The Exploring Functional Role of Ammonium Transporters of Aspergillus oryzae in Nitrogen Metabolism: Challenges towards Cell Biomass Production

Ammonium is a source of fermentable inorganic nitrogen essential for the growth and development of filamentous fungi. It is involved in several cellular metabolic pathways underlying nitrogen transport and assimilation. Ammonium can be transferred into the cell by an ammonium transporter. This study explored the role of ammonium transporters in nitrogen metabolism and cell biomass production in Aspergillus oryzae strain BCC 7051. Specific sequences encoding ammonium transporters (Amts) in A. oryzae were identified using genomic analysis. Four of the identified ammonium transporter genes, aoamt1-aoamt4, showed similarity in deduced amino acid sequences to the proteins in the ammonium transporter/methylammonium permease (AMT/MEP) family. Transcriptional analysis showed that the expression of aoamt2 and aoamt3 was ammonium-dependent, and was highly upregulated under ammonium-limited conditions. Their functional roles are characterized by genetic perturbations. The gene disruption and overexpression of aoamt3 indicated that the protein encoded by it was a crucial ammonium transporter associated with nitrogen metabolism and was required for filamentous growth. Compared with the wild type, the aoamt3-overexpressing strain showed superior growth performance, high biomass yield, and low glucose consumption. These results shed light on further improvements in the production of potent bioproducts by A. oryzae by manipulating the ammonium uptake capacity and nitrogen metabolism.


