•The autophagy related gene Acatg11 is involved in the selective autophagy of A. chrysogenum.•Acatg11 is also related with the nonselective autophagy of A. chrysogenum.•Disruption of Acatg11 reduces cephalosporin production, but enhances conidiation.Autophagy is a highly conserved degradation system in eukaryotes. Selective autophagy is used for the degradation of selective cargoes. Selective autophagic processes of yeast include pexophagy, mitophagy, and cytoplasm-to-vacuole targeting (Cvt) pathway in which particular vacuolar proteins, such as aminopeptidase I (Ape1), are selectively transported to vacuoles. However, the physiological role of selective autophagy remains elusive in filamentous fungi. ATG11 family proteins as a basic scaffold are essential for most selective autophagy pathways in yeast. Here, Acatg11, encoding a putative ATG11 family protein, was identified and cloned from the cephalosporin producing strain Acremonium chrysogenum based on the sequence similarity of ATG11 superfamily proteins. Disruption of Acatg11 inhibited the maturation of preApe1 during fermentation indicating that Acatg11 is involved in Cvt pathway. In addition, pexophagy and mitophagy were blocked in the Acatg11 disruption mutant (ΔAcatg11). Intriguingly, the nonselective autophagy was deficient in ΔAcatg11 under starvation induction or during fermentation. Disruption of Acatg11 significantly enhanced fungal conidiation, but reduced cephalosporin production. These results indicated that Acatg11 is required for both selective and nonselective autophagy during fermentation and has a strong impact on morphological differentiation and cephalosporin production of A. chrysogenum.

Streptomyces coelicolor is the soil-dwelling bacterium with a complex life cycle and a strong ability to produce plenty of secondary metabolites which are strictly regulated by a variety of regulators. Amino acid alignment shows that the deduced protein of SCO2140 belongs to the family of Leucine-responsive regulatory proteins (Lrps). Disruption of SCO2140 significantly decreased the yields of actinorhodin and calcium-dependent antibiotics, and the complemented strain restored the antibiotic productions to the wild-type level. In contrast, overexpression of SCO2140 increased the actinorhodin production. In agreement with it, the transcriptions of actII-ORF4 and cdaR remarkably reduced in the SCO2140 disruption mutant. The aerial mycelium formation of the SCO2140 disruption mutant was clearly delayed in R2YE medium due to the decrease of ramS expression while its complemented strain could restore the normal formation of aerial mycelia. These results indicated that SCO2140 was involved in antibiotic biosynthesis and morphological differentiation of Streptomyces coelicolor A3(2).

Myo-inositol is important for Streptomyces growth and morphological differentiation. Genomic sequence analysis revealed a myo-inositol catabolic gene cluster in Streptomyces coelicolor. Disruption of the corresponding genes in this cluster abolished the bacterial growth on myo-inositol as a single carbon source. The transcriptions of these genes were remarkably enhanced by addition of myo-inositol in minimal medium. A putative regulatory gene SCO6974, encoding a GntR family protein, is situated in the cluster. Disruption of SCO6974 significantly enhanced the transcription of myo-inositol catabolic genes. SCO6974 was shown to interact with the promoter regions of myo-inositol catabolic genes using electrophoretic mobility shift assays. DNase I footprinting assays demonstrated that SCO6974 recognized a conserved palindromic sequence (A/T)TGT(A/C)N(G/T)(G/T)ACA(A/T). Base substitution of the conserved sequence completely abolished the binding of SCO6974 to the targets demonstrating that SCO6974 directly represses the transcriptions of myo-inositol catabolic genes. Furthermore, the disruption of SCO6974 was correlated with a reduced sporulation of S. coelicolor in mannitol soya flour medium and with the overproduction of actinorhodin and calcium-dependent antibiotic. The addition of myo-inositol suppressed the sporulation deficiency of the mutant, indicating that the effect could be related to a shortage in myo-inositol due to its enhanced catabolism in this strain. This enhanced myo-inositol catabolism likely yields dihydroxyacetone phosphate and acetyl-CoA that are indirect or direct precursors of the overproduced antibiotics.

Neonectrolide A (1), an oxaphenalenone spiroketal with the previously undescribed (5,8′-dimethyl-5′-oxo-3a′,4,5,5′-tetrahydro-3H,3′H-spiro[furan-2,2′-isochromeno[3,4,5-def]chromene]-3′-yl)but-3-enoic acid skeleton, was isolated from cultures of the fungus Neonectria sp. Its absolute configuration was assigned by electronic circular dichroism (ECD) calculations. The skeleton of an oxaphenalenone fused with a 1,6-dioxaspiro[4.5]decane moiety in 1 could be derived from the coisolated putative precursors, corymbiferan lactone E (2) and 3-dehydroxy-4-O-acetylcephalosporolide C (3).

