Rational engineering of a novel pathway for producing the aromatic compounds p-hydroxybenzoate, protocatechuate, and catechol in Escherichia coli

Shawn Pugh, Rebekah McKenna, Marwan Osman, Brian Thompson, David Nielsen

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

p-Hydroxybenzoate, protocatechuate, and catechol represent fine and/or commodity chemicals useful as antioxidants and building-block molecules. To date, however, these species have been largely overlooked as focal end-products. An existing route employing protocatechuate and catechol as intermediates suffers from the need for multiple auxotrophies to preserve precursor (3-dehydroshikimate) availability. A novel, modular route from endogenous p-hydroxybenzoate has been engineered in Escherichia coli for the individual biosynthesis of all three products from renewable glucose while minimizing auxotrophy generation. To enhance endogenous biosynthesis of p-hydroxybenzoate, native chorismate pyruvate lyase (ubiC) was over-expressed. p-Hydroxybenzoate was converted to protocatechuate by a hydroxylase (pobA) from Pseudomonas aeruginosa. Catechol was produced by the additional co-expression of protocatechuate decarboxylase from Enterobacter cloacae. Systematic expression of appropriate pathway elements in phenylalanine overproducing E. coli enabled initial titers of 32 ± 4, 110 ± 8, and 81 ± 15 mg/L for p-hydroxybenzoate, protocatechuate, and catechol, respectively. Disruption of chorismate mutase/prephenate dehydratase (pheA) to preserve endogenous chorismate then allowed maximum titers of 277 ± 2, 454 ± 11, and 451 ± 44 mg/L, respectively, at glucose yields of 5.8, 9.7, and 14.3% of their respective theoretical maxima. Catechol titers were further improved to 630 ± 37 mg/L in a batch bioreactor study. The proposed pathway can furthermore serve as a platform for other bioproducts, including the bioplastics precursor cis,cis-muconate.

Original languageEnglish (US)
Pages (from-to)1843-1850
Number of pages8
JournalProcess Biochemistry
Volume49
Issue number11
DOIs
StatePublished - 2014

Fingerprint

Aromatic compounds
Biosynthesis
Escherichia coli
Glucose
Bioreactors
Antioxidants
Availability
Prephenate Dehydratase
Molecules
Enterobacter cloacae
Carboxy-Lyases
Mixed Function Oxygenases
Phenylalanine
Pseudomonas aeruginosa
catechol
4-hydroxybenzoic acid

Keywords

  • Aromatics
  • Catechol
  • Chorismate
  • p-Hydroxybenzoate
  • Protocatechuate

ASJC Scopus subject areas

  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

Rational engineering of a novel pathway for producing the aromatic compounds p-hydroxybenzoate, protocatechuate, and catechol in Escherichia coli. / Pugh, Shawn; McKenna, Rebekah; Osman, Marwan; Thompson, Brian; Nielsen, David.

In: Process Biochemistry, Vol. 49, No. 11, 2014, p. 1843-1850.

Research output: Contribution to journalArticle

@article{572970d211e749faa650c6e76ce7aae5,
title = "Rational engineering of a novel pathway for producing the aromatic compounds p-hydroxybenzoate, protocatechuate, and catechol in Escherichia coli",
abstract = "p-Hydroxybenzoate, protocatechuate, and catechol represent fine and/or commodity chemicals useful as antioxidants and building-block molecules. To date, however, these species have been largely overlooked as focal end-products. An existing route employing protocatechuate and catechol as intermediates suffers from the need for multiple auxotrophies to preserve precursor (3-dehydroshikimate) availability. A novel, modular route from endogenous p-hydroxybenzoate has been engineered in Escherichia coli for the individual biosynthesis of all three products from renewable glucose while minimizing auxotrophy generation. To enhance endogenous biosynthesis of p-hydroxybenzoate, native chorismate pyruvate lyase (ubiC) was over-expressed. p-Hydroxybenzoate was converted to protocatechuate by a hydroxylase (pobA) from Pseudomonas aeruginosa. Catechol was produced by the additional co-expression of protocatechuate decarboxylase from Enterobacter cloacae. Systematic expression of appropriate pathway elements in phenylalanine overproducing E. coli enabled initial titers of 32 ± 4, 110 ± 8, and 81 ± 15 mg/L for p-hydroxybenzoate, protocatechuate, and catechol, respectively. Disruption of chorismate mutase/prephenate dehydratase (pheA) to preserve endogenous chorismate then allowed maximum titers of 277 ± 2, 454 ± 11, and 451 ± 44 mg/L, respectively, at glucose yields of 5.8, 9.7, and 14.3{\%} of their respective theoretical maxima. Catechol titers were further improved to 630 ± 37 mg/L in a batch bioreactor study. The proposed pathway can furthermore serve as a platform for other bioproducts, including the bioplastics precursor cis,cis-muconate.",
keywords = "Aromatics, Catechol, Chorismate, p-Hydroxybenzoate, Protocatechuate",
author = "Shawn Pugh and Rebekah McKenna and Marwan Osman and Brian Thompson and David Nielsen",
year = "2014",
doi = "10.1016/j.procbio.2014.08.011",
language = "English (US)",
volume = "49",
pages = "1843--1850",
journal = "Process Biochemistry",
issn = "0032-9592",
publisher = "Elsevier BV",
number = "11",

