The arabinose transporter MtLat-1 is involved in hemicellulase repression as a pentose transceptor in Myceliophthora thermophila
Biotechnology for Biofuels and Bioproducts volume 16, Article number: 51 (2023)
Filamentous fungi possess an array of secreted enzymes to depolymerize the structural polysaccharide components of plant biomass. Sugar transporters play an essential role in nutrient uptake and sensing of extracellular signal molecules to inhibit or trigger the induction of lignocellulolytic enzymes. However, the identities and functions of transceptors associated with the induction of hemicellulase genes remain elusive.
In this study, we reveal that the l-arabinose transporter MtLat-1 is associated with repression of hemicellulase gene expression in the filamentous fungus Myceliophthora thermophila. The absence of Mtlat-1 caused a decrease in l-arabinose uptake and consumption rates. However, mycelium growth, protein production, and hemicellulolytic activities were markedly increased in a ΔMtlat-1 mutant compared with the wild-type (WT) when grown on arabinan. Comparative transcriptomic analysis showed a different expression profile in the ΔMtlat-1 strain from that in the WT in response to arabinan, and demonstrated that MtLat-1 was involved in the repression of the main hemicellulase-encoding genes. A point mutation that abolished the l-arabinose transport activity of MtLat-1 did not impact the repression of hemicellulase gene expression when the mutant protein was expressed in the ΔMtlat-1 strain. Thus, the involvement of MtLat-1 in the expression of hemicellulase genes is independent of its transport activity. The data suggested that MtLat-1 is a transceptor that senses and transduces the molecular signal, resulting in downstream repression of hemicellulolytic gene expression. MtAra-1 protein directly regulated the expression of Mtlat-1 by binding to its promoter region. Transcriptomic profiling indicated that the transcription factor MtAra-1 also plays an important role in expression of arabinanolytic enzyme genes and l-arabinose catabolism.
M. thermophila MtLat-1 functions as a transceptor that is involved in l-arabinose transport and signal transduction associated with suppression of the expression of hemicellulolytic enzyme-encoding genes. The data presented in this study add to the models of the regulation of hemicellulases in filamentous fungi.
Non-edible plant biomass is recognized as a potential sustainable source for production of biofuels and commodity chemicals in biorefinery processes . However, the costs involved in conversion of insoluble plant lignocellulose into fermentable sugar remain a significant barrier to commercialization . Filamentous fungi are able to secrete a series of hydrolytic enzymes to synergistically deconstruct plant polysaccharides. Several fungi, such as Trichoderma reesei, Myceliophthora thermophila, and Penicillium oxalicum, have been developed as the platforms to produce cellulolytic enzymes for industrial applications [3,4,5].
In fungi, lignocellulolytic enzyme-encoding genes are regulated by the complex signaling networks. Sugar transporters play an essential role in substrate uptake and sensing extracellular signal molecules to inhibit or trigger enzyme induction [6,7,8]. The transporter with signaling activity is called as transceptor. In Neurospora crassa, cellodextrin transporters CDT-1 and CDT-2, and cellobionic acid transporter CLP1 are involved in the induction of expression and secretion of cellulases. The function of CDT-1 and CDT-2 on signal transduction for the activation of cellulolytic gene expression is not dependent on the transporting activities [8,9,10]. In T. reesei, a dual cellobiose/glucose transporter Stp1 is involved in the carbon catabolite repression response (CCR) and repressed induction of cellulase and hemicellulase genes . Similarly, glucose high-affinity transporters HGT-1/2 inhibit cellulase gene expression by both CCR and the cyclic adenosine monophosphate-protein kinase A pathway in N. crassa . Some proteins that possess multiple transmembrane domains but have lost the capacity for sugar transport, also serve as sugar sensors, such as Saccharomyces cerevisiae Snf3 and Rgt2 , and N. crassa RCO3 [12, 13]. Snf3 and Rgt2 sense extracellular low and high glucose levels, respectively, and transduced molecular signal with cytoplasmic C-terminal tail to mediate a glucose signaling pathway . However, the transceptor involved in induction of hemicellulase genes remains to be identified and characterized.
Fungal cellulase and hemicellulase gene expression is tightly regulated at the transcriptional level upon appropriate nutrient sensing . Several essential transcription factors have been studied, mostly in saprobic fungi, including CreA/CRE-1, XYR1 (XLR-1/XInR), CLR-1/A, and CLR-2/B (ManR) [15,16,17,18,19]. CLR-2 and its orthologs are the major regulators of genes encoding cellulolytic enzymes in N. crassa, Aspergillus nidulans, and P. oxalicum [18,19,20]. Transcription factors associated with hemicellulose deconstruction include Xyr1/XlnR, CreA/CRE-1, and Ara-1. The transcriptional activator XlnR/XYR1 is a major positive activator of the expression of hemicellulase genes in a number of filamentous fungi, although the enzymes that are actually regulated by it differ in different species. In T. reesei and Aspergillus niger [21,22,23], Xyr1/XlnR regulates both cellulase and hemicellulase genes, whereas in species such as N. crassa, M. thermophila, and Fusarium graminearum, XlnR homologs regulate xylanase expression and d-xylose utilization [16, 24, 25]. Similarly, in Fusarium oxysporum, a strain carrying a deletion of xlnR lacked transcriptional activation of structural xylanase genes and exhibited dramatically reduced extracellular xylanase activity . CreA/CRE-1 not only inhibits the expression of the major hemicellulase/cellulase genes, but also suppresses key sugar catabolic genes and sugar transporters in N. crassa and Aspergillus spp. [27, 28]. In addition, another transcription factor, Ara1, modulates the use of arabinose and arabinan in N. crassa , Magnaporthe oryzae  and T. reesei , while a different transcription factor (AraR) functions in an analogous manner in A. niger .
