Identification of a (+)-cubenene synthase from filamentous fungi Acremonium chrysogenum.

Sesquiterpene synthases convert farnesyl diphosphate into various sesquiterpenes, which find wide applications in the food, cosmetics and pharmaceutical industries. Although numerous putative sesquiterpene synthases have been identified in fungal genomes, many lack biochemical characterization. In this study, we identified a putative terpene synthase AcTPS3 from Acremonium chrysogenum. Through sequence analysis and in vitro enzyme assay, AcTPS3 was identified as a sesquiterpene synthase. To obtain sufficient product for NMR testing, a metabolic engineered Saccharomyces cerevisiae was constructed to overproduce the product of AcTPS3. The major product of AcTPS3 was identified as (+)-cubenene (55.46%) by GC-MS and NMR. Thus, AcTPS3 was confirmed as (+)-cubenene synthase, which is the first report of (+)-cubenene synthase. The optimized S. cerevisiae strain achieved a biosynthesis titer of 597.3 mg/L, the highest reported for (+)-cubenene synthesis.

Seed Infection Rate, but Not Pathogen Titer, Positively Correlates with Disease Index of Cephalosporium Stripe in Winter Wheat.

Cephalosporium gramineum survives primarily in colonized plant residue but is also transmitted by seed at a low frequency. The purpose of this study was to correlate disease intensity in the field with percentage of infected seed and amount of pathogen DNA using a high-throughput PCR method. Field-grown seed of three wheat cultivars was collected over 4 years from plots with a known disease index. The culture-based seed infection rate was determined by isolation of C. gramineum from 2,016 seeds per seed lot. DNA of 380 seeds from each seed lot was extracted individually, and a PCR assay with a fluorescent-labeled forward primer for detecting C. gramineum was performed on each seed. C. gramineum was isolated from 0.12% of the seed on average (range 0 to 0.74%), whereas it was detected in 3.7% on average (range 1.3 to 7.6%) using PCR detection. The single-seed PCR assay was more sensitive than either the culture-based method or conventional PCR. DNA of 674 seeds that tested positive by this PCR was quantified using a real-time PCR with newly designed primers for the amount of pathogen per seed. Seed contained 0.017 to 77.1 pg/seed of C. gramineum DNA (mean 3.0 pg/seed). Disease index was positively correlated with seed infection rate but not with pathogen titer in seed. This fluorescent-labeled PCR, along with quantitative PCR, improved our understanding of seed transmission of C. gramineum in wheat.

Cephalosporin C biosynthesis and fermentation in Acremonium chrysogenum.

Cephalosporins are currently the most widely used antibiotics in clinical practice. The main strain used for the industrial production cephalosporin C (CPC) is Acremonium chrysogenum. CPC has the advantages of possessing a broad antibacterial spectrum and strong antibacterial activity. However, the yield and titer of cephalosporins obtained from A. chrysogenum are much lower than penicillin, which is also a ß-lactam antibiotic produced by Penicillium chrysogenum. Molecular biology research into A. chrysogenum has focused on gene editing technologies, multi-omics research which has provided information on the differences between high- and low-yield strains, and metabolic engineering involving different functional genetic modifications and hierarchical network regulation to understand strain characteristics. Furthermore, optimization of the fermentation process is also reviewed as it provides the optimal environment to realize the full potential of strains. Combining rational design to control the metabolic network, high-throughput screening to improve the efficiency of obtaining high-performance strains, and real-time detection and controlling in the fermentation process will become the focus of future research in A. chrysogenum. This minireview provides a holistic and in-depth analysis of high-yield mechanisms and improves our understanding of the industrial value of A. chrysogenum. KEY POINTS: • Review of the advances in A. chrysogenum characteristics improvement and process optimization • Elucidate the molecular bases of the mechanisms that control cephalosporin C biosynthesis and gene expression in A. chrysogenum • The future development trend of A. chrysogenum to meet industrial needs.

Knockout and functional analysis of BSSS-related genes in Acremonium chrysogenum by novel episomal expression vector containing Cas9 and AMA1.