}

TY - JOUR

T1 - Rational engineering of a novel pathway for producing the aromatic compounds p-hydroxybenzoate, protocatechuate, and catechol in Escherichia coli

AU - Pugh, Shawn

AU - McKenna, Rebekah

AU - Osman, Marwan

AU - Thompson, Brian

AU - Nielsen, David

PY - 2014

Y1 - 2014

N2 - p-Hydroxybenzoate, protocatechuate, and catechol represent fine and/or commodity chemicals useful as antioxidants and building-block molecules. To date, however, these species have been largely overlooked as focal end-products. An existing route employing protocatechuate and catechol as intermediates suffers from the need for multiple auxotrophies to preserve precursor (3-dehydroshikimate) availability. A novel, modular route from endogenous p-hydroxybenzoate has been engineered in Escherichia coli for the individual biosynthesis of all three products from renewable glucose while minimizing auxotrophy generation. To enhance endogenous biosynthesis of p-hydroxybenzoate, native chorismate pyruvate lyase (ubiC) was over-expressed. p-Hydroxybenzoate was converted to protocatechuate by a hydroxylase (pobA) from Pseudomonas aeruginosa. Catechol was produced by the additional co-expression of protocatechuate decarboxylase from Enterobacter cloacae. Systematic expression of appropriate pathway elements in phenylalanine overproducing E. coli enabled initial titers of 32 ± 4, 110 ± 8, and 81 ± 15 mg/L for p-hydroxybenzoate, protocatechuate, and catechol, respectively. Disruption of chorismate mutase/prephenate dehydratase (pheA) to preserve endogenous chorismate then allowed maximum titers of 277 ± 2, 454 ± 11, and 451 ± 44 mg/L, respectively, at glucose yields of 5.8, 9.7, and 14.3% of their respective theoretical maxima. Catechol titers were further improved to 630 ± 37 mg/L in a batch bioreactor study. The proposed pathway can furthermore serve as a platform for other bioproducts, including the bioplastics precursor cis,cis-muconate.

AB - p-Hydroxybenzoate, protocatechuate, and catechol represent fine and/or commodity chemicals useful as antioxidants and building-block molecules. To date, however, these species have been largely overlooked as focal end-products. An existing route employing protocatechuate and catechol as intermediates suffers from the need for multiple auxotrophies to preserve precursor (3-dehydroshikimate) availability. A novel, modular route from endogenous p-hydroxybenzoate has been engineered in Escherichia coli for the individual biosynthesis of all three products from renewable glucose while minimizing auxotrophy generation. To enhance endogenous biosynthesis of p-hydroxybenzoate, native chorismate pyruvate lyase (ubiC) was over-expressed. p-Hydroxybenzoate was converted to protocatechuate by a hydroxylase (pobA) from Pseudomonas aeruginosa. Catechol was produced by the additional co-expression of protocatechuate decarboxylase from Enterobacter cloacae. Systematic expression of appropriate pathway elements in phenylalanine overproducing E. coli enabled initial titers of 32 ± 4, 110 ± 8, and 81 ± 15 mg/L for p-hydroxybenzoate, protocatechuate, and catechol, respectively. Disruption of chorismate mutase/prephenate dehydratase (pheA) to preserve endogenous chorismate then allowed maximum titers of 277 ± 2, 454 ± 11, and 451 ± 44 mg/L, respectively, at glucose yields of 5.8, 9.7, and 14.3% of their respective theoretical maxima. Catechol titers were further improved to 630 ± 37 mg/L in a batch bioreactor study. The proposed pathway can furthermore serve as a platform for other bioproducts, including the bioplastics precursor cis,cis-muconate.

KW - Aromatics

KW - Catechol

KW - Chorismate

KW - p-Hydroxybenzoate

KW - Protocatechuate

UR - http://www.scopus.com/inward/record.url?scp=84908273805&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84908273805&partnerID=8YFLogxK

U2 - 10.1016/j.procbio.2014.08.011

DO - 10.1016/j.procbio.2014.08.011

M3 - Article

AN - SCOPUS:84908273805

VL - 49

SP - 1843

EP - 1850

JO - Process Biochemistry

JF - Process Biochemistry

SN - 0032-9592

IS - 11

ER -