Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum.

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

Enzyme-mediated transglycosylation of rutinose (6-O-α-l-rhamnosyl-d-glucose) to phenolic compounds by a diglycosidase from Acremonium sp. DSM 24697.

The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-ß-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-l-rhamnopyranosyl-ß-d-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ≤6.0. The structure of 4-hydroxyphenyl-ß-rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4-hydroxyphenyl-ß-rutinoside (38%, regarding hesperidin) was achieved in a 2-h process at pH 5.0 and 30 °C, with 36 mM OH-acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pKa value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.

Expression, Purification, and Activity of ActhiS, a Thiazole Biosynthesis Enzyme from Acremonium chrysogenum.

Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid (ADT) using HPLC and 1H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD+ and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety.

Soluble inhibitors generated during hydrothermal pretreatment of oil palm mesocarp fiber suppressed the catalytic activity of Acremonium cellulase.

Oil palm mesocarp fiber was subjected to hydrothermal pretreatment under isothermal and non-isothermal conditions. The pretreated slurries were separated by filtration, pretreated liquids and solids were characterized. An enzymatic digestibility study was performed for both pretreated slurries and solids to understand the effect of soluble inhibitors generated during the pretreatment process. The highest glucose yield obtained from pretreated slurries was 70.1%, and gradually decreased with higher pretreatment severities. The highest glucose yield obtained in pretreated solids was 100%, after pretreatment at 210°C for 20min. In order to study the inhibitory effects of compounds generated during pretreatment with cellulase, technical grade solutions that mimic the pretreated liquid were prepared and their effect on Acremonium cellulase activity was monitored using Avicel. Xylo-oligomers and tannic acid were identified as powerful inhibitors of Acremonium cellulase, and the lowest hydrolysis rate of Avicel of 0.18g/g-glucose released/L/h was obtained from tannic acid.

Ball milling pretreatment of oil palm biomass for enhancing enzymatic hydrolysis.

Oil palm biomass, namely empty fruit bunch and frond fiber, were pretreated using a planetary ball mill. Particle sizes and crystallinity index values of the oil palm biomass were significantly reduced with extended ball mill processing time. The treatment efficiency was evaluated by the generation of glucose, xylose, and total sugar conversion yields from the pretreatment process compared to the amount of sugars from raw materials. Glucose and xylose contents were determined using high-performance liquid chromatography. An increasing trend in glucose and xylose yield as well as total sugar conversion yield was observed with decreasing particle size and crystallinity index. Oil palm frond fiber exhibited the best material yields using ball milling pretreatment with generated glucose, xylose, and total sugar conversion yields of 87.0, 81.6, and 85.4%, respectively. In contrast, oil palm empty fruit bunch afforded glucose and xylose of 70.0 and 82.3%, respectively. The results obtained in this study showed that ball mill-treated oil palm biomass is a suitable pretreatment method for high conversion of glucose and xylose.

Direct electrochemistry and intramolecular electron transfer of ascorbate oxidase confined on L-cysteine self-assembled gold electrode.

A direct electrochemistry and intramolecular electron transfer of multicopper oxidases are of a great importance for the fabrication of these enzyme-based bioelectrochemical-devices. Ascorbate oxidase from Acremonium sp. (ASOM) has been successfully immobilized via a chemisorptive interaction on the l-cysteine self-assembled monolayer modified gold electrode (cys-SAM/AuE). Thermodynamics and kinetics of adsorption of ASOM on the cys-SAM/AuE were studied using cyclic voltammetry. A well-defined redox wave centered at 166±3mV (vs. Ag│AgCl│KCl(sat.)) was observed in 5.0mM phosphate buffer solution (pH7.0) at the fabricated ASOM electrode, abbreviated as ASOM/cys-SAM/AuE, confirming a direct electrochemistry, i.e., a direct electron transfer (DET) between ASOM and cys-SAM/AuE. The direct electrochemistry of ASOM was further confirmed by taking into account the chemical oxidation of ascorbic acid (AA) by O2 via an intramolecular electron transfer in the ASOM as well as the electrocatalytic oxidation of AA at the ASOM/cys-SAM/AuE. Thermodynamics and kinetics of the adsorption of ASOM on the cys-SAM/AuE have been elaborated along with its direct electron transfer at the modified electrodes on the basis of its intramolecular electron transfer and electrocatalytic activity towards ascorbic acid oxidation and O2 reduction. ASOM saturated surface area was obtained as 2.41×10(-11)molcm(-2) with the apparent adsorption coefficient of 1.63×10(6)Lmol(-1). The ASOM confined on the cys-SAM/AuE possesses its essential enzymatic function.

[Viability, taxonomic confirmation and enzymatic detection of Acremonium species preserved under mineral oil in the URM Culture Collection]. / Viabilidade, confirmação taxonômica e detecção enzimática de espécies de Acremonium preservadas sob óleo mineral na Coleção de Culturas University Recife Mycology.

