Cephalosporin Biosynthesis Research Articles

Diamine Fungal Inducers of Secondary Metabolism: 1,3-Diaminopropane and Spermidine Trigger Enzymes Involved in β-Alanine and Pantothenic Acid Biosynthesis, Precursors of Phosphopantetheine in the Activation of Multidomain Enzymes.

The biosynthesis of antibiotics and other secondary metabolites (also named special metabolites) is regulated by multiple regulatory networks and cascades that act by binding transcriptional factors to the promoter regions of different biosynthetic gene clusters. The binding affinity of transcriptional factors is frequently modulated by their interaction with specific ligand molecules. In the last decades, it was found that the biosynthesis of penicillin is induced by two different molecules, 1,3-diaminopropane and spermidine, but not by putrescine (1,4-diaminobutane) or spermine. 1,3-diaminopropane and spermidine induce the expression of penicillin biosynthetic genes in Penicillium chrysogenum. Proteomic studies clearly identified two different proteins that respond to the addition to cultures of these inducers and are involved in β-alanine and pantothenic acid biosynthesis. These compounds are intermediates in the biosynthesis of phosphopantetheine that is required for the activation of non-ribosomal peptide synthetases, polyketide synthases, and fatty acid synthases. These large-size multidomain enzymes are inactive in the "apo" form and are activated by covalent addition of the phosphopantetheine prosthetic group by phosphopantetheinyl transferases. Both 1,3-diaminopropane and spermidine have a similar effect on the biosynthesis of cephalosporin by Acremonium chrysogenum and lovastatin by Aspergillus terreus, suggesting that this is a common regulatory mechanism in the biosynthesis of bioactive secondary metabolites/natural products.

Co-occurrence network and functional profiling of the bacterial community in the industrial pile fermentation of Qingzhuan tea: Understanding core functional bacteria

The distinct quality of Qingzhuan tea is greatly influenced by the bacterial community but was poorly characterized. Therefore, this study investigated the Co-occurrence network and functional profiling of the bacterial community, with special attention paid to core functional bacteria in the industrial pile fermentation. Microbiomics analysis indicated that Klebsiella and Pantoea dominated raw tea leaves, and were rapidly replaced by Pseudomonas in pile fermentation, but substituted mainly by Burkholderia and Saccharopolyspora in final fermented tea. Bacterial taxa were grouped into 7 modules with the dominant in module I, III, and IV, which were involved in flavor formation and biocontrol production. Functional profiling revealed that “penicillin and cephalosporin biosynthesis” increased in pile fermentation. Twelve bacterial genera were identified as core functional bacteria, in which Klebsiella, Pantoea, and Pseudomonas also dominated the pile fermentation. This work would provide theoretical basis for its chemical biofortification and quality improvement by controlling bacterial communities.

Association of gut microbiota composition and craniosynostosis.

Gut microbiota has been reported to be associated with a series of metabolic diseases including metabolic bone disease. However, study about gut microbiota and craniosynostosis (CS) is very rare. We aim to investigate the gut microbiota composition in CS patients and assess the possible relationship. A total of 30 infants with CS and 30 infants with non-CS treated in Children's Hospital of Nanjing Medical University of Jiangsu Province from June 2021 to March 2022 were finally included in this study. All processing and analysis are carried out using 16S ribosomal RNA (rRNA) high-throughput gene sequencing. The CS group have significantly lower levels of family, genus, and species than non-CS group (all P<0.05). Furthermore, Staphylococcales and Lactobacillales at the order level, Enterococcaceae and Staphylococcaceae at the family level, and Enterococcus and Staphylococcus at the genus level were significantly enriched in the CS group (all P<0.05). Additionally, functional prediction showed that six metabolic pathways significantly differed between the two groups (all P<0.05). Of those, pathways involving polycyclic aromatic hydrocarbon degradation (P=0.030) and penicillin and cephalosporin biosynthesis (P=0.027) were more abundant in CS group than in non-CS group. Gut microbiota was statistically associated with the development of CS, and several taxa and specific functional pathways with significantly altered abundance have been identified in CS patients. These findings can provide clues for the study on the mechanism and early diagnosis of CS.

