Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts.

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus. It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A. fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species.

The secondary metabolite regulator, BbSmr1, is a central regulator of conidiation via the BrlA-AbaA-WetA pathway in Beauveria bassiana.

The filamentous fungus Beauveria bassiana, an insect fungal pathogen, is widely used for pest biocontrol. Aerial conidia are infectious propagules, and their yield and viability greatly affect the field application of this fungus; however, little is known about the molecular regulatory mechanism of the triggered conidiation. In the present study, we find that the secondary metabolite regulator BbSmr1 is involved in the regulation of asexual conidiation development and stress response in B. bassiana. A deficiency in Bbsmr1 results in a prominent fluffy-like phenotype on solid medium, decreased conidial yield, accelerated conidial germination, as well as increased tolerance to H2 O2 stress and cell wall inhibitors. The deletion of Bbsmr1 also leads to thickened conidial cell walls and changed cell epitopes. Overexpressing either BbbrlA or BbabaA in the ∆Bbsmr1 strain can rescue the phenotypes of conidial development and stress response. BbSmr1 activates BbbrlA transcription by directly binding to the A4GA3 sequence of the BbbrlA promoter. BbBrlA in turn binds to the promoter of Bbsmr1 and negatively regulates the expression of Bbsmr1. These results indicate that BbSmr1 positively regulates conidial development in B. bassiana by activating the central development pathway BrlA-AbaA-WetA and provides insights into the developmental regulatory mechanism of entomopathogenic fungi.

Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death.

The regulatory network and biological functions of the fungal secondary metabolite oosporein have remained obscure. Beauveria bassiana has evolved the ability to parasitize insects and outcompete microbial challengers for assimilation of host nutrients. A novel zinc finger transcription factor, BbSmr1 (B. bassiana secondary metabolite regulator 1), was identified in a screen for oosporein overproduction. Deletion of Bbsmr1 resulted in up-regulation of the oosporein biosynthetic gene cluster (OpS genes) and constitutive oosporein production. Oosporein production was abolished in double mutants of Bbsmr1 and a second transcription factor, OpS3, within the oosporein gene cluster (ΔBbsmr1ΔOpS3), indicating that BbSmr1 acts as a negative regulator of OpS3 expression. Real-time quantitative PCR and a GFP promoter fusion construct of OpS1, the oosporein polyketide synthase, indicated that OpS1 is expressed mainly in insect cadavers at 24-48 h after death. Bacterial colony analysis in B. bassiana-infected insect hosts revealed increasing counts until host death, with a dramatic decrease (∼90%) after death that correlated with oosporein production. In vitro studies verified the inhibitory activity of oosporein against bacteria derived from insect cadavers. These results suggest that oosporein acts as an antimicrobial compound to limit microbial competition on B. bassiana-killed hosts, allowing the fungus to maximally use host nutrients to grow and sporulate on infected cadavers.

The LsrB Protein Is Required for Agrobacterium tumefaciens Interaction with Host Plants.

Agrobacterium tumefaciens infects and causes crown galls in dicot plants by transferring T-DNA from the Ti plasmid to the host plant via a type IV secretion system. This process requires appropriate environmental conditions, certain plant secretions, and bacterial regulators. In our previous work, a member of the LysR family of transcriptional regulators (LsrB) in Sinorhizobium meliloti was found to modulate its symbiotic interactions with the host plant alfalfa. However, the function of its homolog in A. tumefaciens remains unclear. In this study, we show that the LsrB protein of A. tumefaciens is required for efficient transformation of host plants. A lsrB deletion mutant of A. tumefaciens exhibits a number of defects, including in succinoglycan production, attachment, and resistance to oxidative stress and iron limitation. RNA-sequencing analysis indicated that 465 genes were significantly differentially expressed (upregulation of 162 genes and downregulation of 303 genes) in the mutant, compared with the wild-type strain, including those involved in succinoglycan production, iron transporter, and detoxification enzymes for oxidative stress. Moreover, expression of the lsrB gene from S. meliloti, Brucella abortus, or A. tumefaciens rescued the defects observed in the S. meliloti or A. tumefaciens lsrB deletion mutant. Our findings suggest that a conserved mechanism of LsrB function exists in symbiotic and pathogenic bacteria of the family Rhizobiaceae.

Sinorhizobium meliloti Glutathione Reductase Is Required for both Redox Homeostasis and Symbiosis.

Glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH), one of the key antioxidants in Sinorhizobium meliloti, is required for the development of alfalfa (Medicago sativa) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione (GSH) or oxidized glutathione (GSSG), and its content is regulated by two pathways in S. meliloti The first pathway is the de novo synthesis of glutathione from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The second pathway is the recycling of GSSG via glutathione reductase (GR). However, whether the S. meliloti GR functions similarly to GshA and GshB1 during symbiotic interactions with alfalfa remains unknown. In this study, a plasmid insertion mutation of the S. melilotigor gene, which encodes GR, was constructed, and the mutant exhibited delayed alfalfa nodulation, with 75% reduction in nitrogen-fixing capacity. The gor mutant demonstrated increased accumulation of GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective growth in rich broth and minimal broth and was more sensitive to the oxidants H2O2 and sodium nitroprusside. Interestingly, the expression of gshA, gshB1, katA, and katB was induced in the mutant. These findings reveal that the recycling of glutathione is important for S. meliloti to maintain redox homeostasis and to interact symbiotically with alfalfa.IMPORTANCE The antioxidant glutathione is regulated by its synthetase and reductase in cells. In the symbiotic bacterium S. meliloti, the de novo synthesis of glutathione is essential for alfalfa nodulation and nitrogen fixation. In this study, we observed that the recycling of glutathione from GSSG not only was required for redox homeostasis and oxidative stress protection in S. meliloti cells but also contributed to alfalfa nodule development and competition capacity. Our findings demonstrate that the recycling of glutathione plays a key role in nitrogen fixation symbiosis.

Regulation of cysteine residues in LsrB proteins from Sinorhizobium meliloti under free-living and symbiotic oxidative stress.

The development of legume nitrogen-fixing nodules is regulated by reactive oxygen species (ROS) produced by symbionts. Several regulators from Rhizobium are involved in ROS sensing. In a previous study, we found that Sinorhizobium meliloti LsrB regulates lipopolysaccharide production and is associated with H2 O2 accumulation in alfalfa (Medicago sativa) nodules. However, its underlying regulatory mechanism remains unclear. Here, we report that the cysteine residues in LsrB are required for adaptation to oxidative stress, gene expression, alfalfa nodulation and nitrogen fixation. Moreover, LsrB directly activated the transcription of lrp3 and gshA (encoding γ-glutamylcysteine synthetase, responsible for glutathione synthesis) and this regulation required the cysteine (Cys) residues in the LsrB substrate-binding domain. The Cys residues could sense oxidative stress via the formation of intermolecular disulfide bonds, generating LsrB dimers and LsrB-DNA complexes. Among the Cys residues, C238 is a positive regulatory site for the induction of downstream genes, whereas C146 and C275 play negative roles in the process. The lsrB mutants with Cys-to-Ser substitutions displayed altered phenotypes in respect to their adaptation to oxidative stress, nodulation and nitrogen fixation-related plant growth. Our findings demonstrate that S. meliloti LsrB modulates alfalfa nodule development by directly regulating downstream gene expression via a post-translational strategy.

Overexpression of phytosulfokine-α induces male sterility and cell growth by regulating cell wall development in Arabidopsis.

KEY MESSAGE: Over-production of functional PSK-α in Arabidopsis caused increases in both plant cell growth and biomass and induced male sterility by regulating cell wall development. Phytosulfokine-α (PSK-α) is a novel disulfated pentapeptide hormone that is involved in promoting plant cell growth. Although a role for PSK-α in stimulating protoplast expansion has been suggested, how PSK-α regulates cell growth in planta remains poorly understood. In this study, we found that overexpression of the normal PSK-α precursor gene AtPSK4, which resulted in high levels of PSK-α, caused longer roots and larger leaves with enlarged cells. As expected, these changes were not observed in transgenic plants overexpressing mutated AtPSK4, which generated unsulfated PSK-α. These findings confirmed the role of PSK-α in promoting plant cell growth. Furthermore, we found that overexpressing AtPSK4, but not mutated AtPSK4, induced a phenotype of male sterility that resulted from the failure of fibrous cell wall development in the endothecium. In addition, overexpressing AtPSK4 enhanced expression of a number of genes encoding expansins, which are involved in cell wall loosening. Accordingly, in addition to its role in cell growth, we propose a novel function for PSK-α signaling in the modulation of plant male sterility via regulation of cell wall development.

Transcriptional regulator LsrB of Sinorhizobium meliloti positively regulates the expression of genes involved in lipopolysaccharide biosynthesis.

