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.

Fungal β-mannanases: Mannan hydrolysis, heterologous production and biotechnological applications

“…Several examples of ␤-mannanase production in excess of 1 g/l through heterologous expression in fungal systems have been reported (see Table 1), both for the expression of fungal genes [66][67][68] and for expression of a bacterial B. subtilis ␤-mannanase in Pichia pastoris [69]. Expression of the Aspergillus aculeatus man1 gene in Yarrowia lipolytica and cultivation of the recombinant strain in fed batch culture has resulted in 5.9 g/l enzyme (1575 U/ml), the highest levels reported to date for a fungal or bacterial ␤-mannanase.…”

“…Expression of the Aspergillus aculeatus man1 gene in Yarrowia lipolytica and cultivation of the recombinant strain in fed batch culture has resulted in 5.9 g/l enzyme (1575 U/ml), the highest levels reported to date for a fungal or bacterial ␤-mannanase. Such production levels are partly due to high biomass densities attainable with the Y. lipolytica yeast expression system, which is a considerable advantage compared to the use of filamentous fungi such as A. niger [67,68]. ␤-Mannanase with a purity of up to 97% in the culture supernatant has been obtained by heterologous expression in P. pastoris [70], requiring little or no further purification prior to industrial application.…”

Cloning and bioinformatics analysis of a novel acidophilic β-mannanase gene, Auman5A, from Aspergillus usamii YL-01-78

“…P13 (Zhao et al 2010), Bispora sp. MEY-1 (Luo et al 2009), A. sulphureus MAFIC001 (Chen et al 2007), A. niger BK01 (Cuong et al 2009), A. aculeatus MRC11624 (Roth et al 2009) and Bacillus sp. JAMB-750 (Hatada et al 2005).…”

Secretory expression of organophosphorus hydrolase OPHC2 inYarrowia lipolyticaPolg

“…[5] Recombinant protein expression levels in the Y. lipolytica system can also be improved using highcell-density fermentation. Roth et al [28] reported that the enzyme activity of an Aspergillus aculeatus endo-1, 4-bmannanase obtained in Braun C 15 L fermenters was increased by 100% compared to a shake-flask trial. Additionally, Nicaud et al [29] reported a 7.9-fold increase in volumetric lipase activity when produced in fed-batch fermentation compared to batch fermentation.…”