The Biosynthesis of Penicillin and Cephalosporin C are Regulated by ROS at Transcriptional Level.

In a recent work we showed that, besides lovastatin, ROS also accumulate during the production phase in Pencillium chrysogenum and in Acremonium chrysogenum, and that these ROS regulate the biosynthesis of penicillin and cephalosporin C. In the present study, we investigated the level at which this positive regulation is exerted. Internal ROS levels were manipulated, i.e., increased or decreased, in the production phase of the respective fermentations. Penicillin production decreased by 51.2% when internal ROS concentration was diminished by 50%, while a 62% production increase was observed when ROS were increased (62%). Similarly, Cephalosporin production decreased (35%) with antioxidants and increased (54.1%) with exogenous ROS. Expression analysis of the respective pcbAB genes, encoding the non-ribosomal peptide synthetase enzymes, was performed. Results showed down regulation of these genes in fermentations with lower ROS content, and upregulation in the cultures with higher ROS content, in both species. This showed that ROS regulation of penicillin in P. chrysogenum and of cephalosporin C in A. chrysogenum, is exerted at transcriptional level. In silico analysis of the pcbAB gene promoters in both species, suggested that this regulation could be mediated by stress-response transcription factors like Yap1,SrrA and/or MsnA, and/or by the Hap complex.

Single‐Cell Transcriptomic Analysis Demonstrates the Regulation of Peach Polysaccharides on Circadian Rhythm Disturbance

Plant polysaccharides are thought to have a prebiotic effect, promoting the growth of probiotics, which may regulate circadian rhythms. This study evaluates the regulation of peach polysaccharides (PPS) on circadian rhythm disturbance through intestinal microbiota by a mouse model. PPS is administered to mice with circadian rhythm disturbance for 4 weeks. The study finds that PPS ameliorated the structural disorder of intestinal microbiota induced by continuous darkness, decreasing the ratio of Firmicutes/Bacteroidetes (F/B), thereby regulating furfural degradation, penicillin and cephalosporin biosynthesis, and antibiotic biosynthesis. Single-cell transcriptomics is used to determine the type of hypothalamus cells and the expression of clock genes in mice, showing that the number of astrocytes and oligoendrocytes cells in the hypothalamus of the transplanted mice is up-regulated, and the expression of neuroprotective genes such as Sox9 and Mobp increased. In addition, clock genes such as Cry2 and Per3 show significant callback. This study shows that PPS can ameliorate the imbalance of intestinal microbiota and cell dysfunction caused by circadian rhythm disorder, suggesting that PPS is a feasible strategy for the prevention and treatment of circadian rhythm disorder and related cognitive impairment.

Natural Dietary Compound Xanthohumol Regulates the Gut Microbiota and Its Metabolic Profile in a Mouse Model of Alzheimer's Disease.

Discovering new and effective drugs for the treatment of Alzheimer’s disease (AD) is a major clinical challenge. This study focuses on chemical modulation of the gut microbiome in an established murine AD model. We used the 16S rDNA sequencing technique to investigate the effect of xanthohumol (Xn) on the diversity of intestinal microflora in 2-month- and 6-month-old APP/PS1 mice, respectively. APP/PS1 and wild-type mice were treated by gavage with corn oil with or without Xn every other day for 90 days. Prior to and following treatment, animals were tested for spatial learning, cognitive and memory function. We found Xn reduced cognitive dysfunction in APP/PS1 mice and significantly regulated the composition and abundance of gut microbiota both in prevention experiments (with younger mice) and therapeutic experiments (with older mice). Differential microflora Gammaproteobacteria were significantly enriched in APP/PS1 mice treated with Xn. Nodosilineaceae and Rikenellaceae may be the specific microflora modulated by Xn. The penicillin and cephalosporin biosynthesis pathway and the atrazine degradation pathway may be the principal modulation pathways. Taken together, oral treatment with Xn may have a neuroprotective role by regulating the composition of intestinal microflora, a result that contributes to the scientific basis for a novel prophylactic and therapeutic approach to AD.

