Author Correction: MIG1 as a positive regulator for the histidine biosynthesis pathway and as a global regulator in thermotolerant yeast Kluyveromyces marxianus

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cells were pre-cultured in 5 ml of YPG medium at 30 °C under a shaking condition at 160 rpm for 18 h. The pre-culture was inoculated into a 300-ml flask containing 100 ml of YNBG and 0.01% (w/v) histidine to adjust the initial optical density at 660 nm (OD 660 ) to 0.1, followed by incubation at 30 °C for 24 h under a shaking condition at 160 rpm. Cell density was measured turbidimetrically at 660 nm on a spectrophotometer (U-2000A, Hitachi, Japan). To observe growth characteristics of yeast strains on agar plates, cells were streaked on YPG or YNBG and 0.01% (w/v) histidine and incubated at 30 °C for 24 h and 48 h.
RNA preparation for RNA-Seq. Cells were pre-cultured in 5 ml of YPG at 30 °C under a shaking condition at 160 rpm for 18 h. The pre-culture was inoculated into a 300-ml flask containing 100 ml of YNBG and 0.01% (w/v) histidine at 30 °C under a shaking condition at 160 rpm for 12 h (in the case of wild type) and for 18 h (in the case of Kmmig1). At the mid-log phase, cells were harvested by centrifugation at 5,000 rpm for 5 min at 4 °C. The different pre-culture times were due to the fact that the growth of the latter was slower than that of the former. The cells were washed with YNBG and transferred to 100 ml of YNBG, followed by incubation at 30 °C for 1 h. The cells were harvested by centrifugation at 5,000 rpm for 5 min at 4 °C and subjected to an RNA preparation process. RNA was prepared by a modified procedure on the basis of the procedure reported previously 27 . The RNA samples then were subjected to RNase-free DNase treatment. All RNA samples were purified by using an RNeasy plus mini kit (QIAGEN) according to the protocol provided by supplier.

RNA-Seq-based transcriptomic analysis. The purified RNA samples were analysed on an Illumina
MiniSeq at the Research Center of Yamaguchi University. The detailed procedure for RNA-Seq has been described previously 29 . All these data were deposited under accession number DRA008595 in DDBJ Sequence Read Archive (https://www.ddbj.nig.ac.jp/dra/index-e.html). The sequencing results were analysed using CLC genomic workbench version 10.1.1. All mapped reads at exons were counted, and the numbers were converted to unique exon reads. The unique exon reads from three biological replicates of Kmmig1 were compared to those of the parental strain.
Gene expression profiles of Kmmig1 and the parental strain were compared to find differentially expressed genes (DEGs) based on unique exon read values from CLC genomic workbench outputs using DESeq2R package 30 . The resulting P-values were adjusted using Benjamin-Hochberg's method for controlling the false discovery rate. Genes with adjusted P values less than 0.01 (P adj < 0.01) and log 2 (fold change) values greater than 1 or lower than −1 were assigned as significant DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping with these significant DEGs was performed by KEGG web tools (http://www.genome.jp/keg/tool/map_ pathway1.html). Gene ontology (GO) enrichment analysis of significant DEGs was performed using topGO R package 31 . GO terms with P values less than 0.01 were considered significantly enriched.
