Abstract
Endophytic fungi are an important source of novel antitumour substances.
Previously, we isolated an endophytic fungus, Alternaria
alstroemeria , from the medicinal plant Artemisia artemisia ,
whose crude extracts strongly inhibited A549 tumour cells. We obtained a
transformant, namely AaLaeA OE26, which
completely loses its antitumour activity due to overexpression of the
global regulator AaLaeA. Re-sequencing analysis of the genome revealed
that the insertion site was in the non-coding region and did not destroy
any other genes. Metabolomics analysis revealed that the level of
secondary antitumour metabolic substances was significantly lower inAaLaeA OE26 compared to the wild strain, in
particular flavonoids were more downregulated according to the
metabolomics analysis. A further comparative transcriptome analysis
revealed that a gene encoding FAD -binding domain protein (Fla1) was
significantly downregulated. On the other hand, overexpression of AaFla1
led to significant enhancement of antitumor activity against A549 with a
7-fold higher inhibition ratio than the wild strain. At the same time,
we also found a significant increase in the accumulation of antitumour
metabolites including quercetin, gitogenin, rhodioloside, liensinin,
ginsenoside Rg2 and cinobufagin. Our data suggest that the global
regulator AaLaeA negatively affects the production of antitumour
compounds via controlling the transcription of AaFla1 in endophyticAlternaria alstroemeria .
Keywords: Alternaria alstroemeria , global regulator
AaLaeA, FAD-binding domain protein, Antitumor substances, Metabolomics
Introduction
As one of the leading causes of death worldwide, cancer poses a serious
threat to human health, and the number of cancer cases is increasing
every year [1]. Although we have a variety of means to treat
tumours, including surgery, radiation and chemotherapy, all these
treatment approaches have certain limitations. Therefore, there is a
great need to find new strategies to fight cancer and discover new
anti-cancer drugs. Many natural products from fungi have pronounced
antitumour activity [2]. Therefore, the search for new structures
and active natural products from microorganisms has become an important
research direction [3] and has attracted much attention. For
example, actinomycin D, daunorubicin, epirubicin, rapamycin and
gelanamycin are all microbial derivatives that are used clinically as
anticancer drugs [4]. The endophytic fungi as microbial biota from
certain niches have also been shown to be able to produce lead compounds
and drug candidates for cancer. For example, the crude extract of the
ethyl acetate layer of the endophytic fungus isolated from Annona
muricata L. was found to have a potent inhibitory effect on MCF -7 cells
[5]. Glyconic acid isolated from the endophytic fungus Bionectria
sp. showed good cytotoxic activity against the cancer cell line A2780
[6]. Trichodermin terpenoids from Nalanthamala psidii were found to
inhibit tumour growth [7]. A p-benzophenone compound cytorhizine
isolated from Cytospora rhizophorae showed some inhibitory effect on
tumour cells MCF -7, HePG-2, NCI-H460 and SF -268 [8].
With its abundant secondary metabolites and their antitumor activity,
fungi in Alternaria genus have also attracted the attention of
researchers. For example, the secondary metabolites of the fungusAlternaria alternarta Y-4-2 isolated from the leaves ofCephalotaxus oliveri showed significant antitumor activity
against a variety of tumor cell lines including A549, NCI-H460, HL60,
NCI-H929 and RPMI8226, including one new dimeric xanthone compound
[9]. Two cyclopentaisochromenone enantiomers isolated fromAlternaria sp. TNXY-P-1 showed antitumor activity against HL-60
cell line [10]. Dibenzo-α-pyrones isolated from Alternariasp. Samif01 possess antibacterial and antioxidant activities [11].
Under laboratory conditions, mostly biosynthetic gene clusters
synthesizing secondary metabolites are silenced [12], and low levels
of gene expression or expression silencing of secondary metabolic gene
clusters affect the production of the corresponding metabolites. LaeA is
involved in the biosynthesis of a variety of fungal secondary
metabolites [13, 14]. In Penicillium chrysogenum ,
overexpression of LaeA increased penicillin production [15]. DNA
microarray analysis of polyketide synthase genes in LaeA deletion mutant
strains of Fusarium verticillioides indicated that the gene
expression of BIK1, FUM1, FUB1, and fusarin was significantly reduced
[16]. CglaeA overexpression in Chaetomium globosumresulted in an increment of
chaetoglobos in A production [17]. These suggest that LaeA could
involve in regulating the synthesis of secondary metabolites and its
overexpression might increase secondary metabolites including new
compounds as well as bio-active substances.
FAD-binding domain protein (Fla1)
belongs to the FAD-binding domain gene superfamily and is a flavoenzyme
that is widely found in nature [18]; with its ability to catalyze a
variety of biochemical reactions, it has been widely used as a
biocatalyst for multifarious reactions [19, 20]. About 1% of
eukaryotic and prokaryotic proteins are expected to encode flavin
adenine dinucleotide (FAD) binding domains [21].
An endophytic fungus viz Alternaria alstroemeria was isolated
from the medicinal plant Artemisia annua early in this study, and
its crude extract showed a strong inhibitory effect on A549.
Overexpression of the secondary metabolic global regulator AaLaeA
discovered a transformant AaLaeA OE26 with
complete loss of antitumor activity. Via further analyses of the
metabolome and transcriptome of mutant strainAaLaeA OE26, we found that the key gene AaFla1
was overexpressed and affecting antitumor activity, meanwhile, the
production of antitumor active substance was regulated by the
FAD-binding protein in Alternaria alstroemeria .
Materials and methods
Identification of AaFla1
The endophytic fungus ZZ-HY-03-02 was identified as Alternaria
alstroemeria by our group in the early stage, The gene encoding the
Fla1 protein of A. alternata was used as a template to amplify
its ORF frame with Fla1-F and Fla1-R and to identify AaFla1.
Construction of genetic transformation system
A Fla1 overexpression vector was constructed by double digestion
(Xba1, HindIII) strategy. Fla1-F and Fla1-R were used as primers to
amplify the Fla1 gene using A. alternata genomic DNA as
the template. PtrpC-F and PtrpC-R were used to amplify the promoter
PtrpC with the plasmid PtrpC as the template, and the hyg resistance
element was amplified with the pK2hyg vector plasmid as the template
(Table S1). The two fragments were fused by the overlapping PCR method,
and the fusion fragment was ligated into the pK2hyg vector andelectroporated into Agrobacterium . The pK2hyg-PtrpC::Fla1
was transformed into A. alternata viaAgrobacterium -mediated genetic transformation [22].