Enhanced Production of Bovine Chymosin by Autophagy Deficiency in the Filamentous Fungus Aspergillus oryzae

Abstract

Aspergillus oryzae has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, A. oryzae often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production. In the present study, we examined the production of heterologous protein in several autophagy (Aoatg) gene disruptants of A. oryzae. We transformed A. oryzae gene disruptants of Aoatg1, Aoatg13, Aoatg4, Aoatg8, or Aoatg15, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the Aoatg gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed Aoatg gene-conditional expression strains in which the promoter region of the Aoatg gene was replaced with the thiamine-controllable thiA promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the Aoatg gene disruptants. Consequently, we succeeded in the construction of A. oryzae strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing heterologous protein production in A. oryzae.

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

“…In particular, reducing protease activity is necessary to limit the degradation of heterologous proteins, as was demonstrated by the 3-fold increase in the level of heterologous proteins in the culture supernatant of an Aspergillus oryzae strain with 10 protease genes deleted (2). Heterologous protein production by A. oryzae was also effectively improved by the repression of vacuolar protein sorting and autophagy (3,4). The genetic fusion of a target protein with an endogenous protein carrier is a commonly used strategy to increase heterologous protein yields in filamentous fungi.…”

Stress Biology of Yeasts and Fungi

“…Advances in molecular genetic techniques for generating multiple gene disruptions (Maruyama and Kitamoto 2008 ;Maruyama and Kitamoto 2011 ) have facilitated the molecular breeding of hyperproducing A. oryzae host strains (Yoon et al 2009 ;Yoon et al 2011 ;Zhu et al 2013 ). As recently reported, inhibition of vacuolar degradation machineries, such as vacuolar protein sorting and autophagy, enhanced the ability of heterologous protein production in A. oryzae (Yoon et al 2010 ;Yoon et al 2013 ). This fi nding indicates that cell biology-based approaches are important for optimizing the potential of A. oryzae as a host for industrial protein production.…”

Autophagy is dispensable to overcome ER stress in the filamentous fungus Aspergillus niger

“…Increased heterologous protein production has been reported upon the overexpression of genes encoding cellular foldases and chaperones in several Aspergillus species (reviewed by Heimel ) and upon the deletion of some autophagy genes in A. oryzae (Yoon et al. ). Deletion of ERAD components derA and hrdC in A. niger resulted in an increase in the intracellular accumulation of the heterologous Gla::Gus fusion protein, but had no severe effect on the growth phenotype (Carvalho et al.…”

“…Besides the extracellular activity of efficiently produced secreted proteases (van den Hombergh et al 1997;Punt et al 2008;Braaksma et al 2009;Yoon et al 2009Yoon et al , 2011, low product yields can also be the result of intracellular degradation of the protein, as a consequence of the strict quality control in the ER which eventually eliminates proteins that fail proper folding or processing (Jacobs et al 2009). Increased heterologous protein production has been reported upon the overexpression of genes encoding cellular foldases and chaperones in several Aspergillus species (reviewed by Heimel 2015) and upon the deletion of some autophagy genes in A. oryzae (Yoon et al 2013). Deletion of ERAD components derA and hrdC in A. niger resulted in an increase in the intracellular accumulation of the heterologous Gla::Gus fusion protein, but had no severe effect on the growth phenotype (Carvalho et al 2011a).…”