Myceliophthora thermophila is a thermophilic fungus known for its capability to efficiently secrete a complete set of thermostable carbohydrate-active enzymes [31, 32]. Recently, this fungus has been developed as an important platform for the production of industrial enzymes  and as a cell factory to produce biochemicals and biofuels by directly conversing plant biomass [5, 33,34,35]. A few lignocellulose degradation-related transcription regulators have been identified and characterized in this fungus, including XYR1, CRE-1, CLR-1/2, and CLR-4 [25, 36, 37]. We previously characterized the l-arabinose transporter MtLat-1 and demonstrated that MtLat-1 exhibits specific transport activity for l-arabinose . In the present study, we investigated the role of MtLat-1 in the repression of hemicellulase genes of M. thermophila. On the basis of comparative transcriptomic analysis and the assays of a non-transporting MtLat-1 mutant, we found that MtLat-1 functions as a transceptor, which is involved in l-arabinose transportation, sensing, and downstream signaling cascades in M. thermophila. In addition, we also demonstrated that Mtlat-1 expression is directly regulated by transcription factor MtAra-1, which was involved in l-arabinose release and metabolism.
The role of sugar transporter MtLat-1 in the growth of M. thermophila on l-arabinose
Our previous study revealed that sugar transporter MtLat-1 (Mycth_95427) showed a high uptake activity and specificity for l-arabinose . To determine the induction of Mtlat-1 on various carbon sources, we conducted comparative transcriptomic analysis of M. thermophila grown on plant biomass-derived monosaccharides, including d-glucose, d-xylose, and l-arabinose. When responding to l-arabinose, 26 putative sugar transporter genes (54% of the total sugar transporter genes in M. thermophila) showed high expression levels (RPKM > 20), including five putative glucose transporter genes (Mycth_112491, Mycth_2308157, Mycth_2295230, Mycth_109194, Mycth_108044), four putative glucose and pentose transporter genes (Mycth_2305795, Mycth_96047, Mycth_108924, Mycth_2302949), and two putative cellodextrin transporter genes (Mycth_114107 and Mycth_43941) (Fig. 1a and Additional file 1: Table S3), similar to observations in N. crassa, where the major sugar transporter genes were highly induced by l-arabinose . Consistent with its substrate specificity, dramatic induction of MtLat-1 (RPKM > 1000) was only observed on l-arabinose. In addition, previous kinetic property assay revealed that MtLat-1 has a great maximum velocity (Vmax) of l-arabinose uptake. Therefore, we were interested in the role of the MtLat-1 in the growth of M. thermophila on plant biomass-derived monosaccharides.
The Mtlat-1-null strain was constructed via homologous replacement with a neomycin resistance gene (neo)-inclusive cassette using CRISPR/Cas9 system . The correct deletion and recombination events in the resulting mutant were confirmed by PCR. When grown on d-glucose or the three most relevant hemicellulose side-chain sugars (d-xylose, l-arabinose, and d-galactose), the consumption rates of d-glucose, d-xylose, and d-galactose in the ΔMtlat-1 mutant were similar to those of the wild-type (WT) strain (Additional file 2: Fig. S1), but a clear retardation of l-arabinose consumption was observed in culture of strain ΔMtlat-1 (Fig. 1). To support these data, we conducted sugar uptake assays using the mycelia pre-induced by l-arabinose. The l-arabinose concentration in the culture supernatants was measured over time. Analysis of the initial uptake rate (20 min) indicated that l-arabinose uptake was reduced by approximately 15% in the ΔMtlat-1 mutant compared with that in the WT (15.4 nmol/min/mg_DCW) (Fig. 1d). In the lat-1 deletion strain of N. crassa, l-arabinose uptake was almost completely abolished . However, the deletion of MtLat-1 ortholog (Trire2_104072) in T. reesei did not cause an altered growth phenotype at high (1%) l-arabinose level, but led to a growth defect at low l-arabinose level .
Negative influence of MtLat-1 on the induction of hemicellulase in M. thermophila
In lignocellulolytic fungi, pentose (d-xylose and l-arabinose) can act as inducers of the expression of genes encoding plant biomass-degrading enzymes [39, 43]. We hypothesized that the MtLat-1 protein, showing l-arabinose-specific transport activity, participates in sensing and transducing an l-arabinose signal that is involved in the induction of hemicellulolytic enzymes in M. thermophila. To test this hypothesis, physiological phenotypes of the strain ΔMtlat-1 were assayed when it was grown on arabinan or xylan. Unexpectedly, the strain ΔMtlat-1 exhibited an approximately 14% increase in dry cell weight compared with the WT when grown on arabinan for 4 days (Fig. 2a), which was different from N. crassa, where lat-1 deletion attenuated the growth on arabinan . In addition, compared with the WT strain, the secreted protein, arabinanase and xylanase activities of the Mtlat-1 mutant increased by 108%, 149%, and 85%, respectively (Fig. 2b–d), indicating that the hemicellulase expression machinery was stimulated by disruption of Mtlat-1. However, when grown on xylan, the Mtlat-1 mutant was similar to the WT in the phenotypes, including biomass accumulation, protein production and hemicellulolytic activity (Additional file 2: Fig. S2), consistent with the specific induction of Mtlat-1 by l-arabinose.
