Enhancing L-malate production of Aspergillus oryzae by nitrogen regulation strategy.

Regulating morphology engineering and fermentation of Aspergillus oryzae makes it possible to increase the titer of L-malate. However, the existing L-malate-producing strain has limited L-malate production capacity and the fermentation process is insufficiently mature, which cannot meet the needs of industrial L-malate production. To further increase the L-malate production capacity of A. oryzae, we screened out a mutant strain (FMME-S-38) that produced 79.8 g/L L-malate in 250-mL shake flasks, using a newly developed screening system based on colony morphology on the plate. We further compared the extracellular nitrogen (N1) and intracellular nitrogen (N2) contents of the control and mutant strain (FMME-S-38) to determine the relationship between the curve of nitrogen content (N1 and N2) and the L-malate titer. This correlation was then used to optimize the conditions for developing a novel nitrogen supply strategy (initial tryptone concentration of 6.5 g/L and feeding with 3 g/L tryptone at 24 h). Fermentation in a 7.5-L fermentor under the optimized conditions further increased the titer and productivity of L-malate to 143.3 g/L and 1.19 g/L/h, respectively, corresponding to 164.9 g/L and 1.14 g/L/h in a 30-L fermentor. This nitrogen regulation-based strategy cannot only enhance industrial-scale L-malate production but also has generalizability and the potential to increase the production of similar metabolites.Key Points• Construction of a new screening system based on colony morphology on the plate.• A novel nitrogen regulation strategy used to regulate the production of L-malate.• A nitrogen supply strategy used to maximize the production of L-malate.

Engineering growth phenotypes of Aspergillus oryzae for L-malate production.

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.