Introduction
Nitrogen is a crucial nutrient element for all living organisms, involved in the biosynthesis of basic biomolecules, including proteins, nucleic acids, and carbohydrates, and plays a role in the secondary metabolism of several fungi [1]. Nitrogen metabolism involves the incorporation of inorganic nitrogen into organic compounds to further synthesize the abovementioned molecules. The metabolic pathways involved include nitrogen transport and assimilation, resulting in glutamic acid and glutamine production [2]. Glutamic acid plays a central role in the nitrogen metabolism in cells. Generally, microorganisms have a mechanism called nitrogen metabolite repression (NMR) to choose the best nitrogen source(s) and prevent the use of non-preferred nitrogen. In filamentous fungi, NMR is manipulated during growth on preferred nitrogen sources by the global nitrogen transcription factor (AreA) [2]. It is considered the major activator involved in utilizing alternative nitrogen duced amino acid sequences of these aoamt genes were designated as Aoamt1 (OOO04540.1), Aoamt2 (OOO09170.1), Aoamt3 (OOO 606.1), and Aoamt4 (OOO14013.1). Based on amino acid sequence similarity analysis, the identified proteins were categorized as AMT/MEP family members containing the ammonium transporter family domain (PF00909). The amino acid sequences of Aoamt1 and Aoamt2 shared 89% (93% similarity) and 87% identity (92% similarity) with the MeaA and MepA proteins of A. nidulans, respectively. The amino acid sequences of Aoamt3 and Aoamt4 displayed 70% (85% similarity) and 86% identity (91% similarity) with MepB and MepC of A. nidulans, respectively. Among these, Aoamt1, Aoamt2, and Aoamt3 contain 11 transmembrane helices with an N (out) -C (in) topology ( Figure 1) based on the TMHMM program [24] in agreement with the conserved characteristic of other fungal ammonium transporters [18,25,26]. However, Aoamt4 is a distinctive AMT/MEP that contains nine transmembrane helices with an N (out) -C (in) topology. Based on the maximum-likelihood method, a phylogenetic tree of Aoamt1, Aoamt2, Aoamt3, and Aoamt4 of A. oryzae and other members of the AMT/MEP family of yeasts and fungi, including 79 sequences from 28 species, is shown in Figure 2. The consensus tree, measured by bootstrap analysis of 500 random sequence replicates, revealed that the yeast and fungal AMT/MEP sequences were grouped into four clusters (Clusters I-IV). The amino acid sequences of A. oryzae BCC 7051 were clustered based on the phylogenic results and were distributed into four clusters. Aoamt1, Aoamt2, Aoamt3, and Aoamt4 are presumed to be MeaA-like, MepA-like, MepB-like, and MepC-like proteins, respectively. Aoamt1 belonging to Cluster I was highly similar to MeaA (XP_680732.1), Ump1 (XP_011390849.1), and Mep1 (NP_011636) and Mep3 (NP_015464.1) of A. nidulans [15], Ustilago maydis [27], and Saccharomyces cerevisiae [28], respectively. The Aoamt2 sequence was similar to that of the protein members in Cluster II, which were discovered in A. nidulans (MepA; XP_658785.1) [15], U. maydis (Ump2; XP_011392234.1) [27], and two species of yeast-like fungi, S. cerevisiae (Mep2; NP_014257.1) [28] and Candida albicans (Mep2; XP_713353) [29]. Aoamt3 was categorized in Cluster III. The protein members of this cluster were identified in 13 fungal strains and one yeast strain. Interestingly, Aoamt4, which was categorized in Cluster IV, was found particularly in Aspergillus spp. Members of this cluster rendered a unique characteristic, in which the length of amino acid sequences was shorter than that of other clusters resulting in nine transmembrane helices, as illustrated in Figure 1. Alignment of the deduced amino acid sequences of the four putative ammonium transporters of A. oryzae BCC 7051 indicated that Aoamt4 shared only 34-40% identity and 52-58% similarity with Aoamt1, Aoamt2, and Aoamt3. This result suggests that Cluster IV is more divergent than Clusters I, II, and III, which may have resulted from an early duplication independently lost in several fungal lineages as previously described [19]. Our data showed that AMT/MEP proteins in each cluster of Aspergillus spp. were varied in length and composition of amino acid residues in the C-terminal region (CTR). The CTR has been proposed as a regulatory region for ammonium transporters. So far, the structure-function relationship of this region has been reported. The crystal structure of plant (Arabidopsis thaliana) and prokaryotic (Archaeoglobus fulgidus) ammonium transporters suggests that the C-terminus interacts physically with the cytosolic loops of neighboring subunits in a phosphorylation-dependent manner [30,31]. In addition, the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation [32][33][34]. Therefore, the variable length and amino acid composition of this region may affect the mechanism of action or transform the transporter into an ion channel and regulate the protein mediated by the CTR. Phylogenetic dendrogram of the ammonium transporters of A. oryzae BCC 7051, yeasts, and fungi. The number over the nodes represents the bootstrap coefficient calculated from 500 replicates. Four AMT/MEP clusters, Cluster I, Cluster II, Cluster III, and Cluster IV, are highlighted in red, blue, orange, and green, respectively. (♦) indicates ammonium transporter sequence obtained from A. oryzae BCC 7051. The phylogenetic tree was constructed by the maximum-likelihood method using MEGA11 software.
The conserved Aoamts motifs of A. oryzae BCC 7051 were identified by multiple sequence alignment of Aspergillus AMT/MEP proteins. Three consensus motifs, the signature motif (DxAGGxPVxIxSG), twin histidine (twin-his), and GxxxG motifs, were present in all clusters, as shown in Figure 3. It has been postulated that the signature motif might be a region conferring a particular feature in the Aspergillus AMT/MEP family. The twin-his motif contained two conserved histidine residues with six non-specific amino acid residue (x) intervals. This core structure has also been proposed as a regulatory region responsible for the function and substrate conductance by mediating substrate deprotonation in AMT/MEP pores [35][36][37]. Together, the twin-his motif may have a significant impact on the kinetics of the AMT/MEP family transporters and a deleterious effect on selectivity, resulting in a switch from a specific transporter (NH4 + or NH3/H + ) to an unspecific ion channel (K + ) [26,35,38]. However, the signaling mechanisms involved remain unclear.
The GxxxG conserved motif has been proposed to be another regulatory region involved in protein folding that is essential for membrane channel formation [39,40]. Accordingly, this motif may play a critical role in regulating transporter pore size and is vital for generating the correct architecture for substrate binding and transport. The sequence logo was generated by WebLogo 3 (http://weblogo.threeplusone.com/, accessed on 11 March 2022) [41], showing signature features, including twin-his, GxxxG, MFS-like, and InDel motifs as indicated by blue, red, yellow, green, and pink boxes, respectively. The height of each letter is proportional to its frequency of occurrence. Hydrophobic residues are indicated by black letters. Acidic and basic amino acids are represented by red and blue letters, respectively. Polar and neutral residues are depicted by green and purple letters, respectively. "x" in the twin-his motif is defined as a non-specific amino acid residue. The number of amino acids (aa) in each cluster is shown.
As shown in Figure 3, a major facilitator superfamily-like (MFS-like) motif with highly conserved amino acid residues of "GAVAERGR" (green boxes) was present only in Clusters I and II of the Aspergillus AMT/MEP family. This motif has been proposed to play a role in the translocation of ammonium across the membrane [16]. Amino acid substitution of the MFS-like motif found in Clusters III and IV presumably indicates an alternative conformation of the ammonium transporter to regulate its activity. Based on these results, we hypothesized that the MFS-like motif may refer to the evolutionary divergence of the Aspergillus AMT/MEP family. Multiple sequence alignment of the Aspergillus AMT/MEP members revealed short insertion/deletion residues (InDel motifs), in which InDel motifs 1 and 3 are present in Cluster III, while Indel motif 2 is found in Cluster IV ( Figure 3, pink boxes). These conserved InDel motifs are observed only in the AMT/MEP members of Aspergilli, which may be involved in the function of the ammonium transporter.