OBJECTIVE: The target sorB gene, related to sorbicillinoid production, and the free expression element, AMA1, were used to verify the methodological approach in Acremonium chrysogenum. RESULT: CRISPR-Cas9 episomal expression system was used to introduce a point mutation into the sorB gene and the addition of sorB donor DNA achieved complete knockout of target genes. Four BSSS (yeast bud site selection system)-related genes, axl1, axl2, bud3, and bud4 were knocked out without impact on yield, dry weight, or pH. Relationships between morphology and stress tolerance in knockout strains were analyzed. CONCLUSION: The gene-editing system used in the current study exceeded 80% efficiency and arthrospores development was found to differ from that in wild-type strain.

Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum.

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

Spermidine and 1,3-Diaminopropane Have Opposite Effects on the Final Stage of Cephalosporin C Biosynthesis in High-Yielding Acremonium chrysogenum Strain.

The addition of exogenous polyamines increases the production of antibiotic cephalosporin C (CPC) in Acremonium chrysogenum high-yielding (HY) strain during fermentation on a complex medium. However, the molecular basis of this phenomenon is still unknown. In the current study, we developed a special synthetic medium on which we revealed the opposite effect of polyamines. The addition of 1,3-diaminopropane resulted in an increase in the yield of CPC by 12-15%. However, the addition of spermidine resulted in a decrease in the yield of CPC by 14-15% and accumulation of its metabolic pathway precursor, deacetylcephalosporin C (DAC); the total amount of cephems (DAC and CPC) was the same as after the addition of DAP. This indicates that spermidine, but not 1,3-diaminopropane, affects the final stage of CPC biosynthesis, associated with the acetylation of its precursor. In both cases, upregulation of biosynthetic genes from beta-lactam BGCs occurred at the same level as compared to the control; expression of transport genes was at the control level. The opposite effect may be due to the fact that N1-acetylation is much more efficient during spermidine catabolism than for 1,3-diaminopropane. The addition of spermidine, but not 1,3-diaminopropane, depleted the pool of acetyl coenzyme A by more than two-fold compared to control, which could lead to the accumulation of DAC.

Establishment of conidial fusion in the asexual fungus Verticillium dahliae as a useful system for the study of non-sexual genetic interactions.

Cell-to-cell fusion is a fundamental biological process across the tree of life. In filamentous fungi, somatic fusion (or anastomosis) is required for the normal development of their syncytial hyphal networks, and it can initiate non-sexual genetic exchange processes, such as horizontal genetic transfer and the parasexual cycle. Although these could be important drivers of the evolution of asexual fungi, this remains a largely unexplored possibility due to the lack of suitable resources for their study in these puzzling organisms. We thus aimed at the characterization of cell fusion in the important asexual fungus Verticillium dahliae via Conidial Anastomosis Tubes (CATs), which can be useful for the analysis of parasexuality. We optimized appropriate procedures for their highly reproducible quantification and live-cell imaging, which were used to characterize their physiology and cell biology, and to start elucidating their underlying genetic machinery. Formation of CATs was shown to depend on growth conditions and require functional Fus3 and Slt2 MAP kinases, as well as the NADPH oxidase NoxA, whereas the GPCR Ste2 and the mating-type protein MAT1-2-1 were dispensable. We show that nuclei and other organelles can migrate through CATs, which often leads to the formation of transient dikaryons. Their nuclei have possible windows of opportunity for genetic interaction before degradation of one by a presumably homeostatic mechanism. We establish here CAT-mediated fusion in V. dahliae as an experimentally convenient system for the cytological analysis of fungal non-sexual genetic interactions. We expect that it will facilitate the dissection of sexual alternatives in asexual fungi.

Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum.

Acremonium chrysogenum has been employed in the industrial production of cephalosporin C (CPC). However, there are still some impediments to understanding the regulation of CPC biosynthesis and improving strains due to the difficulty of genetic manipulation in A. chrysogenum, especially in the CPC high-producing strain C10. Here, an improved CRISPR-Cas9 system was constructed based on an U6/tRNA chimeric promoter. Using this system, high efficiency for single gene disruption was achieved in C10. In addition, double loci were simultaneously targeted when supplying with the homology-directed repair templates (donor DNAs). Based on this system, large DNA fragments up to 31.5 kb for the yellow compound sorbicillinoid biosynthesis were successfully deleted with high efficiency. Furthermore, CPC production was significantly enhanced when the sorbicillinoid biosynthetic genes were knocked out. This study provides a powerful tool for gene editing and strain improvement in A. chrysogenum.

