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Title: Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoringmore » and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Altogether, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases.« less
Authors:
 [1] ;  [1] ;  [2] ;  [3] ;  [1]
  1. Michigan State Univ., East Lansing, MI (United States)
  2. Michigan State Univ., East Lansing, MI (United States); Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)
  3. National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
Report Number(s):
NREL/JA-2700-66896
Journal ID: ISSN 0006-3592
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
Biotechnology and Bioengineering
Additional Journal Information:
Journal Volume: 114; Journal Issue: 4; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; computational protein design; cellulase; lignin; biomass deconstruction; protein engineering
OSTI Identifier:
1345115

Haarmeyer, Carolyn N., Smith, Matthew D., Chundawat, Shishir P. S., Sammond, Deanne, and Whitehead, Timothy A.. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein. United States: N. p., Web. doi:10.1002/bit.26201.
Haarmeyer, Carolyn N., Smith, Matthew D., Chundawat, Shishir P. S., Sammond, Deanne, & Whitehead, Timothy A.. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein. United States. doi:10.1002/bit.26201.
Haarmeyer, Carolyn N., Smith, Matthew D., Chundawat, Shishir P. S., Sammond, Deanne, and Whitehead, Timothy A.. 2016. "Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein". United States. doi:10.1002/bit.26201. https://www.osti.gov/servlets/purl/1345115.
@article{osti_1345115,
title = {Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein},
author = {Haarmeyer, Carolyn N. and Smith, Matthew D. and Chundawat, Shishir P. S. and Sammond, Deanne and Whitehead, Timothy A.},
abstractNote = {Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Altogether, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases.},
doi = {10.1002/bit.26201},
journal = {Biotechnology and Bioengineering},
number = 4,
volume = 114,
place = {United States},
year = {2016},
month = {10}
}

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