Löwe Hannes, Schmauder Lukas, Hobmeier Karina, Kremling Andreas, Pflüger-Grau Katharina
Fachgebiet für Systembiotechnologie, Fakultät für Maschienenwesen, Technische Universität München, Garching, Germany.
Microbiologyopen. 2017 Aug;6(4). doi: 10.1002/mbo3.473. Epub 2017 Mar 27.
Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose.
蔗糖是一种重要的二糖，在许多工业应用中用作底物。它是糖蜜的主要成分，糖蜜是制糖工业的一种廉价副产品。不幸的是，并非所有与工业相关的生物，包括恶臭假单胞菌，都能够代谢蔗糖。我们选择了一种代谢工程方法来绕过这一障碍，并使恶臭假单胞菌具备消耗蔗糖所需的活性。因此，我们构建了一对广宿主范围的微型转座子（pSST - 蔗糖分解转座子），其中一个携带编码能够将蔗糖分解为葡萄糖和果糖的转化酶的cscA，另一个额外携带编码蔗糖通透酶的cscB。引入cscA足以实现蔗糖消耗，尽管构建了蔗糖通透酶并使其定位于膜上，但cscB的额外存在并没有进一步影响。蔗糖通过从细胞中泄漏出来的转化酶CscA的活性在细胞外被分解。这些转座子还被用于赋予食酸铜绿假单胞菌蔗糖消耗能力。有趣的是，在这种菌株中，CscB起到了葡萄糖转运蛋白的作用，使得食酸铜绿假单胞菌也获得了在葡萄糖上生长的能力。因此，pSST转座子是将革兰氏阴性细菌菌株的底物谱扩展到蔗糖的功能性工具。