Molecular detection of fungal and bacterial DNA from pustules in patients with palmoplantar pustulosis: special focus on Malassezia species.

BACKGROUND: Palmoplantar pustulosis (PPP) is a distinct, chronic skin disorder characterized by intraepidermal pustules on the palms and soles. It is hypothesized that microorganisms on the skin might induce the symptoms of PPP via inflammatory cell activation. However, the microbiota has not been studied in detail because of the assumption that the pustules in PPP are sterile. AIM: To elucidate the role of microorganisms in pathogenesis of PPP. METHODS: PCR analysis was performed of microbial DNA fragments in the pustules of patients with PPP. The sequence of the D1/D2 LSU 26s rRNA gene and that of the 16S rRNA gene was used for fungal and bacterial DNA detection, respectively. RESULTS: In total, 71 samples were carefully collected from the pustules of patients with PPP. Fungal DNA bands were detected in 68 samples, and fungi including Malassezia spp. were identified in 30 of 71 samples (42.3%). Malassezia restricta was the most frequently encountered fungus (14/71; 19.7%). However, bacterial DNA was not detected by the methods used. Furthermore, identical fungal DNA was not detected in the outer lid of the pustules, suggesting that the fungi detected within the pustule did not derive from contamination via the skin surface. CONCLUSIONS: In the present study, we demonstrated for the first time that certain pustules in patients with PPP contain fungal DNA fragments, especially those of Malassezia spp. Our findings provide new insights on the role of skin microbiota in the pathogenesis of PPP.

The flavonoid degrading fungus Acremonium sp. DSM 24697 produces two diglycosidases with different specificities.

AbstractDiglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-ß-glucosidase I and II (αRßG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-ß-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRßG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRßG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRßG I. On the other hand, αRßG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRßG I showed exclusive specificity toward 7-O-ß-rutinosyl flavonoids, whereas αRßG II hydrolyzed both 7-O-ß-rutinosyl- and 3-O-ß-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-ß-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRßG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.

Enzyme-mediated transglycosylation of rutinose (6-O-α-l-rhamnosyl-d-glucose) to phenolic compounds by a diglycosidase from Acremonium sp. DSM 24697.

The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-ß-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-l-rhamnopyranosyl-ß-d-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ≤6.0. The structure of 4-hydroxyphenyl-ß-rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4-hydroxyphenyl-ß-rutinoside (38%, regarding hesperidin) was achieved in a 2-h process at pH 5.0 and 30 °C, with 36 mM OH-acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pKa value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.

Identification and Characterization of an Autophagy-Related Gene Acatg12 in Acremonium chrysogenum.

Autophagy is a highly conserved mechanism to overcome various stresses and recycle cytoplasmic components and organelles. Ubiquitin-like (UBL) protein Atg12 is a key protein involved in autophagosome formation through stimulation of Atg8 conjugation to phosphatidylethanolamine. Here, we describe the identification of the autophagy-related gene Acatg12, encoding an Atg12 homologous protein in the cephalosporin C producing fungus Acremonium chrysogenum. Disruption of Acatg12 impaired the delivery and degradation of eGFP-Atg8, indicating that the autophagic process was blocked. Meanwhile, conidiation was dramatically reduced in the Acatg12 disruption mutant (∆Acatg12). In contrast, cephalosporin C production was increased twofold in ∆Acatg12, but fungal growth was reduced after 6 days fermentation. Consistent with these results, the transcriptional level of the cephalosporin biosynthetic genes was increased in ∆Acatg12. The results extend our understanding of autophagy in filamentous fungi.

The High Diversity and Variable Susceptibility of Clinically Relevant Acremonium-Like Species in China.

