A Convenient Fluorescence-Based Assay for the Detection of Sucrose Transport and the Introduction of a Sucrose Transporter from Potato into Clostridium Strains

Abstract

A convenient and effective sucrose transport assay for Clostridium strains is needed. Traditional methods, such as 14C-sucrose isotope labelling, use radioactive materials and are not convenient for many laboratories. Here, a sucrose transporter from potato was introduced into Clostridium, and a fluorescence assay based on esculin was used for the analysis of sucrose transport in Clostridium strains. This showed that the heterologously expressed potato sucrose transporter is functional in Clostridium. Recombinant engineering of high-level sucrose transport would aid sucrose fermentation in Clostridium strains. The assay described herein provides an important technological platform for studying sucrose transporter function following heterologous expression in Clostridium.

Enhanced sucrose fermentation by introduction of heterologous sucrose transporter and invertase into Clostridium beijerinckii for acetone–butanol–ethanol production

“…Open Sci. 8: 201858 competes with esculin for transport into recombinant strains [22]. The fermentation results in the present study confirmed that StSUT1 induced sucrose transport in C. beijerinckii.…”

“…The StSUT1 gene, encoding a sucrose transporter from potato, was expressed in C. beijerinckii . The sucrose transport activity in the engineered strains was assayed using a previously described method [ 22 ], in which highly fluorescent esculin, a structural analogue of sucrose, was used as a probe to characterize sucrose transport activity [ 29 , 30 ]. Strain 13-4 was wild-type strain screened from molasses medium.…”

“…From our previous study and the results shown in figure 2 , it was evident that StSUT1 induced high esculin transfer ability in C. beijerinckii and C . acetobutylicum strains [ 22 ]. Previous sucrose competition experiments demonstrated that sucrose competes with esculin for transport into recombinant strains [ 22 ].…”

“…Escherichia coli strain JM109 (Takara Bio Inc., Dalian, China) was used for routine DNA cloning and was cultivated in Luria–Bertani (LB) broth or on LB agar supplemented with antibiotics as needed. Clostridium beijerinckii strain 13-4 and derived strains were cultivated anaerobically at 30°C in tryptone yeast extract acetate (TYA) medium [ 22 ] or on TYA agar, and 50 µg ml −1 erythromycin was added to the medium when required.…”

“…A sucrose transporter from potato ( Solanum tuberosum ) was previously introduced into a Clostridium strain, which transported a sucrose analogue into cells [ 22 ]. The aim of the present study was to explore whether this heterologous sucrose transporter could induce a sucrose transport function, thereby enhancing sucrose fermentation by Clostridium for n -butanol production.…”