Expression of the transporter encoded by the cefT gene of Acremonium chrysogenum increases cephalosporin production in Penicillium chrysogenum

By introduction of the cefEF genes of Acremonium chrysogenum and the cmcH gene of Streptomyces clavuligerus, Penicillium chrysogenum can be reprogrammed to form adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA), a carbamoylated derivate of adipoyl-7-aminodeacetoxy-cephalosporanic acid. The cefT gene of A. chrysogenum encodes a cephalosporin C transporter that belongs to the Major Facilitator Superfamily. Introduction of cefT into an ad7-ACCCA-producing P. chrysogenum strain results in an almost 2-fold increase in cephalosporin production with a concomitant decrease in penicillin by-product formation. These data suggest that cephalosporin production by recombinant P. chrysogenum strains is limited by the ability of the fungus to secrete these compounds.     

Expression of cefF significantly decreased deacetoxycephalosporin C formation during cephalosporin C production in Acremonium 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.     

Specific cephalosporin C production of Acremonium chrysogenum is independent of the culture density

Specific cephalosporin C production of Acremonium chrysogenum grown on a glucose-based minimal medium using conventional batch and dialysis membrane reactor systems was independent of the cell density in the range of 0.4 to 40 g biomass l^–1.     

Solid-state and submerged fermentations show different gene expression profiles in cephalosporin C production by Acremonium chrysogenum

Despite the importance of Acremonium chrysogenum as the only cephalosporin C (CPC) producer, there is still a limited understanding about the molecular mechanisms regulating antibiotic biosynthesis in this fungus. Based on the previously described relationship between environmental pH and antibiotic production in numerous filamentous fungi, we studied the expression of genes related to CPC production in A. chrysogenum. We report for the first time similarities and differences, characterizing CPC production by A. chrysogenum under a variable pH environment, in submerged and solid-state fermentation. This characterization is supported by measurements of parameters, like CPC production, pH, growth, and expression levels of several genes involved, directly or indirectly, in CPC production. Interesting differences in intermediate (Pen N) and certain biosynthetic gene expression levels were observed. Our results point out some relationships between physiological features and gene expression that open important improvement perspectives for both culture systems.     

Chromosomal polymorphism of Acremonium chrysogenum strains producing cephalosporin C

Using pulse electrophoresis in controlled homogenous electric field we performed molecular karyotyping of cephalosporin C-producing industrial and laboratory strains of Acremonium chrysogenum. Differences in size of several chromosomes of high-producing strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the high-producing strain was not associated with alteration of localization and copy number of cephalosporin C (CPC) biosynthesis and transport genes. A cluster of ??early?? CPC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the ??late genes??, on chromosome II (2.3 Mb). Both clusters are presented as a single copy per A. chrysogenum genome in the wild-type and in CB26/8 high-producing strains. Based on comparative analysis of laboratory and industrial CPC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.     

Influence of pH regulation and nutrient content on cephalosporin C production in solid-state fermentation by Acremonium chrysogenum C10

Aims: To investigate the effect of pH regulation and nutrient concentration on cephalosporin C (CPC) production in solid‐state fermentation (SSF), using sugarcane bagasse as inert support, impregnated with liquid medium. Methods and Results: Solid‐state fermentation using different initial pH values, buffer and nutrient concentrations were performed. Results revealed pH as a key parameter in CPC SSF, as it hampered the antibiotic production not only above 7·8, but also under 6·4. Using initial pH lower than 6·8 and PB in the solid medium, it was possible to keep pH within the production range, increase the production period (from 1 to 3 days) and hence the CPC yield from 468 to 3200 μg gdm^?1 (g^?1 of dry matter). Conclusion: Parameters that help to keep pH in adequate values for CPC production in SSF, such as initial pH, buffering system and nutrient concentration, can greatly increase the production time and CPC yields in this fermentation technique. Significance and Impact of the Study: This is the first work on CPC production on impregnated support, and the only one revealing pH as a key parameter; it is also shown that high nutrient concentration can improve CPC yields in SSF as long as pH is kept under control.     