Acremonium-like fungi are emerging as important opportunistic pathogens in cutaneous, subcutaneous and serious invasive infections, especially in immunocompromised and debilitated individuals, and Acremonium infections are usually resistant to antifungal therapy. Several molecular studies have demonstrated that many species in the genus Acremonium are polyphyletic, and currently, the genus is restricted to the family Bionectriaceae (Hypocreales). Molecular identification and in vitro antifungal susceptibility tests of Acremonium-like fungi isolated from human clinical specimens in China were performed in this study. Three genetic loci: the large subunit ribosomal RNA gene (LSU), ribosomal internal transcribed spacer and elongation factor 1-α (EF1-α), were used to assess their taxonomic position for correct identification among various species. The multilocus study of twenty-eight strains showed that these strains were distributed in three main lineages: egyptiacum, Cordycipitaceae and Sarocladium; Acremonium egyptiacum and Sarocladium kiliense were the main species of these strains, and three isolates were too phylogenetically distant to be considered undescribed species. Relatively low minimum inhibitory concentrations (MICs) of 0.25-2 and 0.031-0.5 µg/mL were found for voriconazole and terbinafine for most species, respectively. Varied antifungal activities of ciclopirox olamine, amorolfine and posaconazole were found in our study. However, no antifungal effect of sertaconazole, itraconazole or fluconazole was observed against most strains. This is the first study on Acremonium-like species diversity by multilocus sequence analyses and antifungal susceptibility of clinically relevant isolates in China.

AcAxl2 and AcMst1 regulate arthrospore development and stress resistance in the cephalosporin C producer Acremonium chrysogenum.

The filamentous fungus Acremonium chrysogenum is the primordial producer of the ß-lactam antibiotic cephalosporin C. This antibiotic is of major biotechnological and medical relevance because of its antibacterial activity against Gram-positive and Gram-negative bacteria. Antibiotic production during the lag phase of fermentation is often accompanied by a typical morphological feature of A. chrysogenum, the fragmentation of the mycelium into arthrospores. Here, we sought to identify factors that regulate the hyphal septation process and present the first comparative functional characterization of the type I integral plasma membrane protein Axl2 (axial budding pattern protein 2), a central component of the bud site selection system (BSSS) and Mst1 (mammalian Sterile20-like kinase), a septation initiation network (SIN)-associated germinal center kinase (GCK). Although an Acaxl2 deletion strain showed accelerated arthrospore formation after 96 h in liquid culture, deletion of Acmst1 led to a 24 h delay in arthrospore development. The overexpression of Acaxl2 resulted in an arthrospore formation similar to the A3/2 strain. In contrast to this, A3/2::Acmst1 OE strain displayed an enhanced arthrospore titer. Large-scale stress tests revealed an involvement of AcAxl2 in controlling osmotic, endoplasmic reticulum, and cell wall stress response. In a similar approach, we found that AcMst1 plays an essential role in regulating growth under osmotic, cell wall, and oxidative stress conditions. Microscopic analyses and plating assays on media containing Calcofluor White and NaCl showed that arthrospore development is a stress-dependent process. Our results suggest the potential for identifying candidate genes for strain improvement programs to optimize industrial fermentation processes.

A Myb transcription factor represses conidiation and cephalosporin C production in Acremonium chrysogenum.

Acremonium chrysogenum is the industrial producer of cephalosporin C (CPC). We isolated a mutant (AC554) from a T-DNA inserted mutant library of A. chrysogenum. AC554 exhibited a reduced conidiation and lack of CPC production. In consistent with it, the transcription of cephalosporin biosynthetic genes pcbC and cefEF was significantly decreased in AC554. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed and sequence analysis indicated that a T-DNA was inserted upstream of an open reading frame (ORF) which was designated AcmybA. On the basis of sequence analysis, AcmybA encodes a Myb domain containing transcriptional factor. Observation of red fluorescent protein (RFP) tagged AcMybA showed that AcMybA is naturally located in the nucleus of A. chrysogenum. Transcriptional analysis demonstrated that the AcmybA transcription was increased in AC554. In contrast, the AcmybA deleted mutant (ΔAcmybA) overproduced conidia and CPC. To screen the targets of AcmybA, we sequenced and compared the transcriptome of ΔAcmybA, AC554 and the wild-type strain at different developmental stages. Twelve differentially expressed regulatory genes were identified. Taken together, our results indicate that AcMybA negatively regulates conidiation and CPC production in A. chrysogenum.