To confirm that these phenotypes for growth on arabinan were attributable to the disruption of Mtlat-1 rather than some other unknown genetic mutations, we complemented the mutant ΔMtlat-1 mutant with an ectopic copy of Mtlat-1 under the control of the native promoter. The complementation strain Pn-Mtlat-1 exhibited completely restored l-arabinose uptake activity and showed similar phenotypes to the WT strain in protein secretion, and arabinanase and xylanase activities, when grown on arabinan (Additional file 2: Fig. S3). These results indicated that the l-arabinose transporter MtLat-1 negatively affects the production of hemicellulolytic enzymes when M. thermophila grown on arabinan.
Transcriptomic analysis of the Mtlat-1 deletion mutant on arabinan
To further investigate the effect of MtLat-1 on hemicellulase production, comparative transcriptomic profiles of the WT and ΔMtlat-1 strains grown in arabinan medium for 2 and 4 days, were determined using RNA-Seq analysis (Additional file 1: Table S2). Analysis of differential gene expression revealed that 617 genes were differentially expressed between the WT and ΔMtlat-1 strains after 4 days, of which 307 genes were significantly downregulated and 310 genes were markedly upregulated in strain ΔMtlat-1 at 4 days (Fig. 3a and Additional file 1: Table S4). Gene Ontology (GO) analysis of the up-regulated genes indicated that carbohydrate metabolic process, transmembrane transport, cellular amino acid metabolic process, l-arabinose metabolic process, carbohydrate transport and so on, were significantly enriched functional categories (Fig. 3b and Additional file 1: Table S6). Consistent with the rapid growth of strain ΔMtlat-1, seven putative sugar transporter genes (Mycth_2305795, Mycth_43941, Mycth_96047, Mycth_104673, Mycth_2299846, Mycth_38796, Mycth_54010) exhibited markedly up-regulated expression levels in ΔMtlat-1 compared with the WT, which is a prerequisite for efficient sugar utilization and mycelium growth. An analysis of the transcriptional profiles of Carbohydrate-Active enzymes (CAZyme) genes (GH62, GH51, GH43, GH11, GH10, and GH3) indicated that genes encoding hemicellulolytic enzymes were dramatically induced in strain ΔMtlat-1 relative to the WT (Fig. 3c, Additional file 2: Fig. S4A and Additional file 1: Table S5). Eight of nine genes encoding arabinanolytic enzymes exhibited significantly up-regulated expression levels, of which four (Mycth_98003, arabinofuranosidase; Mycth_83019, arabinofuranosidase; Mycth_55982, arabinofuranosidase; and Mycth_2303298, arabinanase) with RPKM values > 1000 were up-regulated by more than tenfold in strain ΔMtlat-1 compared with the WT when growth on arabinan for 4 days (Fig. 2b). In plant biomass, arabinan is the component of side-chains of pectin and hemicellulose . We also observed that four pectinolytic enzyme genes (Mycth_90594, pyl-1; Mycth_51766, pyl-2; Mycth_55717, pyl-3; and Mycth_55717, gh28-1) and three xylanolytic enzyme genes (Mycth_2301869, gh43-5; Mycth_104628, gh3-7; Mycth_89603, gh11-1) showed significantly increased expression levels in the ΔMtlat-1 mutant compared with the WT. In addition, the genes associated with the pentose catabolism were significantly up-regulated in ΔMtlat-1, including xylose reductase (Xr, Mycth_43671), l-arabinitol 4-dehydrogenase (Ard, Mycth_62052), xylitol dehydrogenase (Xdh, Mycth_2293953), l-xylulose reductase (Lxr, Mycth_2302811), and d-xylulose kinase (Xks, Mycth_67060), which might attributed to increased l-arabinose release resulting from the enhancement of hemicellulolytic enzyme activity in ΔMtlat-1.
A non-transporting MtLat-1 mutant also participates in the induction of arabinanolytic enzymes
In yeast species and some filamentous fungi, mutation to lysine of a conserved arginine residue (R to K) located in a cytoplasmic loop just preceding the fifth transmembrane domain of several hexose sensors, leads to the loss of substrate uptake activity, but constitutive glucose signaling [6, 11, 45, 46]. To determine whether MtLat-1 repressed the induction of arabinanolytic enzymes when the corresponding residue was altered, a mutant form of the protein, MtLat-1(R136K), was constructed. S. cerevisiae strain BSW5AP with a heterogenous arabinose metabolic pathway and native l-arabinose transporter gene deleted  was used for assay of substrate uptake. MtLat-1(R136K) fused to green-fluorescent protein (GFP) displayed correct plasma membrane localization of MtLat-1(R136K) in the recombinant BSW5AP strain (Fig. 4a). As expected, MtLat-1(R136K) could not support the growth of strain BSW5AP on l-arabinose, showing that the mutation abolished the transport function of MtLat-1 (Fig. 4b). When the mutant protein fused with GFP protein was introduced into the WT strain of M. thermophila, a GFP signal was detected in the cellular membrane of the fungal hyphae (Fig. 5a). Subsequently, the genes encoding MtLat-1 and MtLat-1(R136K) were separately overexpressed in the ΔMtlat-1 mutant under the control of the constitutive tef-1 promoter to generate the resultant strains Ptef-Mtlat-1 and Ptef-Mtlat-1(R136K), respectively. The initial l-arabinose uptake rate of strain Ptef-Mtlat-1(R136K) was lower than that of mutant Ptef-Mtlat-1 (Fig. 5b), but the two mutants displayed a similar final cell yield when grown on arabinan (Additional file 2: Fig. S5). Consistently, the secreted protein content and hemicellulolytic activities of the strain Ptef-Mtlat-1(R136K) reached the similar levels to those of mutant Ptef-Mtlat-1 (Fig. 5c–e). These data indicated that hemicellulase gene expression was correlated with the signaling function of MtLat-1, but not its transport activity. The results supported the hypothesis that MtLat-1 protein senses and transduces the l-arabinose signal, which controls the downstream components of the hemicellulase induction pathway.
