Genome analysis of each strain and identification of mutations in the genome sequence of MT
We previously reported that the riboflavin over-producing mutant (MT strain) was isolated by disparity mutagenesis in the presence of H2O2, itaconate and oxalate and phenotypes of this MT strain were characterized by transcriptomic, proteomic and metabolic flux analyses [13, 16]. In this study, to reveal the genotype of MT strain, genome resequencing and SNP analysis were carried out. Whole-genome shotgun sequencing for WT and MT generated 1,083,909 and 1,519,777 high-quality read pairs totaling approximately 593 and 836 Mb, respectively. The high-quality reads of WT and MT were aligned to the reference genome of A. gossypii ATCC10895, resulting in sequence coverages of 41.9–43.4 and 46.7–53.6, respectively, for chromosome I–VII. Among the variants identified by the GATK based on the aligned reads for WT and MT, mutations in open reading frames (ORFs), missense mutations, frameshift mutations and nonsense mutations were analyzed. In WT, which is same as the original strain A. gossypii ATCC10895, amino acid sequences encoded by all ORFs were the same as those of strain ATCC10895, except for the SEN2 gene (AGOS_AGR073C), which encodes a subunit of the tRNA splicing endonuclease in S. cerevisiae (Supplementary material Table S1). This result indicates that this WT, which has been maintained in our laboratory, could have gained this heterozygous mutation. However, this WT was used in this study because this gene may not be involved in riboflavin production, given the function of the gene product. Additionally, some silent mutations were also detected (data not shown).
From the single-nucleotide variant (SNV) analysis between the genome sequences of WT and MT, we detected 33 homozygous and 1377 heterozygous mutations in the genome sequence of MT (Supplementary materials Tables S1 and S2), which cause missense, nonsense and frameshift mutations, in addition to silent mutations. These heterozygous mutations suggest that nuclei of the MT strain are polyploid. In the 1377 heterozygous mutants, the proportion of mutations in each gene was different. The highest proportion was 75% (chromosome VI:799,900 in AgOCT1, AGOS_AFR198W), and the lowest proportion was 21% (chromosome VII:198,537 and 198541 in AgATP1, AGOS_AGL272C) (Fig. 1). Most heterozygous mutants were found to have ratios of 40–60%. These results suggest that the MT strain may contain multiple nuclei containing different mutations. To prove its ploidy, haploid spores of the MT strain were isolated. However, we never isolated haploid spores of the MT strain. This result indicates that the MT strain lost the ability to sporulate even though it was previously reported that the riboflavin production in A. gossypii is related with its spore production [21]. A. gossypii is a naturally multinucleate fungus, but this fungus may be haploid, and the spores of this fungus produced by asexual sporulation are also haploid [2, 22]. However, Anderson et al. reported that ploidy variation was observed in A. gossypii with minor aneuploidy [23]. In this study, the proportion of heterozygous mutations in each gene ranged from 75% to 21%, and most heterozygous mutations were found at 40–60%. This result may be caused by the polyploidy and multinucleate cells of this organism. They also discussed the low germination frequency of spores produced from variable polypoid nuclei. Two possibilities are suggested: a reduction in ploidy to uninucleate haploid spores and the formation of spores with variable ploidy. In this study, the MT strain never produced haploid spores. The MT strain may survive due to its multinucleation.
In addition, interestingly, we found a region representing ~2-fold sequence coverage compared to other regions in chromosome VII of the MT strain, which correspond to the rRNA gene repeats (Chr VII:441,317-762,344) (Fig. 2). In yeasts, the number of rRNA gene repeats is normally maintained for genome stability and determination of life span [24, 25]. Moreover, the rRNA gene controls chromosome homeostasis [26]. When the number of rRNA gene repeats increases, rRNA gene instability and aging phenotypes are observed. Silva et al showed that riboflavin-overproducing Ashbya mutants are vulnerable to photoinduced oxidative DNA damage and accumulate reactive oxygen species (ROS) [27]. The ROS is largely involved in the aging of cells, suggesting that the riboflavin production in A. gossypii may be associated with the aging of cells.
