4. Discussion
Currently, anti-neoplastic drugs are more or less deficient in terms of weak targeting, toxic side effects, and drug resistance [39]. The search for novel antitumour drugs with low side effects and high targeting is one of the important topics of research in the field of medicinal chemistry. Plant endophytic fungi are an important source of novel antitumour compounds due to their unique ecological niche, which harbours unique and novel compound structures. A strain ofAlternaria alstroemeria was discovered and isolated by our group, and its crude extract showed a strong inhibitory effect on A549 [24]. To investigate the molecular basis of the antitumour activity of this strain, the global regulator of secondary metabolism viz AaLaeA was overexpressed. It was found that AaLaeA OE29crude extract inhibited A549 significantly more than WT and increased the total apoptosis rate by 1.56-fold and AaLaeA mediated the antitumour activity of the crude extract [24]. The function of LaeA has been reported to be conserved in previous studies and is involved in the biosynthesis of a variety of fungal secondary metabolites [13]. Overexpression of LaeA or its homologs in Penicillium chrysogenum[15], Chaetomium globosum [17], and Aspergillus nidulans [40] all resulted in a large increment in fungal secondary metabolites; and these reports are consistent with the results of our group’s previous studies. Interestingly, when we screened for AaLaeA overexpression strains, we found a transformant numbered 26 with 10-fold higher expression of AaLaeA than WT, but with a significant loss of antitumour activity. To clarify whether the insertion was caused by a disruption of the relevant gene at the insertion site, the transformant was subjected to genome re-sequencing, which revealed that no coding gene was present upstream or downstream of the insertion site and that the insertion was a nonsense insertion. It is hypothesized that the AaLaeA-mediated loss of antitumour activity is not directly correlated with the insertion site, but may be quantitatively related to the expression of AaLaeA itself. Metabolomic analysis revealed thatAaLaeA OE26 contained significantly lower levels of secondary metabolites of antitumour activity compared to A. alstroemeria wild type. These differential metabolites were carboxylic acids and their derivatives, fatty acids, phospholipid glycerols, benzene and its substituents, flavonoids, phenols, indole derivatives, and other intermediate derivatives which were reported to have the potential for antitumour activity. In particular, the flavonoids were more significantly down-regulated, with the most significant down-regulation and the largest fold difference being in the flavonoid epicatechin gallate. In combination with comparative transcriptome analyses, a significant down-regulation of the gene encoding the FAD-binding domain-containing protein (Fla1) was found; Fla1 belongs to the FAD-binding domain superfamily and is a flavoenzyme that is widely found in nature [18]; It is capable to catalyze a variety of biochemical reactions and has been widely used as a biocatalyst for a variety of reactions, and many biochemical processes take advantage of the versatility of flavoenzymes and extremely such as flavin cofactors [19, 20]. It is hypothesized that AaFla1 may be negatively regulated by AaLaeA, thereby affecting the synthesis of the antitumour compounds in A. alstroermeria . To further verify the relationship between AaLaeA and AaFla1 genes, the transcript levels of Fla1 in wild-type andAaLaeA OE26 strains were analyzed by qRT-PCR. The results showed that overexpression of AaLaeA resulted in a significant down-regulation of Fla1 transcription. In contrast, overexpression of Fla1 caused a significant increment in the antitumour activity of the strain, with a reduction in IC50 from 445 μg/mL to 368 μg/mL and a significant 7-fold increment in apoptosis. Antitumor-related compounds have been reported to accumulate in Fla1 overexpressing transformants, for example, 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], with the flavonoid Quercetin being the most significantly up-regulated. This result is in contrast to the significant reduction in flavonoid content ofAaLaeA OE26, which also confirms in terms of compound accumulation that AaFla1 is negatively regulated by AaLaeA, and therefore affecting the antitumour activity of the strain.