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Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster.

ABSTRACT: We report on the presence of a functional hydroxyectoine biosynthesis gene cluster, ectABCD-ask, in Pseudomonas stutzeri DSM5190(T) and evaluate the suitability of P. stutzeri DSM5190(T) for hydroxyectoine production. Furthermore, we present information on heterologous de novo production of the compatible solute hydroxyectoine in Escherichia coli. In this host, the P. stutzeri gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in E. coli under the conditions employed exceeded that of the natural producer Pseudomonas stutzeri and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes.

SUBMITTER: Seip B

PROVIDER: S-EPMC3067233 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster.

Seip Britta B Galinski Erwin A EA Kurz Matthias M

Applied and environmental microbiology 20101217 4

We report on the presence of a functional hydroxyectoine biosynthesis gene cluster, ectABCD-ask, in Pseudomonas stutzeri DSM5190(T) and evaluate the suitability of P. stutzeri DSM5190(T) for hydroxyectoine production. Furthermore, we present information on heterologous de novo production of the compatible solute hydroxyectoine in Escherichia coli. In this host, the P. stutzeri gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose whe ...[more]

PMID: 21169432

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Project description:UnlabelledThe gene cluster encoding the 2-chloronitrobenzene (2CNB) catabolism pathway in Pseudomonas stutzeri ZWLR2-1 is a patchwork assembly of a Nag-like dioxygenase (dioxygenase belonging to the naphthalene dioxygenase NagAaAbAcAd family from Ralstonia sp. strain U2) gene cluster and a chlorocatechol catabolism cluster. However, the transcriptional regulator gene usually present in the Nag-like dioxygenase gene cluster is missing, leaving it unclear how this cluster is expressed. The pattern of expression of the 2CNB catabolism cluster was investigated here. The results demonstrate that the expression was constitutive and not induced by its substrate 2CNB or salicylate, the usual inducer of expression in the Nag-like dioxygenase family. Reverse transcription-PCR indicated the presence of at least one transcript containing all the structural genes for 2CNB degradation. Among the three promoters verified in the gene cluster, P1 served as the promoter for the entire catabolism operon, but the internal promoters P2 and P3 also enhanced the transcription of the genes downstream. The P3 promoter, which was not previously defined as a promoter sequence, was the strongest of these three promoters. It drove the expression of cnbAcAd encoding the dioxygenase that catalyzes the initial reaction in the 2CNB catabolism pathway. Bioinformatics and mutation analyses suggested that this P3 promoter evolved through the duplication of an 18-bp fragment and introduction of an extra 132-bp fragment.ImportanceThe release of many synthetic compounds into the environment places selective pressure on bacteria to develop their ability to utilize these chemicals to grow. One of the problems that a bacterium must surmount is to evolve a regulatory device for expression of the corresponding catabolism genes. Considering that 2CNB is a xenobiotic that has existed only since the onset of synthetic chemistry, it may be a good example for studying the molecular mechanisms underlying rapid evolution in regulatory networks for the catabolism of synthetic compounds. The 2CNB utilizer Pseudomonas stutzeri ZWLR2-1 in this study has adapted itself to the new pollutant by evolving the always-inducible Nag-like dioxygenase into a constitutively expressed enzyme, and its expression has escaped the influence of salicylate. This may facilitate an understanding of how bacteria can rapidly adapt to the new synthetic compounds by evolving its expression system for key enzymes involved in the degradation of a xenobiotic.

Dataset Information

Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster.

Publications

Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster.

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