It is reasonable that homozygous mutations have more crucial effects on riboflavin production in the MT strain compared to heterozygous mutations. We selected candidate mutations among 33 homozygous mutations, as shown in Table 1. Among the 33 homozygous mutations, the SEN2 gene (AGOS_AGR073C) has one homozygous mutation in the MT strain, in contrast to the WT strain used in this study, which has one heterozygous mutation at the same nucleotide. Four homozygous mutations in the amino acid metabolism of A. gossypii were detected. First, a frameshift mutation in the AgSHM2 gene (AGOS_ACR215C) was detected in the genome of the MT strain. This gene encodes serine hydroxymethyltransferase 2 (SHMT), and it was previously reported that disruption of this gene enhanced the productivity of riboflavin in A. gossypii, although the growth of the organism was compromised [7]. The frameshift mutation causes the deletion of 25 amino acid residues at the C-terminus of AgSHM2 and the addition of 6 extra amino acid residues in the deletion mutant. This C-terminal region may not be directly involved in catalytic activity [28]. However, the L474F mutation in this region of human and rabbit SHMT causes a decrease in the binding of this protein to co-factors [29]. Therefore, this frameshift mutation in the MT strain may lead to a decrease in the SHMT activity of AcSHM2. In addition to the homozygous frameshift mutation, one heterozygous mutation (593G→A), which causes a missense mutation, R198Q, was also detected in the AcSHM2 gene.
Second, a missense mutation (206C→T) in the AgARO2 gene (AGOS_ADL287C), which produces the T69M mutant, was detected. In S. cerevisiae, this gene encodes chorismate synthase, which produces chorismate, a building block of aromatic compounds. Because T69 in the chorismate synthase of S. cerevisiae is distant from the catalytic site, this residue may not be directly involved in catalytic activity [30]. In addition, this enzyme also exhibits flavin reductase activity for the synthesis of reduced flavin mononucleotide (FMN), which is required for chorismate synthase activity.
Third, a missense mutation (1365G→T) in the AgILV2 gene (AGOS_AEL305C), which produces the Q455H mutant, was detected. In S. cerevisiae, this gene encodes the large subunit of acetohydroxyacid synthase (AHAS), which solely catalyzes the synthesis of 2-acetolactate and 2-aceto-2-hydroxybutyrate. This reaction is the first step of branched-chain amino acid biosynthesis. This mutation may not have considerable effects on enzymatic activity because Q455 is not in the co-factor-binding sites [31]. This enzyme requires flavin adenine dinucleotide (FAD) as a co-factor, even though this reaction does not require oxidation and reduction. A small subunit of AHAS encoded by the ScILV6 gene regulates the AHAS activity of ScILV2 in yeast [32]. A. gossypii also has AgILV2 and AgILV6 genes. In AgILV6 genes, three heterozygous missense mutations (140G→A, S47N; 155G→A, S52N; 673G→T, G225C) were detected.
Fourth, a missense mutation (365G→A) in the AgLYS5 gene (AGOS_AGR382W), which produces the R122H mutant, was detected. In S. cerevisiae, ScLYS5 (4’-phosphopantetheinyl transferase, PPTase) converts the apo-form of ScLYS2 (a-aminoadipate reductase) to the active holo-form by the transfer of phosphopantetheine and is present in the lysine biosynthetic pathway [33]. In addition to modification, PPTase is involved in fungal growth, the biosynthesis of secondary metabolites and asexual and sexual development [34, 35].
In pyrimidine metabolism in A. gossypii, one homozygous mutation was detected in the AgCDD1 gene, which encodes cytosine deaminase in S. cerevisiae. This enzyme catalyzes the conversion of cytidine to uridine in the pyrimidine salvage pathway in S. cerevisiae [36]. In A. gossypii, in the pyrimidine salvage pathway, uracil phosphoribosyltransfrase, encoded by the AgFUR1 gene, controls the amount of phosphoribosyl pyrophosphate (PRPP), which is one of the precursors of riboflavin in this organism [37].
