Description:
<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>Pseudomonas putida,</jats:italic> are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip <jats:italic>P. putida</jats:italic> with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad‐host range mini‐transposons (p<jats:styled-content style="fixed-case">SST</jats:styled-content> – <jats:styled-content>s</jats:styled-content>ucrose <jats:styled-content>s</jats:styled-content>plitting <jats:styled-content>t</jats:styled-content>ransposon), carrying either <jats:italic>cscA</jats:italic>, encoding an invertase able to split sucrose into glucose and fructose, or additionally <jats:italic>cscB</jats:italic>, encoding a sucrose permease. Introduction of <jats:italic>cscA</jats:italic> was sufficient to convey sucrose consumption and the additional presence of <jats:italic>cscB</jats:italic> 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 <jats:italic>Cupriavidus necator</jats:italic>. Interestingly, in this strain, CscB acted as a glucose transporter, such that <jats:italic>C. necator</jats:italic> also gained the ability to grow on glucose. Thus, the p<jats:styled-content style="fixed-case">SST</jats:styled-content> transposons are functional tools to extend the substrate spectrum of Gram‐negative bacterial strains toward sucrose.</jats:p>