Increased expression of HIS4 in Kmmig1. For increased expression of HIS4 in Kmmig1, a TDH3-HIS4-ble DNA fragment was constructed as follows. The TDH3 promoter fragment was amplified by PCR using genomic DNA of S. cerevisiae BY4741 as a template and primers prTDH3-5′-F and prTDH3-3′-R (Table 1). The primers were designed to amplify the fragment corresponding to the region from the TDH3 start codon to 993-bp www.nature.com/scientificreports www.nature.com/scientificreports/ upstream of the start codon. The HIS4 fragment was amplified by PCR using genomic DNA of K. marxianus DMKU 3-1042 as a template and primers HIS4TD-5′-F and HIS4BL-3′-R. The primers were designed to amplify the fragment corresponding to the region from the start codon of HIS4 (2,409 bp). The ble gene (zeomycin resistance gene) was amplified by PCR from pSH65 plasmid DNA as a template with primers BLE-5′-F and BLE-3′-R 32 . Linear pUC19 DNA (Takara Bio, Japan) was prepared by PCR amplification with primers pUC19-5′-F and pUC19-3′-R ( Table 1). The four amplified fragments were purified using a QIAquick gel extraction kit, connected by the In-fusion cloning method (Takara Bio, Japan), introduced into E. coli DH5α by using the heat shock method 33 , and screened on LB plates containing ampicillin, IPTG and X-Gal. Transformants harboring pUC19 containing a TDH3-HIS4-ble fragment (3,905 bp) were confirmed by colony PCR. The TDH3-HIS4-ble fragment was amplified by PCR and directly introduced into Kmmig1 by the lithium acetate method 34,35 . Transformants were obtained on YPD plates containing zeomycin (100 μg ml −1 ), and recombinants were then examined by PCR to check the existence of the TDH3-HIS4-b1e fragment, generating Kmmig1 TDH3-HIS4-ble. Physiological confirmation tests were carried out on YNBD or YNBG plates in the absence or presence of 0.01% (w/v) histidine. ethics statement. This article does not contain any studies with human participants or animals performed by any of the authors.

Results
Effect of MIG1-disrupted mutation on expression of genes for histidine biosynthesis. K. marxianus became histidine-auxotrophic when MIG1 was disrupted 8 , but the possibility that MIG1 is not directly involved in the regulation of histidine biosynthesis but that other genes are directly involved could not be excluded. We thus decided not to examine only genes for histidine biosynthesis but to perform genome-wide expression analysis by RNA-Seq. RNA-Seq analysis was performed with RNAs prepared from Kmmig1 and parental cells that had been incubated for 1 h after shifting from a minimal medium in the presence of histidine to that in the absence of histidine. After sequencing and removing the adaptors and the low quality reads, more than 0.8 Gb clean data qualified for follow-up analysis were acquired from each sample, being equivalent to more than 75-fold genome coverage. Unique exon reads of each gene were determined as transcript abundance. The difference in expression of each gene in Kmmig1 from that in the parental strain was shown as the ratio of unique exon reads in Kmmig1 to that in the parental strain. To further explore the transcriptional changes in Kmmig1 compared to those in the parental strain, we conducted analysis of DEGs based on the ratio of unique exon reads. Significant DEGs showed changes in the transcription level with log 2 (fold change) > 1 and log 2 (fold change) < −1 (P adj < 0.01). Kmmig1 was found to have 1,150 DEGs including 689 up-regulated and 461 down-regulated genes (Supplementary Information Fig. S1 and File S1).
In order to explore the gene(s) responsible for a histidine-auxotrophic phenotype in Kmmig1, the unique exon reads of seven HIS genes for histidine biosynthesis were compared in Kmmig1 and the parental strain (Fig. 1a). Analysis of DEGs indicated that the expression level of HIS4 in Kmmig1 was 2.4-times lower than that in the parental strain (Fig. 2). There was almost no difference between the expression levels of other HIS genes. Therefore, these findings indicated the possibility that the histidine-auxotrophic phenotype of Kmmig1 was due to reduction in the expression of HIS4.
To confirm the significance of down-regulation of HIS4, the consistency of the RNA-Seq data and previous RT-PCR data for INU1, RAG1 and GLK1 was examined. A comparison of the RT-PCR data for Kmmig1 and the parental strain 8 revealed that the MIG1-disrupted mutation increased INU1 expression by 3 fold, decreased RAG1 expression by more than 2 fold and had almost no effect on GLK1 expression. Consistently, the unique exon reads of INU1 and RAG1 in Kmmig1 were 2.2-times higher and 5.3-times lower, respectively, than those in the parental strain, and the unique exon reads of GLK1 in Kmmig1 were not different from those in the parental strain (Fig. 1b). Therefore, the RNA-Seq data confirmed the previous conclusion that Mig1 is involved in the negative regulation of INU1 and in the positive regulation of RAG1 8 and suggested positive regulation of HIS4 by Mig1. Notably, although the RNA samples were prepared from cells grown under different medium conditions, YPD for RT-PCR analysis and histidine-free YNBG for RNA-Seq analysis, data obtained from the different medium conditions showed good consistency in expression of the three genes. These facts may indicate that incubation in histidine-free YNBG for 1 h has almost no effect on cell metabolism and that the data therefore reflect only the effects of MIG1-disrupted mutation on the expression of genomic genes and that the influence of histidine-free YNBG is limited and is specific to some pathways, for example, histidine biosynthesis.