In the MT strain, 1377 heterozygous mutations were also detected (Supplementary material Table S2). Heterozygous mutations do not appear to have critical effects on riboflavin production in the MT strain compared to homozygous mutations [38, 39]. However, heterozygous mutations sometimes have negative effects on protein functions as well as haploinsufficiency [40, 41]. In addition, some mutated proteins that form multimers exhibit dominant-negative effects on functions [42, 43]. Therefore, it is possible that heterozygous mutations also have some effect on riboflavin production in the MT strain. Among the 1377 heterozygous mutations, unusual heterozygous mutations were detected (Table 2). Most genes in the TCA cycle have heterozygous mutations. In particular, three genes, namely, AgSDH1 (AGOS_ACR052W), AgSDH2 (AGOS_ACL065C), and AgSDH3 (AGOS_AFR207C), encoding subunits of succinate dehydrogenase in S. cerevisiae, have heterozygous mutations. In addition, several genes encoding flavoproteins in the mitochondria also have heterozygous mutations. AgSDH1 is also a flavoprotein. Flavoproteins in mitochondria of yeasts function in redox processes via the transfer of electrons [44]. In addition, the flavin in flavoproteins participates in the reduction of heme iron or iron-sulfur clusters. In this study, we detected several homozygous mutations (AgARO2, AgILV2) and heterozygous mutations {AgSDH1, AgNDI1 (AGOS_AFR447C), AgDLD1 (AGOS_AER321W), AgCBR1 (AGOS_ADL087W), AgGLR1 (AGOS_AGR196W), AgMTO1 (AGOS_AGR196W), AgMET5 (AGOS_ABL077W), AgPUT1 (AGOS_AGL165W), AgFAS1 (AGOS_AER085C), AgHEM14 (AGOS_AAR021W), AgERV2 (AGOS_ACR175W), and AgERO1 (AGOS_ADL348W)} in genes encoding flavoproteins in S. cerevisiae. It is possible that the riboflavin overproduction in the MT strain is associated with these mutations of genes encoding flavoproteins and dysfunction of the TCA cycle. MT strain is hypothesized to have mitochondrial dysfunction because many homozygous and heterozygous mutations in genes encoding mitochondrial proteins and flavoproteins were detected in this study (Tables 2 and 3). In humans, riboflavin supplementation rescues the mitochondrial disorders associated with the deficiencies of some flavoproteins and respiratory chains [45]. This result suggests that the overproduction of riboflavin in the MT strain may also be associated with mitochondrial dysfunction.
Related to the heterozygous mutations in flavoprotein genes, a heterozygous mutation in the AgFMN1 gene (AGOS_ABL109W) was detected (Table 2). In S. cerevisiae, this gene encodes riboflavin kinase, which catalyzes the synthesis of FMN from riboflavin. FMN is converted to FAD by FAD synthase. The downregulation of AgFMN1 gene expression prevented riboflavin consumption in this fungus, and the ribC-deleted mutant deregulated riboflavin production in B. subtilis by preventing FMN and FAD accumulation [46, 47]. Therefore, this mutation may partially contribute to riboflavin overproduction in the MT strain by partial restriction of the riboflavin flow to FMN. Additionally, heterozygous mutations were also detected in genes involved in heme biosynthesis and sulfur metabolism (Table 2).
We detected homozygous mutations in the AgSHM2, AgARO2, AgILV2, and AgLYS5 genes involved in amino acid biosynthesis (Table 1). Heterozygous mutations in genes involved in amino acid metabolism were concentrated in glycine, serine, and threonine metabolism; branched-chain amino acid biosynthesis; and aromatic amino acid biosynthesis (Table 2). In our previous study, the increased expression of AgTRP2 (ABR209W) and AgTRP5 (AFR485C) was observed in MT strain by a proteomic analysis. AgTRP2 and AgTRP5 are annotated as anthranilate synthase and tryptophan synthase, respectively, which belong to the tryptophan biosynthetic pathway. These results suggest that these amino acid metabolic pathways may be linked to riboflavin production in A. gossypii.
Several heterozygous mutations were detected in genes involved in sulfur amino acid metabolism. In particular, the sulfur amino acid biosynthesis pathway contains heterozygously mutated genes in the MT strain {AgMET5 (AGOS_ABL077W), AgMET6 (AGOS_ABR212C), AgSTR3 (AGOS_ACL059C), AgMET17 (AGOS_ADL031W), AgMET2 (AGOS_AFR682C), AgSAM2 (AGOS_AFR692C), AgMET10 (AGOS_AGR237C)}. Mainly, genes encoding all enzymes that catalyze homocysteine in S. cerevisiae, except the adenosylhomocysteinase encoded by the AgSAH1 gene, were heterozygously mutated. These results suggest that methionine metabolism, which consists of one-carbon metabolism together with folate metabolism, may be associated with riboflavin production in A. gossypii. The AgMET10 and AgMET5 genes encode alpha and beta subunits of sulfite reductase in S. cerevisiae, respectively, which are both flavoproteins.
