Separation of penicillin G from phenylacetic acid in a supported liquid membrane system

The separation of penicillin G (Pen G) from phenylacetic acid (PAA) by use of a supported liquid membrane (SLM) system with Amberlite LA-2 dissolved in 1-decanol, supported on a microporous polypropylene membrane, was studied. The results show that the individual permeability of each component in mixture was lower than that in a single compartment system and, it suggests a strong transport competition between Pen G and PAA. The SLM system in this study proved to be a promising process for the selective separation of Pen G from PAA. The maximum separation factor was found to be 1.8 under a liquid membrane resistance controlled mechanism. (c) 1994 John Wiley & Sons, Inc.     

Gas-liquid chromatographic determination of total phenylacetic acid in urine

A gas-liquid chromatographic procedure to measure total phenylacetic acid in urine is described. The method is simple, rapid, and reliable. Normal subjects (N = 48) excreted 141.1 ± 10.1 mg/24 h. Untreated depressed patients (N = 42) excreted 102.77 ± 15.9 mg/24 h. The difference in the means is significant and supports the role of phenylacetic acid as a biological marker in certain kinds of mental illnesses.     

3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants

Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused β-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.     

Improvement of penicillin yields in solid-state and submerged fermentation of Penicillium chrysogenum by amplification of the penicillin biosynthetic gene cluster

Penicillium chrysogenum low and high penicillin producing strains were transformed with a cosmid containing the whole penicillin biosynthetic gene cluster. The cosmid library was constructed in a newly developed cosmid vector, IztapaCos, which allows cloning and direct introduction of large DNA fragments in fungal recipients using phleomycin resistance as selection marker. The effect of increased gene dosage on penicillin production was evaluated both in submerged (SmF) and solid-state fermentation (SSF). Transformants from the low-producing strain Wis 54-1255, showed a 67.3 and 28.3% increased penicillin titer in SSF and SmF, respectively. In transformants from the high-producing strain P2-32 the increase was 92.9 and 158.4% respectively. Strain P2-32 already contains originally about 14 copies of the penicillin biosynthetic cluster, which shows that the strategy of increasing the gene dosage is still valid for high copy-number strains. The different behavior of the two strains in each type of culture is discussed, along with the practical implications for industrial penicillin production.     

Analysis of the regulation of penicillin biosynthesis in Aspergillus nidulans by targeted disruption of the acvA gene

To analyse the regulation of the biosynthesis of the secondary metabolite penicillin in Aspergillus nidulans, a strain with an inactivated acvA gene produced by targeted disruption was used. acvA encodes -(l--aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), which catalyses the first step in the penicillin biosynthetic pathway. To study the effect of the inactivated acvA gene on the expression of acvA and the second gene, ipnA, which encodes isopenicillin N synthase (IPNS), A. nidulans strain XEPD, with the acvA disruption, was crossed with strain AXB4A carrying acvA-uidA and ipnA-lacZ fusion genes. Ascospores with the predicted non-penicillin producing phenotype and a hybridization pattern indicating the presence of the disrupted acvA gene, and the fusion genes integrated in single copy at the chromosomal argB locus were identified. Both fusion genes were expressed at the same level as in the non-disrupted strain. Western blot analysis (immunoblotting) revealed that similar amounts of IPNS enzyme were present in both strains from 24 to 68 h of a fermentation run. In the acvA disrupted strain, IPNS and acyl-CoA: 6-aminopenicillanic acid acyltransferase (ACT) specific activities were detected, excluding a sequential induction mechanism of regulation of the penicillin biosynthesis gene ipnA and the third gene aat.     

