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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 β-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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In an earlier work on lovastatin production by Aspergillus terreus, we found that reactive oxygen species (ROS) concentration increased to high levels precisely at the start of the production phase (idiophase) and that these levels were sustained during all idiophase. Moreover, it was shown that ROS regulate lovastatin biosynthesis. ROS regulation has also been reported for aflatoxins. It has been suggested that, due to their antioxidant activity, aflatoxins are regulated and synthesized like a second line of defense against oxidative stress. To study the possible ROS regulation of other industrially important secondary metabolites, we analyzed the relationship between ROS and penicillin biosynthesis by Penicillium chrysogenum and cephalosporin biosynthesis by Acremonium chrysogenum. Results revealed a similar ROS accumulation in idiophase in penicillin and cephalosporin fermentations. Moreover, when intracellular ROS concentrations were decreased by the addition of antioxidants to the cultures, penicillin and cephalosporin production were drastically reduced. When intracellular ROS were increased by the addition of exogenous ROS (H 2 O 2 ) to the cultures, proportional increments in penicillin and cephalosporin biosyntheses were obtained. It was also shown that lovastatin, penicillin, and cephalosporin are not antioxidants. Taken together, our results provide evidence that ROS regulation is a general mechanism controlling secondary metabolism in fungi.

The co-occurrence of polycyclic aromatic hydrocarbons (PAHs) with heavy metals (HMs) is very common in contaminated soils, but the influence of HMs on fungal-bacterial synergism during PAH bioremediation has not been investigated. The bioremediation of fluoranthene-contaminated sand using co-cultures of Acremonium sp. P0997 and Bacillus subtilis showed increases of 109.4 and 9.8 % in degradation compared to pure bacterial and fungal cultures, respectively, removing 64.1 ± 1.4 % fluoanthene in total. The presence of Cu(2+) reduced fluoranthene removal to 53.7 ± 1.7 %, while inhibiting bacterial growth, and reducing translocation of bacteria on fungal hyphae by 49.5 %, in terms of the bacterial translocation ratio. Cu(2+) reduced bacterial diffusion by 46.8 and 31.9 %, as reflected by D (a bulk random motility diffusional coefficient) and D eff (the effective one-dimensional diffusion coefficient) compared to the control without HM supplementation, respectively. However, Mn(2+) resulted in a 78.2 ± 1.9 % fluoranthene degradation, representing an increase of 21.9 %, while enhancing bacterial growth and bacterial translocation on fungal hyphae, showing a 12.0 % increase in translocation ratio, with no observable impact on D and D eff. Hence, the presence of HMs has been shown to affect fungal-bacterial synergism in PAH degradation, and this effect differs with HM species.

The co-occurrence of polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) is very common in contaminated environments. It is of paramount importance and great challenge to exploit a bioremediation to remove PAHs in these environments with combined pollution. We approached this question by probing the influence of HMs coexisting with PAHs on the removal of PAHs by Acremonium sp. P0997 possessing metal resistance. A removal capability for naphthalene, fluorene, phenanthrene, anthracene, and fluoranthenepresentalone (98.6, 99.3, 89.9, 60.4, and 70 %, respectively) and in a mixture (96.9, 71.8, 67.0, 85.0, and 87.9 %, respectively) was achieved in mineral culture inoculated with Acremonium sp. P0997, and this strain also displayed high resistance to the individual HMs (Mn(2+), Fe(2+), Zn(2+), Cu(2+), Al(3+), and Pb(2+)). The removal of individual PAHs existing in a mixture was differently affected by the separately tested HMs. Cu(2+)enhanced the partition process of anthracene to dead or alive mycelia and the contribution of the biosorption by this strain but imposed a little negative influence on the contribution of biodegradation to the total removal of anthracene individually in a culture. However, Mn(2+) had an inhibitory effect on the partition process of anthracene to dead or alive mycelia and decreased the contributions of both biosorption and biodegradation to the total anthracene removal. This work showcased the value of fungi in bioremediation for the environments with combined pollution, and the findings have major implications for the bioremediation of organic pollutants in metal-organic mixed contaminated sites.