Rab GTPase is required for vesicle–vacuolar fusion during the vacuolar biogenesis in fungi. Rab GTPase-encoding gene, pcvA, was cloned from Penicillium chrysogenum: it contained five introns and its predicted protein contained the conserved Rab GTPase domain involved in GTP-binding and hydrolysis. Over-expression of pcvA significantly stimulated the vesicle–vacuolar fusion but repressed the conidiation and decreased conidial tolerance against thermal stress. Penicillin production was decreased in the pcvA over-expressed strain suggesting that pcvA is involved in vesicle–vacuolar fusion participates in the penicillin biosynthesis in P. chrysogenum.

Deacetoxycephalosporin C (DAOC) is not only the precursor but also one of the by-products during cephalosporin C (CPC) biosynthesis. One enzyme (DAOC/DAC synthase) is responsible for the two-step conversion of penicillin N into deacetylcephalosporin C (DAC) in Acremonium chrysogenum, while two enzymes (DAOC synthase and DAOC hydroxylase) were involved in this reaction in Streptomyces clavuligerus and Amycolatopsis lactamdurans (Nocardia lactamdurans). In this study, the DAOC hydroxylase gene cefF was cloned from Streptomyces clavuligerus and introduced into Acremonium chrysogenum through Agrobacterium tumefaciens-mediated transformation. When cefF was expressed under the promoter of pcbC, the ratio of DAOC/CPC in the fermentation broth significantly decreased. These results suggested that introduction of cefF could function quite well in Acremonium chrysogenum and successfully reduce the content of DAOC in the CPC fermentation broth. This work offered a practical way to improve the CPC purification and reduce its production cost.

•AcstuA encodes an APSES family regulator, and involved in conidiation of A. chrysogenum.•Disruption of AcstuA drastically reduced cephalosporin production.•Deficiency of AcstuA has an influence on cell wall integrity.A transcriptional regulatory gene AcstuA was identified from Acremonium chrysogenum. AcstuA encodes a basic helix-loop-helix protein with similarity to StuA which regulates the core developmental processes of Aspergillus nidulans. Like disruption of stuA in A. nidulans, deficiency of AcstuA blocked the conidiation of A. chrysogenum through severely down-regulating the expression of AcbrlA and AcabaA which encode orthologs of the key fungal developmental regulators BrlA and AbaA. Disruption of AcstuA also drastically reduced cephalosporin production of A. chrysogenum. In agreement, the transcriptions of pcbAB, pbcC, cefD1, cefD2, cefEF and cefG were remarkably decreased in the AcstuA disruption mutant (ΔAcstuA). In addition to defects in conidiation and cephalosporin biosynthesis, ΔAcstuA produced abnormal swollen and fragmented hyphal cells during fermentation. The phenotypic alterations of hyphal cells caused by AcstuA deletion were restored by supplementation of NaCl in the medium, indicating that the deficiency of AcstuA has an influence on the cell wall integrity of A. chrysogenum. The transcriptions of two putative mannoprotein encoding genes Acmp2 and Acmp3 significantly reduced in ΔAcstuA, further indicating that cell wall integrity of the mutant is impaired. These results strongly suggested that AcstuA is involved in conidiation, cephalosporin production, hyphal fragmentation and cell wall integrity in A. chrysogenum.

•The verticillin biosynthesis cluster (ver) was identification in C. rogersoniana.•The verP gene is essential for the dimeric ETP verticillin biosynthesis.•Disruption of verP inhibited the conidiation of C. rogersoniana.Verticillin is one of the dimeric epipolythiodioxopiperazines (ETPs) which are toxic secondary metabolites produced only by fungi. ETPs have received substantial attention since its complex molecular architecture and a wide range of biological activities. Although biosynthesis of the monomeric gliotoxin has been studied extensively, the biosynthetic pathway of dimeric ETPs is far from being studied. To investigate the biosynthesis of dimeric ETPs and expand our understanding of their dimerization, the verticillin biosynthetic gene cluster (ver) was identified and cloned from a genomic DNA fosmid library of the Cordyceps-colonizing fungus Clonostachys rogersoniana with the designed primers based on the sequence of a nonribosomal peptide synthetase (NRPS) ChaP which was predicted to be responsible for chaetocin biosynthesis in Chaetomium virescens. To validate it, the chaP homologous gene verP in the ver cluster was disrupted. HPLC-MS analysis demonstrated that the verP disruption mutant (ΔverP) completely abolished verticillin production, and it could be restored by introducing a copy of the wild-type verP gene. Further gene disruptions and chemical analysis demonstrated that most genes of this ver cluster were essential for verticillin biosynthesis. Intriguingly, disruption of verP almost abolished the conidiation of Clonostachys rogersoniana and it was partially restored by addition of the fermentation extract which contains verticillin, implying that verticillin or its intermediate plays a role in the Cordyceps-colonizing fungal morphological differentiation.