Enhancing the production of cephalosporin C through modulating the autophagic process of Acremonium chrysogenum.

BACKGROUND: Autophagy is used for degradation of cellular components and nutrient recycling. Atg8 is one of the core proteins in autophagy and used as a marker for autophagic detection. However, the autophagy of filamentous fungi is poorly understood compared with that of Saccharomyces cerevisiae. Our previous study revealed that disruption of the autophagy related gene Acatg1 significantly enhanced cephalosporin C yield through reducing degradation of cephalosporin biosynthetic proteins in Acremonium chrysogenum, suggesting that modulation of autophagic process is one promising way to increase antibiotic production in A. chrysogenum. RESULTS: In this study, a S. cerevisiae ATG8 homologue gene Acatg8 was identified from A. chrysogenum. Acatg8 could complement the ATG8 mutation in S. cerevisiae, indicating that Acatg8 is a functional homologue of ATG8. Microscope observation demonstrated the fluorescently labeled AcAtg8 was localized in the cytoplasm and autophagosome of A. chrysogenum, and the expression of Acatg8 was induced by nutrient starvation. Gene disruption and genetic complementation revealed that Acatg8 is essential for autophagosome formation. Disruption of Acatg8 significantly reduced fungal conidiation and delayed conidial germination. Localization of GFP-AcAtg8 implied that autophagy is involved in the early phase of conidial germination. Similar to Acatg1, disruption of Acatg8 remarkably enhanced cephalosporin C yield. The cephalosporin C biosynthetic enzymes (isopenicillin N synthase PcbC and isopenicillin N epimerase CefD2) and peroxisomes were accumulated in the Acatg8 disruption mutant (∆Acatg8), which might be the main reasons for the enhancement of cephalosporin C production. However, the biomass of ΔAcatg8 decreased drastically at the late stage of fermentation, suggesting that autophagy is critical for A. chrysogenum cell survival under nutrition deprived condition. Disruption of Acatg8 also resulted in accumulation of mitochondria, which might produce more reactive oxygen species (ROS) which promotes fungal death. However, the premature death is unfavorable for cephalosporin C production. To solve this problem, a plasmid containing Acatg8 under control of the xylose/xylan-inducible promoter was introduced into ∆Acatg8. Conidiation and growth of the recombinant strain restored to the wild-type level in the medium supplemented with xylose, while the cephalosporin C production maintained at a high level even prolonged fermentation. CONCLUSIONS: Our results demonstrated inducible expression of Acatg8 and disruption of Acatg8 remarkably increased cephalosporin C production. This study provides a promising approach for yield improvement of cephalosporin C in A. chrysogenum.

Expression of the 5-enoylpyruvylshikimate-3-phosphate synthase domain from the Acremonium sp. aroM complex enhances resistance to glyphosate.

OBJECTIVE: To discover and isolate a glyphosate-resistant gene from a microorganism through gene mining. RESULTS: The full aroM gene from Acremonium sp. (named aroMA.sp.) was cloned using rapid amplification of cDNA ends. The transcriptional expression level of each domain increased significantly after glyphosate treatment in the aroMA.sp. complex and reached its maximum at 48 h. The aroA domain of the aroMA.sp. (named aroA A.sp.) was expressed in Escherichia coli BL21 (DE3) and the product was purified through Ni-NTA affinity chromatography. Furthermore, 45 KDa was indicated by SDS-PAGE and its enzyme activity was optimal at 30 °C and PH 7.0. The Ki/Km value of aroAA.sp. was 0.106, and the E. coli BL21 harboring aroAA.sp. could grow in the M9 minimal medium with 100 mM glyphosate. CONCLUSION: The aroAA.sp. from the aroMA.sp. complex had high enzyme activity and glyphosate resistance. Therefore, this research offers a new strategy for improving glyphosate resistance using the aroA domain of the aroM complex in the fungi.

Transcriptome analysis of the two unrelated fungal ß-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs.

