Production of functionalized biopolyester granules by recombinant Lactococcus lactis

Abstract

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.

FIG. 1.

TEM analysis of L. lactis NZ9000 accumulating PHB and isolated PHB granules. (A) Cells of L. lactis NZ9000(pNZ-AB) not showing any PHB accumulation (negative control); (B) cells of L. lactis NZ9000(pNZ-CAB) filled with small PHB granules (diameter, 100 to 200 nm); (C) PHB granules isolated from L. lactis NZ9000(pNZ-CAB), demonstrating the retention of size and shape during the isolation process; (D) cells of L. lactis NZ9000(pNZ-ZZCAB).

FIG. 2.

ELISA demonstrating specific binding of IgG to ZZ domain-displaying beads isolated from L. lactis; ZZ beads L. lactis indicates beads isolated from L. lactis NZ9000(pNZ-ZZCAB) that produce ZZ-PhaC; control beads L. lactis indicates granules isolated from L. lactis NZ9000(pNZ-CAB) that produce PhaC only; and ZZ beads E. coli indicates granules isolated from E. coli producing ZZ-PhaC. Isolated PHB granules were bound to ELISA plates, and HRP-conjugated rabbit anti-mouse IgG was used to detect the functional display of the ZZ domain. o-Phenylenediamine was used as the substrate for HRP. L. lactis-derived wild-type granules not displaying the ZZ domain and ZZ domain-displaying granules from E. coli were used as negative and positive controls, respectively. The increasingly darker gray shading of the columns indicates decreasing amounts of granule protein used in the assays (5 μg, 2.5 μg, 1.25 μg, 625 ng, 312.5 ng, and 156.25 ng). Measurements were performed in triplicate, and the mean values and standard deviations are indicated.

FIG. 3.

SDS-PAGE analysis of human serum proteins bound in vitro to ZZ domain-displaying beads isolated from L. lactis or E. coli and released after elution, demonstrating IgG purification. Lane 1, human serum; lanes 2 and 6, molecular size standard; lane 3, proteins eluted at pH 2.7 from L. lactis ZZ beads; lane 4, proteins eluted at pH 2.7 from E. coli ZZ beads; and lane 5, proteins eluted at pH 2.7 from L. lactis control beads not displaying the ZZ domain. Beads were washed as described in Materials and Methods, incubated with human serum for 30 min, and washed again, and bound proteins were eluted with glycine, pH 2.7. The heavy (asterisk) and light chains (circle) of the strongly enriched Igs are indicated.