a similar mechanism of solubilization that proceeded at different rates. Fermentation metabolome testing did not reveal the release of known biomass-derived alcohol and aldehyde inhibitors that could explain observed differences in microbial hydrolytic activity. Biomass-derived p-hydroxybenzoic acid was up to nine-fold higher in low S/G ratio biomass fermentations, but was not found to be inhibitory in subsequent test fermentations. Cellulose crystallinity and degree of polymerization did not vary between Populus lines and had minor changes after fermentation. However, lignin molecular weights and cellulose accessibility determined by Simons staining were positively correlated to the S/G content. Conclusions: Higher S/G ratios in Populus biomass lead to longer and more linear lignin chains and greater access to surface cellulosic content by microbe-bound enzymatic complexes. Substrate access limitation is suggested as a primary bottleneck in solubilization of minimally processed Populus, which has important implications for microbial deconstruction of lignocellulose biomass. Our findings will allow others to examine different Populus lines and to test if similar observations are possible for other plant species.« less
  • The bacterium Clostridium thermocellum offers a distinct and integrated approach to ethanol production through consolidated bioprocessing (CBP). The Simons’ stain technique, which assays the accessibility of lignocellulosic biomass, has been traditionally applied to fungal cellulase systems; however, its application to CBP has not been fully explored. For this reason, the structural properties of eight Populus trichocarpa with either high or low biomass densities were compared in this paper to determine bioconversion differences during separate hydrolysis and fermentation (SHF) and CBP with C. thermocellum. Simons’ staining generally identifies low density poplar as more accessible than high density poplar. Additionally, low densitymore » P. trichocarpa generally contained less Klason lignin than high density poplar. SHF and CBP treatments consistently identified BESC-7 (high density, low accessibility, low surface roughness) as a low ethanol yielding biomass and GW-9914 (low density, high accessibility, high surface roughness) as a high ethanol yielding biomass. Upon further investigation, BESC-7 also contained a high Klason lignin content (~25%), while GW-9914 had a low lignin content (~20%). Cellulose degree of polymerization (DP) measurements exhibited a weak linear correlation with accessibility (r 2 = 0.17). Finally, therefore, the ethanol yields were correlated with accessibility and lignin content extremes but not cellulose DP.« less