Asexual cephalosporin C producer Acremonium chrysogenum carries a functional mating type locus

Acremonium chrysogenum, the fungal producer of the pharmaceutically relevant beta-lactam antibiotic cephalosporin C, is classified as asexual because no direct observation of mating or meiosis has yet been reported. To assess the potential of A. chrysogenum for sexual reproduction, we screened an expressed sequence tag library from A. chrysogenum for the expression of mating type (MAT) genes, which are the key regulators of sexual reproduction. We identified two putative mating type genes that are homologues of the alpha-box domain gene, MAT1-1-1 and MAT1-1-2, encoding an HPG domain protein defined by the presence of the three invariant amino acids histidine, proline, and glycine. In addition, cDNAs encoding a putative pheromone receptor and pheromone-processing enzymes, as well as components of a pheromone response pathway, were found. Moreover, the entire A. chrysogenum MAT1-1 (AcMAT1-1) gene and regions flanking the MAT region were obtained from a genomic cosmid library, and sequence analysis revealed that in addition to AcMAT1-1-1 and AcMAT1-1-2, the AcMAT1-1 locus comprises a third mating type gene, AcMAT1-1-3, encoding a high-mobility-group domain protein. The alpha-box domain sequence of AcMAT1-1-1 was used to determine the phylogenetic relationships of A. chrysogenum to other ascomycetes. To determine the functionality of the AcMAT1-1 locus, the entire MAT locus was transferred into a MAT deletion strain of the heterothallic ascomycete Podospora anserina (the PaDeltaMAT strain). After fertilization with a P. anserina MAT1-2 (MAT(+)) strain, the corresponding transformants developed fruiting bodies with mature ascospores. Thus, the results of our functional analysis of the AcMAT1-1 locus provide strong evidence to hypothesize a sexual cycle in A. chrysogenum.     

Fatty acids reduce the tensile strength of fungal hyphae during cephalosporin C production in Acremonium chrysogenum

Fragmentation rate constants, which can be used to estimate the tensile strength of fungal hyphae, were used to elucidate relationships between morphological changes and addition of fatty acids during cephalosporin C production in Acremonium chrysogenum M35. The number of arthrospores increased gradually during fermentation, and, in particular, was higher in the presence of rice oil, oleic acid or linoleic acid than in their absence. Because supplementation of rice oil or fatty acids increased cephalosporin C, we concluded that differentiation to arthrospores is related to cephalosporin C production. To estimate the relative tensile strengths of fungal hyphae, fragmentation rate constants (k _frag) were measured. When rice oil, oleic acid, or linoleic acid were added into medium, fragmentation rate constants were higher than for the control, and hyphal tensile strengths reduced. The relative tensile strength of fungal hyphae, however was not constant presumably due to differences in physiological state.     

The concentration dynamics of inorganic polyphosphates during the cephalosporin C synthesis by Acremonium chrysogenum

The contents of five fractions of energy-rich inorganic polyphosphates (polyPs), ATP, and H⁺-ATPase activity in the plasma membrane were determined in a low-activity cephalosporin C (cephC) producer Acremonium chrysogenum ATCC 11550 and selected highly efficient producer strain 26/8 grown on glucose or a synthetic medium providing for active synthesis of this antibiotic. It was shown that strain 26/8 on the synthetic medium produced 26-fold higher amount of cephC as compared with strain ATCC 11550. This was accompanied by a drastic decrease in the cell contents of ATP and the high-molecular-weight fractions polyP2, polyP3, and polyP5 with a concurrent increase in the low-molecular-weight fraction polyP1. These data suggest that polyPs are involved in the cephC synthesis as a source of energy. H⁺-ATPase activity insignificantly changed at both low and high levels of cephC production. This confirms the assumption that A. chrysogenum has other alternative antibiotic transporters in addition to cefT. The obtained results can be used for optimizing commercial-scale cephC biosynthesis.     

Analysis of the relationship between growth, cephalosporin C production, and fragmentation in Acremonium chrysogenum.