It was previously reported that riboflavin production in A. gossypii was improved by disruption of the AgURA3 gene, which leads to blockage of the pyrimidine biosynthetic pathway in this organism [37]. In the MT strain, several genes in the pyrimidine biosynthetic pathway have heterozygous mutations (Table 2). These results suggest that pyrimidine metabolism, including the pyrimidine de novo and salvage pathways, may be associated with riboflavin production in A. gossypii. In the purine biosynthetic pathway, the AgRKI1 (AGOS_ACL077C), AgRPS1 (AGOS_AER083C), AgADE5,7 (AGOS_AFR254C), AgADE8 (AGOS_AAR120C), and AgADE2 (AGOS_ACR210C) genes have heterozygous mutations in the MT strain. Moreover, the AgBAS1 gene (AGOS_AFR297W), which encodes the transcription factor for regulation of the purine and glycine biosynthesis pathways in A. gossypii [48], also has one heterozygous mutation. These heterozygous mutations may partially force the restriction of purine biosynthesis, which is important for riboflavin production in A. gossypii. This limited purine biosynthesis in A. gossypii was also reported by Ledesma-Amaro et al., who showed the downregulation of purine biosynthesis during riboflavin production [46].
In addition to mutations in genes involved in metabolic pathways in A. gossypii, 17 heterozygous mutations in genes involved in DNA repair were detected (Table 4). In particular, genes involved in mismatch DNA repair {AgMSH2 (AGOS_AAL093C), AgMSH3 (AGOS_ADR168C), AgMSH6 (AGOS_AGR116W), AgMLH1 (AGOS_AFL199C), AgMLH2 (AGOS_AFR226C), AgMLH3 (AGOS_AAL093C), and AgPMS1 (AGOS_AER421W)} were heterozygously mutated. These proteins function cooperatively to repair DNA mismatches in S. cerevisiae. Among MutS homologs, genes encoding AgMSH2, AgMSH3 and AgMSH6 had heterozygous mutations, but no mutation was detected in genes encoding AgMSH1, AgMSH4 and AgMSH5. ScMSH2, ScMSH3 and ScMSH6 of S. cerevisiae function to maintain nuclear genome stability [49]. In contrast, ScMSH1 functions in mitochondria, and ScMSH4 and ScMSH5 function during meiosis [50, 51]. These results suggest that the heterologous mutations in AgMSH2, AgMSH3 and AgMSH6 may compromise the DNA mismatch repair pathway and contribute to the maintenance of DNA mismatches and accumulation of heterologous mutations in the genome of A. gossypii during disparity mutagenesis and rapid evolution of A. gossypii to the riboflavin-overproducing mutant MT strain. Previous papers have shown that heterologous mutations of the ScMSH2 gene showed mutator phenotypes in diploid yeasts and suppression of the mismatch repair pathway and proofreading-deficient DNA polymerase e in human cells, leading to the accumulation of numerous mutations [52, 53]. However, the riboflavin production level in MT was stable during 14 passages [13].
As mentioned above, MT strain never produced its haploid spores. Some heterozygous mutations were found in genes involved in the sporulation (Table 5). Putative two 1,3-b-D-glucan synthase genes (AGOS_ACL181C, AGOS_AAR053W) had heterozygous mutations. Especially, AGOS_AAR053W had one frameshift mutation which may have great influences on the protein function. In S. cerevisiae, FKS2 is a 1,3-b-D-glucan synthase during its sporulation and FKS2 and FKS3 works in spore wall assembly [54]. In addition, FKS2 binds to a sporulation-specific kinase, SMK1 [55]. Heterozygous mutations of AGOS_ACL181C and AGOS_AAR053W may have some influences on the sporulation in MT strain. Moreover, we found heterozygous mutations in AgIME2 (AGOS_AFR076W) and AgKAR4 (AGOS_AFR736C) genes. Disruption of AgIME2 gene or AgKAR4 gene leads to the deficiency of its sporulation in A. gossypii [56]. These heterozygous mutations may also be one of the reasons for the sporulation deficiency in MT strain.