No.
Primer name Nucleotide sequences www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ Increased expression of HIS4 in Kmmig1. RNA-Seq analysis indicated the possibility that down-regulation of the expression of HIS4 was responsible for the histidine-auxotrophic phenotype in Kmmig1. Interestingly, His4 is involved in 4 catalytic steps of the histidine biosynthesis pathway (Fig. 2) and down-regulation (58% reduction) at each step thus led to a large effect (97% reduction) on the entire histidine biosynthesis. Increased expression of HIS4 in Kmmig1 was thus tested (Fig. 3). A DNA fragment of TDH3-HIS4-ble, in which HIS4 was under the control of the promoter of TDH3 as one of the strong promoters from S. cerevisiae, was constructed and introduced into the genome of Kmmig1. The growth of the recombinant on YNBG without histidine was compared with that of Kmmig1 (Fig. 3a). Kmmig1 exhibited almost no growth as expected, but the recombinant grew well like the wild type. Similarly, the recombinant showed growth equivalent to that of the wild type in the liquid minimal medium, but Kmmig1 showed greatly retarded growth even with the addition of 0.01% (w/v) histidine to the medium (Fig. 3b). These results and the down-regulation of HIS4 in Kmmig1 (MIG1-disruption mutation) suggest that the down-regulation of HIS4 in Kmmig1 caused the defect of growth in the minimal medium and that Mig1 positively regulates HIS4 expression. However, we cannot exclude the possibility that Mig1 regulates His4 expression via another regulator(s).

Effects of MIG1-disrupted mutation on expression of genomic genes.
Since there were many significant DEGs caused by the MIG1-disrupted mutation, suggesting its global influence on the genomic genes in K. marxianus, these DEGs were subjected to a GO term enrichment test ( Supplementary Information File S2). In the 689 up-regulated DEGs, the enriched GO terms for biological processes were related to the lipid catabolic process, cellular lipid catabolic process, fatty acid catabolic process, fatty acid oxidation, lipid oxidation, fatty acid beta-oxidation, fatty acid metabolic process, organic acid catabolic process, carboxylic acid catabolic process, small molecule catabolic process, monocarboxylic acid catabolic process, lipid modification, antibiotic metabolic process, glutamate metabolic process, and other processes. The enriched GO terms for cellular components included an integral component of the membrane, intrinsic component of the membrane, membrane part, peroxisome organelle, integral component of the peroxisome, peroxisomal matrix and intrinsic component of the peroxisome, cell wall, and other cellular components. The GO terms for molecular functions included oxidoreductase activity, catalytic activity, transmembrane transporter activity, hydrolase activity, transporter activity, coenzyme binding, and other molecular functions ( Supplementary Information File S2).
On the other hand, in the 461 down-regulated DEGs, the enriched GO terms for biological processes included ribosome biogenesis, rRNA processing, ribonucleoprotein complex biogenesis, rRNA metabolic process, ncRNA www.nature.com/scientificreports www.nature.com/scientificreports/ processing, ncRNA metabolic process, glycolytic process, ATP generation from ADP, pyruvate biosynthetic process, nucleoside diphosphate metabolic process, purine nucleoside diphosphate metabolic process, purine ribonucleoside diphosphate metabolic process, and pyridine nucleotide biosynthetic process. The enriched GO terms for cellular components included the preribosome, nucleolus, small-subunit processome, ribonucleoprotein complex, nucleolar part, 90S preribosome, preribosome, large subunit precursor, nuclear lumen, cytosolic ribosome, cytosolic large ribosomal subunit, nucleolus, and nucleus. The enriched GO terms for molecular functions included snoRNA binding, RNA binding, rRNA binding, oxidoreductase acitivity, oxidoreductase activity, organic cyclic compound binding, heterocyclic compound binding, iron ion binding, coenzyme binding, and nucleic acid binding ( Supplementary Information File S2).
