Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene

doi:10.1016/S0168-1656(00)00367-9

Journal of Biotechnology

Volume 85, Issue 2, 13 February 2001, Pages 103-113

https://doi.org/10.1016/S0168-1656(00)00367-9 Get rights and content

Abstract

In this article, we illustrate the challenges and bottlenecks in the metabolic engineering of bacteria destined for environmental bioremediation, by reporting current efforts to construct Pseudomonas strains genetically designed for degradation of the recalcitrant compound 2-chlorotoluene. The assembled pathway includes one catabolic segment encoding the toluene dioxygenase of the TOD system of Pseudomonas putida F1 (todC1C2BA), which affords the bioconversion of 2-chlorotoluene into 2-chlorobenzaldehyde by virtue of its residual methyl-monooxygenase activity on o-substituted substrates. A second catabolic segment encoded the entire upper TOL pathway from pWW0 plasmid of P. putida mt-2. The enzymes, benzyl alcohol dehydrogenase (encoded by xylB) and benzaldehyde dehydrogenase (xylC) of this segment accept o-chloro-substituted substrates all the way down to 2-chlorobenzoate. These TOL and TOD segments were assembled in separate mini-Tn5 transposon vectors, such that expression of the encoded genes was dependent on the toluene-responsive Pu promoter of the TOL plasmid and the cognate XylR regulator. Such gene cassettes (mini-Tn5 [UPP2] and mini-Tn5 [TOD2]) were inserted in the chromosome of the 2-chlorobenzoate degraders Pseudomonas aeruginosa PA142 and P. aeruginosa JB2. GC-MS analysis of the metabolic intermediates present in the culture media of the resulting strains verified that these possessed, not only the genetic information, but also the functional ability to mineralise 2-chlorotoluene. However, although these strains did convert the substrate into 2-chlorobenzoate, they failed to grow on 2-chlorotoluene as the only carbon source. These results pinpoint the rate of the metabolic fluxes, the non-productive spill of side-metabolites and the physiological control of degradative pathways as the real bottlenecks for degradation of certain pollutants, rather than the theoretical enzymatic and genetic fitness of the recombinant bacteria to the process. Choices to address this general problem are discussed.

Section snippets

Introduction: ventures and limitations of metabolic engineering

It is now 25 years since the European Federation of Biotechnology established a Working Party to identify key factors that limit the usefulness of micro-organisms in biotechnology. Microbial physiology is at the interface between biological discovery and genetic engineering on the one hand and environmental biotechnology, biochemical engineering and the exploitation of microbial productivity on the other. The development of novel organisms for use in biodegradation has been a key challenge

Rationale for a hybrid pathway for metabolism of 2-chlorotoluene

The strategy of choice to produce Pseudomonas strains able to degrade 2-chlorotoluene is summarised in Fig. 1. The most salient feature of the scheme is the assembly of a hybrid upper pathway able to convert 2-chlorotoluene into 2-chlorobenzoate. This involves the mono-oxydation of the methyl group of 2-chlorotoluene by the broad substrate range toluene dioxygenase from P. putida F1, encoded by todC1C2BA genes. The resulting 2-chlorobenzylalcohol is then expected to be further oxydised to the

Construction and gross characterisation of P. aeruginosa strains bearing TOL and TOD genes

The metabolic engineering strategies described above were used to construct strains AH001 and AH002 as follows. The transposable elements mini-Tn5 [TOD2] and mini-Tn5 [UPP2] are shown in Fig. 2. Plasmid pUPP2 (Table 1) is the delivery plasmid for hybrid transposon mini-Tn5 [UPP2], which bears the upper TOL genes driven by the Pu promoter adjacent to a selectable tellurite resistance marker. pTOD2 bears mini-Tn5 [TOD2], in which the todC1C2BA genes expressed from an upstream Pu promoter in the

Metabolism of 2-chlorotoluene by citrate-fed P. aeruginosa AH001 and P. aeruginosa AH002

In view of the results above, we set out to examine the performance of the hybrid pathway for degradation of 2-chlorotoluene under growth conditions that did not require its utilisation as sole growth substrate. Because XylR regulated the expression of the tod and xyl genes, the maximum expression was expected at stationary phase of growth (Cases et al., 1996). Thus, we cultured separately P. aeruginosa AH001 and P. aeruginosa AH002 in a minimal medium containing 0.2% citrate and let it grow

Conclusion: genetic, enzymatic and physiological bottlenecks in degradation of chloro-aromatic compounds

In this work we have reported the performance of two Pseudomonas strains equipped with a set of genes and enzymatic activities which allow them to metabolise 2-chlorotoluene, along with a suitable regulatory circuit. Although the strains were separately able to convert 2-chlorotoluene via 2-chlorobenzylalcohol to 2-chlorobenzoate and to grow as well on 2-chlorobenzoate as the only carbon source, they failed to use the initial substrate as the only C and energy source. On the contrary, when

Acknowledgements

The authors are indebted to R.P. Hausinger and D.D. Focht for kindly providing strains, to Alicia Prieto and Angel Martínez's group for help with GC-MS analyses and to D. Pieper for essential advice on the work and useful discussions. This research was supported by Contracts BIO4-CT97-2040 and QLRT-99-00041 of the EU and by Grant BIO98-0808 of the Comisión Interministerial de Ciencia y Tecnología. MAH was a predoctoral Fellow of the Spanish Ministry of Education and Culture.

