Methionine control of cephalosporin C formation

DL -Norleucine, a nonsulfur analogue of methionine was found to markedly stimulate synthesis of cephalosporin C by Cephalosporium acremonium strain CW19 in three different chemically defined media. Methionine, but not norleucine, stimulated cephalosporin C biosynthesis in a crude medium. The lack of stimulation by norleucine in complex medium was shown to be due to lack of uptake of this amino acid by mycelia growing in such a medium. In defined media containing a suboptimal methionine concentration, norleucine stimulated antibiotic production up to the level reached by optimal methionine. At an optimal dose of methionine, norleucine elicited no further increase in cephalosporin C production, indicating that these two amino acids act by the same mechanism. The data strongly indicate that stimulation by methionine is not a function of its ability to donate sulfur for antibiotic formation. Methionine was found to neither repress nor inhibit cysteine metabolism.     

Synthesis of cephalosporin C by a methionine analogue resistant mutant of Cephalosporium acremonium

Summary DL-seleno-methionine resistant mutants of Cephalosporium acremonium were isolated which have an enhanced capacity to utilized sulfate for the synthesis of cephalosporin C. Of these mutants, one designated as SMR-I3 produced three-fold more cephalosporin C from sulfate than its parent CW19. Mutant SMR-I3 required less dl-methionine for maximal synthesis of cephalosporin C, but an excess of dl-methionine inhibited the synthesis of the antibiotic. Furthermore, the mutant accumulated excessive methionine in the amino acid pool and possessed superior activity for sulfate uptake. These observations indicate that in the mutant SMR-I3, the biosynthesis of methionine from sulfate is very active and excess methionine becomes available for the synthesis of cephalosporin C.     

Role of Acetyl CoA: Deacetylcephalosporin C Acetyltransferase in Cephalosporin C Biosynthesis by Cephalosporium acremonium

Existence of an acetyltransferase, which catalizes acetylation of deacetylcephalosporin C to cephalosporin C, was demonstrated for the first time in cell-free extracts of Cephalosporium acremonium. The pH optimum of the enzyme appeared to be 7.0 to 7.5 and the enzyme required essentially Mg²⁺ as a cofactor for its reaction. The activity of this enzyme was not observed in the cell-free extracts of deacetylcephalosporin C-producing mutants Nos. 20, 29, 36 and 40, but was recovered in a revertant obtained from the mutant No. 40. These results indicate that deacetylcephalosporin C accumulation by these mutants was due to the lack of the acetyltransferase and made it reasonable that the terminal reaction of cephalosporin C biosynthesis in Cephalosporium acremonium proceeded by the catalytic action of acetyltransferase.     

Production of cephalosporin C using crude glycerol in fed-batch culture of <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> M35

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.     

Methionine induction of ACV synthetase inCephalosporium acremonium

Summary Methionine markedly stimulates the biosynthesis of penicillin N and cephalosporin C inCephalosporium acremonium. Examination of intra- and extracellular ACV tripeptide in non-producing mutant N-2 showed that growth in the presence of methionine increased ACV accumulation. Direct measurement of ACV synthetase activity in a cell-free system indicated that the methionine effect was mainly due to induction of this first enzyme of the -lactam biosynthetic pathway, resulting in a corresponding increase in -lactam production in both a low-producing strain and a high-producing mutant.     

Investigations of cephalosporin C production in an airlift tower loop reactor

Summary Cephalosporin C was produced by Cephalosporium acremonium in a 60 l airlift loop reactor on complex medium (with 30 kg/m³ peanut flour) in fed-batch operation. A final product concentration of 5 kg/m³ and a maximum productivity of 45 g/m³ h were attained. On-line analysis was used to determine ammonia, methionine, phosphate, reducing sugar and cephalosporin C by an autoanalyser, glucose by a flow injection analyser and cephalosporin C, penicillin N, deacetoxycephalosporin C, deacetylce-phalosporin C and methionine by HPLC. The volumetric productivity of the stirred tank reactor was higher than that of the airlift reactor because of differences in cell concentration. Specific productivities in relative to cell mass were similar in the two reactors. The substrate yield coefficient in the airlift reactor was twice that in the stirred tank reactor.Nomenclature E _o2 efficiency of oxygen transfer with regard to the specific power input - K _La volumetric mass transfer coefficient - OTR oxygen transfer rate - P power input - PR volumetric productivity of CPC - q _a volumetric aeration rate/broth volume (vvm) - SPR specific productivity with regard to RNA - V _L broth volume in reactor - z relative height of the aerated reactor     

Cybernetic modeling of the cephalosporin C fermentation process by Cephalosporium acremonium

A cybernetic mathematical model has been developed to describe the production of cephalosporin C. In developing the model, diauxic behavior of substrate consumption, morphological differentiation of cells, and catabolite repression of cephalosporin C production by the preferred substrate, glucose, were considered. The proposed model was tested on the experimental data from the literature and could adequately describe the morphological differentiation of cells, the sequential utilization of carbon sources and the production of cephalosporin C. It could be a useful tool to optimize the production of cephalosporin C by Cephalosporium acremonium in batch, fed-batch or continuous operations.     