Solea senegalensis Bacterial Intestinal Microbiota Is Affected by Low Dietary Inclusion of Ulva ohnoi.

The inclusion of macroalgae in the diets of farmed fish offers the opportunity for an added-value dietary ingredient to the nutraceutical feed. The composition of algae varies greatly among species. Several Ulva species have been considered in aquafeed formulations for different farmed fish, and Ulva ohnoi is being applied recently. However, the effects of seaweed dietary inclusion on the host must be evaluated. Considering the important role of the host intestinal microbiota, the potential effects of U. ohnoi dietary inclusion need to be studied. In this study, the characterization of the intestinal microbiome of Solea senegalensis, a flatfish with high potential for aquaculture in South Europe, receiving U. ohnoi (5%)-supplemented diet for 90 days has been carried out. In addition, the functional profiles of bacterial communities have been determined by using PICRUSt, a computational approach to predict the functional composition of a metagenome by using marker gene data and a database of reference genomes. The results show that long-term dietary administration of U. ohnoi (5%)-supplemented feed modulates S. senegalensis intestinal microbiota, especially in the posterior intestinal section. Increased relative abundance of Vibrio jointly with decreased Stenotrophomonas genus has been detected in fish receiving Ulva diet compared to control-fed fish. The influence of the diet on the intestinal functionality of S. senegalensis has been studied for the first time. Changes in bacterial composition were accompanied by differences in predicted microbiota functionality. Increased abundance of predicted genes involved in xenobiotic biodegradation and metabolism were observed in the microbiota when U. ohnoi diet was used. On the contrary, predicted percentages of genes associated to penicillin and cephalosporin biosynthesis as well as beta-lactam resistance were reduced after feeding with Ulva diet.

Deacetoxycephalosporin C synthase (expandase): Research progress and application potential

Cephalosporins play an indispensable role against bacterial infections. Deacetyloxycephalosporin C synthase (DAOCS), also called expandase, is a key enzyme in cephalosporin biosynthesis that epoxides penicillin to form the hexavalent thiazide ring of cephalosporin. DAOCS in fungus Acremonium chrysogenum was identified as a bifunctional enzyme with both ring expansion and hydroxylation, whereas two separate enzymes in bacteria catalyze these two reactions. In this review, we briefly summarize its source and function, improvement of the conversion rate of penicillin to deacetyloxycephalosporin C through enzyme modification, crystallography features, the prediction of the active site, and application perspective.

Comparative Metagenomics Reveals Microbial Communities and Their Associated Functions in Two Types of Fuzhuan Brick Tea.

Fuzhuan brick tea (FBT) is a unique post-fermented tea product, naturally co-fermented by microorganisms, and has gained global popularity due to its potential health benefits for humans. Considerable efforts have been made toward elucidating the microbial diversity within FBT, but an understanding of the underlying FBT community interactions and functions remains poorly studied. Consequently, the microbial communities of two types of FBT, originating from Hunan and Shaanxi provinces, were investigated using comparative shotgun metagenomic sequencing and functional annotations. Metagenomic analysis indicated that two communities shared similar taxonomic and functional attributes. Two samples shared 486 genera, in which Pseudomonas contributed most to the abundant functions within the two samples. The carbohydrate active enzyme functions of the communities primarily comprised GH (32.92%), GT (26.8%), CEs (20.43%), and AAs (18.04%). Furthermore, the overall metabolic pathways encoded by the metagenomes were largely associated with carbohydrate and amino acid metabolism, with nine metabolic pathways that were differential between two groups including penicillin and cephalosporin biosynthesis. Significantly, a total of 35 potential probiotics were inferred, with Pseudomonas putida being the most abundant inferred probiotic (80%) within the FBT communities. This study provides new insights into FBT microbial communities on their potential functions and roles in FBT characteristics.

Characterization of gut microbiota and short-chain fatty acid in breastfed infants with or without breast milk jaundice.