Previous studies have shown that C-terminal cytoplasmic sequence extension of several transceptors related to the CCR trigger or the induction of cellulase was required for their interaction with a component of the molecular signal transduction pathway [11, 48]. To investigate the effects of deletion of the cytoplasmic C-terminal tail of MtLat-1 on the induction of hemicellulase, a truncation mutant was constructed by deleting the C-terminal extension peptide (63 amino acids) of MtLat-1M, and this variant was introduced into the ΔMtlat-1 strain. When grown on arabinan, the resultant strain Ptef-Mtlat-1M showed mycelium yield, secreted protein content, and hemicellulolytic activities similar to those of the strain Ptef-Mtlat-1(R136K), revealing that the C-terminal cytosolic region of MtLat-1 was dispensable for the function of the protein in transmitting the l-arabinose signal.
The expression of Mtlat-1 is directly regulated by transcription factor MtAra-1
To search for transcription factors essential for expression of Mtlat-1, we determined transcriptomic profiles of all transcription factor genes in M. thermophila when grown on mono-saccharides (d-glucose, d-xylose, or l-arabinose) or under starvation (no carbon) conditions. As shown in Fig. 6a, a total of 24 genes were significantly induced by monosaccharides, compared with starvation condition. Given that a high expression level of Mtlat-1 was specific to the presence of l-arabinose, we considered the four genes (Mycth_2121737; MtPdr-1, Mycth_53224; MtPdr-2, Mycth_46266; and Mycth_2300935) that were only highly induced by l-arabinose as the candidate regulators of Mtlat-1. Next, single-gene mutants of these genes were constructed and the expression level of Mtlat-1 in response to l-arabinose was detected by real-time quantitative PCR (RT-qPCR) assay. Three of the mutants showed no significant change in Mtlat-1 expression compared with the WT strain. However, the ΔMycth_2121737 strain showed dramatically decreased expression of Mtlat-1 (Fig. 6b). Mycth_2121737 is a zinc binuclear cluster [Zn(II)2Cys6] DNA-binding protein and its orthologs in N. crassa (Ara-1) and T. reesei (Ara1) were related to utilization of arabinan, l-arabinose, and d-galactose [14, 27]. Therefore, in this study, Mycth_2121737 was named as MtAra-1.
To investigate whether MtAra-1 directly regulates Mtlat-1 expression, we conducted electromobility shift assays (EMSAs), involving the DNA-binding domain (DBD) of MtAra-1 and the promoter region of Mtlat-1. A glutathione S-transferase (GST)-fused MtAra-1-DBD was expressed in and purified from Escherichia coli. Two probe fragments of the promoter region of Mtlat-1 (nucleotide positions − 396 to − 1 and − 860 to − 396, respectively) were amplified by PCR. In the EMSAs, the recombinant MtAra-1 bound the Mtlat-1 promoter regions in a typical protein concentration-dependent manner. Retardation occurred upon addition of 10 nM recombinant MtAra-1 protein (Fig. 6c).
Transcription factor MtAra-1 substantially affects l-arabinose release and catabolism
The deletion of Mtara-1 abolished growth of M. thermophila on l-arabinose (Fig. 6d), indicating that MtAra-1 was not related only to the expression of Mtlat-1. A comparative transcriptomic analysis revealed that the genes involved in pentose catabolism, including xr, lad, xdh, lxr, and xks, showed a significant decrease in expression levels in the ΔMtara-1 mutant compared with those in the WT strain when grown on l-arabinose (Fig. 6e). Six putative pentose transporter genes (Mtlat-1, Mycth_2305795, Mycth_96047, Mycth_2302949, Mycth_112491, and Mycth_108924) were also dramatically downregulated in ΔMtara-1 grown on l-arabinose (Additional file 2: Fig. S6). However, strain ΔMtara-1 grew similar to the WT on xylose (Additional file 2: Fig. S1B), indicating that the expression of the genes xr, xdh, and xks, which are shared by l-arabinose and d-xylose metabolism, was induced independently of MtAra-1 in the presence of xylose (Additional file 1: Table S2). In M. thermophila, Xlr1 is the main regulator of the expression of xylanolytic genes and the genes involved in xylose utilization . In filamentous fungi, Ard (l-arabinitol-4-dehydrogenase), also catalyzes the third reaction of the oxidoreductive catabolism of d-galactose. Consequently, we also found that the ΔMtara-1 strain showed a severely decreased rate of d-galactose consumption (Additional file 2: Fig. S2), consistent with observations in N. crassa and T. reesei [14, 27]. Unexpectedly, growth phenotype analysis indicated that Mtara-1 disruption significantly increased the d-glucose consumption rate of M. thermophila, indicating a complex regulatory network of d-glucose and pentose metabolism.