The up-regulated and down-regulated DEGs were also mapped to the terms in the KEGG database ( Supplementary Information File S3). The mapping analysis revealed that pathways related to the 689 up-regulated DEGs included metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, carbon metabolism, autophagy, MAPK signaling pathway, biosynthesis of amino acids, meiosis, peroxisome, glyoxylate and dicarboxylate metabolism, spliceosome, fatty acid degradation, glycolysis/gluconeogenesis, glycerolipid metabolism, endocytosis, arginine and proline metabolism, citrate cycle (TCA cycle), glycine, serine, and threonine metabolism, tyrosine metabolism, phenylalanine metabolism, autophagy, pyruvate metabolism, purine metabolism, and pyrimidine metabolism. Pathways related to the 461 down-regulated DEGs included metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, ribosome biogenesis in eukaryotes, biosynthesis of amino acids, carbon metabolism, glycolysis/gluconeogenesis, ribosome, purine metabolism, RNA transport, methane metabolism, starch and sucrose metabolism, cysteine and methionine metabolism, MAPK signaling pathway, galactose metabolism, RNA polymerase, cell cycle, glycine, serine and threonine metabolism, steroid biosynthesis, amino sugar and nucleotide sugar metabolism, pentose phosphate pathway, pyruvate metabolism, fatty acid metabolism, and pyrimidine metabolism.
To further understand the possible downstream relationship from Mig1, we explored significant DEGs for transcription factors (TFs) that are orthologues to those of S. cerevisiae, from the lists in Supplementary Information File S1 [36][37][38][39][40][41][42][43][44][45][46][47] . As a result, three down-regulated genes corresponding to SFP1, RGT1, and MTH1 in S. cerevisiae and four up-regulated genes corresponding to KAR4, ADR1, GSM1, and SIP4 in S. cerevisiae were found, and they were subjected to GO and KEGG analyses but no item in KEGG pathway was found for all TFs (Supplementary Information Table S1). Based on the physiological functions of these TFs in S. cerevisiae (Supplementary Information Table S2), it is assumed that KLMA_60316 (Rgt1) and KLMA_30237 (Mth1) function under a glucose-rich condition, whereas KLMA_60316 (Rgt1), KLMA_20117 (Adr1), KLMA_20140 (Gsm1), and KLMA_30166 (Sip4) function under a glucose-starved condition. Such glucose level-specific manners might indicate the link with Mig1. The remaining two, KLMA_40457 (Sfp1) and KLMA_10029 (Kar4), presumably regulate cognate genes under a condition unrelated to glucose level. These putative TFs could be involved in regulation of the expression of genes included in terms of GO (Supplementary Information Table S1). Notably, in S. cerevisiae, Sip4 expression is negatively regulated by Mig1 via Cat8 48 , Sip4 activates the expression of many genes for gluconeogenesis 49 , and the expression of 108 genes is significantly decreased in the absence of Adr1 50 . Taken together, the results indicate the possibility that Mig1 regulates many genes directly or indirectly via various TFs including the seven putative TFs described above.