Effect of phenylacetic acid on the growth and production of penicillin G acylase of recombinant and host strains derived from Escherichia coli W

The industrial production strain Escherichia coli RE3(pKA18) for penicillin G acylase (PGA) bears simultaneously the pga gene on the chromosome as an inducible gene pgaⁱ, (the inductor is phenylacetic acid, PAA) and on the recombinant plasmid pKA18 as a constitutively expressed gene pga^c. Under non-selective conditions, plasmid-less strains (P⁻) appeared in 17th successive batch culture. However, the population was over taken by P⁻ cells already in fourth culture if the medium was supplemented with PAA. The rate of plasmid loss from the culture depends on the PAA concentration and on the expression of pgaⁱ, not on PGA overproduction from pga^c. PAA at inducing concentration has a negative effect on PGA expression and plasmid stability in the high-expression self-cloning system RE3(pKA18) which results in the reduction of: (1) the specific growth rate of a culture and biomass concentration, (2) the synthesis of PGA (e.g. the specific activity of the strain) and (3) the copy number of the recombinant plasmid and promotion of the plasmid loss from the culture. Segregational stability of pKA18 increases in P⁺ persisting clones and in re-transformed P⁻ clones segregated during the selection in the presence of PAA.     

Biooxidation of 2-phenylethanol to phenylacetic acid by whole-cell Gluconobacter oxydans biocatalyst immobilized in polyelectrolyte complex capsules

A high-performance biocatalyst in the form of encapsulated cells of Gluconobacter oxydans have been developed for production of phenylacetic acid (PAA) as a natural flavor component. Polyelectrolyte complex (PEC) capsules consisting of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl₂, and NaCl were used for highly controlled and mild encapsulation of cells. Utilization of encapsulated G. oxydans cells was a significant improvement on existing data on operational stability of cells and cumulative product concentration during biocatalytic production of PAA from 2-phenylethanol. Concerning operational stability, encapsulated cells were active over 12 cycles with a high biotransformation rate, while free cells were inactive after 7 cycles of use. The biocatalytic properties of encapsulated G. oxydans were tested in a bubble column reactor over 7 days with a final cumulative product concentration of 25 g/L. High cell viability (90%) was observed within PEC capsules by confocal laser scanning microscopy, performed before and after repetitive PAA production in the bubble column reactor. The surface microstructure of fully hydrated capsules with and without G. oxydans cells was investigated and compared using an environmental scanning electron microscope.     

Promotion of elongation and acid invertase activity in Phaseolus vulgaris L. internode segments by phenylacetic acid

Phenylacetic acid (PAA) significantly stimulated the elongation of isolated Phaseolus vulgaris internodal segments and prevented the decline in acid invertase specific activity observed in segments incubated in the absence of growth substances. Unlike IAA, which stimulated both elongation and invertase activity over a very wide range of concentrations (<10^-4 - 1 mol.m^-3; optimum 10^-2 mol.m^-3), the response to PAA was restricted to a much narrower range of concentrations (3 × 10^-2 - 1 mol.m^-3; optimum ca. 1–2 × 10^-1mol.m^-3). At the optimum concentration of PAA, the stimulation of both responses was about 63–75% of that induced by the optimum concentration of IAA. The differences in the concentration range and magnitude of the responses to IAA and PAA were not due to differences in uptake of the two compounds. The stimulation of elongation by both compounds was prevented by 3.6 × 10^-2mol.m^-3 cycloheximide (CH), and acid invertase activites were greatly reduced compared with samples treated with growth substances alone. A saturating concentration of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA) slightly promoted the growth of control segments, probably by reducing the loss of residual endogenous auxin to the incubation medium. The elongation induced by PAA at its optimum concentration was considerably greater than the elongation induced by NPA, indicating that PAA did not cause growth by preventing the loss of endogenous auxin from the segments. Elongation responses to combinations of IAA and PAA suggested that the compounds were acting additively and that they were affecting growth by the same mechanism.     

l-Lysine repression of penicillin biosynthesis and the expression of penicillin biosynthesis genes acvA and ipnA in Aspergillus nidulans

The addition of 0.1 M L-lysine to the fermentation medium reduced the production of penicillin by about 50% in Aspergillus nidulans. To analyse this effect at the molecular level, the expression of the penicillin biosynthesis genes acvA and ipnA, encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, was studied by using translational fusions with different reporter genes (strain AXB4A, acvA-uidA, ipnA-lacZ fusions; AXB4B, acvA-lacZ, ipnA-uidA fusions) integrated in single copy at the chromosomal argB locus of Aspergillus nidulans. Irrespective of the reporter genes used the expression of acvA and ipnA fusion genes was repressed in L-lysine grown cultures. The expression of a fusion gene of an A. nidulans primary metabolism gene (oliC-lacZ) was not affected by L-lysine.     

