In-lab produced maltodextrin
Maltodextrins are linear polymers of D-glucose linked by α1-4 glycosidic bonds ranging from degree of polymerization (DP) 2 to 20. They are metabolizable by E. coli but can’t diffuse innately through bacterial membranes if their DP is higher than 3. These two properties were of paramount importance in the first step of our selection process. Indeed, maltodextrins, as the sole carbon source in minimal medium, were only accessible by mutants expressing an ˝open˝ porin phenotype. We particularly focused on obtaining maltodextrins of DP 5, ˝maltopentaose˝ because it best combined selectivity and rapid cell growth in comparison to other oligomers (Marciano et al., 1999 ; Spagnuolo et al., 2010).
Figure 1 : Growth differences exhibited by E. coli expressing wt PIV porin and PIVS324G leaky porin, on different maltodextrins after 36h of incubation at 37°C (Marciano et al. 1999)
Nonetheless, we quickly faced a determining obstacle: industrial maltopentaose is extremely expensive, easily reaching 1500€/g, and large amounts of it were required. It was inconceivable to allow our whole budget to buy a simple substrate, so we looked for a way to produce it by ourselves. We managed to achieve this by precipitating corn syrup (kindly given by our sponsor Cargill) in ethanol. Another scientific paper (Balto et al., 2015) helped us to establish an efficient purification protocol
Corn syrup contains D-glucose and maltodextrins from DP 2 to more than 9. Our objective here was to remove glucose, maltose and triose, which can diffuse through bacterial membranes, and ideally, fractions of DP higher than 6 that are unable to pass through our porin. This way, we could use the corn syrup fraction of DP 4 to 6, which is assumed to be close enough to industrial hexamaltose composition to achieve our selection goal.
In a few words, we proceeded by selecting the corn syrup fraction which was insoluble in 90% ethanol, but also soluble in 70% ethanol. This fraction was obtained by following a sequence of 5 operations, repeated 4 times:
- Dissolving corn syrup in water
- Adding ethanol to reach the desired concentration (three times 90% and once 70%)
- Stirring
- Decantation
- Retrieving the liquid or solid phase, depending on the stage of the protocol using a centrifuge.
After the final retrieving, we had to dry our product. To proceed, we needed a freeze-dryer, device which is not in possession of the BBGM lab. We asked Professor Benjamin Elias, who runs another lab of the university, if we could use their own, under supervision, what he accepted. Thanks to this support, we could dry our “homemade pentamaltose” as we called it and continue our experiments.
Before using this maltopentaose in our experiments, we first had to test each of the produced batches to be sure only our positive control, with the leaky porin phenotype, ˝pIV*˝ could grow in its presence. To do so, we developed a very simple test: we prepared seven petri dishes with our M63 medium. The plan of the test was as follows:
- 1 dish received nothing (control dish)
- 1 dish received homemade maltodextrin and was inoculated with pIV phenotype
- 1 dish received industrial maltodextrin and was inoculated with pIV phenotype
- 1 dish received homemade maltodextrin and was inoculated with pIV* phenotype
- 1 dish received industrial maltodextrin and was inoculated with pIV* phenotype
- 1 dish just received industrial maltodextrin
- 1 dish just received homemade maltodextrin
After an overnight incubation at 37°C, the seven dishes were checked and the batch was accepted if only the 2 dishes with incoulated pIV* expressing bacteria showed colonies and only them. The 5 others had to stay blank.
Even though this quick test was regarded as enough to pursue our experiments, we wanted to know the exact composition of our homemade product. So we sent samples to our Cargill contact who made chromatography analysis of them. The results are shown in the chromatograms at figures 2, 3 and 4:
Figure 2 : Corn Syrup chromatography analysis
Figure 3 : in-lab produced maltodextrin chromatography analysis
Figure 4 : Industrial hexamaltaose chromatography analysis
First, we can see that the industrial maltopentaose is closer to hexamaltose than maltopentaose. The homemade one is particularly rich in tetramaltose. By comparing homemade maltopentaose to corn syrup, we can assert that the purification we conducted is an enrichment in tetramaltose. We can also see that there are still some DP 2 and 3 in our homemade product. This could be a problem regarding our selection goal, but we observed that petri dishes with maltopentaose (both kinds) never showed growth of our negative control with the wt pIV. We concluded that these DP 2 and 3 were not in high enough concentrations to allow growth of false positives on our selection media. Traces of DP 2 and 3 might even have helped our bacteria to implant on their medium before facing restrictive media with low resources concentrations. Considering these results, we judged this step of maltopentaose production by ourselves as a success.
Bibliography
Balto, A., Lapis, T.J., Silver, R.K., Ferreira, A.J., Beaudry, C.M., Lim, J., Penner, M.H. (2016). On the use of differential solubility in aqueous ethanol solutions to narrow the DP range of food-grade starch hydrolysis products. Food Chemistry, 197, 872-880. dx.doi.org/10.1016/j.foodchem.2015.10.120.
Marciano, Denise K., Marjorie Russel, et Sanford M. Simon. 1999. « An Aqueous Channel for Filamentous Phage Export ». Science 284 (5419): 1516‑19. doi:10.1126/science.284.5419.1516.
Spagnuolo, J., Opalka, N., Wen, W. X., Gagic, D., Chabaud, E., Bellini, P., Rakonjac, J. (2010). Identification of the gate regions in the primary structure of the secretin pIV. Molecular Microbiology, 76(1), 133–150. doi:10.1111/j.1365-2958.2010.07085.x