References (36)

R. Blasco et al.
From xenobiotic to antibiotic, formation of protoanemonin from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway
J. Biol. Chem.
(1995)
U. Brinkmann et al.
Degradation of chlorotoluenes by in vivo constructed hybrid strains: problems of enzyme specificity, induction and prevention of meta-pathway
FEMS Microbiol. Lett.
(1992)
V. de Lorenzo et al.
Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons
Methods Enzymol.
(1994)
V. de Lorenzo et al.
Engineering of alkyl- and haloaromatic-responsive gene expression with mini-transposons containing regulated promoters of biodegradative pathways of Pseudomonas
Gene
(1993)
H.J. Knackmuss
Basic knowledge and perspectives of bioelimination of xenobiotic compounds
J. Biotech.
(1996)
G.J. Zylstra et al.
Toluene degradation by Pseudomonas putida F1
J. Biol. Chem.
(1989)
M.A. Abril et al.
Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway
J. Bacteriol.
(1989)
G.M. Brazil et al.
Construction of a rhizosphere Pseudomonads with potential to degrade polychlorinated biphenyls an detection of bph gene expression in the rhizosphere
Appl. Environ. Microbiol.
(1995)
I. Cases et al.
Expression systems and physiological control of promoter activity in bacteria
Curr. Opin. Microbiol.
(1998)
I. Cases et al.
Involvement of σ⁵⁴ in exponential silencing of the Pseudomonas putida TOL plasmid Pu promoter
Mol. Microbiol.
(1996)

W.R. Farmer et al.

Improving lycopene production in Escherichia coli by engineering metabolic control

Nature Biotechnol.

(2000)

S. Fernández et al.

Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains

Mol. Microbiol.

(1995)

B.E. Haigler et al.

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6

Appl. Environ. Microbiol.

(1989)

S. Harayama et al.

Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products

J. Bacteriol.

(1989)

W.J. Hickey et al.

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2

Appl. Environ. Microbiol.

(1990)

T. Kumamaru et al.

Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase

Nature Biotech.

(1998)

P.C.K. Lau et al.

A bacterial basic region leucine zipper histidine kinase regulating toluene degradation

Proc. Natl. Acad. Sci. USA

(1997)

A. Lehning et al.

Metabolism of chlorotoluenes by Burkholderia sp. strain PS12 and toluene dioxygenase of Pseudomonas putida F1: Evidence for monooxygneation by toluene and chlorobenzene dioxygenases

Appl. Environ. Microbiol.

(1997)

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Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene

Abstract

Section snippets

Introduction: ventures and limitations of metabolic engineering

Rationale for a hybrid pathway for metabolism of 2-chlorotoluene

Construction and gross characterisation of P. aeruginosa strains bearing TOL and TOD genes

Metabolism of 2-chlorotoluene by citrate-fed P. aeruginosa AH001 and P. aeruginosa AH002

Conclusion: genetic, enzymatic and physiological bottlenecks in degradation of chloro-aromatic compounds

Acknowledgements

J. Biol. Chem.

FEMS Microbiol. Lett.

Methods Enzymol.

Gene

J. Biotech.

J. Biol. Chem.

Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway

J. Bacteriol.

Construction of a rhizosphere Pseudomonads with potential to degrade polychlorinated biphenyls an detection of bph gene expression in the rhizosphere

Appl. Environ. Microbiol.

Expression systems and physiological control of promoter activity in bacteria

Curr. Opin. Microbiol.

Involvement of σ54 in exponential silencing of the Pseudomonas putida TOL plasmid Pu promoter

Mol. Microbiol.

Improving lycopene production in Escherichia coli by engineering metabolic control

Nature Biotechnol.

Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains

Mol. Microbiol.

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6

Appl. Environ. Microbiol.

Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products

J. Bacteriol.

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2

Appl. Environ. Microbiol.

Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase

Nature Biotech.

A bacterial basic region leucine zipper histidine kinase regulating toluene degradation

Proc. Natl. Acad. Sci. USA

Metabolism of chlorotoluenes by Burkholderia sp. strain PS12 and toluene dioxygenase of Pseudomonas putida F1: Evidence for monooxygneation by toluene and chlorobenzene dioxygenases

Appl. Environ. Microbiol.

Involvement of σ⁵⁴ in exponential silencing of the Pseudomonas putida TOL plasmid Pu promoter