Relationship between ß-d-fructofuranosidase activity, fructooligosaccharides and pullulan biosynthesis in Aureobasidium melanogenum P16.

It has been thought that when different strains of Aureobasidium spp. were grown in sucrose, the produced fructooligosaccharides (FOSs) by ß-d-fructofuranosidase were beneficial for their cell growth and pullulan biosynthesis. However, it is still unknown about how ß-d-fructofuranosidases activity and synthesized FOSs influence on pullulan biosynthesis. It was found that the genomic DNA of Aureobasidium melanogenum P16, a high pullulan producing yeast, contained three genes encoding ß-d-fructofuranosidase1, ß-d-fructofuranosidase2 and ß-d-fructofuranosidase3. The FTR1 factor, a transcriptional activator, activated expression of the three ß-d-fructofuranosidase genes and invertase gene. Disruption of the FTR1 gene rendered a disruptant DF3 to produce less FOSs (12.1 ±â€¯0.4 g/L), less ß-d-fructofuranosidase activity (1.1 ±â€¯0.2 U/mL), lower Mw (3.8 × 105) of the pullulan and more pullulan titer (77.0 ±â€¯2.6 g/L) than the yeast strain P16. Similarly, removal of both the two genes encoding ß-d-fructofuranosidase1 and ß-d-fructofuranosidase3 resulted in a double mutant DF4-7 producing 77.5 ±â€¯3.1 g/L pullulan with Mw of 3.4 × 105, 0.2 ±â€¯0.0 U/mL of ß-d-fructofuranosidase activity and the trace amount of FOSs while its wild type strain P16 yielded 65.7 ±â€¯3.5 g/L pullulan with Mw of 4.4 × 105, 6.8 ±â€¯0.0 U/mL of ß-d-fructofuranosidase activity and 6.2 ±â€¯0.5 g/L of FOSs. These confirmed that high ß-d-fructofuranosidase activity, the presence of high level of FOSs negatively influenced pullulan biosynthesis, but positively increased Mw of the produced pullulan. However, the ß-d-fructofuranosidase2 had no such function. Furthermore, complementation of the FTR1 gene, ß-d-fructofuranosidase1 gene and ß-d-fructofuranosidase3 gene enabled the corresponding transformants to restore ß-d-fructofuranosidase activity, FOSs and pullulan biosynthesis and Mw of the pullulan.