Engineering growth phenotypes of Aspergillus oryzae for L-malate production

Abstract

Improving the growth status of Aspergillus oryzae is an efficient way to enhance L-malate production. However, the growth mechanism of filamentous fungi is relatively complex, which limits A. oryzae as a cell factory to produce L-malate industrially. This study determined the relationship between growth status and L-malate production. The optimal ranges of colony diameter, percentage of vegetative mycelia, and pellet number of A. oryzae were determined to be 26-30 mm, 35-40%, and 220-240/mL, respectively. To achieve this optimum range, adaptive evolution was used to obtain the evolved strain Z07 with 132.54 g/L L-malate and a productivity of 1.1 g/L/h. Finally, a combination of transcriptome analysis and morphological characterization was used to identify the relevant pathway genes that affect the growth mechanism of A. oryzae. The strategies used in this study and the growth mechanism provide a good basis for efficient L-malate production by filamentous fungi.

Keywords: Aspergillus oryzae; Adaptive evolution; Growth mechanism; Growth phenotypes; L-malate.

Fig. 1

Effect of the A. oryzae growth status on L-malate production. a The mutant library was constructed by ARTP irradiation and LiCl mutagenesis. b Images of the colonies (a1–a5), compactness (b1–b5), percentage of vegetative mycelia (PVM) of colonies(c1–c5) and pellet number (d1–d5) of the five mutants. c The relationship between the colony diameter and L-malate titer meet $Y=-0.15X^2+7.06X-10.72\left(R^2=0.95\right)$. d The relationship between PVM and L-malate titer meet $Y=-0.05X^2+4.92X-53.89\left(R^2=0.94\right)$. e The relationship between pellet number and L-malate titer meet $Y=-0.002X^2+0.82X-12.94\left(R^2=0.97\right)$

Fig. 2

Assessment of the adaptive evolution of Aspergillus oryzae strains. a Inhibition of Aspergillus oryzae by different concentrations of 2-PE. b Influence of different concentrations of 2-PE on changes in mycelial dry weight and spores germination rate. c Images of colony morphology, compactness and PVM values during the evolution of strain Z05, Z06 and Z07. d-f Growth parameters: colony diameter, PVM value and pellet number of strain Z05, Z06 and Z07. g-i Fermentation performance parameters: L-malate titer, DCW and glucose consumption of strain Z05, Z06 and Z07 in the 7.5-L fermenter. All data are presented as mean values from three independent experiments. Error bars indicate the standard deviations

Fig. 3

Transcriptome analysis of strain Z05, Z06 and Z07. a1–a3 Differently expressed genes in strain Z05, Z06 and Z07. b Venn diagrams depicting the numbers of commonly regulated genes in strain Z05, Z06 and Z07. c Statistical analysis of the metabolic pathways in which the identified significant differentially expressed genes in strain Z05, Z06 and Z07 are involved

Fig. 4

Transcriptome analysis and morphological characterization to clarify the mechanism of hyphal branching formation in Aspergillus oryzae. a Diagram of the mechanism of hyphal branching formation. b Heat map of the hyphal branching formation mechanism following comparison between the strain Z06 and Z07. c The morphological characteristics of hyphal branching of the strain Z05, Z06 and Z07 under light microscopy. d Mycelial dry weight of the strain Z05, Z06 and Z07. All data are presented as mean values from three independent experiments. Error bars indicate the standard deviations

Fig. 5

Transcriptome analysis and morphological characterization to clarify the mechanism of conidiophore formation in Aspergillus oryzae. a Diagram of the mechanism of conidiophore formation. b Heat map of the conidiophore formation mechanism following comparison between the strain Z06 and Z07. c The morphological characteristics of conidiophore of the strain Z05, Z06 and Z07 under scanning electron microscope. d Spore concentration of the strain Z05, Z06 and Z07. All data are presented as mean values from three independent experiments. Error bars indicate the standard deviations

Fig. 6

Transcriptome analysis and morphological characterization to clarify the mechanism of mycelial pellets formation in Aspergillus oryzae. a Diagram of the mechanism of mycelial pellets. b Heat map of the mycelial pellets formation mechanism following comparison between strain Z06 and Z07. c The morphological characteristics of mycelial pellets of the strain Z05, Z06 and Z07 under scanning electron microscope. d Mycelial coverage rate of strain Z05, Z06 and Z07. All data are presented as mean values from three independent experiments. Error bars indicate the standard deviations