To further dissect the effects of the deletion, we compared protein secretion and the hemicellulolytic/cellulolytic activities of strains WT and ΔMtara-1 when grown on plant biomass. As shown in Fig. 7a, the ΔMtara-1 mutant showed a dramatical reduction in total secreted protein production, xylanase activity and arabinase activity compared with the WT strain when grown on arabinan. Similar results were also obtained with xylan (Additional file 2: Fig. S7A). However, only a slight effect on protein production and cellulase activity of strainΔMtara-1 was observed for growth on Avicel (cellulose microcrystal) (Additional file 2: Fig. S7B). Next, to further investigate the role of MtAra-1 in arabinan utilization, we transcriptionally profiled the response of the WT and ΔMtara-1 strains to arabinan induction for 4 h. In the presence of arabinan, the expression levels of 308 genes were significantly downregulated inΔMtara-1 compared with the WT (Additional file 1: Table S8). GO enrichment analysis revealed that the downregulated genes in the ΔMtara-1 mutant were mainly involved in l-arabinose metabolism, amino acid biosynthetic process, transmembrane transport, and arabinofuranosidase activity (Fig. 7b). When transcription profiles of CAZyme genes were analyzed, we found that all the genes encoding arabinanolytic enzymes exhibited markedly decreased expression levels in strain ΔMtara-1 compared with the WT (Fig. 7c), showing their high dependence on MtAra-1. However, only one gene involved in xylan degradation (Mycth_52904) was included among the downregulated genes, suggesting that MtAra-1 is mainly involved in inducing expression of arabinanolytic enzyme genes. The reduced secreted protein production and xylanase activity in the ΔMtara-1 mutant when grown on xylan may be attributable to the low purity of the xylan used.
Filamentous fungi are able to secrete a series of lignocellulosic enzymes to degrade the plant biomass to fermentable sugars, and have been exploited as the main source of commercial hydrolases for biotechnological applications . During the degradation of lignocellulose, the released mono/oligo-saccharides enter the cytoplasm via membrane sugar transporters to provide energy and building blocks, or serve as molecular signals that regulate hydrolytic enzyme production. Several membrane proteins that serve as molecular sensors and sugar transporters play important roles in nutrient acquisition and the induction of lignocellulolytic enzyme genes, such as Hgt-1/2, CDT-1/2 and CLP1 from N. crassa, and Crt1 and Stp1 from T. reesei [6,7,8,9].
Our previous study revealed that sugar transporter MtLat-1 of M. thermophila displays a high uptake activity and specificity for l-arabinose . LAT-1 is conserved among ascomycete fungi. The closest relatives of LAT-1 that have been biochemically characterized include Lat-1 (Trire2_104072) from T. reesei, NcLat-1 (NCU02188) from N. crassa, LAT-1 from Ambrosiozyma monospora , and AraT from Pichia stipitis  and Penicillium chrysogenum . Although all these proteins function as l-arabinose transporters, their effects on physiological phenotypes differ in various fungi when grown on l-arabinose and arabinan. In T. reesei, Lat-1 is a high-affinity symporter and shows high specificity for l-arabinose. The absence of Trire2_104072 did not result in an altered growth phenotype or different total protein secretion when grown on in high (1%) concentration of l-arabinose, lactose, or spent grain extract with a high content of arabinoxylan . However, a severe growth defective of an N. crassa lat-1 mutant was observed on arabinan. Moreover, in N. crassa, lat-1 disruption caused a remarkably reduced l-arabinose uptake, which abolished the induction of ard-1 (gene encoding l-arabinitol-4-dehydrogenase) at low (2 μM) l-arabinose concentrations . Herein, the expression of Mtlat-1 was specifically induced by l-arabinose and its absence resulted in a decrease in l-arabinose uptake and consumption rates, but not those of d-glucose, d-xylose, or d-galactose, which is consistent with the specific l-arabinose transport activity of MtLat-1 . However, mycelium growth, secreted protein production, and hemicellulolytic enzyme activities were markedly pronounced in the ΔMtlat-1 mutant when grown on arabinan. Comparative transcriptomic analysis revealed that deletion of Mtlat-1 enhanced the expression of genes encoding xylanolytic enzymes and arabinanolytic enzymes. These results suggested that l-arabinose transporter MtLat-1 repressed the expression of hemicellulolytic enzymes on arabinan but not on xylan. Furthermore, a point mutant of MtLat-1 in which the transport activity was abolished exhibited similar effects to the wild-type MtLat-1 on the protein secretion and hemicellulolytic activities on arabinan, clearly demonstrating that the involvement of MtLat-1 in M. thermophila hemicellulase gene expression was independent of its transport activity. These experiments have helped to separate nutrient sensing from substrate transport, and indicate that MtLat-1 protein serves as a transceptor, which might sense and transduce molecular signal of l-arabinose, one of the end products of hemicellulose degradation, to repress the induction of hemicellulase genes. The role of transceptor in the induction of genes involved in plant biomass degradation has also been demonstrated for CDT-1/2 in N. crassa and Crt1 in T. reesei [9, 52].
The C-terminal cytoplasmic fragments of several transporters and sensors have been reported to be essential for signal transduction . Truncation of some C-terminal amino acids did not alter their transport activities, but abolished their signaling function [48, 52]. In T. reesei, a Crt1 mutant retaining only the first five amino acids of C-terminus still had the lactose transport activity but lost the cellulase induction activity . In M. thermophila, MtLat-1 has a C-terminal extension of 63 amino acids, which is markedly shorter than those of Snf3 (303 amino acids) and Rgt2 (220 amino acids), but longer than that of Crt1 (44 amino acids) [11, 52]. However, an MtLat-1 mutant with truncation of the entire C-terminus restored the growth phenotype and hemicellulolytic activities of strain ΔMtlat-1 to similar levels to those of the WT on arabinan, showing that the C-terminal cytosolic region of MtLat-1 is not required for its signaling function. Similarly, in Pichia pastoris, deletion of the C-terminal fragment (150 amino acids) of glucose sensor Gss1 slightly affected glucose catabolite repression and pexophagy, but the signaling function of the protein was maintained . Signal transduction by transporters and sensors can be mediated by their relocation from the cell membrane to intracellular compartments, including endosomes, the Golgi apparatus, and nuclear membranes . In T. reesei, the internalization of Crt1 was observed on cellulose, although it was also found under glucose or glycerol and may not be correlated with the induction of cellulases . Herein, MtLat-1 was mainly localized in the cellular membrane and dual localization of MtLat-1–GFP was not observed. The precise mechanism by which MtLat-1 mediates the signal transduction involved in hemicellulase induction requires further investigation.