Differential Expression of Ammonium Transporter Genes (Aoamts) in A. oryzae by Ammonium Treatment
Elucidating the differentially expressed genes (DEGs) involved in ammonium transport provides insights into how A. oryzae cells respond to the presence of ammonium. The transcriptional response of aoamt genes to ammonium-containing conditions was investigated in relation to ammonium uptake by fungal cells. After transferring the logarithmic culture of A. oryzae grown in modified Czapek Dox (mCD) medium to nitrogen-lacking medium followed by medium containing ammonium at different concentrations (1 mM and 20 mM NH 4 Cl), the residual ammonium concentrations in the culture broth at different cultivation times were quantified. The ammonium concentration declined to an undetectable level after transferring the culture to a 1 mM NH 4 Cl-containing medium for 1 h ( Figure S1). The ammonium deficient stage was prolonged to 8 h on using 20 mM NH 4 Cl. Differential activation of aoamt genes during cultivation in the presence of 1 mM and 20 mM ammonium was observed and the transfer point of the cultures was used as a control (0 h). As shown in Figure 4, the transcript levels of the four aoamt genes were significantly affected by ammonium. Gene expression profiles can be divided into two groups: ammonium-sensitive and non-sensitive genes. Transcript analysis showed that aoamt2, aoamt3, and aoamt4 expressions were sensitive to ammonium exposure. The expression level of aoamt2 was detectable in the presence of ammonium and was significantly upregulated when ammonium was deficient, in contrast to the ammonium starvation condition. This was clearly observed after transferring the culture to a 1 mM NH 4 Cl-containing medium for 8 h ( Figure 4A). The transcript level of aoamt3 was also upregulated, similar to the aoamt2 expression profile. However, the relative expression of aoamt3 was maintained at an equivalent level during starvation, which was clearly observed in both media containing different NH 4 Cl concentrations ( Figure 4A,B). These results do not coincide with the previous findings for A. nidulans, in which the mepA (aoamt2-like) expression continually increased when encountering ammonium starvation, and the expression of mepB (aoamt3-like) was detected only under complete nitrogen starvation conditions [19]. As shown in Figure 4, the ammonium starvation periods of the cultures using 1 mM and 20 mM NH 4 Cl were different after cultivation for 1 and 8 h, respectively. At the defined starvation stage, the different transcription profiles of aoamt2 and aoamt3 were found in both cultures. These results indicate that gene expression is not only dependent on the starvation stage but is also indirectly influenced by cultivation time, during which metabolic alteration might occur along with growth development, especially on prolonged cultivation (ammonium and other nutrient starvation). Moreover, it was found that the expression of aoamt4 decreased during ammonium starvation. However, this result did not coincide with the mepC (aoamt4-like) of A. nidulans, whose expression increased under ammonium starvation conditions [19]. Our results revealed that the Aoamt4 protein encoded by aoamt4 is an ammonium transporter required for cell growth and metabolism that is specific to ammonium availability.
It was found that the transcript levels of aoamt1 in the cultures grown under ammoniumavailable and ammonium-limited conditions were not significantly different. However, a slight increase in aoamt1 expression was observed under nitrogen starvation conditions. It is likely that this gene does not participate directly in ammonium uptake. This result was similar to that previously described for A. nidulans, in which meaA (aoamt1-like) was constitutively expressed in the presence and absence of ammonium [19]. Our study revealed the differential expression responses of aoamt genes in A. oryzae to ammonium-sufficient, ammonium-deficient, and ammonium-starvation conditions. The two ammonium-induced genes in A. oryzae, aoamt2, and aoamt3 were selected for further study. oryzae. Expression analysis of aoamt1, aoamt2, aoamt3, and aoamt4 genes of the wild-type cultures grown in modified Czapek Dox (mCD) medium in the presence of 1 mM NH 4 Cl (A) and 20 mM NH 4 Cl (B) was carried out by RT-qPCR. Total RNA was extracted from the cultures after medium transfer for 0, 1, 8, and 10 h (black, gray, white, and horizontal bars, respectively). The expression level of each gene at 0 h (black bars) was adjusted to 1. Different letters (a, b, c, and d) above the bars indicate a statistically significant difference in the transcript levels of each gene at various time points analyzed by Duncan's multiple range test (MRT) (p-value < 0.05). The mean ± standard deviation (mean ± SD) of the expression level is derived from triplicate analysis.

