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Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated

N Hoffmann1, A Steinbüchel, B H Rehm

  • 1Institut für Mikrobiologie, Westfälischen Wilhelms-Universität Münster, Germany.

Applied Microbiology and Biotechnology
|December 29, 2000
PubMed

Insights

Pseudomonas oleovorans possesses a phaG gene homologue, but it is not functionally expressed, preventing polyhydroxyalkanoate (PHA) synthesis from simple carbon sources. Introducing this gene under a different promoter enabled PHA production.

Area of Science:

  • Microbiology
  • Biochemistry

Background:

  • Various pseudomonads synthesize polyhydroxyalkanoates (PHA), a type of biopolymer, from simple carbon sources.
  • The transacylase PhaG links fatty acid synthesis and PHA production in Pseudomonas putida.
  • Homologues of the phaG gene are found in other pseudomonads, including Pseudomonas oleovorans.

Purpose of the Study:

  • To investigate the functional expression of the phaG homologue in Pseudomonas oleovorans.
  • To determine why P. oleovorans does not accumulate PHA from non-related carbon sources despite possessing a phaG homologue.

Main Methods:

  • Southern hybridization to detect phaG homologues.
  • Polymerase chain reaction (PCR) to amplify the P. oleovorans phaG gene (phaG(Po)).
  • DNA sequencing, reverse transcriptase PCR (RT-PCR), and complementation assays.

Main Results:

  • A phaG homologue (phaG(Po)) was identified in P. oleovorans with high sequence identity to P. putida's phaG (phaG(Pp)).
  • phaG(Po) was not transcribed under inducing conditions in P. oleovorans, unlike phaG(Pp) in P. putida.
  • Complementation of a phaG-negative mutant with phaG(Po) and subsequent reintroduction into P. oleovorans under a lac promoter enabled PHA synthesis.

Conclusions:

  • The phaG gene in P. oleovorans is not functionally expressed in its native context.
  • The lack of functional phaG expression prevents PHA accumulation from simple carbon sources in P. oleovorans.
  • PHA synthesis can be restored in P. oleovorans by expressing its phaG homologue under an inducible promoter.

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