Enhancement of growth and antibiotic titre inCephalosporium acremonium induced by sesame oil

The role of sesame oil as part of the carbon source on growth and cephalosporin C production byCephalosporium acremonium was studied in shake-flask fermentation. The growth and antibiotic production were maximum on the fifth and sixth day, respectively, irrespective of the presence of sesame oil. Sesame oil enhanced cephalosporin C production by 54%. Analysis of fatty acid profile indicated that C_18∶1, C_18∶2 and C_18∶3 are the major fatty acids inC. acremonium. The percentage of C_18∶2 was higher in the culture grown with sesame oil.     

Optimization of feed conditions in a 2.5-l fed-batch culture using rice oil to improve cephalosporin C production by <Emphasis Type="Italic">Cephalosporium acremonium</Emphasis> M25

Summary Rice oil significantly affected cephalosporin C production in a 2.5-l bioreactor culture of Cephalosporium acremonium M25. To improve cephalosporin C production, the feed conditions of rice oil were optimized. Reducing the feed rate of rice oil improved cephalosporin C production to 1.01 g/l when the consumption rate of rice oil decreased. Overall, under optimal feed conditions in the 2.5-l fed-batch culture, cephalosporin C production increased about four times compared to before optimization.     

Constant dissolved oxygen concentrations in cephalosporin C fermentation: Applicability of different controllers and effect on fermentation parameters

Summary The behaviour and applicability of several controllers for maintaining a constant dissolved oxygen concentration (DO) during the cephalosporin C production with Cephalosporium acremonium in a laboratory fermentor is described. The process controllers were realized on a MC 68000 based process computer using the real-time language PEARL. The discrete signum integral controller showed the best control action. In addition some derived fermentation data were calculated on-line by the process computer.The results obtained by comparison of fermentations carried out at DO between 10% and 40% saturation during ideophase indicate that high DO leads to a high specific production rate for cephalosporin C and a low specific production rate for penicillin N and vice versa. In the range of DO investigated the production of deacetyl and deacetoxy cephalosporin C is not affected by DO. A direct correlation between DO and the yield coefficients Y _P/S and Y _P/X could be established. The yield coefficient Y _P/O for cephalosporin C is constant in the DO range from 10%–40%.Dedicated to Prof. Dr. H. J. Rehm on the occasion of his 60th birthday     

Effect of Tenuazonic Acid on In Vitro Amino Acid Incorporation by Rice Embryo Ribosomes

Deacetylcephalosporin C negative mutants, lacking a certain step in the pathway of deacetylcephalosporin C biosynthesis, were obtained from the deacetylcephalosporin C producing mutant No. 40 of Cephalosporium acremonium by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. Among these mutants, the strain No. 40-20 was found to mainly accumulate a cephalosporin compound other than deacetylcephalosporin C and cephalosporin C. The cephalosporin was isolated as crystals from the culture broth of the mutant No. 40-20, and identified as deacetoxycephalosporin C, possessing a D-a-aminoadipyl side chain at C-7, by physical, chemical and biological methods. The profile of deacetoxycephalosporin C fermentation and the examination of the biochemical reduction of deacetylcephalosporin C led us to the conclusion that deacetoxycephalosporin C would be produced through de novo synthesis by this mutant.     

CysK2 from Mycobacterium tuberculosis Is an O-Phospho-l-Serine-Dependent S-Sulfocysteine Synthase

Mycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to l-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), have been described in M. tuberculosis, but the function of a third putative sulfhydrylase in this pathogen, CysK2, has remained elusive. We present biochemical and biophysical evidence that CysK2 is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. The enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as k_max/K_s = (3.97 × 10³) ± 619 M⁻¹ s⁻¹, which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a k_max/K_s of (1.34 × 10³) ± 48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant k_cat/K_m for thiosulfate is 40-fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine. Hypothetically, S-sulfocysteine could also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy.     