Assessing the Role of Ammonium Transporters in Phenotypic Growth on Solid Medium and Nitrogen Metabolism of A. oryzae
To study the role of Aoamt protein in nitrogen metabolism, particularly in relation to ammonium transporting capacity and ammonium assimilation to glutamic acid/glutamine biosynthesis, genetic perturbation of aoamt2 and aoamt3 was performed. For gene disruption, the aopyrG auxotrophic marker was used for gene replacement with homologous recombination resulting in the prototrophic aoamt disruptants. The genetic modification of both transporter genes was verified by reverse transcription-polymerase chain reaction (RT-PCR) ( Figure S2). The growth phenotypes of the disruptant strains cultivated on solid agar media containing various NH 4 Cl concentrations (1, 5, 10, and 20 mM) were assessed and compared to that of the recipient strain (prototroph). As seen in Figure 5, the aoamt2 and aoamt3 disruptant strains showed defective growth on mCD agar, and the increase in ammonium concentration did not support aerial growth development, in contrast to previous studies. The deletion of amt3 and mepB (aoamt3-like) genes in Schizosaccharomyces pombe and A. nidulans, respectively, had no effect on cell growth at a wide range of ammonium concentrations tested [19,42]. Significantly, the loss of aoamt3 gene function led to severe growth retardation, which is different from the observations in ∆aoamt2 growth. The existing function of the Aoamt3 transporter may explain the survival of the aoamt2 strain in ammonium. We suggest that the Aoamt3 transporter encoded by the aoamt3 gene is responsible for the ammonium transport system in A. oryzae, which is significantly involved in the ammonium transport capacity required for cell growth. It seems that other ammonium transporters cannot compensate for the activity of Aoamt3 in the uptake of extracellular ammonium. These results also suggest that Aoamt2 might not mediate ammonium transport. Notably, only 1 mM NH 4 Cl was sufficient to differentiate the phenotypic growth of the AoT16 recipient, and ∆aoamt2 and ∆aoamt3 strains on a solid agar medium. Therefore, this concentration was used in subsequent experiments.
Using RT-qPCR, the transcriptional profiles of genes involved in the nitrogen metabolism, namely aoamts, aogdhA, aoglnA, aogltA, aocrnA, and aoareaA, of the disrupted and overexpressed strains were investigated after transferring the cultures to the medium broth containing 1 mM NH 4 Cl (Figures 6 and 7). Ammonium deficient and starvation conditions for all tested strains were defined at 1 and 8 h of cultivation, respectively (Figure S1B-F). Compared with the recipient strain, upregulation of the aoamt3 gene was found only in the ∆aoamt2 strain under ammonium-deficient condition (gray bar in Figure 6A). Under starvation condition, the growth of the ∆aoamt2 strain was also sustained because of the expression of genes involved in glutamic/glutamine biosynthesis and transcription factor AreA in regulation of nitrogen-associated metabolism (gray bars in Figure 6B). It was observed that the expression profiles of all tested genes were upregulated under deficient condition and downregulated equivalent to or lower than the expression level at 0 h (starvation condition) ( Figure S4A). Defect in the aoamt3 gene led to significant repression of all genes compared with that found in the AoT16 strain (white bars in Figure 6A,B and Figure S4B), particularly under ammonium starvation condition, leading to a lack of growth ( Figure 5). This evidence confirms the importance of the aoamt3 gene encoding Aoamt3 in ammonium uptake for cell growth and metabolism. The mepB (aoamt3-like) gene deletion in S. pombe showed no detectable effect on cell growth at all the ammonium concentrations tested [42]. The function of the aoamt3 gene involved in ammonium transport was explored using gene disruption analysis. Gene complementation studies are also a potent approach for verifying gene function.   The overexpression of aoamt2 and aoamt3 was investigated at different time points during cultivation. RT-qPCR analysis showed constitutive expression of both genes in the oeaoamt2 and oeaoamt3 cultures as a result of the control of toxA promoter ( Figure 7B,C). Interestingly, the expression of the aoamt3 gene in the oeaoamt3 strain was strongly upregulated, particularly under ammonium starvation condition ( Figure 7C) compared with the AoT16 recipient ( Figure 7A). It has been suggested that aoamt3 is a key gene involved in nitrogen metabolism. It is likely that there was a cooperative regulation of several genes involved in ammonium transport at least at the transcription level to sustain cell growth and metabolism, that is aoamt1-4, aoglnA, aogltA, aocrnA, and aoareA. Reduced expression of the aogdhA gene was observed in the oeaoamt3 strain grown under ammonium starvation condition, which may explain by the metabolic function of an alternative route of glutamic acid biosynthesis via the activity of AogltA [2]. Therefore, our results suggest the significance of the aoamt3 gene, which is expressed in all stages of ammonium existing cultivation. The encoded protein transporter contributes to ammonium uptake. In contrast, the absence of the aoamt3 gene resulted in severe growth defects. Taken together, the Aoamt3 transporter encoded by the aoamt3 gene plays a significant role in ammonium transport and nitrogen metabolism in A. oryzae.