Modifications of the C6-substituent of penicillin sulfones with the goal of improving inhibitor recognition and efficacy

In order to evaluate the importance of a hydrogen-bond donating substituent in the design of β-lactamase inhibitors, a series of C6-substituted penicillin sulfones, lacking a C2′ substituent, and having an sp³ hybridized C6, was prepared and evaluated against a representative classes A and C β-lactamases. It was found that a C6 hydrogen-bond donor is necessary for good inhibitory activity, but that this feature alone is not sufficient in this series of C6β-substituted penicillin sulfones. Other factors which may impact the potency of the inhibitor include the steric bulk of the C6 substituent (e.g., methicillin sulfone) which may hinder recognition in the class A β-lactamases, and also high similarity to the natural substrates (e.g., penicillin G sulfone) which may render the prospective inhibitor a good substrate of both classes of enzyme. The best inhibitors had non-directional hydrogen-bonding substituents, such as hydroxymethyl, which may allow sufficient conformational flexibility of the acyl-enzyme for abstraction of the C6 proton by E166 (class A), thus promoting isomerization to the β-aminoacrylate as a stabilized acyl-enzyme.     

Pipecolic acid in microbes: biosynthetic routes and enzymes

Pipecolic acid is an important precursor of many useful microbial secondary metabolites. Pipecolic acid-derived moieties are often crucial for the biological activities of some microbial natural products with pharmaceutical applications. Understanding the biogenesis of pipecolic acid in microorganisms would be a significant step toward the mutasynthesis of novel analogs of choice. This review focuses on various microbial pathways and enzymes for pipecolic acid synthesis, especially those related to the origination of pipecolic acid moieties in secondary metabolites.     

Biosynthesis of propyl cannabinoid acid and its biosynthetic relationship with pentyl and methyl cannabinoid acids

Biosynthesis of propyl cannabinoid acid has been determined by in vitro incubation with a crude enzyme solution from three strains of Cannabis sativa using ¹⁴C-labelled cannabinoid acid. Biosynthetic relationships between methyl, propyl and pentyl cannabinoid acids have been demonstrated.     

In vivo transport of the intermediates of the penicillin biosynthetic pathway in tailored strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Penicillium chrysogenum npe10 (Δpen; lacking the 56.8-kbp amplified region containing the penicillin gene cluster), complemented with one, two, or three penicillin biosynthetic genes, was used for in vivo studies on transport of benzylpenicillin intermediates. 6-Aminopenicillanic acid (6-APA) was taken up efficiently by P. chrysogenum npe10 unlike exogenous δ(l-α-aminoadipyl)-l-cysteinyl-d-valine or isopenicillin N (IPN), which were not taken up or were taken up very poorly. Internalization of exogenous IPN and 6-APA inside peroxisomes was tested by quantifying their peroximal conversion into benzylpenicillin in strains containing only the penDE gene. Exogenous 6-APA was transformed efficiently into benzylpenicillin, whereas IPN was converted very poorly into benzylpenicillin due to its weak uptake. IPN was secreted to the culture medium. IPN secretion decreased when increasing levels of phenylacetic acid were added to the culture medium. The P. chrysogenum membrane permeability to exogenous benzylpenicillin was tested in the npe10 strain. Penicillin is absorbed by the cells by an unknown mechanism, but its intracellular concentration is kept low. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inhibition of O-acetylserine sulfhydrylase by fluoroalanine derivatives

