Functional analysis of MFS protein CefT involved in the transport of beta-lactam antibiotics in Acremonium chrysogenum and Saccharomyces cerevisiae

Vectors for the expression of MFS transporter CefT of Acremonium chrysogenum—a producer of beta-lactam antibiotic cephalosporin C—and in Saccharomyces cerevisiae as a fusion with the cyan fluorescent protein (CFP) have been generated. The subcellular localization of the CefT-CFP hybrid protein in yeast cells has been investigated. It was shown that the CefT-CFP hybrid protein is capable of complementation of the qdr3, tpo1, and tpo3 genes encoding for orthologous MFS transporters of Saccharomycetes, making the corresponding strains resistant to spermidine, ethidium bromide, and hygromycin B. High-producing strain A. chrysogenum VKM F 4081D, expressing the cefT-cfp fusion construct, was obtained by an agrobacteria mediated transformation. It was also shown that the constitutive expression of cefT in A. chrysogenum VKM F-4081D led to a change in the biosynthetic profiles of cephalosporin C and its precursors. This resulted in a 25–35% decrease in the amount of the final product accumulated in the cultural liquid with a simultaneous increase in the concentration of its intermediates.     

Isolation and Characterization of a Pseudomonas Strain Producing Glutaryl-7-Aminocephalosporanic Acid Acylase

Several screening methods were developed for the selection of Pseudomonas strains capable of hydrolyzing glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid. An isolate exhibiting high acylase activity, designated BL072, was identified as a strain of Pseudomonas diminuta. It grew optimally at pH 7 to 8 and at a temperature of 32 to 40°C, but acylase activity was highest when the strain was grown at 28°C. Mutants of BL072 were generated by nitrosoguanidine treatment and screened for increased production of glutaryl-7-aminocephalosporanic acid acylase. A superior mutant gave a fourfold increase in acylase titer. The cell-associated acylase had similar activities against various glutaryl-cephems but had undetectable activity against cephalosporin C. This acylase may prove useful for the conversion of cephalosporin C to 7-aminocephalosporanic acid.     

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.     

Genetic transformation of the mycelium fungi <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>

The system of transformation of heterologous genes under the method of agrobacterial transfer into Acremonium chrysogenum ATCC 11550 wild-type strains, natural producents of beta-lactam antibiotic cephalosporin C, and strains highly producing cephalosporin C no. 26/8 revealed by the multistage selection on its basis were developed. Vectors for agrobacterial transformation of A. chrysogenum containing expression cassettes of genes encoding resistance to geneticin (G418) and bleomicin (Zeocin^TM) antibiotics under control of Ashbya gossypii and Saccharomyces cerevisiae TEF1 promoters were constructed. A comparable assessment of agrotransformation methods while co-cultivating fungi and agrobacterial cells on filters and in deep culture was conducted. Transformants, selected by resistance to geneticin and bleomicin, were characterized by PCR and Southern blot analyses.     

Comparative characterization of new glutaryl 7-ACA and cephalosporin C acylases

We performed a comparative characterization of three new cephalosporin acylases which were prepared from E. coli recombinant strains and found originally from Pseudomonas sp. A14, Bacillus laterosporus J1 and Pseudomonas diminuta N176. Both A14 and N176 acylases consisted of two non-identical subunits (α, β) whose molecular weights were 28,000 (α), 61,000 (β) and 26,000 (α), 58,000 (β), respectively, whereas J1 acylase consisted of a single peptide with molecular weight of 70,000. The maximum specific activities of A14, J1 and N176 acylases for glutaryl 7-ACA were 7.1, 5.3 and 100 units/mg, respectively, and that of N176 acylase for cephalosporin C was 3.1 units/mg. The K_m values of glutaryl 7-ACA for A14, J1 and N176 acylases were 2.1, 3.2 and 2.6 mM, respectively, and that of cephalosporin C for N176 acylase was 4.8 mM. A14, J1 and N176 acylases exhibited differential activities for cephalosporins having an aliphatic dicarboxylic acid in the acyl side chain and only N176 acylase showed an activity for cephalosporin C. N176 acylase as well as A14 acylase also showed a weak activity for a cephalosporin derivative having a heterocyclic carboxylic acid in the side chain. A14, J1 and N176 acylases catalyzed the reverse reaction to synthesize glutaryl 7-ACA from 7-ACA and glutaric acid, although the rate of the synthesis was 10 to 10⁵ fold slower than that of hydrolysis. The activities of the cephalosporin acylases were considerably inhibited by the reaction products, 7-ACA and glutaric acid. The types of the inhibition by 7-ACA and glutaric acid were both competitive. A14, J1 and N176 acylases were thermostable, their residual activities exceeding more than 90% after treatment at 50°C for 1 h at their optimal pHs.     

Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum

Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Here we describe a new industrially amenable bioprocess for the production of the important intermediate 7-ADCA that can replace the expensive and environmentally unfriendly chemical method classically used. The method is based on the disruption and one-step replacement of the cefEF gene, encoding the bifunctional expandase/hydroxylase activity, of an actual industrial cephalosporin C production strain of Acremonium chrysogenum. Subsequent cloning and expression of the cefE gene from Streptomyces clavuligerus in A. chrysogenum yield recombinant strains producing high titers of deacetoxycephalosporin C (DAOC). Production level of DAOC is nearly equivalent (75-80%) to the total beta-lactams biosynthesized by the parental overproducing strain. DAOC deacylation is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase (DAO) and glutaryl acylase (GLA) yielding 7-ADCA. In contrast to the data reported for recombinant strains of Penicillium chrysogenum expressing ring expansion activity, no detectable contamination with other cephalosporin intermediates occurred.     

