Evidence of medium-chain-length polyhydroxyoctanoate accumulation in transgenic potato lines expressing the Pseudomonas oleovorans Pha-C1 polymerase in the cytoplasm

Abstract

The phaC1 gene from Pseudomonas oleovorans, coding for the Pha-C1 polymerase, was introduced into the potato genome. Transgenic callus and plant lines which transcribed and translated the transgene were selected and cell suspension cultures from the wild type and transgenic lines were established. The substrate for the Pha-C1 polymerase, 3-(R)-hydroxyoctanoate, was provided to the growth medium. In the transgenic lines, but not in the wild type or in transgenic cell suspension cultures without Pha-C1 expression, evidence of medium-chain-length polyhydroxyalkanoate accumulation ranging from 0.02 to 9.7 mg of polymer per gram of dry weight was observed after feeding in the growth medium the substrate.

Introduction

Fluorescent pseudomonads belonging to the rRNA group I accumulate granules that contain medium-chain-length polyhydroxyalkanoate (mcl-PHA), consisting of 3-(R)-hydroxyalkanoic acids with carbon chains ranging from C6 to C14 [1], [2]. The PHA-polymerase is the key enzyme for the synthesis of mcl-PHA using 3-(R)-hydroxy-acyl-CoA derived from fatty acid metabolism. In these pseudomonads, the pha-operon [3], [4] comprises the phaC1 and phaC2 genes coding for the Pha-C1 and Pha-C2 polymerases, respectively. These PHA-polymerases are classified as type II and are specifically active on mcl-alkanoic acids. The two PHA-polymerases share 50–54% identity at the protein sequence level and have a molecular weight of 62–64 kDa. Several additional genes are present [4], [5], [6], [7] in the operon which are involved in PHA degradation or in structural aspects of the PHA granules [8], [9]. A high degree of identity (69–80%) at the amino acid level is observed between corresponding mcl-PHA-polymerases from different species and 40% identity is observed with the Ralstonia eutropha polyhydroxybutyrate (PHB)-polymerase, [3], [4], which is active on short-chain-length alkanoates (C4 and C5 carbon units). PHB-polymerase (type I) and type II PHA-polymerase contain a lipase box [10] and it has been postulated that the catalytic mechanism involves residues Cys-296 and Cys-430 (Pseudomonas oleovorans numbering [3], [10]).

Both Pha-C1 and Pha-C2 polymerases are functional proteins which are able to catalyse mcl-PHA formation independently from each other when expressed in heterologous hosts [11], [12] including plants [13]. Overproduction of the mcl-PHA-polymerase in E. coli did not result in a corresponding increase of PHA yield, and most of the protein was recovered as inactive protein in inclusion bodies [14]. However, solubilisation of the trapped protein resulted in active polymerase, and this enzyme was capable of in vitro synthesis of mcl-PHA without any additional component.

In this work, the Pha-C1 polymerase of P. oleovorans has been expressed in the cytoplasm of transgenic potato lines. Although plant cytoplasm does not contain any PHA precursors due to the compartmentation of fatty acid metabolisms in plants (β-oxidation occurs mainly in peroxisomes and fatty acid biosynthesis (FAB) in the plastids), the ability of potato to express the Pha-C1 polymerase and to accumulate mcl-PHA was tested using a feeding approach, exogenously providing the substrate (3-(R)-hydroxyoctanoate) to cell suspensions of the selected transgenic lines. Evidence for mcl-PHA accumulation in transgenic lines expressing the phaC1 gene, but not in the wild type line nor in transgenic lines not expressing the Pha-C1 polymerase, was obtained.