O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5′-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is pursued as a strategy for the identification of novel antibiotics that, targeting dispensable proteins, holds a great promise for circumventing resistance development. In the present study, we have investigated the reactivity of Salmonella enterica serovar Typhimurium OASS-A and OASS-B isozymes with fluoroalanine derivatives. Monofluoroalanine reacts with OASS-A and OASS-B forming either a stable or a metastable α-aminoacrylate Schiff’s base, respectively, as proved by spectral changes. This finding indicates that monofluoroalanine is a substrate analogue, as previously found for other beta-halogenalanine derivatives. Trifluoroalanine caused different and time-dependent absorbance and fluorescence spectral changes for the two isozymes and is associated with irreversible inhibition. The time course of enzyme inactivation was found to be characterised by a biphasic behaviour. Partially distinct inactivation mechanisms for OASS-A and OASS-B are proposed.     

Stereoselectivity in the biosynthetic conversion of xanthoxin into abscisic acid

All stereoisomers of xanthoxin (XAN) and abscisic aldehyde (ABA-aldehyde) were prepared from (R) and (S)-4-hydroxy--cyclogeraniol via asymmetric epoxidation. Their stomatal closure activities were measured on epidermal strips of Commelina communis L. Natural (S)-ABA-aldehyde showed strong activity comparable to that of (S)-abscisic acid (ABA). Natural (1S, 2R, 4S)XAN and (1S, 2R, 4R)-epi-XAN also induced stomatal closure at high concentrations. On the other hand, unnatural (1R)-enantiomers of XAN, epi-XAN, and ABA-aldehyde were not effective. To further examine the Stereoselectivity on the biosynthetic pathway to ABA, deuterium-labeled substrates were prepared and fed to Lycopersicon esculentum Mill, under non-stressed or water-stressed conditions. Substantial incorporations into ABA were observed in the cases of natural (1S, 2R, 4S)-XAN, (1S, 2R, 4R)-epi-XAN and both enantiomers of ABA-aldehyde, leading to the following conclusions. The negligible effect of unnatural (1R)-enantiomers of XAN, epi-XAN and ABA-aldehyde can be explained by their own biological inactivity and/or their conversion to inactive (R)-ABA. Even in the isolated epidermal strips, putative aldehyde oxidase activity is apparently sufficient to convert ABA-aldehyde to ABA while the activity of XAN dehydrogenase seems very weak. The stereochemistry of the 1, 2-epoxide is very important for the XAN-dehydrogenase while this enzyme is less selective regarding the 4-hydrdxyl group of XAN and converts both (1S, 2R, 4S)-XAN and (1S, 2R, 4R)-epi-XAN to (S)-ABA-aldehyde. Abscisic aldehyde oxidase can nonstereoselectively convert both (S) and (R)-ABA-aldehyde to biologically active (S) and inactive (R)-ABA, respectively.Abbreviations ABA abscisic acid - ABA-aldehyde abscisic aldehyde - DET diethyl tartrate - epi-XAN xanthoxin epimer - FCC flash column chromatography - GC-EI-MS gas chromatography-electron impact-mass spectrometry - MeABA abscisic acid methyl ester - IR infrared - NMR nuclear magnetic resonance - PCC pyridinium chlorochromate - THF tetrahydrofuran - XAN xanthoxin The authors are very grateful to Mr J.K. Heald (Department of Biological Sciences, University of Wales, Aberystwyth, UK) and Dr. R. Horgan for carrying out GC-EI-MS analyses and advice, respectively.This work was supported by the Japan Society for the Promotion of Science (Fellowship for Young Japanese Researcher No. 0040672).     

Determination of the rate-limiting step(s) in the biosynthetic pathways leading to penicillin and cephalosporin

Summary This paper is a review of strategies that have been used, or that could be used, to determine the rate-limiting step(s) in the biosynthetic pathways leading to penicillin or cephalosporin. Information is summarized from published material that involves studies with low-producing strains ofPenicillium chrysogenum andCephalosporium acremonium. We also summarize information derived from some high-producing production strains. Identification of the rate-limiting step(s) was of great interest to us as the first step in a rational program to further improve antibiotic titers of these highly developed strains. A number of approaches that could be used to elucidate the rate-limiting step(s) are described herein.