BACKGROUND: Cephalosporins and penicillins are the most frequently used ß-lactam antibiotics for the treatment of human infections worldwide. The main industrial producers of these antibiotics are Acremonium chrysogenum and Penicillium chrysogenum, two taxonomically unrelated fungi. Both were subjects of long-term strain development programs to reach economically relevant antibiotic titers. It is so far unknown, whether equivalent changes in gene expression lead to elevated antibiotic titers in production strains. RESULTS: Using the sequence of PcbC, a key enzyme of ß-lactam antibiotic biosynthesis, from eighteen different pro- and eukaryotic microorganisms, we have constructed a phylogenetic tree to demonstrate the distant relationship of both fungal producers. To address the question whether both fungi have undergone similar genetic adaptions, we have performed a comparative gene expression analysis of wild-type and production strains. We found that strain improvement is associated with the remodeling of the transcriptional landscape in both fungi. In P. chrysogenum, 748 genes showed differential expression, while 1572 genes from A. chrysogenum are differentially expressed in the industrial strain. Common in both fungi is the upregulation of genes belonging to primary and secondary metabolism, notably those involved in precursor supply for ß-lactam production. Other genes not essential for ß-lactam production are downregulated with a preference for those responsible for transport processes or biosynthesis of other secondary metabolites. Transcriptional regulation was shown to be an important parameter during strain improvement in different organisms. We therefore investigated deletion strains of the major transcriptional regulator velvet from both production strains. We identified 567 P. chrysogenum and 412 A. chrysogenum Velvet target genes. In both deletion strains, approximately 50% of all secondary metabolite cluster genes are differentially regulated, including ß-lactam biosynthesis genes. Most importantly, 35-57% of Velvet target genes are among those that showed differential expression in both improved industrial strains. CONCLUSIONS: The major finding of our comparative transcriptome analysis is that strain improvement programs in two unrelated fungal ß-lactam antibiotic producers alter the expression of target genes of Velvet, a global regulator of secondary metabolism. From these results, we conclude that regulatory alterations are crucial contributing factors for improved ß-lactam antibiotic titers during strain improvement in both fungi.

Deactivation of the autotrophic sulfate assimilation pathway substantially reduces high-level ß-lactam antibiotic biosynthesis and arthrospore formation in a production strain from Acremonium chrysogenum.

The filamentous ascomycete Acremonium chrysogenum is the only industrial producer of the ß-lactam antibiotic cephalosporin C. Synthesis of all ß-lactam antibiotics starts with the three amino acids l-α-aminoadipic acid, l-cysteine and l-valine condensing to form the δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine tripeptide. The availability of building blocks is essential in every biosynthetic process and is therefore one of the most important parameters required for optimal biosynthetic production. Synthesis of l-cysteine is feasible by various biosynthetic pathways in all euascomycetes, and sequencing of the Acr. chrysogenum genome has shown that a full set of sulfur-metabolizing genes is present. In principle, two pathways are effective: an autotrophic one, where the sulfur atom is taken from assimilated sulfide to synthesize either l-cysteine or l-homocysteine, and a reverse transsulfuration pathway, where l-methionine is the sulfur donor. Previous research with production strains has focused on reverse transsulfuration, and concluded that both l-methionine and reverse transsulfuration are essential for high-level cephalosporin C synthesis. Here, we conducted molecular genetic analysis with A3/2, another production strain, to investigate the autotrophic pathway. Strains lacking either cysteine synthase or homocysteine synthase, enzymes of the autotrophic pathway, are still autotrophic for sulfur. However, deletion of both genes results in sulfur amino acid auxotrophic mutants exhibiting delayed biomass production and drastically reduced cephalosporin C synthesis. Furthermore, both single- and double-deletion strains are more sensitive to oxidative stress and form fewer arthrospores. Our findings provide evidence that autotrophic sulfur assimilation is essential for growth and cephalosporin C biosynthesis in production strain A3/2 from Acr. chrysogenum.

Functional analysis of the selective autophagy related gene Acatg11 in Acremonium chrysogenum.

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 asaminopeptidase I (Ape1), are selectively transported to vacuoles. However, the physiological role of selective autophagy remains elusive in filamentous fungi. ATG11 family proteins asa 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.