Transcription factors play an essential role in regulation of the expression of CAZyme genes and genes involved in sugar transport and intracellular catabolism. Several essential transcription factors associated with hemicellulose deconstruction and utilization have been studied in fungi, including CreA/CRE-1, Xyr1/XlnR, and Ara-1. CreA/CRE-1 is a major regulator of CCR, a process through which the expression of genes involved in the utilization of non-preferred carbon sources, including hemicellulose/cellulose, are inhibited [18, 55]. In T. reesei, XlnR/XYR1 regulates both cellulase and hemicellulase genes, while it is a major positive activator of hemicellulase genes in N. crassa and F. graminearum [16, 21, 24]. Similarly, in M. thermophila, Xlr1 controls the expression of xylanolytic genes and genes involved in pentose transport and catabolism, but it has a smaller impact on l-arabinose catabolism . In several fungi, Ara-1 is essential for expression of d-galactose and l-arabinose releasing and catabolic genes. In N. crassa and T. reesei, deletion of ara-1 dramatically downregulated the expression of l-arabinose transporter Lat-1 [27, 42]. In M. thermophila, the induced expression of Mtara-1 was observable only on l-arabinose, as also observed for Mtlat-1. Transcriptional analysis and EMSAs demonstrated that MtAra-1 directly regulates Mtlat-1 expression by binding to its promoter region. Consistent with observations in N. crassa [14, 27],
Mtara-1 disruption abolished the growth of M. thermophila on l-arabinose and d-galactose, but no effect on the growth of strain ΔMtara-1 was observed on d-xylose. Moreover, the ΔMtara-1 mutant exhibited a remarkable reduction in secreted protein production and hemicellulolytic activities when grown on arabinan. Our transcriptomic data reflected the significantly downregulated expression of all arabinanolytic enzyme-encoding genes in the ΔMtara-1 mutant when it was grown on arabinan. These results demonstrated that MtAra-1 is mainly involved in regulation of the expression of arabinanolytic genes and the genes associated with l-arabinose and d-galactose catabolism in M. thermophila.
In this study, we demonstrated that MtLat-1 is involved in l-arabinose transport and signal transduction associated with the suppression of expression of hemicellulolytic enzyme-encoding genes in M. thermophila. The absence of Mtlat-1 caused a decrease on l-arabinose uptake and consumption rates, but led to increases in mycelium growth, secreted protein production, and hemicellulolytic enzyme activities on arabinan. Furthermore, point mutation of MtLat-1 revealed that the involvement of MtLat-1 in the expression of hemicellulase genes is independent of its transport activity. Moreover, transcription factor MtAra-1 played an important role in the induction of arabinanolytic genes and l-arabinose catabolism, and directly regulated Mtlat-1 expression by binding to its promoter region.
Materials and methods
Strains and growth conditions
M. thermophila ATCC 42464 was obtained from the American Type Culture Collection (ATCC). This WT strain and its mutants were grown on Vogel’s minimal medium (VMM) with 2% (w/v) glucose at 35 °C to obtain mature conidia. Antibiotic was added when needed to screen for transformants. For media shift experiments, M. thermophila strains were precultured in 100 mL of 1 × VMM with 2% glucose for 16 h. Subsequently, the mycelia were collected, washed three times with 1 × VMM, and then transferred to 100 mL of fresh 1 × VMM with 2% l-arabinose (Sigma) or arabinan (Megazyme) for continued incubation for 2 h. For fungal growth assays, mature conidia were inoculated into 100 mL of 1 × VMM with 2% carbon source (glucose, d-xylose, l-arabinose, arabinan, xylan, or Avicel) at a concentration of 2.5 × 105 conidia/mL in 250-mL Erlenmeyer flasks. The culture was incubated at 45 °C with rotary shaking at 150 rpm.
S. cerevisiae BSW5AP with heterogenous arabinose metabolic pathway , was cultured in YPD medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose) at 30 °C. The medium was supplemented with 400 g/mL Geneticin (G418; Amresco) when required to maintain a plasmid with the KanMX4 marker. For assay of growth properties, a single colony from a fresh agar plate was precultured in 10 mL of YPD medium supplemented with 400 g/mL G418. Cells in the late exponential phase were washed with sterile water, and then inoculated into 30 mL 2% l-arabinose medium in 250-mL Erlenmeyer flasks at an initial OD600 value of 1.0. Cells were cultivated in an orbital shaker at 200 rpm at 30 °C and samples were taken at intervals.
E. coli Mach-T1 cells were used for vector manipulation and propagation, and were cultivated in Luria–Bertani medium with 100 µg/mL ampicillin or 50 µg/mL kanamycin for plasmid selection.