T-DNA inserted mutants of Acremonium chrysogenum were constructed by Agrobacterium tumefaciens-mediated transformation (ATMT). One mutant 1223 which grew slowly was selected. TAIL-PCR and sequence analysis indicated that a putative septation protein encoding gene AcsepH was partially deleted in this mutant. AcsepH contains nine introns, and its deduced protein AcSEPH has a conserved serine/threonine protein kinase catalytic (S_TKc) domain at its N-terminal region. AcSEPH shows high similarity with septation H proteins from other filamentous fungi based on the phylogenetic analysis of S_TKc domains. In sporulation (LPE) medium, the conidia of AcsepH mutant was only about one-seventh of the wild-type, and more than 20% of conidia produced by the mutant contain multiple nuclei which were rare in the wild-type. During fermentation, the AcsepH disruption mutant grew slowly and its cephalosporin production was only about one quarter of the wild-type, and the transcription analysis showed that pcbC expression was delayed and the expressions of cefEF, cefD1 and cefD2 were significantly decreased. The vegetative hyphae of AcsepH mutant swelled abnormally and hardly formed the typical yeast-like cells. The amount of yeast-like cells was about one-tenth of the wild-type after fermentation for 5 days. Comparison of hyphal viabilities revealed that the cells of AcsepH mutant died easily than the wild-type at the late stage of fermentation. Fluorescent stains revealed that the absence of AcsepH in A. chrysogenum led to reduction of septation and formation of multinucleate cells. These data indicates that AcsepH is required for the normal cellular septation and differentiation of A. chrysogenum, and its absence may change the cellular physiological status and causes the decline in cephalosporin production.Highlights► A septation related gene AcsepH was identified by T-DNA insertion mutagenesis in Acremonium chrysogenum. ► Characterization of AcsepH. ► Discovered the important role of AcsepH in cellular differentiation and cephalosporin production in A. chrysogenum.

Glutathione is a ubiquitous thiol in eukaryotic cells, and its high intracellular ratio of reduced form (GSH) to oxidized form (GSSG) is largely maintained by glutathione reductase (GR) using NADPH as electron donor. glrA, a glutathione reductase encoding gene, was found and cloned from Acremonium chrysogenum by searching its genomic sequence based on similarity. Its deduced protein exhibits high similarity to GRs of other eukaryotic organisms. Disruption of glrA resulted in lack of GR activity and accumulation of a high level of GSSG in A. chrysogenum. Overexpression of glrA dramatically enhanced GR activity and the ratio of GSH/GSSG in this fungus. The spore germination and hyphal growth of glrA disruption mutant was strongly reduced in chemical defined medium. Meanwhile, the mutant was more sensitive to hydrogen peroxide than the wild-type strain. We found that the glrA mutant recovered normal germination and growth by adding exogenous methionine (Met). Exogenous Met also enhanced the antioxidative ability of both the mutant and wild-type strain. GSH determination indicated that the total GSH and ratio of GSH/GSSG in the mutant or wild-type strain were significantly increased when addition of Met into the medium. The glrA mutant grew poorly and could not produce detectable cephalosporin in the fermentation medium without Met. However, its growth and cephalosporin production was restored with addition of exogenous Met. These results indicate that glrA is required for the normal growth and protection against oxidative damage in A. chrysogenum, and its absence can be complemented by exogenous Met.Highlights► Characterization of a glutathione reductase encoding gene glrA in Acremonium chrysogenum. ► Discovered the important role of glrA in growth and antioxidative activity. ► Found methionine could complement the function of glrA. ► Revealed methionine could regulate intracellular glutathione level and redox balance.

•AcAreA directly controls the nitrogen metabolism in Acremonium chrysogenum.•AcAreA regulates cephalosporin biosynthesis positively.•Ammonium represses cephalosporin biosynthesis through AcAreA.AcareA, encoding a homologue of the fungal nitrogen regulatory GATA zinc-finger proteins, was cloned from Acremonium chrysogenum. Gene disruption and genetic complementation revealed that AcareA was required for nitrogen metabolism and cephalosporin production. Disruption of AcareA resulted in growth defect in the medium using nitrate, uric acid and low concentration of ammonium, glutamine or urea as sole nitrogen source. Transcriptional analysis showed that the transcription of niaD/niiA was increased drastically when induced with nitrate in the wild-type and AcareA complemented strains but not in AcareA disruption mutant. Consistent with the reduction of cephalosporin production, the transcription of pcbAB, cefD2, cefEF and cefG encoding the enzymes for cephalosporin production was reduced in AcareA disruption mutant. Band shift assays showed that AcAREA bound to the promoter regions of niaD, niiA and the bidirectional promoter region of pcbAB-pcbC. Sequence analysis showed that all the AcAREA binding sites contain the consensus GATA elements. These results indicated that AcAREA plays an important role both in the regulation of nitrogen metabolism and cephalosporin production in A. chrysogenum.

