The discovery of the AMA1 sequence derived from Aspergillus nidulans was the only element reported that could replicate autonomously in Aspergillus, and its discovery enabled the episomal expression of plasmids in filamentous fungi. The efficient utilization of this sequence in the plasmid overcame the metabolic burden of mutation strains, caused by the integration of the overexpressed gene into the genome, especially when the Crispr-Cas9 system was employed, the continuous expression of Cas9 was unnecessary. The plasmid pAN7-1-sorB constructed by Chen (Chen and Chu 2019) contains multiple PgpdA promoters, which may affect the integration process and the stability of the integrated fragment in the genome. In addition, we also tried to replace the Cas9 expression cassette and hygromycin expression cassette in pFC332 with the original parts in pAN7-1-sorB to construct a new integrated expression plasmid, but its point mutation efficiency was far less than that of this newly constructed system.
PCR validation on the sorB point mutant strain showed that the mutation sites of all transformants were deletions at sites 4–5 of the gRNA (Fig. 1b), and the conserved mutation sites could be used for a more accurate gene-editing process. The selection of the left and right arms of the five target genes was between 600 and 1000 bp, and the length of the homology arm had no obvious effect on the gene-editing efficiency. During homologous recombination in filamentous fungi, the length of the homology arm was usually chosen to be around 1 kb. In Aspergillus niger, homologous recombination could be accomplished even if the homology arm length was only 39 bp (Dong et al. 2019). Whether this system could use shorter left and right arms to achieve homologous recombination remains to be further verified in A. chrysogenum.
The special morphogenesis process of filamentous fungi was considered to be closely related to the metabolic process. During the fermentation process of A. niger, the morphology of hyphal was more favorable to the production of enzymes, and the uniform mycelial spherical shape was more favorable for the production of citric acid (Papagianni 2004). There was a certain correlation between the production of CPC and the formation of arthrospores in A. chrysogenum, and the overexpression of the axl2 gene of the industrial strain FC3-5-23 significantly affected the proportion of arthrospores and the yield of CPC (Xu et al. 2021). However, the knockout of the four genes Δaxl1, Δaxl2, Δbud3, and Δbud4 in the wild strain CGMCC 3.3795, which affected the formation of arthrospores, did not significantly affect the formation of CPC. This result might suggest that the other limiting factors rather than morphology hindered the CPC production in the wild strain, so even if the morphology of knockout strains had been changed, the yield of CPC had little impact. In fact, we also identified the key gene for valine production (acetolactate synthase, GME5735_g, homologous to ACRE_004720 in ATCC 11550) and key genes in the PPP pathway involved NADPH formation (Glucose-6-phosphate 1-dehydrogenase: GME3969_g homologous to ACRE_033950 in ATCC 11550; 6-phosphogluconate dehydrogenase: GME8565_g and GME7826_g, homologous to ACRE_056380 in ATCC 11550 and XM_018280118.1 in Pochonia chlamydosporia 170, respectively in the high-yielding strain FC3-5-23 (whole genome sequenced, unpublished). Overexpression of these key genes in the wild strain CGMCC 3.3795 can increase the CPC production. This phenomenon suggested that if we anticipate to increase the CPC production of CGMCC 3.3795, we need to start from the perspective of global metabolic engineering rather than morphological regulation. This study also tried to measure the oxidative stress ability of the knockout strain and found that neither the starting strain nor the knockout strains could tolerate 0.0025% (v/v) hydrogen peroxide. 1 µL 104 CGMCC 3.3795 spores could not grow on 1.8mM (0.0055%) hydrogen peroxide plates. This phenomenon was different from A. chrysogenum A3/2 (Kluge and Kuck 2018) and industrial production strain FC3-5-23 (Xu et al. 2021). Both of them could grow on the MM medium containing 0.0075% (v/v) hydrogen peroxide, indicating that the oxidative stress ability of A. chrysogenum CGMCC 3.3795 was relatively weak compared with other reported strains.
The morphological changes of hyphae and the changes of stress response in the knockout strains of axl2, bud3, and bud4 were similar, indicating that the gene functions of the three genes were similar. The Δaxl1 strain showed higher degrees of yellow color in the colonies under ER and osmotic pressure conditions, suggesting that although Δaxl1 did not increase CPC production, it might increase other secondary metabolites such as yellow pigment sorbicillinoids under certain stress conditions. Although there was a correlation between the morphology and the antioxidant capacity of the strains, the growth and stress resistance levels of different strains after knocking out the same gene were different due to the differences in the growth, morphogenesis process, and the capacity of stress tolerance.