Effect of Aoamt3 Overexpression of on Cell Biomass Production of A. oryzae
From an industrial perspective, more speculation was then given to gene overexpression and the utilization of simple and inexpensive nitrogen sources to investigate the growth phenotypic alteration in terms of biomass production. The increase in the uptake capacity of basic and cheap nitrogen sources, such as ammonium and mixed nitrogen into cells with efficient glutamic acid biosynthesis, is a prospect for enhanced biomass production that would be beneficial in the development of the fungal production process, particularly in the production of growth-associated metabolites of a target. Using a mixture of nitrogen sources (NH 4 Cl and yeast extract) for submerged cultivation, the fungal growth and biomass titer of the oeaoamt3 strain in the early phase were comparable to those of the wild type ( Figure 8). However, biomass production was sustained and was higher than that of the wild type as clearly observed in the stationary phase. The maximum biomass titer was observed in the oeaoamt3 culture grown for four days. Moreover, the period of complete glucose consumption of the oeaoamt3 strain was prolonged compared with that of the wild type. This may be a result of constitutive overexpression of aoamt3 leading to a continual increase in biomass until the starvation stage. The growth parameters of the oeaoamt3 as well as the wild type strains were calculated from the cultures grown for two days, as shown in Table 1. Even though the biomass titer and productivity of both strains were not significantly different, the glucose consumption and glucose consumption rate of the overexpressing strain reduced by 26 and 24%, respectively, and the biomass yield increased by 24%. These results indicate that Aoamt3 is an attributive transporter for ammonium uptake, which is involved in cell growth and biomass production. Therefore, overexpression of aoamt3 increases the ammonium transport capacity to sustain cell growth and biomass production with less glucose consumption.

