Relationship between β-d-fructofuranosidase activity, fructooligosaccharides and pullulan biosynthesis in...

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