The thioredoxin system including thioredoxin and thioredoxin reductase (TrxR) is used for oxidative stress defenses in fungi. Based on the genomic sequence, a thioredoxin reductase-encoding gene (ActrxR1) was isolated from Acremonium chrysogenum CGMCC3.3795. Like other TrxRs, AcTrxR1 contains FAD binding domain, Redox domain, and NADPH binding domain. Disruption of ActrxR1 in A. chrysogenum led to the formation of smaller colonies and hyphal swelling in Tryptic soy agar (TSA). In chemically defined medium, the spore germination of ActrxR1 disruption mutant was strongly inhibited, which was recovered by the addition of DL-methionine. The disruption mutant grew slowly on TSA compared with the wild-type strain, but it did not show to be more sensitive to exogenous hydrogen peroxide or menadione. In defined medium of fermentation supplemented with DL-methionine, the ActrxR1 disruption mutant grew normally, and its cephalosporin C production increased by about onefold compared with the wild type (73 μg/ml for wild-type strain and 136 μg/ml for the mutant at 5 days of fermentation). Real-time polymerase chain reaction (RT-PCR) showed that the transcriptional levels of pcbC, cefEF, and cefG were obviously enhanced in the ActrxR1 mutant at the early stage of fermentation. These results indicate that ActrxR1 is required for the normal growth of A. chrysogenum and related with cephalosporin C production in methionine-supplemented medium.

Preparative-scale fermentation of ginsenoside Rb(1) (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12beta-hydroxydammar-3-one-20 (S)-O-beta-D -glucopyranoside (7), 12beta, 25-dihydroxydammar-(E)-20(22)-ene-3-O-beta-D -glucopyranosyl-(1-->2)-beta-D -glucopyranoside (8), and 12beta, 20 (R), 25-trihydroxydammar-3-O-beta-D -glucopyranosyl-(1-->2)-beta-D -glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg(3) (4), ginsenoside F(2) (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2-6 are also the metabolites of ginsenoside Rb(1) in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.

Cultivation processes involving filamentous fungi have been optimised for decades to obtain high product yields. Several bulk chemicals like citric acid and penicillin are produced this way. A simple adaptation of cultivation parameters for new production processes is not possible though. Models explaining the correlation between process-dependent growth behaviour and productivity are therefore necessary to prevent long-lasting empiric test series. Yet, filamentous growth consists of a complex microscopic differentiation process from conidia to hyphae resulting in various macroscopically visible appearances. Early approaches to model this morphologic development are recapitulated in this review to explain current trends in this area of research. Tailoring morphology by adjusting process parameters is one side of the coin, but an ideal morphology has not even been found. This article reviews several reasons for this fact starting with nutrient supply in a fungal culture and presents recent advances in the investigation of fungal metabolism. It illustrates the challenge to unfold the relationship between morphology and productivity.

The nucleotide sequence of a 3240-bp genomic fragment including the gamma-actin-encoding gene from Acremonium chrysogenum has been determined, showing an open reading frame of 1691 bp, interrupted by five introns with fungal consensus splice-site junctions. The untranslated regions of the actA gene contain a consensus TATA box, a CCAAT motif, pyrimidine stretches and the polyadenylation sequence AATAA. The predicted protein (375 amino acids) revealed high identity to gamma-actins from fungi (> 90%). Gene phylogenies constructed using DNA and protein sequences support the grouping of A. chrysogenum actin close to those from the majority of the filamentous fungi. The actA gene is present as a single copy in the genome of A. chrysogenum; and its expression level, opposite to pcbC and cefEF cephalosporin biosynthetic genes, was steady during cephalosporin fermentation, showing a single 1.4-kb transcript.

In the filamentous fungus Acremonium chrysogenum, biosynthesis of the beta-lactam antibiotic cephalosporin C is repressed by glucose. A wild-type strain grown in the presence of glucose shows a reduction of transcripts derived from the pcbC and cefEF biosynthesis genes. Interestingly, the amount of the pcbC transcript is not affected in another strain with enhanced cephalosporin C production, suggesting a correlation between strain improvement and deregulation of glucose repression. The function of the glucose repressor CRE1 in this regulation was further analyzed by transforming both A. chrysogenum strains with multiple copies of the cre1 gene. The molecular analysis of transformants revealed that additional copies of the cre1 gene restore wild-type-like regulation of pcbC gene expression in the semi-producer strain, while repression of the cefEF gene expression is increased. Overall, our data indicate a regulation of the pcbC and the cefEF gene expression by CRE1.

Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the stimulation of cephalosporin C production and the role of isocitrate lyase are discussed.

Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of L-alpha-aminoadipic acid, L-cysteine and L-valine to form the tripeptide delta(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase and isopenicillin N (IPN) synthase respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms, biosynthesis of the alpha-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes involved in methoxylation at C-7 and carbamoylation at C-3' of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P7 and P8) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative to form the methoxyl group. Carbamoylation at the C-3'-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase encoded by the gene cmcH. Finally, genes for a beta-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the beta-lactam biosynthetic pathways such as a channelling precursors and directed removal of bottlenecks in the beta-lactam biosynthetic pathways. Several new beta-lactam antibiotics have been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new molecules.

The conversion of deacetylcephalosporin C to cephalosporin C is inefficient in most Acremonium chrysogenum strains. The cefG gene, which encodes deacetylcephalosporin C acetyltransferase, is expressed very poorly in A. chrysogenum as compared to other genes of the cephalosporin pathway. Introduction of additional copies of the cefG gene with its native promoter (in two different constructions with upstream regions of 1056 bp and 538 bp respectively) did not produce a significant increase of the steady-state level of the cefG transcript. Expression of the cefG gene from the promoters of (i) the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of Aspergillus nidulans, (ii) the glucoamylase (gla) gene of Aspergillus niger, (iii) the glutamate dehydrogenase (gdh) and (iv) the isopenicillin N synthase (pcbC) genes of Penicillium chrysogenum, led to very high steady-state levels of cefG transcript and to increased deacetylcephalosporin-C acetyltransferase protein concentration (as shown by immunoblotting) and enzyme activity in the transformants. Southern analysis showed that integration of the new constructions occurred at sites different from that of the endogenous cefG gene. Cephalosporin production was increased two- to threefold in A. chrysogenum C10 transformed with constructions in which the cefG gene was expressed from the gdh or gpd promoters as a result of a more efficient acetylation of deacetylcephalosporin C.

A kinetic model describing the biosynthesis of cephalosporin C in Cephalosporium acremonium has been developed to identify the rate-limiting step(s). Using this model and in-vitro kinetic data of the biosynthetic enzymes, the production kinetics of cephalosporin C were examined theoretically. The predicted time profile of the specific production rate during batch culture is in good agreement with that of experimental results published previously. Sensitivity analysis indicates that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase is the rate-limiting enzyme. Our analysis also predicts that increasing ACV synthetase enhances the production rate initially until expandase/hydroxylase becomes rate-limiting. Furthermore, increasing expandase/hydroxylase reduces the accumulation of penicillin N, and thus, enhances the production of cephalosporin C. Based on our analysis, amplifying both ACV synthetase and expandase/hydroxylase concurrently should enhance the production rate to a great extent.

Single and multisensor field effect transistors (FET) with a pH-sensitive Si/SiO2/Si3N4/Ta2O5-gate and reference electrode (for single sensor) were developed and used for manufacturing the following biological (Bio)-FETs: for glucose analysis, glucose oxidase-FET (GOD-FET); for urea analysis, urease-FET; and for cephalosporin C analysis, cephalosporinase-FET. The GOD-FETs were integrated into flow injection analysis (FIA) of the Eppendorf variables analyser (EVA) system and used for monitoring the glucose concentration in microbial cultivation and production processes with recombinant Escherichia coli K12 MF, recombinant E. coli JM103, Saccharomyces cerevisiae H620, and Candida boidinii. Urease-FET-FIA was used to monitor the urea concentration in a simulated cultivation of Cephalosporium acremonium and urease-FET-FIA and GOD-FET-FIA for the monitoring of urea and glucose concentrations in simulated S. cerevisiae cultivations.