3.4 FAD-binding domain proteins mediate the production of
antitumour active substances
To investigate the material basis of AaFla1 mediated antitumour
activity, differential metabolites between WT andAaFla1 OE23 strains were analyzed using LC/MS
non-targeted metabolomics. Principal component analysis (PCA) showed
that there was a significant separation between the WT andAaFla1 OE23 samples, with PC1 and PC2 accounting
for 48.34% and 14.72% of the total variation, respectively (Fig. 5A).
Hierarchical cluster analysis showed that the 2 sets of samples shared
two branches, with both the AaFla1 OE23 and WT
repeats forming a separate branch, indicating significant metabolic
differences between them (Fig. S5A); Analysis of metabolite differences
between WT and AaFla1 OE23 from partial least
squares discriminant analyses (PLS-DA) also showed the difference (Fig.
S5B and C). The results of the above analyses showed that there were
significant differences in secondary metabolites between WT andAaFla1 OE23 (Fig. 5B).
Screening of differential metabolites (Log2FC
> 0.5, P value < 0.05, and VIP > 1)
for the top 10 up-regulated differential metabolites (Table 3) and
Cluster analysis of the differential substances (Fig.5C) revealed that
the differential metabolites were alkaloids, carbohydrates, lipids,
organic acids, peptides, PK polyketides, steroids, and terpenoids.
Further enrichment by the KEGG pathway revealed that the metabolic
pathways involved in the differential substances included flavonoids,
cofactors, unsaturated fatty acids, various secondary metabolites, and
amino acid metabolism as biosynthetic pathways were enriched (Fig. 5D).
Some compounds with anti-tumor activity were found to be significantly
up-regulated (Table 3). Quercetin [26], Strychnopentamine [27],
Gitogenin [28], Rhodioloside [29], Liensinine [30],
L-Selenomethionine [31], Compactin [32], Tonantzitlolone B
[33], Ginsenoside Rg2 [34], Campesterol [35], Pristimerin
derivative [36], Cinobufagin [37] and Anisomycin [38] have
been shown to have antitumour activity. Among them, quercetin, a
flavonoid, was the most significantly up-regulated.
To investigate the effect of overexpression of AaFla1 on antitumour
active substances, the top ten differential metabolites of WT andAaFla1 OE23 metabolic groups (Table 2) and WT
and AaLaeA OE26 metabolic groups (Table 3) were
compared (Table 4). Overexpression of LaeA resulted in down-regulation
of Fla1 expression and reduced antitumour activity inAaLaeA OE26, with the most pronounced
down-regulation of antitumour flavonoids in the analyzed metabolome.
Overexpression of Fla1 elevated the antitumour activity ofAaFla1 OE23, and the most up-regulated
antitumour compounds in the AaFla1 OE23metabolome were flavonoids.
Taken together, the results indicate that the global regulator AaLaeA
negatively regulates the AaFla1 gene, that AaFla1 regulates the
production of antitumour active substances in the strain, and that theA. alstroemeria FAD-binding domain protein regulates the
production of antitumour active substances.