Nonbiological Conversion of Cephalosporin C to a New Antibiotic by Sodium Thiosulfate

The apparent stimulation of cephalosporin C biosynthesis by sodium thiosulfate is due to a nonbiological conversion of cephalosporin C to a new derivative, cephalosporin C_x. The new compound is more active than cephalosporin C against the assay organism, Escherichia coli W-208. Cephalosporin C_x retains the properties of ultraviolet absorption and resistance to penicillinase, but migrates more slowly than cephalosporin C in the paper-chromatographic system used.     

Cannabinoid formation in Cannabis sativa grafted inter-racially,and with two Humulus species

Inter-racial grafts between high and low Δ¹-THC strains of Cannabis sativa, as well as cross-grafts with two Humulus (hop) species have been effected. C. sativa strains continue to produce essentially their own characteristic mixtures of cannabinoids, with undiminished vigour, whatever part of the graft system they form. There is no evidence of transport of intermediates or factors critical to cannabinoid formation across the grafts.     

An Efficient Fungal RNA-Silencing System Using the DsRed Reporter Gene

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.     

The transporter CefM involved in translocation of biosynthetic intermediates is essential for cephalosporin production

The cluster of early cephalosporin biosynthesis genes (pcbAB, pcbC, cefD1, cefD2 and cefT of Acremonium chrysogenum) contains all of the genes required for the biosynthesis of the cephalosporin biosynthetic pathway intermediate penicillin N. Downstream of the cefD1 gene, there is an unassigned open reading frame named cefM encoding a protein of the MFS (major facilitator superfamily) with 12 transmembrane domains, different from the previously reported cefT. Targeted inactivation of cefM by gene replacement showed that it is essential for cephalosporin biosynthesis. The disrupted mutant accumulates a significant amount of penicillin N, is unable to synthesize deacetoxy-, deacetyl-cephalosporin C and cephalosporin C and shows impaired differentiation into arthrospores. Complementation of the disrupted mutant with the cefM gene restored the intracellular penicillin N concentration to normal levels and allowed synthesis and secretion of the cephalosporin intermediates and cephalosporin C. A fused cefM-gfp gene complemented the cefM-disrupted mutant, and the CefM-GFP (green fluorescent protein) fusion was targeted to intracellular microbodies that were abundant after 72 h of culture in the differentiating hyphae and in the arthrospore chains, coinciding with the phase of intense cephalosporin biosynthesis. Since the dual-component enzyme system CefD1-CefD2 that converts isopenicillin N into penicillin N contains peroxisomal targeting sequences, it is probable that the epimerization step takes place in the peroxisome matrix. The CefM protein seems to be involved in the translocation of penicillin N from the peroxisome (or peroxisome-like microbodies) lumen to the cytosol, where it is converted into cephalosporin C.     

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)₂, similar to the transposition intermediates described for a number of IS elements.     

Isolation and Characterization of Thermophilic Bacilli Degrading Cinnamic, 4-Coumaric, and Ferulic Acids

Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60°C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The β-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-β-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-β-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.     

In Vitro Antimicrobial Activity and Human Pharmacology of Cephalexin, a New Orally Absorbed Cephalosporin C Antibiotic

Concentrations of cephalexin (an orally absorbed derivative of cephalosporin C) in serum and urine were determined in normal volunteers and patients. The in vitro antibacterial activity was also studied. All strains of group A β-hemolytic streptococci and Diplococcus pneumoniae were inhibited by 3.1 μg/ml. Of the Staphylococcus aureus strains, 88% were inhibited by 6.3 μg/ml, and 12.5 μg/ml was inhibitory for all S. aureus, 80% of Escherichia coli, 72% of Klebsiella-Aerobacter, and 56% of Proteus mirabilis strains. About 90 to 96% of E. coli, Klebsiella Aerobacter, and P. mirabilis strains were inhibited by 25 μg of cephalexin per ml. Pseudomonas and indole-positive Proteus strains proved to be quite resistant to cephalexin. Cephalexin was well absorbed after oral administration. A peak serum concentration of cephalexin of at least 5 μg/ml was achieved in each volunteer with 250 and 500-mg doses. A mean peak serum concentration of 7.7 μg/ml was achieved with 250-mg doses; 12.3μg/ml was achieved with 500-mg doses of antibiotic. Food did not interfere with absorption. Probenecid enhanced both the peak serum concentration and the duration of antibiotic activity in the serum. Over 90% of the administered dose was excreted in the urine within 6 hr. The mean peak serum concentration of cephalexin after an oral dose of 500 mg was adequate to inhibit all group A streptococci, D. pneumoniae, and S. aureus, 85% of E. coli, and about 40 to 75% of Klebsiella-Aerobacter and P. mirabilis strains. Levels of cephalexin in urine were adequate to inhibit over 90% of E. coli, and P. mirabilis and 80 to 96% of Klebsiella-Aerobacter strains.