Rhizobia induce nitrogen-fixing nodules on host legumes, which is important in agriculture and ecology. Lipopolysaccharide (LPS) produced by rhizobia is required for infection or bacteroid survival in host cells. Genes required for LPS biosynthesis have been identified in several Rhizobium species. However, the regulation of their expression is not well understood. Here, Sinorhizobium meliloti LsrB, a member of the LysR family of transcriptional regulators, was found to be involved in LPS biosynthesis by positively regulating the expression of the lrp3-lpsCDE operon. An lsrB in-frame deletion mutant displayed growth deficiency, sensitivity to the detergent sodium dodecyl sulfate, and acidic pH compared to the parent strain. This mutant produced slightly less LPS due to lower expression of the lrp3 operon. Analysis of the transcriptional start sites of the lrp3 and lpsCDE gene suggested that they constitute one operon. The expression of lsrB was positively autoregulated. The promoter region of lrp3 was specifically precipitated by anti-LsrB antibodies in vivo. The promoter DNA fragment containing TN11A motifs was bound by the purified LsrB protein in vitro. These new findings suggest that S. meliloti LsrB is associated with LPS biosynthesis, which is required for symbiotic nitrogen fixation on some ecotypes of alfalfa plants.

Expression of a toll signaling regulator serpin in a mycoinsecticide for increased virulence.

Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The Drosophila melanogaster serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus Beauveria bassiana offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD50) for the wild-type parent, the LD50 of B. bassiana expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ~3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ~24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin>laminarin>lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassianaderived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.

Fungal infection of insects: molecular insights and prospects.

Entomopathogenic fungi (EPF) distribute in different fungal phyla with variable host ranges and play essential role in regulating insect populations by infecting hosts via cuticle penetration. The representative ascomycete EPF of Metarhizium and Beauveria species have been widely used in mechanistic investigations of fungus-insect interactions and as ecofriendly mycoinsecticides. Here, we review the function of diverse genes, pathways, and secondary metabolites associated with EPF stepwise infections. In particular, emerging evidence has shown that EPF have to outcompete insect ectomicrobiotas prior to penetrating cuticles, and subvert or evade host antifungal immunity by using effector-like proteins and chemicals like plant pathogens. Future prospects are discussed for a better understanding of fungal pathobiology, which will provide novel insights into microbe-animal interactions.

Suppression of Drosophila antifungal immunity by a parasite effector via blocking GNBP3 and GNBP-like 3, the dual receptors for ß-glucans.

The tactics used by animal pathogens to combat host immunity are largely unclear. Here, we report the depiction of the virulence-required effector Tge1 deployed by the entomopathogen Metarhizium robertsii to suppress Drosophila antifungal immunity. Tge1 can target both GNBP3 and GNBP-like 3 (GL3), and the latter can bind to ß-glucans like GNBP3, whereas the glucan binding by both receptors can be attenuated by Tge1. As opposed to the surveillance GNBP3, GL3 is inducible in Drosophila depending on the Toll pathway via a positive feedback loop mechanism. Losses of GNBP3 and GL3 genes result in the deregulations of protease cascade, Spätzle maturation, and antimicrobial gene expressions in Drosophila upon fungal challenges. Fly survival assays confirm that GL3 plays a more essential role than GNBP3 in combating fungal infections. In addition to evidencing the gene-for-gene interactions between fungi and insects, our data advance insights into Drosophila antifungal immunity.

The Sinorhizobium meliloti LysR family transcriptional factor LsrB is involved in regulation of glutathione biosynthesis.

Glutathione, a key antioxidant in Sinorhizobium meliloti, is required for the development of alfalfa (Medicago sativa) nitrogen-fixing nodules. This tripeptide can be synthesized by both γ-glutamyl cysteine synthetase (GshA) and glutathione synthetase (GshB) in Escherichia coli and S. meliloti. Genetic evidence has indicated that the null mutant of S. meliloti gshA or gshB1 does not establish efficient symbiosis on alfalfa. However, the transcriptional regulation of gshA and gshB has not been well understood. Here, S. meliloti LsrB, a member of LysR family transcriptional factors, was found to positively regulate glutathione biosynthesis by activating the transcription of gshA and gshB1 under both free-living and symbiotic conditions. The decrease in glutathione production in the lsrB in-frame deletion mutant (lsrB1-2) was determined by using quadrupole time-of-flight liquid chromatography-mass spectrometry. The expression of gshA and gshB1 was correspondingly reduced in the mutant under free-living and symbiotic conditions by analyses of real-time quantitative reverse transcription-polymerase chain reaction and promoter-GUS fusions. Interestingly, LsrB positively regulated the transcription of oxyR, which encodes another member of LysR family regulators and responds to oxidative stresses in S. meliloti. The oxyR null mutant produced less glutathione, in which the transcription of gshA was consistently down-regulated. These findings demonstrate that glutathione biosynthesis is positively regulated by both LsrB and OxyR in S. meliloti.

