Heterologous Expression and Optimized Production of an Aspergillus aculeatus Endo-1,4-β-mannanase in Yarrowia lipolytica

Abstract

The Aspergillus aculeatus MRC11624 man1 gene, encoding an endo-beta-1,4-mannanase, was cloned and expressed in the promising heterologous enzyme producer, the ascomycetous yeast Yarrowia lipolytica. Both single- and multi-copy transformants were constructed, and the secretion of the enzyme was evaluated as an in-frame fusion with the LIP2 secretion signal, as well as with its natural secretion signal. In shake-flask analysis, the highest volumetric enzyme activity (13,073 nkat/ml) and specific enzyme activity (1,020 nkat/(mg dcw)) were obtained with a multi-copy integrant utilizing beta-mannanase's own secretion signal. The best beta-mannanase-producing strain was subsequently evaluated in batch fermentation and resulted in a maximum volumetric enzyme activity of 6,719 nkat/ml. Fed batch fermentations resulted in a 3.9-fold increase in volumetric enzyme activity compared with batch fermentation, and a maximum titre of 26,139 nkat/ml was obtained. The results reported in this study indicate that Y. lipolytica is a promising producer of A. aculeatus beta-mannanase, producing higher beta-mannanase activity than that of recombinant Saccharomyces cerevisiae or Aspergillus niger when cultivated in shake flasks, which is encouraging for the use of the enzyme in industrial processes such as extraction of vegetable oil from leguminous seeds and the reduction in viscosity of coffee extracts.