Hydrolytic enzymes secreted by fungi play an important role in the pathogenesis of infection. With the aim of evaluating the enzymatic activity, 31 isolates of Acremonium stored in the University of Recife Mycology (URM) Culture Collection were tested. Culture fragments were transferred to glycoside broth for reactivation and further growth in potato dextrose agar medium in order to investigate viability and purity and to confirm the taxonomy through observing the macroscopic and microscopic characteristics. To detect enzymes, milk casein and gelatin were used as substrates for proteinase, starch for amylase and soy lecithin for phospholipase. Among the 31 cultures, 26 (83.9%) remained viable and 24 (92.3%) were confirmed taxonomically. Out of these 24 cultures, 12 (50%) presented proteinase activity, of which two (16.7%) were on milk casein, one (8.3%) on gelatin and nine (75%) on both substrates; 16 (66.7%) degraded starch. None of the cultures presented phospholipase activity. It was concluded that Acremonium species are able to produce enzymes that are involved in the pathogenicity of fungal infections.

Extracellular hemicellulolytic enzymes from the maize endophyte Acremonium zeae.

Microorganisms that colonize plants require a number of hydrolytic enzymes to help degrade the cell wall. The maize endophyte Acremonium zeae was surveyed for production of extracellular enzymes that hydrolyze cellulose and hemicellulose. The most prominent enzyme activity in cell-free culture medium from A. zeae NRRL 6415 was xylanase, with a specific activity of 60 U/mg from cultures grown on crude corn fiber. Zymogram analysis following SDS-PAGE indicated six functional xylanase polypeptides of the following masses: 51, 44, 34, 29, 23, and 20 kDa. Xylosidase (0.39 U/mg), arabinofuranosidase (1.2 U/mg), endoglucanase (2.3 U/mg), cellobiohydrolase (1.3 U/mg), and beta-glucosidase (0.85 U/mg) activities were also detected. Although apparently possessing a full complement of hemicellulolytic activities, cell-free culture supernatants prepared from A. zeae required an exogenously added xylosidase to release more than 90% of the xylose and 80% of the arabinose from corn cob and wheat arabinoxylans. The hydrolytic enzymes from A. zeae may be suitable for application in the bioconversion of lignocellulosic biomass into fermentable sugars.

Viabilidade, confirmação taxonômica e detecção enzimática de espécies de Acremonium preservadas sob óleo mineral na Coleção de Culturas University Recife Mycology / Viability, taxonomic confirmation and enzymatic detection of Acremonium species preserved under mineral oil in the URM Culture Collection

Enzimas hidrolíticas secretadas por fungos têm um papel importante na patogenicidade das infecções. Objetivando avaliar a atividade enzimática foram testados 31 isolados de Acremonium mantidos na Coleção de Culturas University Recife Mycology. Fragmentos das culturas foram transferidos para caldo glicosado para reativação e posterior crescimento em meio ágar batata dextrose, para verificar viabilidade, pureza e confirmação taxonômica pela observação das características macroscópicas e microscópicas. Para detecção enzimática foram utilizados substratos de caseína do leite e gelatina para protease, amido para amilase e lecitina de soja para fosfolipase. Das 31 culturas, 26 (83,9 por cento) mantiveram-se viáveis e 24 (92,3 por cento) foram confirmadas taxonomicamente. Das 24 culturas, 12 (50 por cento) apresentaram atividade proteásica, duas (16,7 por cento) em caseína do leite, uma (8,3 por cento) em gelatina e nove (75 por cento) em ambos os substratos; 16 (66,7 por cento) degradaram amido. Nenhuma cultura apresentou atividade fosfolipásica. Conclui-se que espécies de Acremonium são capazes de produzir enzimas envolvidas na patogenicidade das infecções fúngicas.

Hydrolytic enzymes secreted by fungi play an important role in the pathogenesis of infection. With the aim of evaluating the enzymatic activity, 31 isolates of Acremonium stored in the University of Recife Mycology (URM) Culture Collection were tested. Culture fragments were transferred to glycoside broth for reactivation and further growth in potato dextrose agar medium in order to investigate viability and purity and to confirm the taxonomy through observing the macroscopic and microscopic characteristics. To detect enzymes, milk casein and gelatin were used as substrates for proteinase, starch for amylase and soy lecithin for phospholipase. Among the 31 cultures, 26 (83.9 percent) remained viable and 24 (92.3 percent) were confirmed taxonomically. Out of these 24 cultures, 12 (50 percent) presented proteinase activity, of which two (16.7 percent) were on milk casein, one (8.3 percent) on gelatin and nine (75 percent) on both substrates; 16 (66.7 percent) degraded starch. None of the cultures presented phospholipase activity. It was concluded that Acremonium species are able to produce enzymes that are involved in the pathogenicity of fungal infections.