Sensing of a spore surface protein by a Drosophila chemosensory protein induces behavioral defense against fungal parasitic infections.

In addition to innate immunity in a physiological context, insects have evolved behavioral defenses against parasite attacks. Here, we report that Drosophila can sense the CFEM (common in fungal extracellular membrane) protein Mcdc9, which acts as a negative virulence factor of the entomopathogenic fungus Metarhizium robertsii. The individual deletions of 18 CFEM genes in Metarhizium followed by fly infection identified three null mutants that could kill the flies more quickly than the wild-type strain, among which Mcdc9 can coat fungal spores and interact with the fly chemosensory protein CheA75a. The deletion of Mcdc9 in the fungus or the knockdown of CheA75a in flies had a similar effect, in which a greater number of fungal spores were left on flies than on the respective controls after topical infection. Thus, similar to the accelerated death of the wild-type flies treated with ΔMcdc9, the CheA75aRNAi flies succumbed more quickly than the control insects topically challenged with the wild-type strain. The CheA75a gene is highly transcribed in fly legs and wings, and positive electrophysiological responses were evidenced in tarsal sensilla after stimulation with the Mcdc9 protein. The results imply that this CFEM protein could be sensed as a contact elicitor inducing the hygienic behavior of flies against fungal parasitic infection, which reveals a previously unsuspected mechanism of fungus-insect interactions.

Promotion of Arabidopsis immune responses by a rhizosphere fungus via supply of pipecolic acid to plants and selective augment of phytoalexins.

The ascomycete insect pathogenic fungi such as Metarhizium species have been demonstrated with the abilities to form the rhizosphere or endophytic relationships with different plants for nutrient exchanges. In this study, after the evident infeasibility of bacterial disease development in the boxed sterile soils, we established a hydroponic system for the gnotobiotic growth of Arabidopsis thaliana with the wild-type and transgenic strain of Metarhizium robertsii. The transgenic fungus could produce a high amount of pipecolic acid (PIP), a pivotal plant-immune-stimulating metabolite. Fungal inoculation experiments showed that M. robertsii could form a non-selective rhizosphere relationship with Arabidopsis. Similar to the PIP uptake by plants after exogenous application, PIP level increased in Col-0 and could be detected in the PIP-non-producing Arabidopsis mutant (ald1) after fungal inoculations, indicating that plants can absorb the PIP produced by fungi. The transgenic fungal strain had a better efficacy than the wild type to defend plants against the bacterial pathogen and aphid attacks. Contrary to ald1, fmo1 plants could not be boosted to resist bacterial infection after treatments. After fungal inoculations, the phytoalexins camalexin and aliphatic glucosinolate were selectively increased in Arabidopsis via both PIP-dependent and -independent ways. This study unveils the potential mechanism of the fungus-mediated beneficial promotion of plant immunity against biological stresses. The data also highlight the added values of M. robertsii to plants beyond the direct suppression of insect pest populations.

Selection of optimal reference genes for expression analysis in the entomopathogenic fungus Beauveria bassiana during development, under changing nutrient conditions, and after exposure to abiotic stresses.

The selection of suitable reference genes is crucial for accurate quantification of gene expression. To identify suitable reference genes in Beauveria bassiana, the expression of 14 candidates (18S, 28S, ß-Tub, GAPD, γ-Act, TEF, HGPT, His3, His2A, TBP, CypA, CypB, PP1, and CrzA) was measured by quantitative polymerase chain reaction at different development stages and under various nutritional and stress conditions. Expression stability, as evaluated by the geNorm and NormFinder programs, revealed that His2A/γ-Act/CrzA was the most stably expressed set of genes throughout development, while 28S/PP1/CypA and His2A/γ-Act/CypA were the most stably expressed gene sets under a variety of nutritional and stress conditions, respectively. Overall, the most stably expressed genes under all conditions examined were PP1, γ-Act, and CypA.

Activation of microlipophagy during early infection of insect hosts by Metarhizium robertsii.