Plasmid and strain construction
The primer sequences used in this study are listed in Additional file 1: Table S1. For the deletion of target genes using the CRISPR/Cas9 system, the guide RNA (gRNA) expression cassettes were constructed as described previously . Specific sgRNA target sites in target genes (Mtlat-1, Mycth_95427; Mtara-1, Mycth_2121737; MtPdr-1, Mycth_53224; MtPdr-2, Mycth_46266; Mycth_2300935) were identified using the sgRNACas9 tool . Fragments, including the M. thermophila U6 promoter sequence, the synthetic gRNA scaffold sequence, and the target DNA sequence were constructed via overlapping PCR and cloned into the pJET1.2/blunt cloning vector to generate the corresponding plasmids U6p-Mtlat-1-sgRNA, U6p-Mtara-1-sgRNA, U6p-MtPdr-1-sgRNA, U6p-MtPdr-2-sgRNA, and U6p-Mycth_2300935-sgRNA. To construct donor DNA sequences, the 5′- and 3′-flanking fragments of Mtlat-1, Mtara-1, MtPdr-1, MtPdr-2, and Mycth_2300935 were separately amplified by PCR from M. thermophila genomic DNA, fused with the selectable marker cassette PtrpC-neo from plasmid p0380-neo , and cloned into pPK2BarGFP to generate donor DNA sequences Donor-Mtlat-1-neo, Donor-Mtara-1-neo, Donor-MtPdr-1-neo, Donor-MtPdr-2-1-neo, and Donor-Mycth_22300935-neo, respectively.
To generate the complementation strain, a DNA fragment containing the upstream region (1485 bp), the downstream region (853 bp), and full-length Mtlat-1 (1934 bp), was amplified from the M. thermophila genome and ligated between the BglII and BamHI sites of pAN52-PgpdA-bar  to generate the corresponding complementation plasmid pAN52-bar-Mtlat, using a NEB Gibson Assembly Kit. For overexpression of target genes, the genes encoding MtLat-1, MtLat-1(R136K), MtLat-1M, and their GFP fusion proteins were amplified by PCR and inserted into pAN52-Ptef1-TtrpC-bar  at the SpeI and BamHI sites, under the control of the strong constitutive tef1 (Mycth_2298136) promoter of M. thermophila, to generate the corresponding recombinant vectors. Polyethylene glycol (PEG)-mediated protoplast transformation for gene disruption or overexpression in M. thermophila was performed as described previously .
For expression of transporters in S. cerevisiae strain BSW5AP, the genes encoding MtLat-1, MtLat-1–GFP, MtLat-1(R136K), and MtLat-1(R136K)–GFP were separately inserted into the shuttle plasmid p426kanmx4  with the selective marker KanMX4, behind the PGK promoter. S. cerevisiae transformations were carried out as described previously .
GFP fluorescence and confocal fluorescence microscopy
To detect subcellular localization of MtLat-1 and its mutants in M. thermophila, mycelia were pre-grown in VMM with 2% arabinose for 16 h at 45 °C and then the cells were observed using the Olympus BX51 fluorescence microscopy system. Recombinant S. cerevisiae BSW5AP strains expressing sugar transporters tagged with GFP were inoculated into YPD medium and grown to the exponential phase (OD600 ~ 1.0). The cells were collected, washed twice with sterile water, and then resuspended in sterile water. Next, 10 μL of culture was spotted on a cover glass for confocal microscopy. The images were processed using ImageJ software.
Sugar uptake assays of M. thermophila strains
The M. thermophila strains were precultured in 1 × VMM containing 2% glucose for 18 h. Then, mycelia were washed three times in 1 × Vogel’s salts without any carbon source and shifted into 0.5% arabinose medium for the additional 4 h of induction. After that, the mycelia were harvested, washed again as above, and resuspended in uptake buffer (1 × Vogel’s salts plus 10 mM arabinose and 10 μg/mL cycloheximide) for 20 min. The residual sugar in the supernatant was determined using HPLC with an e2695 instrument (Waters, Manchester, United Kingdom), and the fungal biomass was completely dried to determine the dry weight for data normalization.
Protein and enzyme activity assays
The mature conidia of M. thermophila strain were inoculated into 100 mL of 1 × VMM with 2% carbon source (arabinan, xylan, or Avicel) at a concentration of 2.5 × 105 conidia/mL in 250-mL Erlenmeyer flasks and incubated at 45 °C with rotary shaking at 150 rpm. Samples were taken at the indicated time for the assays of secreted protein and enzyme activity. The total extracellular protein in culture supernatants was measured using a Bio-Rad protein assay kit. Endo-1,5-l-arabinanase, endo-1,4-xylanase, and endo-glucanase activities in the supernatants of culture of M. thermophila strains were measured using an Azo-Xylan kit (Megazyme), an endo-1,5-l-arabinanase assay kit (Megazyme), and an Azo-CM-Cellulose assay kit (Megazyme), respectively.
RNA extraction and transcriptional analysis
M. thermophila mycelia were shifted into 1 × VMM with 2% carbon source (glucose, xylose, arabinose, or arabinan) for the induction of 4 h, harvested by vacuum filtration, and immediately ground to a fine powder using a pestle and mortar with liquid nitrogen for the subsequent extraction of total RNA . For transcriptome analysis of the Mtlat-1 deletion mutant grown on arabinan, M. thermophila wild-type strain and Mtlat-1 deletion mutant were cultured in 1 × VMM with 2% arabinan (Sampled at 2 days and 4 days).
RNA integrity and concentration were determined using agarose gel electrophoresis and NanoDrop spectrophotometer. Purified RNA samples, with RNA integrity number > 8.0, determined using an Agilent 2100 Bioanalyzer (Agilent Technologies), were sequenced by Novogene Corporation (Tianjin, China) using the Illumina NovaSeq 6000 platform to generate 150-bp paired-end reads.