This study aims to investigate the gut microbiota and metabolites in breastfed infants with breast milk jaundice (BMJ) using gut microbiome-metabolomics. Breastfed newborns diagnosed with BMJ and those without BMJ (control group) were enrolled. Faecal samples were collected from the participants and subjected to high-throughput sequencing of the 16s rDNA V3 and V4 regions of the gut flora and metabolomics of short-chain fatty acids (SCFAs). Proteobacteria, Fimicutes and Actinobacteria were the main bacteria at the phylum level. Eshcerichia-Shigella and Enterobacteriacea were the main bacteria at the genus level. The difference between the two groups was compared. Compared to the control group, the amount of Streptococcus was significantly increased while the amount of Enterococcus was significantly decreased in the faeces from infants with BMJ. Functional prediction analysis of 16S found that biosynthesis of penicillin and cephalosporin significantly increased in the BMJ group. Gas chromatography-mass spectrometry detection of SCFAs revealed that levels of acetic acid and propionic acid were significantly lower in the BMJ group than in the control group. The reduced levels of acetic acid and propionic acid may be related to the increase in Streptococcus and decrease in Enterococcus, both of which may contribute to BMJ.

Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate–dependent nonheme ferrous enzymes

Determining the requirements for efficient oxygen (O2) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O2) and a sequential mechanism (cofactor binding and reaction with O2 precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the FeII), αKG binding to FeII-DAOCS results in ∼45% five-coordinate sites that selectively react with O2 relative to the remaining six-coordinate sites. However, this reaction produces an FeIII species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to FeII-DAOCS produces five-coordinate sites that rapidly react with O2 to form an FeIV=O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes.

Penicillin and cephalosporin biosyntheses are also regulated by reactive oxygen species.

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 (H2O2) 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.

Studies on the Physical Parameters for Cephalosporin Biosynthesis from Acremonium chrysogenum by Submerged Fermentation

In the present study, cephalosporin was produced through submerged fermentation in 250 ml Erlenmeyer flask. About one hundred fungal strains were isolated and screened for maximum productivity of the antibiotic cephalosporin. Among these, the strain IIB-10 identified as Acremonium chrysogenum was found to have maximum production of cephalosporin when cultivated on M-4 medium containing (%, w/v): Corn meal, 2; Baker’s flour, 1.5; Ammonium sulphate, 0.1; Calcium carbonate, 0.3 and Methyl oleate, 1.6. The nutritional study for maximum cephalosporin productivity was undertaken. Sucrose as a sole carbon source at 2.5% level, peptone at a concentration of 1.5% as an organic nitrogen source and Ammonium sulphate at a concentration of 0.4% as an inorganic nitrogen source supported maximum production of cephalosporin i.e., 721.89 mg/l.

Metabolome profiles of moulds on carton-gypsum board and malt extract agar medium obtained using an AuNPET SALDI-ToF-MS method

The aim of this study was to compare the metabolite profile of the moulds Alternaria, Aspergillus, Cladosporium, Penicillium, Stachybotrys, and Trichoderma under model conditions on MEA (malt extract agar) medium and carton-gypsum board (CGB). This was followed by comparison of the metabolite profile of each particular strain and of a mixed culture of all moulds together. Metabolome analysis was performed using a high-resolution surface assisted laser desorption/ionization time-of-flight mass spectrometry based on a gold nanoparticle-enhanced target (AuNPET SALDI-ToF-MS) imaging method. All water extracts tested showed the presence of compounds with molecular weights in the range of 80–2000 m/z (mass-to-charge ratio). On CGB, the results obtained showed the existence of 299–453 peaks with higher intensity and 351–487 peaks with lower intensity compared to the peaks obtained from MEA, depending on the mould species. A distinct change in the metabolite profile under the influence of the growth medium was observed for the mixed culture on the MEA medium. The profile showed ∼200 peaks with higher intensities of metabolites within m/z ranges 80–300 and 450–600; for the culture growing on CGB, the peak intensity was within m/z 300–450, and the number of peaks was 600–750. A new metabolomic methodology allowed the identification of 89 metabolites belonging to 48 metabolomic pathways. Compounds belonging to the citrate cycle and penicillin and cephalosporin biosynthesis pathways were identified on MEA. On CGB moulds, the metabolism changed to steroid, ubiquinone and other terpenoid-quinone biosynthesis.