The requirement of macroautophagic/autophagic machinery for filamentous fungal development and pathogenicity has been recognized, but the underlying effects and mechanisms remain elusive. The insect pathogenic fungus Metarhizium robertsii infects hosts by cuticular penetration through the formation of the infection structure appressoria. Here, we show that autophagic fluxes were highly activated during the appressorial formation of M. robertsii. Genome-wide deletion of the autophagy-related genes and insect bioassays identified 10 of 23 encoded MrATG genes with requirements for topical fungal infection of insect hosts. Besides the defect in forming appressoria on insects (two null mutants), these virulence-reduced mutants were largely impaired in penetrating cellophane membrane and insect cuticles, suggesting their failures in generating proper appressorium turgor. We found that the conidial storage of lipid droplets (LDs) had no obvious difference between strains, but autophagic LD degradation was impaired in different mutants. After induction of cell autophagy by nitrogen starvation, we found that LD entry into vacuoles was unaffected in the selected mutant cells with potential failures in forming autophagosomes. The finding therefore reveals a microlipophagy machinery employed in this fungus and that the direct engulfment of LDs occurs without inhibition by the downstream defective lipolysis. Our data first unveil the activation and contribution of microlipophagy to fungal infection biology. The obtained technique may benefit future detection of microlipophagy in different organisms by examining vacuolar or lysosomal engulfment of LDs in core autophagic gene deletion mutants.Abbreviations: AIM: Atg8-family interacting motif; ATG: autophagy-related; CM: complete medium; CMAC: 7-amino-4-chloromethylcoumarin; DTT: dithiothreitol; ER: endoplasmic reticulum; GFP: green fluorescent protein; LD, lipid droplet; MM: minimum medium; MM-N: minimum medium without nitrogen source; PDA: potato dextrose agar; PMSF: phenylmethylsulfonyl fluoride; RFP: red fluorescent protein; SDB: Sabouraud dextrose broth; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAG: triacylglycerol; TEM: transmission electron microscopy; WT, wild type.

Identification of a key G-protein coupled receptor in mediating appressorium formation and fungal virulence against insects.

Fungal G-protein coupled receptors (GPCRs) play essential roles in sensing environmental cues including host signals. The study of GPCR in mediating fungus-insect interactions is still limited. Here we report the evolution of GPCR genes encoded in the entomopathogenic Metarhizium species and found the expansion of Pth11-like GPCRs in the generalist species with a wide host range. By deletion of ten candidate genes MrGpr1-MrGpr10 selected from the six obtained subfamilies in the generalist M. robertsii, we found that each of them played a varied level of roles in mediating appressorium formation. In particular, deletion of MrGpr8 resulted in the failure of appressorium formation on different substrates and the loss of virulence during topical infection of insects but not during injection assays when compared with the wild-type (WT) strain. Further analysis revealed that disruption of MrGpr8 substantially impaired the nucleus translocation of the mitogen-activated protein kinase (MAPK) Mero-Fus3 but not the MAPK Mero-Slt2 during appressorium formation. We also found that the defect of AMrGpr8 could not be rescued with the addition of cyclic AMP for appressorium formation. Relative to the WT, differential expression of the selected genes have also been detected in AMrGpr8. The results of this study may benefit the understanding of fungus-interactions mediated by GPCRs.

Improving Hypoxia Adaption Causes Distinct Effects on Growth and Bioactive Compounds Synthesis in an Entomopathogenic Fungus Cordyceps militaris.

Cordyceps militaris is an entomopathogenic fungus producing a variety of bioactive compounds. To meet the huge demand for medicinal and edible products, industrialized fermentation of mycelia and cultivation of stromata have been widely developed in China. The content of bioactive metabolites of C. militaris, such as cordycepin, is higher when cultivated on silkworm pupae than on rice or in broth. However, compared with other cultivation methods, C. militaris grows more slowly and accumulates less biomass. The hypoxic environment in pupa hemocoel is one of environmental factor which is not existed in other cultivation methods. It is suggested that hypoxia plays an important role on the growth and the synthesis of bioactive compounds in C. militaris. Here, we demonstrated that the distinct effects on the growth and synthesis of bioactive compounds employing different strategies of improving hypoxia adaption. The introduction of Vitreoscilla hemoglobin enhanced growth, biomass accumulation, and crude polysaccharides content of C. militaris. However, cordycepin production was decreased to 9-15% of the control group. Meanwhile, the yield of adenosine was increased significantly. Nonetheless, when the predicted bHLH transcription factor of sterol regulatory element binding proteins (SREBPs) was overexpressed in C. militaris to improve the hypoxia adaption of fungal cells, cordycepin content was significantly increased more than two-fold. These findings reveal the role of SREBPs on growth and bioactive compounds synthesis. And it also provides a scientific basis for rationally engineering strains and optimization strategies of air supply in cultivation and fermentation.