Echocardiography
The echocardiographic assessment of left ventricular improvement is based on data obtained at rest and during a repeated short-term, discontinued LVAD support or reduced LVAD speed, while the patient is optimally anticoagulated [4,5,7,8,27,41,[44][45][46][47]. Ideally, such testing at low LVAD speeds should be repeated regularly to assess the underlying cardiac function, to allow for optimization of the LVAD unloading, and to guide the pharmacologic management of patients to promote reverse cardiac remodeling. Pulsatileflow LVADs allow for the assessment of native heart function during complete pump stops, as the inflow and outflow valves prevent regurgitation of blood from the aorta to the left ventricle during device deactivation [20,21,27,41,47,48]. However, in the case of continuous-flow devices, a speed reduction can result in regurgitant blood volume flowing from the aorta to the left ventricle, making the assessment of native left ventricular function less reliable; hence, it is important to identify the reduction in pump speed at which there is no forward or back flow (zero net flow) [7,8,20,29,41,44,49]. Another strategy is to temporarily occlude the outflow graft with a balloon which effectively prevents the back flow (i.e., regurgitant flow) during the speed turn-down [50,51].
The LVAD speed required to achieve a zero net flow varies depending on the type of the device [5,41,46,52]. George et al. performed a prospective study on the blood flow across the HeartMate II™ LVAD (Thoratec Corporation, Pleasanton, CA, USA) in patients with idiopathic dilated cardiomyopathy [46]. After ensuring an INR of ≥2.0, echocardiographic assessment of the left ventricle and peripheral hemodynamics were measured serially at three device speed settings: at a baseline device speed, 15 min after ) represent the AoT16 and oeaoamt3 strains, respectively. Biomass titer and residual glucose concentration are shown by solid and dashed lines, respectively. "*" above the symbols indicates a significant difference in residual glucose concentrations and biomass titers, respectively of the oeaoamt3 and AoT16 strains (p < 0.05). Experiments were performed in triplicates.

Microbial Strains and Cultivation
The wild type A. oryzae strain BCC 7051 was used for expression analysis of the putative aoamt genes identified. A pyrG-deficient strain (auxotroph) of A. oryzae was used to generate recombinant strains by gene disruption and overexpression. The pyrG gene complementation strain (prototroph) was used as a recipient for comparison with the recombinant strains in terms of growth phenotypes on solid cultivation and gene expression. Czapek Dox (CD) medium (BD Difco) was used to maintain the fungal cultures. The deficient strain was cultivated by supplementation with 5.0 g/L uridine and 0.2 g/L uracil. For submerged cultivation, spore inoculum was prepared by cultivating mycelial cells on sterile rice for 5 days. Spores were harvested by suspension with 0.05% (v/v) Tween 80 solution, filtered using Miracloth (MerckMillipore, Darmstadt, Germany), and centrifuged. The spore suspension (10 8 spores) was inoculated into 50 mL of appropriate culture medium.

Identification of Aoamt Homologs of A. oryzae
To obtain the putative genes encoding ammonium transporters of A. oryzae (aoamts), a BLAST search against the genome sequence of strain BCC 7051 [23] was performed using the NCBI database (http://www.ncbi.nlm.nih.gov/, accessed on 30 November 2021) with the amt sequences of Aspergillus flavus as queries [43]. The cDNA cloning of these putative genes was carried out by RT-PCR using 50 ng of total RNA from A. oryzae BCC 7051 as a template, specific primer sets (Table S1), and the Super Script III One-Step RT-PCR with Platinum Taq DNA Polymerase (Invitrogen, Waltham, CA, USA). The amplified cDNA fragments were purified using the QIAquick Gel Extraction kit (Qiagen, Germany) and subcloned into the TOPO 2.1 vector (TOPO ® TA cloning ® kit, Invitrogen). Sequence analysis was performed using by Macrogen (Seoul, South Korea). The four cDNA sequences obtained, aoamt1, aoamt2, aoamt3, and aoamt4, were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov, accessed on 30 March 2022) under the accession numbers ON098152, ON098153, ON098154, and ON098155, respectively.

Transmembrane Topology Prediction
To analyze the transmembrane protein topology of the individual A. oryzae BCC 7051 ammonium transporters, the protein sequences were predicted using TMHMM 2.0 [24] based on a hidden Markov model. For predicting results, the program presents the number and location of predicted transmembrane helix features involved in the charge, polarity, and hydrophobicity of each protein sequence.