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non-Heme Iron Oxidases.

Isopenicillin N synthase (IPNS) catalyses the four‐electron oxidation of a tripeptide, l‐δ‐(α‐aminoadipoyl)‐l‐cysteinyl‐d‐valine (ACV), to give isopenicillin N (IPN), the first‐formed β‐lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co‐substrate. In the absence of substrate, the carbonyl oxygen of the side‐chain amide of the penultimate residue, Gln330, co‐ordinates to the active‐site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C‐terminal residues, which move to take up a conformation that extends the final α‐helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C‐terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side‐chains around the ACV binding pocket is important in catalysis. Deletion of seven C‐terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC‐MS and NMR analyses to be the ene‐thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl β‐carbon of the other. A mechanism for its formation is proposed, supported by an X‐ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl β‐carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non‐heme iron oxidases in general.

A GATA-type transcription factor AcAREB for nitrogen metabolism is involved in regulation of cephalosporin biosynthesis in Acremonium chrysogenum.

In filamentous fungi, nitrogen metabolism is repressed by GATA-type zinc finger transcription factors. Nitrogen metabolite repression has been found to affect antibiotic production, but the mechanism is still poorly understood. AcareB, encoding a homologue of fungal GATA-type regulatory protein, was cloned from Acremonium chrysogenum. Gene disruption and genetic complementation demonstrated that AcareB plays a key role in utilization of ammonium, glutamine and urea. In addition, significant reduction of cephalosporin production in the AcareB disruption mutant indicated that AcareB is important for cephalosporin production. In consistence with it, the transcriptional level of cephalosporin biosynthetic genes was significantly decreased in the AcareB disruption mutant. Electrophoretic mobility shift assay showed that AcAREB directly bound to the intergenic regions of pcbAB-pcbC, cefD1-cefD2 and cefEF-cefG. Sequence analysis showed that all the AcAREB binding sites contained the consensus GATA elements. AcareB is negatively autoregulated during cephalosporin production. Moreover, another GATA zinc-finger protein encoded by AcareA positively regulates the transcription of AcareB. However, AcareB does not regulate the transcription of AcareA. These results indicated that AcAREB plays an important role in both regulation of nitrogen metabolism and cephalosporin production in A. chrysogenum.

Antibiotic biosynthesis and secondary metabolism in high-yielding strains of Streptomyces, Penicillium chrysogenumand Acremonium chrysogenum

Abstract In this article, the secondary metabolism as a basis for antibiotics production by industrial strains of Streptomyces, Penicillium chrysogenum and Acremonium chrysogenum is discussed. Images from transmission electron microscopy reveal some important features of the mycelial cells which are related to antibiotics biosynthesis. This discovery is important for further industrial strain improvement and has economic significance. Possibilities of new strategies for antimicrobial treatment are discussed. 1. Introduction. 2. Industrial strain improvements. 3. The pathways of antibiotic biosynthesis by Streptomyces spp. 4. Compartmentalization in antibiotic biosynthesis by Streptomyces spp. 5. The pathway of penicillin G biosynthesis by Penicillium chrysogenum. 6. Compartmentalization in penicillin G biosynthesis by Penicillium chrysogenum. 7. The pathway of cephalosporin C biosynthesis by Acremonium chrysogenum.8. Compartmentalization in cephalosporin biosynthesis by Acremonium chrysogenum. 9. The future of antibiotic therapy. 10. Conclusions

Modified Deacetylcephalosporin C Synthase for the Biotransformation of Semisynthetic Cephalosporins.