Comparative lipidomic analysis of <Emphasis Type="Italic">Cephalosporium acremonium</Emphasis> insights into industrial and pilot fermentations

Cephalosporium acremonium has been widely applied in industrial cephalosporin C fermentation. However, little is known about the molecular basis of fermentation behavior of this strain. In this study, comparative lipidomic analysis using LC/ESI/MSⁿ technology was employed to investigate responses of Cephalosporium acremonium to multiple environment variations in realistic industrial cephalosporin C fermentation process and provide molecular basis for the discrepancies between industrial and pilot fermentations. Totally 77 phospholipids species were detected and 65 species were further quantified. Score plot revealed that phospholipids metabolism differed in industrial and pilot process. Loading pilot indicated that the main variables responsible for the discrimination of industrial and pilot process were phosphatidylinositols (PIs), phosphatidylserines (PSs) and phosphatic acids (PAs). Higher PIs content in industrial process indicated that cells were more vigorous in industrial process than those in pilot process. Larger increases of PSs, PAs and ratio of oleic acid to linoleic acid coincided well with the earlier and more thorough cellular morphological differentiation in industrial process. The synergetic reaction between cellular behavior and cells living environment led to titer discrepancies between industrial and pilot process. These findings provided lipidomic insights into industrial cephalosporin C production.     

Deacetylcephalosporin C Formation by Cephalosporin C Acetylhydrolase Induced in a Cephalosporium acermonium Mutant

Cephalosporin C acetyl-hydrolase, which had not yet been found in Cephalosporium acremonium cultures, was partially purified from the culture fluid of the mutant No. 81 by ammonium sulfate fractionation, dialysis and DEAE-cellulose column chromatography. The optimum pH and temperature of the enzyme reaction were found to be about 8.0 and 50°C, respectively. The enzyme activity was hardly affected by Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ni²⁺, Na⁺, K⁺, EDTA, PCMB and 2,4-dinitrophenol, but markedly inhibited by diisopropylfluoro-phosphate at 1 mm. The product formed from cephalosporin C by the enzyme reaction was proved to be deacetylcephalosporin C by physical and chemical analyses and chromatographic behaviors.     

The Cysteine Desulfhydrase CdsH Is Conditionally Required for Sulfur Mobilization to the Thiamine Thiazole in Salmonella enterica

Thiamine pyrophosphate is a required coenzyme that contains a mechanistically important sulfur atom. In Salmonella enterica, sulfur is trafficked to both thiamine biosynthesis and 4-thiouridine biosynthesis by the enzyme ThiI using persulfide (R-S-S-H) chemistry. It was previously reported that a thiI mutant strain could grow independent of exogenous thiamine in the presence of cysteine, suggesting there was a second mechanism for sulfur mobilization. Data reported here show that oxidation products of cysteine rescue the growth of a thiI mutant strain by a mechanism that requires the transporter YdjN and the cysteine desulfhydrase CdsH. The data are consistent with a model in which sulfide produced by CdsH reacts with cystine (Cys-S-S-Cys), S-sulfocysteine (Cys-S-SO₃⁻), or another disulfide to form a small-molecule persulfide (R-S-S-H). We suggest that this persulfide replaced ThiI by donating sulfur to the thiamine sulfur carrier protein ThiS. This model describes a potential mechanism used for sulfur trafficking in organisms that lack ThiI but are capable of thiamine biosynthesis.     

Metabolism of 5-methylthioribose to methionine

During ethylene biosynthesis, the H₃CS- group of S-adenosylmethionine is released as 5′-methylthioadenosine, which is recycled to methionine via 5-methylthioribose (MTR). In mungbean hypocotyls and cell-free extracts of avocado, [¹⁴C]MTR was converted into labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB), as intermediates. Incubation of [ribose-U-¹⁴C]MTR with avocado extract resulted in the production of [¹⁴C]formate, indicating the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR. Tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract requires an amino donor. Among several potential donors examined, l-glutamine was the most efficient. Anaerobiosis inhibited only partially the oxidation of MTR to formate, KMB/HMB, and methionine by avocado extract. The role of O₂ in the conversion of MTR to methionine is discussed.     

The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C‐terminal cysteine of the CXXC motif

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N‐terminal cysteine interacts with substrates, whereas the C‐terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C⁸⁶P⁸⁷D⁸⁸C⁸⁹ catalytic motif. In vitro, SdbA single cysteine variants at the N or C‐terminal position (SdbA_C86P and SdbA_C89A) were active but displayed different susceptibility to oxidation, and N‐terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N‐terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C‐terminal cysteine alone (in the SdbA_C86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C‐terminal cysteine.