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Title: Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neither enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.
Authors:
ORCiD logo [1] ; ORCiD logo [2] ; ORCiD logo [2]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Shull Wollan Center - A Joint Institute for Neutron Sciences, Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Grant/Contract Number:
AC05-00OR22725; AC02-05CH11231
Type:
Published Article
Journal Name:
Metabolic Engineering Communications
Additional Journal Information:
Journal Volume: 6; Journal Issue: C; Journal ID: ISSN 2214-0301
Publisher:
Elsevier
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Lignin; Protocatechuate; Experimental evolution
OSTI Identifier:
1433579
Alternate Identifier(s):
OSTI ID: 1436042

Standaert, Robert F., Giannone, Richard J., and Michener, Joshua K.. Identification of parallel and divergent optimization solutions for homologous metabolic enzymes. United States: N. p., Web. doi:10.1016/j.meteno.2018.04.002.
Standaert, Robert F., Giannone, Richard J., & Michener, Joshua K.. Identification of parallel and divergent optimization solutions for homologous metabolic enzymes. United States. doi:10.1016/j.meteno.2018.04.002.
Standaert, Robert F., Giannone, Richard J., and Michener, Joshua K.. 2018. "Identification of parallel and divergent optimization solutions for homologous metabolic enzymes". United States. doi:10.1016/j.meteno.2018.04.002.
@article{osti_1433579,
title = {Identification of parallel and divergent optimization solutions for homologous metabolic enzymes},
author = {Standaert, Robert F. and Giannone, Richard J. and Michener, Joshua K.},
abstractNote = {Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neither enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.},
doi = {10.1016/j.meteno.2018.04.002},
journal = {Metabolic Engineering Communications},
number = C,
volume = 6,
place = {United States},
year = {2018},
month = {4}
}