Deacetylcephalosporin C synthase (DACS), a 2-oxoglutarate-dependent oxygenase synthesized by Streptomyces clavuligerus, transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporin. It is a step which is chemically difficult to accomplish, but its development by use of an enzymatic method with DACS can facilitate a cost-effective technology for the manufacture of semisynthetic cephalosporin intermediates such as 7-amino-cephalosporanic acid (7ACA) and hydroxymethyl-7-amino-cephalosporanic acid (HACA) from cephalosporin G. As the native enzyme showed negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, directed-evolution strategies such as random, semirational, rational, and computational methods were used for systematic engineering of DACS for improved activity. In comparison to the native enzyme, several variants with improved catalytic efficiency were found. The enzyme was stable for several days and is expressed in soluble form at high levels with significantly higher kcat/Km values. The efficacy and industrial scalability of one of the selected variants, CefFGOS, were demonstrated in a process showing complete bioconversion of 18 g/liter of cephalosporin G into deacetylcephalosporin G (DAG) in about 80 min and showed reproducible results at higher substrate concentrations as well. DAG could be converted completely into HACA in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. The experimental findings with several mutants were also used to rationalize the functional conformation deduced from homology modeling, and this led to the disclosure of critical regions involved in the catalysis of DACS. 7ACA and HACA serve as core intermediates for the manufacture of several semisynthetic cephalosporins. As they are expensive, a cost-effective enzyme technology for the manufacture of these intermediates is required. Deacetylcephalosporin C synthase (DACS) was identified as a candidate enzyme for the development of technology from cephalosporin G in this study. Directed-evolution strategies were employed to enhance the catalytic efficiency of deacetylcephalosporin C synthase. One of the selected mutants of deacetylcephalosporin C synthase could convert high concentrations of cephalosporin G into DAG, which subsequently could be converted into HACA completely. As cephalosporin G is inexpensive and readily available, the technology would lead to a substantial reduction in the cost for these intermediates upon commercialization.

AcstuA, which encodes an APSES transcription regulator, is involved in conidiation, cephalosporin biosynthesis and cell wall integrity of Acremonium chrysogenum

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.

Acthi, a thiazole biosynthesis enzyme, is essential for thiamine biosynthesis and CPC production in Acremonium chrysogenum

BackgroundThe filamentous fungus Acremonium chrysogenum is an important industrial fungus and is used in the production of the β-lactam antibiotic cephalosporin C. Little is known regarding the molecular and biological mechanisms of how this industrial strain was improved by mutagenesis and molecular breeding. Comparative proteomics is one of the most powerful methods to evaluate the influence of gene expression on metabolite production.ResultsIn this study, we used comparative proteomics to investigate the molecular mechanisms involved in the biosynthesis of cephalosporin C between a high-producer (HY) strain and a wide-type (WT) strain. We found that the expression levels of thiamine biosynthesis-related enzymes, including the thiazole biosynthesis enzyme (Acthi), pyruvate oxidase, flavin adenine dinucleotide (FAD)-dependent oxidoreductase and sulfur carrier protein-thiS, were up-regulated in the HY strain. An Acthi-silencing mutant of the WT strain grew poorly on chemically defined medium (MMC) in the absence of thiamine, and its growth was recovered on MMC medium supplemented with thiamine. The intracellular thiamine content was changed in the Acthi silencing or over-expression mutants. In addition, we demonstrated that the manipulation of the Acthi gene can affect the hyphal growth of Acremonium chrysogenum, the transcription levels of cephalosporin C biosynthetic genes, the quantification levels of precursor amino acids for cephalosporin C synthesis and the expression levels of thiamine diphosphate-dependent enzymes. Over-expression of Acthi can significantly increase the cephalosporin C yield in both the WT strain and the HY mutant strain.ConclusionsUsing comparative proteomics, four differently expressed proteins were exploited, whose functions may be involved in thiamine diphosphate metabolism. Among these proteins, the thiazole biosynthesis enzyme (ActhiS) may play an important role in cephalosporin C biosynthesis. Our studies suggested that Acthi might be involved in the transcriptional regulation of cephalosporin C biosynthesis. Therefore, the thiamine metabolic pathway could be a potential target for the molecular breeding of this cephalosporin C producer for industrial applications.