TY - JOUR
T1 - Highly Active C 8 -Acyl-ACP Thioesterase Variant Isolated by a Synthetic Selection Strategy
AU - Hernández Lozada, Néstor J.
AU - Lai, Rung Yi
AU - Simmons, Trevor R.
AU - Thomas, Kelsey A.
AU - Chowdhury, Ratul
AU - Maranas, Costas D.
AU - Pfleger, Brian F.
N1 - Funding Information:
Support for this work was provided by the National Science Foundation (CBET-1703504) and Dow Chemical through a project grant to B.F.P. N.H.L. is the recipient of a NIH Chemistry-Biology Interface Training Program fellowship (No. T32 GM008505) and a Graduate Engineering Research Scholars fellowship from the UW−Madison College of Engineering. The authors would like to acknowledge the helpful discussions with Dr. Devon Rosenfeld and Dr. Christopher Stowers during the course of the project and the assistance of Dr. Travis Korosh in the octanoyl-ACP liquid chromatography analysis.
Funding Information:
Support for this work was provided by the National Science Foundation (CBET-1703504) and Dow Chemical through a project grant to B.F.P. N.H.L. is the recipient of a NIH Chemistry-Biology Interface Training Program fellowship (No. T32 GM008505) and a Graduate Engineering Research Scholars fellowship from the UW-Madison College of Engineering. The authors would like to acknowledge the helpful discussions with Dr. Devon Rosenfeld and Dr. Christopher Stowers during the course of the project and the assistance of Dr. Travis Korosh in the octanoyl-ACP liquid chromatography analysis.
Publisher Copyright:
Copyright © 2018 American Chemical Society.
PY - 2018/9/21
Y1 - 2018/9/21
N2 - Microbial metabolism is an attractive route for producing medium chain length fatty acids, e.g., octanoic acid, used in the oleochemical industry. One challenge to this strategy is the lack of enzymes that are both highly active in a microbial host and selective toward substrates with desired chain length. Of the many steps in fatty acid biosynthesis, the thioesterase is the most widely used enzyme for controlling chain length. Thioesterases hydrolyze the thioester bond between fatty acids and the acyl-carrier protein (ACP) or coenzyme A (CoA) cofactor. The functional role of thioesterases varies between organisms (i.e., bacteria vs plant) and therefore so do the substrate specificities. As a result, microbial biocatalysts that utilize a heterologous thioesterase either produce high titers of fatty acids with mixed chain lengths or low titers of products with a narrow chain length distribution. To search for highly active enzymes that selectively hydrolyze octanoyl-ACP, we developed a genetic selection based on the lipoic acid requirement of Escherichia coli. We used the selection to identify variants in a randomly mutagenized library of the C 8 -specific Cuphea palustris FatB1 thioesterase. After optimizing expression of the thioesterase, E. coli cultures produced 1.7 g/L of octanoic acid with >90% specificity from a single chromosomal copy of this thioesterase. In vitro studies confirmed the mutant thioesterase possessed a 15-fold increase in k cat compared to its native sequence. The high level of specific activity allowed for low levels of expression while maintaining fatty acid titer. The low expression requirement will allow metabolic engineers to use more cellular resources to address other limitations in the pathway and maximize overall productivity.
AB - Microbial metabolism is an attractive route for producing medium chain length fatty acids, e.g., octanoic acid, used in the oleochemical industry. One challenge to this strategy is the lack of enzymes that are both highly active in a microbial host and selective toward substrates with desired chain length. Of the many steps in fatty acid biosynthesis, the thioesterase is the most widely used enzyme for controlling chain length. Thioesterases hydrolyze the thioester bond between fatty acids and the acyl-carrier protein (ACP) or coenzyme A (CoA) cofactor. The functional role of thioesterases varies between organisms (i.e., bacteria vs plant) and therefore so do the substrate specificities. As a result, microbial biocatalysts that utilize a heterologous thioesterase either produce high titers of fatty acids with mixed chain lengths or low titers of products with a narrow chain length distribution. To search for highly active enzymes that selectively hydrolyze octanoyl-ACP, we developed a genetic selection based on the lipoic acid requirement of Escherichia coli. We used the selection to identify variants in a randomly mutagenized library of the C 8 -specific Cuphea palustris FatB1 thioesterase. After optimizing expression of the thioesterase, E. coli cultures produced 1.7 g/L of octanoic acid with >90% specificity from a single chromosomal copy of this thioesterase. In vitro studies confirmed the mutant thioesterase possessed a 15-fold increase in k cat compared to its native sequence. The high level of specific activity allowed for low levels of expression while maintaining fatty acid titer. The low expression requirement will allow metabolic engineers to use more cellular resources to address other limitations in the pathway and maximize overall productivity.
UR - http://www.scopus.com/inward/record.url?scp=85053761285&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85053761285&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.8b00215
DO - 10.1021/acssynbio.8b00215
M3 - Article
C2 - 30064208
AN - SCOPUS:85053761285
VL - 7
SP - 2205
EP - 2215
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
SN - 2161-5063
IS - 9
ER -