Discussion
Physiological analysis of the effect of MIG1-disrupted mutation indicated the possibility that Mig1 is required for histidine biosynthesis 8 . In this study, in order to understand the role of Mig1 in histidine biosynthesis, genome-wide expression analysis was performed. Among seven HIS genes for enzymes related to the histidine biosynthesis pathway, only the expression level of HIS4 in the MIG1-disrupted mutant was significantly down-regulated compared to that in the parental strain. The level of reduction of HIS4 expression was only 58%, but it is assumed that such an intermediate level of the effect becomes very strong in total to cause the defect of growth in a minimal medium because His4 catalyzes 4 steps in the histidine biosynthesis pathway. This assumption was examined by increased expression of HIS4 in Kmmig1, resulting in the recovery of growth in the minimal www.nature.com/scientificreports www.nature.com/scientificreports/ medium without the addition of histidine (Fig. 3). Consequently, it is thought that Mig1 is a positive regulator for HIS4 and thus for histidine biosynthesis. It is noteworthy that the regulation of histidine biosynthesis in K. marxianus by Mig1 creates a novel and effective mechanism targeting one gene, HIS4, of which the product is involved in 4 catalytic steps of histidine biosynthesis. Interestingly, S. cerevisiae has three His4-involved steps in the histidine biosynthesis pathway 24 , though its regulation by Mig1 remains to be investigated. On the other hand, Hua et al. reported that Kmmig1 was isolated on a minimal medium (SD medium) 58 . It is possible that they took slowly formed colonies when Kmmig1 was screened because we noticed that Kmmig1 is able to grow on a is shown at the right bottom side. Red-coloured bars: significantly up-regulated genes in Kmmig1; blue-coloured bars: significantly down-regulated genes; black-coloured bars: not significantly changed genes. In the central carbon metabolic network, significantly up-regulated and down-regulated genes in Kmmig1 are represented in red and blue, respectively, and genes that were not significantly changed in Kmmig1 are represented in black. Further details are given in Supplementary Information File S4. www.nature.com/scientificreports www.nature.com/scientificreports/ minimal medium, though very slowly 8 (Fig. 3 in this manuscript). Alternatively, their Kmmig1 might have an additional suppressor mutation that allowed it to grow on the SD medium or the histidine-auxotrophic phenotype of Kmmig1 might be strain-specific.
In S. cerevisiae, Mig1 has been extensively analysed and shown to be a key regulator as a complex with other proteins including Hxk2 for glucose repression 9,10 . Surprisingly, the present study indicated the possibility that Mig1 is a global regulator for genomic genes in K. marxianus. Transcriptome analysis of the MIG1-disrupted mutant and its parental strain was performed with RNAs prepared from cells that were cultivated in a minimal medium containing galactose as a sole carbon source (under a condition with no glucose repression). The analysis suggests that Mig1 acts as a positive regulator for most genes (except GLK1 and FBP1) in glycolysis and as a negative regulator for many genes in the TCA cycle and fatty acid degradation (Fig. 4). In anabolic pathways, Mig1 may activate the expression of genes for biosynthesis of secondary metabolites, antibiotics and amino acids, ribosome biogenesis, rRNA processing, and purine and pyrimidine metabolism and inhibit the expression of genes for biosynthesis of secondary metabolites, antibiotics and amino acids, and for gluconeogenesis. Considering that the medium still contained a sufficient amount of galactose under the condition in which RNA was prepared for RNA-Seq and considering that Mig1 seems to activate genes for the ethanol synthesis pathway in addition to genes for glycolysis and to inhibit genes for the TCA cycle, it is likely that Mig1 is a crucial regulation factor to enhance ethanol production in K. marxianus.
In addition, as shown in KEGG analysis, the down-regulation of genes for ribosome biogenesis, biosynthesis of amino acids, carbon metabolism, ribosome, RNA transport, RNA polymerase and purine and pyrimidine metabolism in Kmmig1 suggests that Mig1 promotes cell proliferation. There are some pathways that seem to be subjected to both positive regulation and negative regulation by Mig1; for example, biosynthesis of secondary metabolites, antibiotics, amino acids, purine and pyrimidine. It is assumed that such a dual regulation by Mig1 contributes to the fine tuning of these pathways or balanced metabolism in cells. Further analysis of the regulation of individual gene expression in these pathways may lead to an understanding of the physiological importance of Mig1-directed regulation in each pathway.
www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ Science and Technology of Laos, and by the Core to Core Program A. Advanced Research Networks, which was granted by the Japan Society for the Promotion of Science, the National Research Council of Thailand, Ministry of Science and Technology in Vietnam, National Univ. of Laos, Univ. of Brawijaya and Beuth Univ. of Applied Science Berlin.