Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material

Abstract

The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

Identification of a Classical Mutant in the Industrial HostAspergillus nigerby Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites

“…Owing to its low production of proteases, the A. niger D15 mutant has been used in various studies of the production of heterologous proteins (Gordon et al 2002; Rose and van Zyl 2002 ; Record et al 2003 ; Benoit et al 2007 ; Chimphango et al 2012 ; Turbe-Doan et al 2013 ; Benghazi et al 2014 ; Piumi et al 2014 ; Zwane et al 2014 ). The D15 strain does not only contain a mutation in the protease regulator gene ( prtT ) ( Punt et al 2008 ) but also a mutation leading to a nonacidifying phenotype and, consequently, low levels of acid-induced proteases in the medium.…”

Enrichment of maize and triticale bran with recombinant Aspergillus tubingensis ferulic acid esterase

“…MSA and MFA) as well as substrates with a hydroxyl group at the C4 position of the aromatic ring (e.g. MpCA), as reported for Aspergillus awamori (Koseki et al 2006) and Aspergillus niger (Faulds and Williamson 1993;Zwane et al 2014). The phenolic acids in maize bran, representing about 4-5% of the cell wall on a mass basis, are mostly ferulic acid and lower concentrations of p-coumaric acid and other phenolic acids esterified to the cell wall polymers (Lapierre et al 2001).…”

“…Of particular importance is that the crude A. niger D15[AtfaeA] extract was able to release additional phenolic acids from maize and triticale bran without any pre-treatment of the substrate, which would support the cost-effective extraction of ferulic acid from plant substrates. Enzyme production by the A. niger D15[AtfaeA] strain was significantly improved when cultured under either batch or fed-batch conditions, relative to shake flasks (Zwane et al 2014), with the glucose-limited conditions in the fed-batch fermentation yielding higher extracellular FAE activity (185.14 vs. 83.48 U/ml) and volumetric productivity (3.86 vs. 1.90 U/ml/h) than the batch fermentation.…”

“…The A. tubingensis AtfaeA gene was previously cloned and successfully expressed in A. niger D15#26 under the constitutive A. niger gpd promoter (Zwane et al 2014). In this study, the recombinant A. niger D15[AtfaeA] strain was cultured in shake-flasks with maize bran as sole carbohydrate source, followed by purification and characterisation of the enzyme.…”

“…A genomic copy of the Aspergillus tubingensis ferulic acid esterase Type A (faeA) gene was previously cloned and expressed in Aspergillus niger D15#26 under control of the A. niger gpdA promoter (Zwane et al 2014). The current study describes the evaluation of the crude A. niger D15[AtfaeA] extract for the release of phytochemicals from triticale and maize bran, as well as the upscaled production of the recombinant A. tubingensis AtFAEA in bioreactors.…”