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Applied and Environmental Microbiology, July 2009, p. 4668-4675, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00487-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Production of Functionalized Biopolyester Granules by Recombinant Lactococcus lactis{triangledown} ,{dagger}

Jun Mifune,1,{ddagger} Katrin Grage,2,{ddagger} and Bernd H. A. Rehm2*

Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-37 4259 Midori-ku, Nagatsuta, Yokohama 226-8501, Japan,1 Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand2

Received 26 February 2009/ Accepted 13 May 2009

Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisin-controlled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granule-associated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.

* Corresponding author. Mailing address: Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand. Phone: 64 6 350 5515, ext. 7890. Fax: 64 6 350 5688. E-mail: B.Rehm{at}massey.ac.nz

{triangledown} Published ahead of print on 22 May 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} These authors contributed equally to this work.

Applied and Environmental Microbiology, July 2009, p. 4668-4675, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00487-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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