Analysis of Phylogenetic Tree and Conserved Motifs of the Aoamt Proteins
Full-length amino acid sequences of the ammonium transporters of A. oryzae BCC 7051, yeast, and other filamentous fungi were retrieved from the NCBI database using BLASTP. Multiple sequence alignment of the amino acid sequences was performed to calculate the genetic distance using the Clustal algorithm with the default parameter settings [44]. For phylogenetic analysis, the alignment results were imported into the Molecular Evolutionary Genetics Analysis software (MEGA, version 11) [45]. The optimal model of phylogenetic relationship was determined using the Find Best Protein Model' option provided by MEGA. Subsequently, the evolutionary tree was generated using the maximum-likelihood method with a different amino acid frequency (+F) model [46], in which a discrete gamma distribution (+G) with five rate categories is under the assumption that a certain fraction of sites is evolutionarily invariable (+I). These sites were selected as the best-fit substitution model for the AMT/MEP family sequence evolution. Additionally, all positions containing gaps and missing data were eliminated from the dataset (complete deletion). The confidence intervals of the phylogenetic trees were tested using the bootstrap statistical method [47] with 500 resampling iterations, in which the initial trees were automatically obtained.
The conserved motifs among the AMP/MEP proteins of Aspergilli were identified by comparing amino acid sequences between these proteins and protein family databases using Pfam release 35.0 at the Sanger Centre, UK (http://pfam.xfam.org/, accessed on 4 February 2022) [48]. The functional regions of the query proteins were searched against the domains Pfam-A and Pfam-B using a default E-value cut-off of 1.0, which was defined based on expert knowledge, sequence similarity, other protein family databases, and the ability of HMM profiles to correctly identify and align the members. Conservative sequence motifs were extracted from the amino acid sequence alignment using the multiple sequence alignment program from the Clustal Omega package (http://www.clustal.org/omega/, accessed on 14 February 2022) [49].

Plasmid Construction, Fungal Transformation, and Verification of Transformants
A scheme summarizing the plasmid construction is presented in Figure S3. All plasmids were constructed using PCR and DNA assembly in yeast cells [50]. To construct the disruption plasmid, pDaoamt2 or pDaoamt3 ( Figure S3A), the specific 5 and 3 DNA fragments with homologous sequences of individual aoamt genes were amplified using the genomic DNA template of A. oryzae BCC 7051, Platinum TM Tag DNA polymerase Hi-Fidelity (Invitrogen) and overlapping primer sets (Table S2). DNA assembly of the homologous fragments and linearized backbone plasmid BPO152-carrying aopyrG marker was performed in S. cerevisiae. Plasmids were extracted from the yeast transformants using the Zymoprep TM Yeast plasmid Miniprep I (Zymo Research, Irvine, CA, USA) and were then shuttled into E. coli cells. The constructed plasmids were confirmed using DNA sequencing. To generate the overexpression plasmids, poeaoamt2 and poeaomt3 ( Figure  S3B), the constitutive toxA promoter and nos3 terminator fragments were amplified from the plasmid pCT74 [51], and the targeted aoamt cDNAs were derived from the cloning plasmids using overlapping primers (Table S2). Then, DNA assembly of these fragments with the linearized backbone plasmid BPO152 was performed in yeast cells. The plasmid or DNA fragment (2-5 µg) was transformed into protoplast cells using the PEG-mediated method (PMT) [52]. Transformants were selected on CD medium after incubation at 30 • C for 3-7 d. Spore re-isolation was performed to obtain a pure culture. To verify gene disruption or overexpression, total RNA extracted from the fungal cultures was subjected to the Super Script III One-Step RT-PCR with Platinum Taq DNA Polymerase (Invitrogen, Waltham, CA, USA) using specific oligonucleotide primers (Table S3).

Submerged Cultivation for Gene Expression Analysis and Determination of Ammonium Consumption
To study differential gene expression and ammonium consumption, fungal cultivation was performed with some modifications from the published method [18], in which the logarithmic culture was transferred to ammonium-lacking medium, followed by ammoniumcontaining medium at different concentrations. The spore suspension (10 8 spores) was inoculated into a modified CD (mCD) medium (10 g/L (w/v) glucose, 5 g/L (w/v) yeast extract, 1 g/L (w/v) K 2 HPO 4 , 0.5 g/L (w/v) MgSO 4 .7H 2 O, 0.5 g/L (w/v) KCl, 15 mg/L (w/v) FeCl 3 ·7H 2 O, 10 mg/L (w/v) MnSO 4 ·H 2 O, and 7.5 mg/L (w/v) ZnSO 4 ·7H 2 O) with initial pH 4.5 as a basal medium. The fungal culture was carried out at 30 • C with shaking for 16 h. Mycelial cells were harvested, washed twice with sterile distilled water, and resuspended in mCD medium without nitrogen (nitrogen-free medium) at 30 • C with shaking for 4 h. Then, the fungal mycelia were transferred to ammonium-containing medium by changing the nitrogen source in the basal mCD medium, in which the yeast extract was replaced with 1 or 20 mM NH 4 Cl (Sigma-Aldrich, St. Louis, MO, USA) as a sole nitrogen source and incubated at 30 • C with shaking. The culture samples were harvested at different time points.