•AcATG1 is essential for autophagy and involved in conidiation of A. chrysogenum.•Conidiation defect in ΔAcatg1 could be suppressed by carbon sources.•Disruption of Acatg1 enhances cephalosporin production.Autophagy is a highly conserved eukaryotic mechanism for degradation of cellular components and nutrient recycling process. A serine/threonine kinase Atg1 is essential for autophagosome formation under starvation. Acatg1, the homologous gene of atg1 was cloned from the cephalosporin producing fungus Acremonium chrysogenum. Disruption of Acatg1 inhibited the autophagosome formation under starvation and significantly reduced conidia formation. However, exogenous supply of glucose, sucrose, mannitol or inositol restored the conidia formation of the Acatg1 disruption mutant to the wild-type level, suggesting that autophagy is involved in the carbon utilization which is required for cell growth and morphological differentiation. Unexpectedly, the Acatg1 disruption mutant showed strong resistance to exogenous hydrogen peroxide comparing with the wild-type strain. Disruption of Acatg1 also enhanced cephalosporin production at the late stage of fermentation. Consistent with cephalosporin production, the transcription of cephalosporin biosynthetic genes was increased in the Acatg1 disruption mutant and Western blotting demonstrated that the isopenicillin N synthase PcbC involved in cephalosporin biosynthesis was retained at the late stage of fermentation in the Acatg1 disruption mutant while it was sharply reduced in the wild-type strain. These results indicated that Acatg1 plays an important role in both autophagosome formation and cephalosporin production of A. chrysogenum.

•AcATG1 is essential for autophagy and involved in conidiation of A. chrysogenum.•Conidiation defect in ΔAcatg1 could be suppressed by carbon sources.•Disruption of Acatg1 enhances cephalosporin production.Autophagy is a highly conserved eukaryotic mechanism for degradation of cellular components and nutrient recycling process. A serine/threonine kinase Atg1 is essential for autophagosome formation under starvation. Acatg1, the homologous gene of atg1 was cloned from the cephalosporin producing fungus Acremonium chrysogenum. Disruption of Acatg1 inhibited the autophagosome formation under starvation and significantly reduced conidia formation. However, exogenous supply of glucose, sucrose, mannitol or inositol restored the conidia formation of the Acatg1 disruption mutant to the wild-type level, suggesting that autophagy is involved in the carbon utilization which is required for cell growth and morphological differentiation. Unexpectedly, the Acatg1 disruption mutant showed strong resistance to exogenous hydrogen peroxide comparing with the wild-type strain. Disruption of Acatg1 also enhanced cephalosporin production at the late stage of fermentation. Consistent with cephalosporin production, the transcription of cephalosporin biosynthetic genes was increased in the Acatg1 disruption mutant and Western blotting demonstrated that the isopenicillin N synthase PcbC involved in cephalosporin biosynthesis was retained at the late stage of fermentation in the Acatg1 disruption mutant while it was sharply reduced in the wild-type strain. These results indicated that Acatg1 plays an important role in both autophagosome formation and cephalosporin production of A. chrysogenum.

Penicillin is historically important as the first discovered drug against bacterial infections in human. Although the penicillin biosynthetic pathway and regulatory mechanism have been well studied in Penicillium chrysogenum, the compartmentation and molecular transport of penicillin or its precursors are still poorly understood. In search of the genomic database, more than 830 open reading frames (ORFs) were found to encode transmembrane proteins of P. chrysogenum. In order to investigate their roles on penicillin production, one of them (penT) was selected and cloned. The deduced protein of penT belongs to the major facilitator superfamily (MFS) and contains 12 transmembrane spanning domains (TMS). During fermentation, the transcription of penT was greatly induced by penicillin precursors phenylacetic acid (PAA) and phenoxyacetic acid (POA). Knock-down of penT resulted in significant decrease of penicillin production, while over-expression of penT under the promoter of trpC enhanced the penicillin production. Introduction of an additional penT in the wild-type strain of P. chrysogenum doubled the penicillin production and enhanced the sensitivity of P. chrysogenum to the penicillin precursors PAA or POA. These results indicate that penT stimulates penicillin production probably through enhancing the translocation of penicillin precursors across fungal cellular membrane.