Mycelial fragmentation in submerged cultures of the cephalosporin C (CPC) producing fungus Acremonium chrysogenum was characterized by image analysis. In both fed-batch and chemostat cultures, the proportion of mycelial clumps seemed to be the most sensitive morphological indicator of fragmentation. In a fed-batch fermentation culture, this declined from roughly 60% at inoculation to less than 10% after 43 h. Subsequent additions of glucose resulted in a sharp increase back to near the initial value, an increase that reversed itself a few hours after glucose exhaustion. Meanwhile CPC production continued to decline steadily. On the other hand, the addition of soybean oil enhanced CPC production, but had no significant effect on the morphology. Although it may sometimes appear that morphology and productivity are related in batch or fed-batch cultures, this study suggests that this is because both respond simultaneously to more fundamental physiological changes, dependent on the availability of carbon. In circumstances, such as supplementary carbon source addition, the relationship is lost. Chemostat cultures supported this belief, as CPC-production rates were hardly affected by the specific growth rate, but the morphology showed significant differences, i.e., lower dilution rates resulted in a lower proportion of clumps and in smaller clumps.     

CefR modulates transporters of beta-lactam intermediates preventing the loss of penicillins to the broth and increases cephalosporin production in Acremonium chrysogenum

The Acremonium chrysogenum cephalosporin biosynthetic genes are divided in two different clusters. The central step of the biosynthetic pathway (epimerization of isopenicillin N to penicillin N) occurs in peroxisomes. We found in the “early” cephalosporin cluster a new ORF encoding a regulatory protein (CefR), containing a nuclear targeting signal and a “Fungal_trans” domain. Targeted inactivation of cefR delays expression of the cefEF gene, increases penicillin N secretion and decreases cephalosporin production. Overexpression of the cefR gene decreased (up to 60%) penicillin N secretion, saving precursors and resulting in increased cephalosporin C production. Northern blot analysis revealed that the CefR protein acts as a repressor of the exporter cefT and exerts a small stimulatory effect over the expression level of cefEF that explains the increased cephalosporin yields observed in transformants overexpressing cefR. In summary, we describe for the first time a modulator of beta-lactam intermediate transporters in A. chrysogenum.     

flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein

We describe the sequence and characterization of the Bacillus subtilis flhF gene. flhF encodes a basic polypeptide of 41 kDa that contains a putative GTP-binding motif. The sequence of FlhF reveals a structural relationship to two Escherichia coli proteins, Ffh and FtsY, as well as to other members of the SRP54 family, in a domain presumed to bind GTP. flhF is located in a large operon consisting of chemotaxis and flagellar genes. Cells deficient in flhF are nonmotile. Through the use of anti-flagellar antibodies we have established that flhF is a flagellar (fla) gene. Thus, flhF is a unique flagellar gene in that it encodes a GTP-binding protein with similarities to members of the SRP54 family of proteins. These data suggest that flagellar biosynthesis in B. subtilis requires GTP.     

Undefined xylose media extracted from biorefinery waste for enhanced and eco-friendly production of cephalosporin C by Acremonium chrysogenum M35

Xylose-rich undefined broth, extracted from the dilute acid pretreatment wastes of barley straw, serves as resourceful media for Acremonium chrysogenum M35 culture and production of cephalosporin C (CPC). Concentrating the extract with proper reprocessing enables to prepare various concentrations of xylose broth (2%–8%). The undefined xylose media were prepared for CPC production from A. chrysogenum M35 by the addition of other nutrients. Cell growth and CPC production were the most effective at 6% xylose and additional 2% glycerol, with maximum CPC production of 9.07 g/L after 6 days, which is higher production than that in defined media prepared with laboratory-level nutrients and reagents. Investigation of autotrophic and reverse trans-sulfuration pathways for cysteine synthesis, a limited element of three precursors for CPC synthesis, supports the enhanced CPC production in undefined media. Abundance of xylose ensures a maintained NADPH concentration required for sulfate reduction and synthesis of amino sulfide such as cysteine. Cystathionine-γ-lyase activity profiling indicated more efficient biosynthesis in undefined media than in other cultures use glycerol and glucose, and the biosynthesis pathway of CPC production by the cephalosporin gene cluster (i.e. pcbC and cefG genes) was investigated. The process using undefined xylose media was designed, and process simulation program confirmed our results.