RT-qPCR Analysis
Total RNA was extracted from harvested mycelial cells using a PureLinkTM RNA Mini kit (Invitrogen). Reverse transcription of total RNA to cDNA was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). RT-qPCR was conducted according to a previously described method [53] using the Luna ® Universal qPCR Master Mix (New England BioLabs, Hitchin, UK) and specific primer sets (Table S3). Relative quantification of gene expression was performed by normalization to 18S rRNA [54]. Individual experiments were independently performed in triplicates.

Radial Growth and Cell Biomass Measurements
To investigate mycelial growth on solid agar, a spore inoculum (5000 spores) of the recipient or disruptant strains was dropped onto basal agar media containing 1, 5, 10, and 20 mM NH 4 Cl and incubated at 30 • C for 3 days. Each experiment was performed with three technical replicates.
To determine biomass production of the oeaoamt3 strain, cultivation was performed by inoculating spores into a semi-synthetic medium, SM (40 g/L (w/v) glucose, 0.  [55]. Mycelial samples were harvested at different time points during cultivation, collected by filtration with gentle suction, washed with sterile water, and dried in a hot-air oven until a constant weight was obtained. The biomass titer was represented as the dry cell weight per liter (DCW/L).

Determination of Residual Glucose and Ammonium Concentrations
The residual glucose concentration in the fermented broth was measured using highperformance liquid chromatography (HPLC). The analysis was performed at 60 • C with a flow rate of 0.6 mL/min on an Aminex HPX-87H column (9 µm particle size, 300 × 7.8 mm, Bio-Rad, Hercules, CA, USA), and 18 mM sulfuric acid was used as the mobile phase. A calibration curve of glucose concentration was generated to calculate the residual glucose concentration in the fermented broth samples.
Ammonium concentration was determined calorimetrically using the phenol-hypochlorite method with some modifications from a published method [56]. The filtration broth (0.1 mL) of the culture was thoroughly mixed with 0.5 mL of phenol (5%, v/v) and nitroprusside (0.005%, w/v), and then 0.5 mL alkaline hypochlorite solution (0.5% (w/v) sodium hydroxide, and 0.8% (w/v) sodium hypochlorite) was added. The mixture was incubated at 37 • C for 20 min and subjected to measure the colorimetric absorbance was measured at 625 nm. A calibration curve was obtained by using the ammonium chloride at various concentrations (0.1-0.5 mM was the standard). The ammonium concentration in the culture broth sample was reported as millimolar (mM).

Statistical Analyses
Statistical analysis was performed using SPSS version 11.5 software for Windows (SPSS Inc., Chacago, IL, USA). Three replicates were established for each experiment. The results are presented as mean values with standard deviation (mean ± SD). The data were subjected to one-way analysis of variance (ANOVA) and compared using Duncan's multiple range test (DMRT) with a statistically significant difference (p-value < 0.05) for multiple data sets or independent sample t-test at a 95% confidence interval for two sample means.

Conclusions
Four ammonium transporters of A. oryzae (aoamt1-4), belonging to the AMT/MEP family, were identified and characterized. Differential expression analysis of these genes indicated that the expression of aoamt2, aoamt3, and aoamt4 was ammonium-dependent. Aoamt2 was highly upregulated in ammonium-deficient, whereas the upregulation of aoamt3 was prolonged under starvation conditions. The role of these transporters in nitrogen metabolism was explored by gene disruption and overexpression, which showed that Aoamt3 is the major ammonium transporter required for cell growth and has an impact on the nitrogen metabolism of A. oryzae. Moreover, overexpression of aoamt3 enhanced the overall biomass production with a lower glucose consumption rate than that of the wild type. These results provide insight into the molecular mechanisms of nitrogen metabolism underlying the ammonium transport capacity of Aoamt3 in A. oryzae, which offers rational improvement in cell biomass production by either physiological control or synthetic biology approaches.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.