Understanding Saccharomyces cerevisiae invertase activity and specificity in the context of breadmaking

Promovendus/a: Jitka Laurent

Promotor(en): Prof. dr. ir. Christophe Courtin , Prof. dr. ir. Kevin Verstrepen

Saccharomyces cerevisiae invertase (ScInv) is a β-fructofuranosidase encoded by different, closely related SUC genes and classified within the family 32 of glycoside hydrolases. ScInv prefers sucrose as a substrate but also hydrolyses raffinose, fructo-oligosaccharide (FOS) and short-chain fructans. Invertase produced by baker’s yeast plays and important role during breadmaking. It is responsible for setting free those sugars that yield most of the CO2 produced during the first stages of fermentation. Despite its importance during breadmaking and several other industrial processes, little is known about the variability in ScInv activity and specificity and the factors determining this variability. Hence, the main goal of this research was to gain more insight into the activity and specificity of ScInv as it is present in yeast, and not just as an isolated enzyme. In the end, these insights can be used to develop yeast-based strategies to modulate fructan hydrolysis during breadmaking.

A set of 28 S. cerevisiae strains was investigated for invertase activity and specificity. The strains displayed high variability in invertase activity towards sucrose and FOS, while the variability in FOS specificity was less pronounced. A comparison of 29 different SUC alleles showed a sequence similarity from 89 to 100%. The phylogenetic tree showed three distinct clades of SUC alleles, indicating a certain variability in SUC gene sequences. Further experiments showed that minor sequence differences impacted yeast invertase activity and specificity towards sucrose, FOS and MCI. Other factors that can influence ScInv activity and specificity were also investigated. A clear correlation between invertase activity and ploidy, or invertase activity and the amount of SUC genes was not observed. However, the impact of the industrial provenance of the strains was more pronounced. ScInv invertase activity increases with an increasing temperature, and an optimum was observed around 60°C. Furthermore, higher incubation temperatures resulted in a higher specificity towards FOS, probably due to higher molecular mobility in the enzyme.

Using the variability in yeast invertase activity and specificity seemed to be a simple yet effective mean to modulate fructan hydrolysis during breadmaking. Strains with high invertase activities towards FOS hydrolysed wheat grain fructans faster and to a greater extent, resulting in a lower final fructan level and thus a lower FODMAP level in bread. Strains with a lower invertase activity could increase the fermentable dietary fibre levels in bread by retaining more fructan during fermentation. The selection of yeast strains for the modulation of fructan hydrolysis during fermentation can also be made based on SUC gene sequence. Indeed, the sequences of yeast SUC genes influence their ability to hydrolyse fructan and sucrose during dough fermentation. These differences in fructan and sucrose hydrolysis could be related to the differences in invertase activity and specificity and to differences in the capacity to hydrolyse FOS and mixed-chain inulin. Another approach is to use these insights to make small adaptations to the SUC gene sequence of a commonly used baker’s yeast to improve or reduce its fructan hydrolysing capacity. Using this approach, the same yeast strain can be used, and the impact on CO2 production, bread texture and flavour is limited.

In a final part, the activity and specificity of Kluyveromyces marxianus inulinase (KmInu) was compared to those of ScInv. The fructosidase activity towards sucrose of the strains expressing INU1 was lower compared to strains expressing SUC alleles. In comparison both fructosidase activity and specificity on FOS and mixed- and long-chain inulin were higher for strains expressing INU1. Furthermore, it was shown that the longer the substrate, the bigger the share of the secreted dimeric enzyme to the total fructosidase activity. Moreover, this free enzyme showed a higher specificity towards longer substrates, which can be explained by its higher accessibility for these substrates, as there is no cell wall barrier. Finally, wheat dough fermentation experiments showed that expressing an INU1 allele in S. cerevisiae might be a good alternative for the use of K. marxianus during breadmaking.

In summary, this PhD study provided more insight into the molecular basis for natural differences in invertase substrate specificity between S. cerevisiae strains and other factors influencing invertase activity and specificity. Hence, it opens the door for the selection or engineering of yeasts and Suc-enzymes with specific activities that allow controlling fructan hydrolysis. New yeast-based strategies to modulate fructan hydrolysis during dough fermentation were developed.