Mifune Jun, Grage Katrin, Rehm Bernd H A
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-37 4259 Midori-ku, Nagatsuta, Yokohama 226-8501, Japan.
Appl Environ Microbiol. 2009 Jul;75(14):4668-75. doi: 10.1128/AEM.00487-09. Epub 2009 May 22.
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.
许多细菌天然能够积累由3-羟基脂肪酸组成的生物聚酯作为细胞内包涵体，这些包涵体充当储存颗粒。最近，这些包涵体被认为是表面附着有蛋白质的纳米/微珠，可以对其进行工程改造以展示各种基于蛋白质的功能，适用于生物技术和生物医学应用。在本研究中，对食品级、一般认为安全的革兰氏阳性菌乳酸乳球菌进行工程改造，使其重组生产生物聚酯聚（3-羟基丁酸酯）及相应的细胞内包涵体。使用乳酸链球菌素控制的基因表达系统表达经密码子优化的来自贪铜菌的聚羟基丁酸酯生物合成操纵子phaCAB。重组乳酸乳球菌积累的聚（3-羟基丁酸酯）可达细胞干重的6%（重量/重量）。分离出聚（3-羟基丁酸酯）颗粒，使用生化方法分析其结合蛋白，并使用透射电子显微镜分析其形状/大小。选择金黄色葡萄球菌蛋白A的免疫球蛋白G（IgG）结合ZZ结构域作为示例性功能，通过将其融合到颗粒相关的聚（3-羟基丁酸酯）合酶的N末端，使其展示在颗粒表面。使用基质辅助激光解吸电离飞行时间（质谱）通过肽指纹图谱确认分离颗粒表面存在融合蛋白。通过酶联免疫吸附测定和IgG亲和纯化证明了展示ZZ结构域的颗粒的功能。在这两种测定中，重组乳酸乳球菌的ZZ珠表现至少与大肠杆菌的ZZ珠相当。总体而言，本研究表明重组乳酸乳球菌可用于制造无内毒素的聚（3-羟基丁酸酯）珠，其表面功能适用于生物医学应用。
