CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 26601, China.
CAS Key Laboratory of Animal Models and Human Disease Mechanisms / Key Laboratory of Bioactive Peptides of Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, China.
Enzyme Microb Technol. 2020 Apr;135:109498. doi: 10.1016/j.enzmictec.2019.109498. Epub 2019 Dec 20.
Whole-cell biocatalysts could be used in wide-ranging applications. In this study, a new kind of whole-cell biocatalyst was successfully constructed by genetically immobilizing soybean seed coat peroxidase (SBP) on the cell surface of Yarrowia lipolytica Po1h, using a new integrative surface display expression vector (pMIZY05). The coding sequence of SBP was optimized and chemically synthesized, then inserted into pMIZY05 to generate expression plasmid pMIZY05-oEp. A DNA fragment corresponding to SBP and selection marker expression cassettes, without bacterial sequences, was released from pMIZY05-oEp by enzyme digestion and used to transform host yeast cells. A transformant (CM11) with a high recombinant SBP activity of 1571.9 U/mL was obtained, and recombinant SBP was proved to be successfully anchored on cell surface by testing the activities of different cellular fractions. After optimization of culture conditions, the recombinant SBP activity of CM11 was increased to 4187.8 U/mL. Afterwards, biochemical properties of the recombinant SBP were determined: optimum catalytic conditions were 37.5℃ at pH 3.5, and recombinant SBP exhibited high stability during thermal or acidic treatment. Recombinant activity of cell-displayed SBP was re-examined at optimum temperature and pH, which promoted an increase up to 4432.5 U/mL. To our knowledge, this represents the highest activity ever reported for heterologous expression of SBP. This study also provides a useful strategy for heterologous expression of proteins which could be toxic to intracellular content of host cells.
全细胞生物催化剂可应用于广泛的领域。在本研究中，通过使用新型整合表面展示表达载体（pMIZY05），将大豆种皮过氧化物酶（SBP）基因固定在解脂耶氏酵母（Yarrowia lipolytica Po1h）细胞表面，成功构建了一种新型的全细胞生物催化剂。优化并化学合成 SBP 的编码序列，然后将其插入 pMIZY05 中，生成表达质粒 pMIZY05-oEp。通过酶切从 pMIZY05-oEp 中释放出与 SBP 和选择标记表达盒相对应的 DNA 片段，该片段不含细菌序列，并用于转化宿主酵母细胞。通过测试不同细胞级分的活性，获得了具有 1571.9 U/mL 高重组 SBP 活性的转化子（CM11），并证明重组 SBP 成功锚定在细胞表面。通过优化培养条件，将 CM11 的重组 SBP 活性提高到 4187.8 U/mL。随后，测定了重组 SBP 的生化特性：最适催化条件为 37.5℃、pH3.5，并且在热或酸性处理下具有很高的稳定性。在最适温度和 pH 下重新检测细胞展示的 SBP 的重组活性，其活性提高了 4432.5 U/mL。据我们所知，这是 SBP 异源表达的最高活性。本研究还为表达对宿主细胞内容物可能有毒性的蛋白质提供了一种有用的策略。