Among both mutations, Gene Ontology (GO) enrichment analysis was performed (Supplementary materials Tables S3–S5). Over-represented GO terms are ATP binding, Protein binding and ATPase activity. Especially, in “ATP binding”, all 22 ATP-dependent helicase genes have a single heterologous mutation, respectively. It was recently reported that RNA helicases have the relationship with aging and life span of cells [57]. Mutations of all RNA helicase genes support the suggestion that riboflavin production in A. gossypii may be associated with the aging of cells. On the other hand, “Ribosome”, “Translation”, “Structural constituent of ribosome” and “Intracellular” were under-represented. These GO terms contain ribosomal proteins involved in translation (Supplementary materials Tables S5). Mutations of genes encoding these proteins are lethal in organisms and, therefore, these GO terms were under-presented.
Effect of temperature on riboflavin production in MT strain
By genomic analysis of the MT strain, one homozygous mutation in the AgHSP104 gene (AGOS_AGL036C), which causes a nonsense mutation, was detected (Table 1). This mutation generates the mutated AgHSP104, composed of 355 amino acid residues at its N-terminus. HSP104 in fungi contributes to the thermotolerance and disaggregation of denatured and aggregated proteins, ethanol tolerance and survival in the stationary phase [58]. We confirmed this nonsense mutation in the MT strain by DNA sequencing (Fig 3a). In addition, other four homozygous mutations in the MT strain were also confirmed by DNA sequencing (Data not shown). These results validate the results of the genomic analysis. The WT and MT strains were cultivated on YD medium at 28 and 37°C. The growth and riboflavin production in WT cultivated at 37°C were slightly lower than those in WT cultivated at 28°C (Fig. 3b). However, the growth of and riboflavin production in the MT strain were dramatically reduced at 37°C compared to those at 30°C, and the MT strain was not able to grow normally. These results reflected the generation of truncated AgHSP104 in the MT strain, leading to loss of thermotolerance, even at 37°C. This result also confirms the presence of the homozygous mutation in the AgHSP104 gene of the MT strain.
Effect of iron for the riboflavin production in MT strain
In Table2 and 3, many heterozygous mutations were detected in genes encoding proteins involved in mitochondrial function and DNA. Iron-sulfur (Fe/S) clusters are required for TCA cycles, the electron transfer chain and fatty acid oxidation in mitochondria and DNA repair in nucleus [59, 60]. Therefore, the addition of iron ion for the MT strain cultivation was investigated. Fe3+ enhanced the growth of mycelia and riboflavin production in the MT strain (Fig. 4A) also in the presence of glycine, which is well-known for the improvement of the riboflavin production in A. gossypii. Addition of Fe3+ and Fe3+ + glycine improved the riboflavin production of MT strain by 1.6 and 2.0 fold, respectively although we were not able to find its significant differences. (Fig. 4B). Specific riboflavin production of MT strain in the presence of Fe3+ and Fe3+ + glycine were also improved by 1.4 and 1.3 fold, respectively although we were not able to find its significant differences. These results indicate that Fe3+ and glycine enhanced the riboflavin production by the improvement of its growth. Glycine is one of the precursors of heme, and activation of heme biosynthesis induced respiration in S. cerevisiae, which increased the synthesis of ATP (61). In the MT strain, we found a relatively high proportion of a mutation (0.632) in the AgHEM1 gene (AGOS_ABL104C), encoding 5-aminolevulinate synthase, which catalyzes the first step in the heme biosynthetic pathway. Moreover, the AgHEM4 gene (AGOS_AER351W), which encodes uroporphyrinogen III synthase in S. cerevisiae, has a heterozygous nonsense mutation. In addition, a heterozygous mutation was also detected in the AgHEM14 gene (AGOS_AAR021W). This gene encodes protoporphyrinogen oxidase in S. cerevisiae, which is a flavoprotein in mitochondria. These results suggest that heme biosynthesis may be compromised in the MT strain. Supplementation with glycine may partially restore heme biosynthesis in the MT strain, leading to enhancement of respiration in the MT strain, and the growth of the MT strain may be improved. (Fig. 5). This result supports the hypothesis. We previously reported that lactate and pyruvate was produced more in MT strain than WT strain in the minimum medium and succinate was decreased in MT strain compared to WT stain [16]. In addition, gene expression of most of genes involved in TCA cycle was down-regulated in MT strain cultivated compared to WT stain [16]. In Fig. 4, the growth and riboflavin production in MT strain were enhanced by the addition of iron ion, which is involved in mitochondrial functions with flavoproteins [44, 59, 60]. This result also supports the relationship of riboflavin production with the mitochondrial dysfunction. The addition of Fe2+ had no effect on the riboflavin production in WT strain (Data not shown).