Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440

Bentley, Gayle; Narayanan, Niju; Jha, Ramesh K.; Salvachua Rodriguez, Davinia; Elmore, Joshua R.; Peabody, George L.; Pleitner, Brenna P.; Kinley, Kelsey J.; De Capite, Annette; Michener, William E.; Werner, Allison J.; Klingeman, Dawn Marie; Schindel, Heidi S.; Nelson, Robert S.; Foust, Lindsey; Guss, Adam M.; Dale, Taraka; Johnson, Christopher W.; Beckham, Gregg T.

doi:10.1016/j.ymben.2020.01.001

Title: Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440

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Abstract

Pseudomonas putida KT2440 has been given increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows strain growth and muconate production. In this work, we employed adaptive evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improvedmore »« less

Authors:

Bentley, Gayle ^[1]; Narayanan, Niju ^[2]; Jha, Ramesh K. ^[2];

^[1];

^[3]; Peabody, George L. ^[3]; Pleitner, Brenna P. ^[1]; Kinley, Kelsey J. ^[1]; De Capite, Annette ^[1]; Michener, William E. ^[1];

^[1];

^[3]; Schindel, Heidi S. ^[3]; Nelson, Robert S. ^[1]; Foust, Lindsey ^[1];

^[3]; Dale, Taraka ^[2];

^[1];

^[1]

National Renewable Energy Lab. (NREL), Golden, CO (United States)
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Publication Date:: Fri Jan 10 00:00:00 EST 2020

Research Org.:: National Renewable Energy Lab. (NREL), Golden, CO (United States); Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office; USDOE Laboratory Directed Research and Development (LDRD) Program; USDOE Office of Energy Efficiency and Renewable Energy (EERE). Bioenergy Technologies Office (EE-3B)

OSTI Identifier:: 1596244

Alternate Identifier(s):: OSTI ID: 1617801; OSTI ID: 1634964; OSTI ID: 1702976

Report Number(s):: NREL/JA-2A00-75899; LA-UR-19-29034
Journal ID: ISSN 1096-7176

Grant/Contract Number:: AC36-08GO28308; 89233218CNA000001; AC05-00OR22725; NL0032182

Resource Type:: Accepted Manuscript

Journal Name:: Metabolic Engineering

Additional Journal Information:: Journal Volume: 59; Journal Issue: C; Journal ID: ISSN 1096-7176

Publisher:: Elsevier

Country of Publication:: United States

Language:: English

Subject:: 09 BIOMASS FUELS; cis,cis-muconic acid; Pseudomonas putida KT2440; glycolysis regulation; HexR; 2-ketogluconate; gluconate; GacS; biosensor; adaptive laboratory evolution; CatM; FACS; high throughput selection; Biological Science; cis,cis-muconic acid, Pseudomonas putida KT2440, glycolysis regulation, HexR, 2-ketogluconate, gluconate, GacS, biosensor, adaptive laboratory evolution, CatM, FACS, high throughput selection

Citation Formats


                    Bentley, Gayle, Narayanan, Niju, Jha, Ramesh K., Salvachua Rodriguez, Davinia, Elmore, Joshua R., Peabody, George L., Pleitner, Brenna P., Kinley, Kelsey J., De Capite, Annette, Michener, William E., Werner, Allison J., Klingeman, Dawn Marie, Schindel, Heidi S., Nelson, Robert S., Foust, Lindsey, Guss, Adam M., Dale, Taraka, Johnson, Christopher W., and Beckham, Gregg T. Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440.  United States: N. p., 2020. 
Web.  doi:10.1016/j.ymben.2020.01.001.

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                    Bentley, Gayle, Narayanan, Niju, Jha, Ramesh K., Salvachua Rodriguez, Davinia, Elmore, Joshua R., Peabody, George L., Pleitner, Brenna P., Kinley, Kelsey J., De Capite, Annette, Michener, William E., Werner, Allison J., Klingeman, Dawn Marie, Schindel, Heidi S., Nelson, Robert S., Foust, Lindsey, Guss, Adam M., Dale, Taraka, Johnson, Christopher W., & Beckham, Gregg T. Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440.  United States.  https://doi.org/10.1016/j.ymben.2020.01.001

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                    Bentley, Gayle, Narayanan, Niju, Jha, Ramesh K., Salvachua Rodriguez, Davinia, Elmore, Joshua R., Peabody, George L., Pleitner, Brenna P., Kinley, Kelsey J., De Capite, Annette, Michener, William E., Werner, Allison J., Klingeman, Dawn Marie, Schindel, Heidi S., Nelson, Robert S., Foust, Lindsey, Guss, Adam M., Dale, Taraka, Johnson, Christopher W., and Beckham, Gregg T. Fri .  
"Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440".  United States.  https://doi.org/10.1016/j.ymben.2020.01.001.  https://www.osti.gov/servlets/purl/1596244.

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@article{osti_1596244,

  title        = {Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440},

  author       = {Bentley, Gayle and Narayanan, Niju and Jha, Ramesh K. and Salvachua Rodriguez, Davinia and Elmore, Joshua R. and Peabody, George L. and Pleitner, Brenna P. and Kinley, Kelsey J. and De Capite, Annette and Michener, William E. and Werner, Allison J. and Klingeman, Dawn Marie and Schindel, Heidi S. and Nelson, Robert S. and Foust, Lindsey and Guss, Adam M. and Dale, Taraka and Johnson, Christopher W. and Beckham, Gregg T.},

  abstractNote = {Pseudomonas putida KT2440 has been given increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows strain growth and muconate production. In this work, we employed adaptive evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, deletion of hexR, gntZ, and gacS improved performance at similar conditions, with the resulting strain achieving 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.},

  doi          = {10.1016/j.ymben.2020.01.001},

  journal      = {Metabolic Engineering},

  number       = C,

  volume       = 59,

  place        = {United States},

  year         = {Fri Jan 10 00:00:00 EST 2020},

  month        = {Fri Jan 10 00:00:00 EST 2020}

}

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