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Improving the autotransporter-based surface display of enzymes in Pseudomonas putida KT2440.
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2019-05-02 , DOI: 10.1111/1751-7915.13419 Iasson E P Tozakidis 1 , Lena M Lüken 1 , Alina Üffing 1 , Annika Meyers 1 , Joachim Jose 1
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2019-05-02 , DOI: 10.1111/1751-7915.13419 Iasson E P Tozakidis 1 , Lena M Lüken 1 , Alina Üffing 1 , Annika Meyers 1 , Joachim Jose 1
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Pseudomonas putida can be used as a host for the autotransporter‐mediated surface display of enzymes (autodisplay), resulting in whole‐cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter‐mediated secretion depends on the N‐terminal signal peptide as well as on the C‐terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole‐cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12‐fold enhanced to 638 mU ml−1 OD−1 compared with the signal peptide of V. cholerae CtxB (52 mU ml−1 OD−1). This positive effect was confirmed with a β‐glucosidase as a second example enzyme. Here, cells expressing the protein with N‐terminal OprF signal peptide showed more than fourfold higher β‐glucosidase activity (181 mU ml−1 OD−1) than with the CtxB signal peptide (42 mU ml−1 OD−1). SDS‐PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.
中文翻译：
改善恶臭假单胞菌KT2440中基于自动转运蛋白的酶的表面展示。
恶臭假单胞菌可用作宿主的自动转运蛋白介导的酶表面展示（自动展示），从而在其细胞膜上产生具有重组功能的全细胞生物催化剂。自转运蛋白介导的分泌效率取决于自转运蛋白融合蛋白的N端信号肽和C端转运蛋白结构域。我们着手通过比较酯酶表面展示的不同信号肽和易位域，来优化恶臭假单胞菌作为宿主细菌的自动展示。易位域对全细胞催化剂的活性没有显着影响。相反，通过使用恶臭假单胞菌的信号肽与霍乱弧菌CtxB的信号肽（52 mU ml -1 OD -1）相比，外膜蛋白OprF的活性增强到638 mU ml -1 OD -1超过12倍。β-葡萄糖苷酶作为第二种酶实例证实了这种积极作用。在这里，表达具有N末端OprF信号肽的蛋白质的细胞显示的β-葡萄糖苷酶活性（181 mU ml -1 OD -1）比CtxB信号肽（42 mU ml -1 OD -1）高四倍以上）。SDS-PAGE和流式细胞仪分析表明，活性增加与外膜中重组蛋白的量增加以及细胞表面可检测到的酶数量增加有关。
更新日期：2019-05-02
中文翻译：
改善恶臭假单胞菌KT2440中基于自动转运蛋白的酶的表面展示。
恶臭假单胞菌可用作宿主的自动转运蛋白介导的酶表面展示（自动展示），从而在其细胞膜上产生具有重组功能的全细胞生物催化剂。自转运蛋白介导的分泌效率取决于自转运蛋白融合蛋白的N端信号肽和C端转运蛋白结构域。我们着手通过比较酯酶表面展示的不同信号肽和易位域，来优化恶臭假单胞菌作为宿主细菌的自动展示。易位域对全细胞催化剂的活性没有显着影响。相反，通过使用恶臭假单胞菌的信号肽与霍乱弧菌CtxB的信号肽（52 mU ml -1 OD -1）相比，外膜蛋白OprF的活性增强到638 mU ml -1 OD -1超过12倍。β-葡萄糖苷酶作为第二种酶实例证实了这种积极作用。在这里，表达具有N末端OprF信号肽的蛋白质的细胞显示的β-葡萄糖苷酶活性（181 mU ml -1 OD -1）比CtxB信号肽（42 mU ml -1 OD -1）高四倍以上）。SDS-PAGE和流式细胞仪分析表明，活性增加与外膜中重组蛋白的量增加以及细胞表面可检测到的酶数量增加有关。
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