Metabolic engineering to expand the substrate spectrum of Pseudomonas putida toward sucrose. Issue 4 (27th March 2017)

• Record Type: Journal Article
• Authors: Löwe, Hannes; Schmauder, Lukas; Hobmeier, Karina; Kremling, Andreas; Pflüger‐Grau, Katharina
• Is Part Of: MicrobiologyOpen. Volume 6:Issue 4(2017:Aug.)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2017-03-27
• Subjects: metabolic engineering -- Pseudomonas putida -- sucrose metabolism; Microbiology -- Periodicals; 579
• DOI: 10.1002/mbo3.473
• Languages: English
• ISSNs: 2045-8827

Abstract

Abstract: 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 –s ucroses plittingt ransposon), 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. Abstract : Sucrose is an important disaccharide used as substrate in many industrial applications. To expand the substrate spectrum of Pseudomonas putida toward this substrate, the pSST transposons were constructed, which were used to successfully add sucrose consumption not only to P. putida but also to other Gram‐negative bacteria such as Cupriavidus necator . The functionality of the added activities (uptake and hydrolysis of sucrose) was analyzed and promising targets for further optimization of the process were identified.