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

Abstract

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.

Keywords: Aureobasidium melanogenum; Fructooligosaccharides; Pullulan; Transcriptional activator; β-d-fructofuranosidase.