Blank Marshal, Schweiger Paul
Biology Department, Missouri State University, Springfield, MO, USA.
Department of Microbiology, University of Wisconsin-La Crosse, La Crosse, WI, USA.
PeerJ. 2018 Apr 6;6:e4626. doi: 10.7717/peerj.4626. eCollection 2018.
Acetic acid bacteria have unique metabolic characteristics that suit them for a variety of biotechnological applications. They possess an arsenal of membrane-bound dehydrogenases in the periplasmic space that are capable of regiospecific and enantioselective partial oxidations of sugars, alcohols, and polyols. The resulting products are deposited directly into the medium where they are easily recovered for use as pharmaceutical precursors, industrial chemicals, food additives, and consumer products. Expression of extracytoplasmic enzymes to augment the oxidative capabilities of acetic acid bacteria is desired but is challenging due to the already crowded inner membrane. To this end, an original surface display system was developed to express recombinant enzymes at the outer membrane of the model acetic acid bacterium . Outer membrane porin F (OprF) was used to deliver alkaline phosphatase (PhoA) to the cell surface. Constitutive high-strength p264 and moderate-strength p452 promoters were used to direct expression of the surface display system. This system was demonstrated for biocatalysis in whole-cell assays with the p264 promoter having a twofold increase in PhoA activity compared to the p452 promoter. Proteolytic cleavage of PhoA from the cell surface confirmed proper delivery to the outer membrane. Furthermore, a linker library was constructed to optimize surface display. A rigid (EAAAK) linker led to the greatest improvement, increasing PhoA activity by 69%. This surface display system could be used both to extend the capabilities of acetic acid bacteria in current biotechnological processes, and to broaden the potential of these microbes in the production of value-added products.
醋酸菌具有独特的代谢特性，使其适用于多种生物技术应用。它们在周质空间拥有一系列膜结合脱氢酶，能够对糖类、醇类和多元醇进行区域特异性和对映选择性部分氧化。生成的产物直接沉积到培养基中，在那里很容易回收，用作药物前体、工业化学品、食品添加剂和消费品。由于内膜已经很拥挤，期望表达胞外酶来增强醋酸菌的氧化能力具有挑战性。为此，开发了一种原始的表面展示系统，用于在模式醋酸菌的外膜上表达重组酶。外膜孔蛋白F（OprF）被用于将碱性磷酸酶（PhoA）递送到细胞表面。组成型高强度p264和中等强度p452启动子被用于指导表面展示系统的表达。在全细胞测定中证明了该系统的生物催化作用，与p452启动子相比，p264启动子的PhoA活性提高了两倍。从细胞表面蛋白水解切割PhoA证实其正确递送到了外膜。此外，构建了一个接头文库以优化表面展示。一个刚性（EAAAK）接头带来了最大的改进，使PhoA活性提高了69%。这种表面展示系统既可以用于扩展醋酸菌在当前生物技术过程中的能力，也可以拓宽这些微生物在生产增值产品方面的潜力。
