Expression of the Escherichia coli fabA gene encoding β-hydroxydecanoyl thioester dehydrase and transport to chloroplasts in transgenic tobacco

Kazuki Saito, Akira Hamajima, Mayumi Ohkuma, Isamu Murakoshi, Shigeru Ohmori, Akihiko Kawaguchi, Teemu H. Teeri, John E. Cronan

Research output: Contribution to journalArticle

Abstract

The fabA gene of Escherichia coli encodes β-hydroxydecanoyl thioester dehydrase (HDDase), a pivotal enzyme in the biosynthesis of the unsaturated fatty acid cis-vaccenic acid, through the anaerobic pathway. This enzyme is specific to bacterial fatty acid biosynthetic pathways, although other enzymes for fatty acid synthesis are very similar in plants and bacteria. We constructed chimaeric plant expression vectors, pfab21 and pfab22, carrying the fabA gene under the transcriptional control of the cauliflower mosaic virus (CaMV) promoter of 35S RNA. In pfab21, fabA was placed directly under the control of the CaMV 35S promoter; whereas in pfab22, the DNA sequence coding for the chloroplast-targeting transit peptide (TP) of the pea ribulose-1,5-bisphosphate carboxylase (RuBisCo) small subunit was fused to the fabA gene in order to allow transport of HDDase to the chloroplast, the organelle responsible for de novo fatty acid biosynthesis in plants. Transgenic plants of Nicotiana tabacum were obtained by Agrobacterium-mediated transformation with pfab21 or pfab 22. Expression of fabA transcripts of sizes expected from the chimaeric constructs was shawn by RNA blot hybridization. The HDDase protein derived from pfab22 was correctly processed and transported to chloroplasts in transformed plants. The enzymatic activity of HDDase was also detected in chloroplasts isolated from the transformants derived from pfab22 (but not pfab21) and in total leaf protein of all transformants. However, no significant changes were observed in the fatty acid compositions, including cis-vaccenic acid, of leaf chloroplasts and self-fertilized seeds. These results are discussed in relation with the possible structural organization of plant fatty acid synthase.

Original languageEnglish (US)
Pages (from-to)60-69
Number of pages10
JournalTransgenic Research
Volume4
Issue number1
DOIs
StatePublished - Jan 1 1995

Keywords

  • chloroplast
  • fabA
  • fatty acid biosynthesis
  • transgenic plant
  • unsaturated fatty acid
  • β-hydroxydecanoyl thioester dehydrase

ASJC Scopus subject areas

  • Biotechnology
  • Animal Science and Zoology
  • Agronomy and Crop Science
  • Genetics

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