Abstract
The plant storage carbohydrate inulin represents an attractive biomass feedstock for fueling industrial scale bioconversion processes due to its low cost, ability for cultivation on arid and semi-arid lands, and amenability to consolidated bioprocessing applications. As a result, increasing efforts are emerging towards engineering industrially relevant microorganisms, such as yeast, to efficiently ferment inulin into high value fuels and chemicals. Although some strains of the industrially relevant yeast model Saccharomyces cerevisiae can naturally ferment inulin, the efficiency of this process is often supplemented through expression of exogenous inulinase enzymes that externally convert inulin into its more easily fermentable component monomeric sugars. Here, the effects of overexpressing the Aspergillus niger InuA inulinase enzyme in an S. cerevisiae strain incapable of endogenously fermenting inulin were evaluated to determine their impact on growth. Expression of the A. niger InuA inulinase enzyme permitted growth on otherwise intractable inulin substrates from both Dahlia tubers and Chicory root. Despite being in the top 10 secreted proteins, growth on inulin was not observed until 120 h post-inoculation and required the addition of 0.1 g fructose/l to initiate enzyme production in the absence of endogenous inulinase activity. High temperature/pressure pre-treatment of inulin prior to fermentation decreased this time to 24 h and removed the need for fructose addition. The pre-growth lag time on untreated inulin was attributed primarily to low enzymatic efficiency, with a maximum value of 0.13 ± 0.02 U InuA/ml observed prior to the peak culture density of 2.65 ± 0.03 g/l. However, a minimum excreted enzymatic activity level of only 0.03 U InuA/ml was found to be required for sustained growth under laboratory conditions, suggesting that future metabolic engineering strategies can likely redirect carbon flow away from inulinase production and reorient it towards product production or cellular growth in order to optimize strain development.