Transformation of L-tyrosine to L-dopa by a novel fungus, Acremonium rutilum, under submerged fermentation.

The present study deals with the transformation of L-tyrosine to L-dopa by Acremonium rutilum, a fungal tyrosinase producer, isolated from decomposed banana stud. This appears to be the first report on A. rutilum as a polyphenoloxidase producer with both cresolase and catecholase activity. Enriched Czapek-Dox agar was used for plate assay screening. Enriched potato dextrose broth was used for optimization studies, which induced high levels of L-dopa under submerged fermentation. A. rutilum gave the maximum L-dopa production (0.89 mg/ml) and tyrosinase activity (1095 U/mg) under the optimized parameters, that is, a temperature of 25 degrees C, pH 5.5, an inoculum size of 2.5 ml, and an incubation time of 72-120 h, with L-tyrosine (5 mg/ml) as substrate. Five resolved bands, with R(f) values of 0.73, 0.60, 0.54, 0.37, and 0.26, were observed, which confirmed the presence of L-dopa. This study involves the elevated profile of L-dopa production. Such study is needed, as L-dopa has the ability to control Parkinson's disease.

Functional roles of the 6-S-cysteinyl, 8alpha-N1-histidyl FAD in glucooligosaccharide oxidase from Acremonium strictum.

The crystal structure of glucooligosaccharide oxidase from Acremonium strictum was demonstrated to contain a bicovalent flavinylation, with the 6- and 8alpha-positions of the flavin isoalloxazine ring cross-linked to Cys(130) and His(70), respectively. The H70A and C130A single mutants still retain the covalent FAD, indicating that flavinylation at these two residues is independent. Both mutants exhibit a decreased midpoint potential of approximately +69 and +61 mV, respectively, compared with +126 mV for the wild type, and possess lower activities with k(cat) values reduced to approximately 2 and 5%, and the flavin reduction rate reduced to 0.6 and 14%. This indicates that both covalent linkages increase the flavin redox potential and alter the redox properties to promote catalytic efficiency. In addition, the isolated H70A/C130A double mutant does not contain FAD, and addition of exogenous FAD was not able to restore any detectable activity. This demonstrates that the covalent attachment is essential for the binding of the oxidized cofactor. Furthermore, the crystal structure of the C130A mutant displays conformational changes in several cofactor and substrate-interacting residues and hence provides direct evidence for novel functions of flavinylation in assistance of cofactor and substrate binding. Finally, the wild-type enzyme is more heat and guanidine HCl-resistant than the mutants. Therefore, the bicovalent flavin linkage not only tunes the redox potential and contributes to cofactor and substrate binding but also increases structural stability.

Manganese(IV) oxide production by Acremonium sp. strain KR21-2 and extracellular Mn(II) oxidase activity.

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; Km, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes.

Structural characterization of glucooligosaccharide oxidase from Acremonium strictum.

Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the Km. Instead, the variants displayed kcat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.

Crystal structure of glucooligosaccharide oxidase from Acremonium strictum: a novel flavinylation of 6-S-cysteinyl, 8alpha-N1-histidyl FAD.

Glucooligosaccharide oxidase from Acremonium strictum has been screened for potential applications in oligosaccharide acid production and alternative carbohydrate detection, because it catalyzes the oxidation of glucose, maltose, lactose, cellobiose and cello- and maltooligosaccharides. We report the crystal structures of the enzyme and of its complex with an inhibitor, 5-amino-5-deoxy- cellobiono-1,5-lactam at 1.55- and 1.98-A resolution, respectively. Unexpectedly, the protein structure demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8alpha-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C(6) atom and the 8alpha-methyl group of the isoalloxazine ring with Cys(130) and His(70), respectively. This sugar oxidase possesses an open carbohydrate-binding groove, allowing the accommodation of higher oligosaccharides. The complex structure suggests that this enzyme may prefer a beta-d-glucosyl residue at the reducing end with the conserved Tyr(429) acting as a general base to abstract the OH(1) proton in concert with the H(1) hydride transfer to the flavin N(5). Finally, a detailed comparison illustrates the structural conservation as well as the divergence between this protein and its related flavoenzymes.

Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage.

alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.