Clean reads were mapped to the M. thermophila ATCC42446 genome sequence  using Tophat (v2.0.8b) . The counts of reads mapped to unique exons were calculated by Htseq-count (V0.13.5) with SAM files and the GFF file as input and used for normalizing transcript abundance (RPKM, reads per kilobase per million mapped reads). DESeq package was used for analysis of differential gene expression . Unless otherwise noted, genes with |log2 fold-change|≥ 1, RPKM ≥ 20 (at least one sample), and DESeq P-adj value < 0.05, were considered significantly differentially expressed between two samples. The detailed data are shown in Additional file 1: Table S2. The raw reads of transcriptomic data were deposited in the Gene Expression Omnibus (accession number: GSE221464) at the National Center for Biotechnology Information.
Real-time quantitative PCR
For RT-qPCR analysis, precultured mycelia of M. thermophila strains were shifted into induction medium containing 2% arabinose as the carbon source and incubated for 4 h, then harvested for the subsequent extraction of total RNA using the method described previously . Quantitative PCR was performed with SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) using a CFX96 real-time PCR detection system (Bio-Rad). Negative controls contained an equal volume of water instead of RNA. The relative transcript level of each gene was calculated by the 2−ΔΔCt method with the actin-encoding gene (Mycth_2314852) as the internal control.
Electrophoretic mobility shift assays
The DNA sequence encoding the DBD of transcription factor MtAra-1 (residues 9–68, identified in NCBI) was amplified from M. thermophila cDNA and inserted between the BamHI and XhoI sites of pGEX-4T-1 (GE Healthcare) to form a GST-tagged protein expression plasmid. The recombinant plasmid was introduced into E. coli BL21 (DE3) cells for protein expression and purification as previously described .
EMSAs were performed as described previously . Briefly, promoter regions of Mtlat-1 (P1, − 396 to − 1; P2, − 860 to − 396) were amplified from M. thermophila genomic DNA using primers shown in Additional file 1: Table S1. In each EMSA, recombinant MtAra-1 were incubated with a constant amount (10 ng) of the DNA probe at 25 °C for 30 min in buffer.
Availability of data and materials
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Green fluorescent protein
Transporter and acceptor
Vogel’s minimal medium
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This work was supported by funding from the Key Project of the Ministry of Science and Technology of China (2018YFA0901400 and 2018YFA0900500), the National Natural Science Foundation of China (31601013 and 32071424), the Chinese Academy of Sciences (XDA21060900), the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-015) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020183).
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: List of PCR primers used in this study. Table S2: Profiles of RNA-Seq reads mapped to the genome of M. thermophila. Table S3: Transcriptomic profiles of 26 sugar transporters with robust expression levels (RPKM > 20) in at least one tested condition. Table S4: Genes showing significantly different transcriptional levels in strain ∆Mtlat-1 compared with the WT when grown on 1 × VMM with 2% arabinan for 4 days. Table S5: Transcriptomic profiles of genes encoding major hemicellulases from RNA-Seq data when grown on 1 × VMM with 2% arabinan. Table S6: Gene ontology (GO) analysis of up-regulated genes in strain ΔMtlat-1 compared with the WT when grown on 1 × VMM with 2% arabinan for 4 days. Table S7: Transcriptomic profiles of transcription factor genes with significantly upregulated expression levels in WT M. thermophila grown on l-arabinose, d-xylose, or d-glucose, compared with that under no carbon. Table S8: Genes showing significantly different transcriptional levels in strain ∆Mtara-1 compared with the WT strain when grown on 2% arabinan for the induction of 4 h.
: Sugar consumption by M. thermophila strains WT, ΔMtlat-1, and ΔMtara-1, when grown in 1 × VMM with 2% d-glucose (A), 2% d-xylose (B), or 2% d-galactose (C). Error bars indicate the SD from at least three biological replicates. Fig. S2: Growth phenotypes of the M. thermophila ΔMtlat-1 mutant under xylan condition. A Cell dry weight of M. thermophila strains WT and ΔMtlat-1 after growth on 2% xylan for 2 d. B Protein concentrations, C xylanase activity, and D arabinanase activity of the culture supernatants for the M. thermophila strains grown in 2% xylanase medium. Error bars indicate the SD from at least three biological replicates. Fig. S3: Growth phenotypes of the complementation strain of the ΔMtlat-1 mutant. A l-arabinose transport rates of mycelia from the complementation strain Pn-Mtlat-1. B Cell dry weight of the M. thermophila complementation strain after growth on 2% arabinan for 4 days. C Protein concentrations, D arabinanase activity, and E xylanase activity of the culture supernatants for M. thermophila grown in 2% arabinan medium. Error bars indicate the SD from at least three biological replicates. Fig. S4: Comparative transcriptomic analysis of the WT and ΔMtlat-1 M. thermophila strains grown in arabinan medium for 2 d. A Total expression of genes encoding major hemicellulases from RNA-Seq data. B Transcriptional profiles of genes encoding arabinanolytic enzymes in the ΔMtlat-1 and WT strains when grown on 2% arabinan for 4 days. Fig. S5: Cell dry weight of M. thermophila strains after growth on 2% arabinan for 4 days. Fig. S6: Heatmap analysis of expression profiles for putative sugar transporter genes with statistically significant differences in transcript levels between ΔMtara-1 and the WT under l-arabinose condition. Log-transformed expression values are color-coded. Fig. S7: Protein concentrations and hemicellulase/cellulase activity of the culture supernatants for M. thermophila strain ΔMtara-1 grown in 1 × VMM with 2% xylan (A) or 2% Avicel (B) for 4 days.
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Gu, S., Zhao, Z., Xue, F. et al. The arabinose transporter MtLat-1 is involved in hemicellulase repression as a pentose transceptor in Myceliophthora thermophila. Biotechnol Biofuels 16, 51 (2023). https://doi.org/10.1186/s13068-023-02305-3
- l-Arabinose transport
- Signal transduction