Nitrate regulation of alpha-aminoadipate reductase formation and lysine inhibition of its activity in Penicillium chrysogenum and Acremonium chrysogenum.

alpha-Aminoadipate reductase (alpha-AAR) is a key enzyme in the branched pathway for lysine and beta-lactam biosynthesis of filamentous fungi since it competes with alpha-aminoadipyl-cysteinyl-valine synthetase for their common substrate L-alpha-aminoadipic acid. The alpha-AAR activity in two penicillin-producing Penicillium chrysogenum strains and two cephalosporin-producing Acremonium chrysogenum strains has been studied. The alpha-AAR activity peaked during the growth-phase preceding the onset of antibiotic production, which coincides with a decrease in alpha-AAR activity, and was lower in high penicillin- or cephalosporin-producing strains. The alpha-AAR required NADPH for enzyme activity and could not use NADH as electron donor for reduction of the alpha-aminoadipate substrate. The alpha-AAR protein of P. chrysogenum was detected by Western blotting using anti-alpha-AAR antibodies. The mechanism of lysine feedback regulation in these two filamentous fungi involves inhibition of the alpha-AAR activity but not repression of its synthesis by lysine. This is different from the situation in yeasts where lysine feedback inhibits and represses alpha-AAR. Nitrate has a strong negative effect on alpha-AAR formation as shown by immunoblotting studies of alpha-AAR. The nitrate effect was reversed by lysine.

L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase: production of dipeptides containing valine residue at its C-terminus.

L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase (ACVS) has been recently studied as a model enzyme for peptide synthetases. It was found that in the absence of alpha-aminoadipic acid but in the presence of several cysteine analogues it was incorporated into several analogue dipeptides upon incubation of the potential cysteine analogues with ACVS. [(14)C]Cysteine was incorporated into the[(14)C]cysteinyl-valine analogue dipeptides. Notably, [(14)C]valine incorporation in the presence of N-acylated cysteine analogues was observed. The alpha-aminoadipic acid activation site is influential, inhibitory or promotive, on the production of these putative dipeptide products. The production of dipeptide analogues, containing valine or analogues at the C-terminus, leads to the speculation that the biosynthetic direction of ACV could be from the C-terminus to the N-terminus.

A moderate amplification of the mecB gene encoding cystathionine-gamma-lyase stimulates cephalosporin biosynthesis in Acremonium chrysogenum.

L-cysteine is a precursor of the penicillin, cephalosporin and cephamycin families of beta-lactam antibiotics. Cystathionine-gamma-lyase (encoded by the mecB gene), an enzyme that splits cystathionine releasing cysteine, is required for high-level cephalosporin production in methionine-supplemented medium. By amplification of the mecB gene in Acremonium chrysogenum C10, several transformants were obtained that produced 10-40% higher levels of cephalosporin. All selected transformants contained at least two or three copies of the mecB gene as shown by Southern hybridization with a probe internal to mecB. Two of these transformants, A. chrysogenum T27 and A. chrysogenum T58, showed 4- to 10-fold higher cystathionine-gamma-lyase activity than the control strain. Northern hybridizations indicated that the levels of the two mecB transcripts of 1.7 and 1.5 kb were greatly increased in transformants T27 and T58. Fermentor studies using controlled conditions confirmed that transformant T27 was a cephalosporin overproducer, reaching titers of nearly 2000 microg/ml of cephalosporin in Shen-defined medium that correlated with two- to fourfold higher cystathionine-gamma-lyase levels than in the control strain. Transformant T58 containing five- to sixfold higher levels of cystathionine-gamma-lyase in fermentor cultures showed a reduced growth rate and a slow cephalosporin accumulation rate. In conclusion, moderately increased levels of cystathionine-gamma-lyase stimulated cephalosporin production but very high levels of this enzyme were deleterious for growth and cephalosporin biosynthesis.

Mutational analysis of conserved glycines 42 and 256 in Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is critical for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N in the penicillin and cephalosporin biosynthetic pathway. Two conserved glycine residues in Cephalosporium acremonium IPNS (cIPNS), namely glycine-42 and glycine-256, were identified by multiple sequence alignment and investigated by site-directed mutagenesis to study the effect of the substitution on catalysis. Our study showed that both the mutations from glycine to alanine or to serine reduced the catalytic activity of cIPNS and affected its soluble expression in a heterologous host at 37 degrees C. Soluble expression was restored at a reduced temperature of 25 degrees C, and thus, it is possible that these glycine residues may have a role in maintaining the local protein structure and are critical for the soluble expression of cIPNS.

Replacement of tyrosine-197 and the corresponding tyrosine-195 to isoleucine in Cephalosporium acremonium and Streptomyces clavuligerus isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyses the conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. The IPNS from Penicillium chrysogenum 23X-80-269-37-2, a high penicillin V-producer, was found to possess an isoleucine residue instead of tyrosine at position 195. An attempt to increase the specific activity of IPNS from Cephalosporium acremonium and Streptomyces clavuligerus was undertaken by altering the corresponding tyrosine residue to an isoleucine at the corresponding location. Unfortunately, no apparent increase in specific activity was encountered when the purified mutant enzymes were analysed and thus, this amino acid difference is likely not responsible for high specific activity in IPNS.