Isolation of proline-based cyclic dipeptides from Bacillus sp. N strain associated with rhabitid entomopathogenic nematode and its antimicrobial properties

Entomopathogenic nematodes (EPN) are well-known as biological control agents and are found to have associated bacteria which can produce a wide range of bioactive secondary metabolites. We report herewith isolation of six proline containing cyclic dipeptides cyclo(d-Pro-l-Leu), cyclo(l-Pro-l-Met), cyclo(d-Pro-l-Phe), cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Tyr) and cyclo(l-Pro-d-Tyr) from ethyl acetate extract of the Luria Broth (LB) cell free culture filtrate of Bacillus sp. strain N associated with a new EPN Rhabditis sp. from sweet potato weevil grubs collected from Central Tuber Crops Research Institute farm. Antimicrobial studies of these 2,5-diketopiperazines (DKPs) against both medicinally and agriculturally important bacterium and fungi showed potent inhibitory values in the range of μg/mL. Cyclic dipeptides showed significantly higher activity than the commercial fungicide bavistin against agriculturally important fungi, viz., Fusarium oxysporum, Rhizoctonia solani, and Pencillium expansum. The highest activity of 2 μg/mL by cyclo(l-Pro-l-Phe) was recorded against P. expansum, a plant pathogen responsible for causing post harvest decay of stored apples and oranges. To our knowledge, this is the first report on the isolation of these DKPs from Rhabditis EPN bacterial strain Bacillus sp.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine production by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach

The l-phenylalanine (l-Phe) production by Escherichia coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem for an improved l-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N’-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in l-Phe production and a remarkable tolerance to 1 × 10¹⁰ pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced l-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH–stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on l-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at μ _set = 0.18 h⁻¹ in the first 20 h and a following linear varying rate feeding with F = (−0.55 × t + 18.6) ml/h, was developed to improve l-Phe production. With this two-stage feeding approach, a maximum l-Phe titer of 57.63 g/l with a high l-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential l-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03).     

The ncgl1108 (PheP (Cg)) gene encodes a new L-Phe transporter in Corynebacterium glutamicum

Corynebacterium glutamicum played a central role in the establishment of fermentative production of amino acids, and it is a model for genetic and physiological studies. The general aromatic amino acid transporter, AroP _Cg , was the sole functionally identified aromatic amino acid transporter from C. glutamicum. In this study, the ncgl1108 (named as pheP _Cg ), which is located upstream of the genetic cluster (ncgl1110 ∼ ncgl1113) for resorcinol catabolism, was identified as a new l-Phe specific transporter from C. glutamicum RES167. The disruption of pheP _Cg resulted in RES167∆ncgl1108, and this mutant showed decreased growth on l-Phe (as nitrogen source) but not on l-Tyr or l-Trp. Uptake assays with unlabeled and ¹⁴C-labeled l-Phe and l-Tyr indicated that the mutants RES167∆ncgl1108 showed significant reduction in l-Phe uptake than RES167. Expression of pheP _Cg in RES167∆ncgl1108/pGXKZ1 or RES167∆(ncgl1108-aroP _Cg )/pGXKZ1 restored their ability to uptake for l-Phe and growth on l-Phe. The uptake of l-Phe was not inhibited by nine amino acids but by l-Tyr. The K _m and V _max values of RES167∆(ncgl1108-aroP _Cg )/pGXKZ1 for l-Phe were determined to be 10.4 ± 1.5 μM and 1.2 ± 0.1 nmol min⁻¹ (mg DW)⁻¹, respectively, which are different from K _m and V _max values of RES167∆(ncgl1108-aroP _Cg ) for l-Phe [4.0 ± 0.4 μM and 0.6 ± 0.1 nmol min⁻¹ (mg DW)⁻¹]. In conclusion, this PheP _Cg is a new l-Phe transporter in C. glutamicum.     

Entomopathogenic potential of fungi isolated from intertidal environments against the cabbage aphid Brevicoryne brassicae (Hemiptera: aphididae)

The use of biopesticides formulated from entomopathogenic fungi is a strategy utilised in integrated pest management programmes. The microorganisms used in these biopesticides are isolated from terrestrial organisms and ecosystems. However, bioprospecting in marine environments may lead to the discovery of promising fungi for pest control. In this study, marine fungi were identified and evaluated for the control of Brevicoryne brassicae. The effects of the most virulent isolate so identified on the mortality of aphids were compared to the effects of bioinsecticides that were formulated from fungal strains of Beauveria bassiana (Bovemax®) and Metarhizium anisopliae (Methamax®). Moreover, lethal and sublethal effects of this isolate on B. brassicae biological parameters were also examined. The isolates were identified as Aspergillus versicolor, Aspergillus sydowii (isolates 1 and 2), Penicillium dipodomyicola, and Trichoderma harzianum. The fungal strain A. versicolor was the most virulent fungal species, causing 85.9% mortality in B. brassicae at 24 h. The mortality rate caused by A. versicolor was similar to that caused by Bovemax® and Methamax® at concentrations of 10⁵ conidia mL^?1, and superior to that caused by Methamax® at a concentration of 10⁹ conidia mL^?1. The exposure of B. brassicae to CL₂₅ (0.32 × 10³) of A. versicolor did not affect the net reproductive rate (R_o), average generation time (T), intrinsic rate of population growth (r_m), and finite rate of population increase (λ). This is the first study to demonstrate that A. versicolor isolated from a marine environment is a promising candidate for the biological control of agricultural pests.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

From scratch to value: engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> wild type cells to the production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine and other fine chemicals derived from chorismate

Recombinant strains of Escherichia coli K-12 for the production of the three aromatic amino acids (l-phenylalanine, l-tryptophan, l-tyrosine) have been constructed. The largest demand is for l-phenylalanine (l-Phe), as it can be used as a building block for the low-calorie sweetener, aspartame. Besides l-Phe, an increasing number of shikimic acid pathway intermediates can be produced from appropriate E. coli mutants with blocks in this pathway. The last common intermediate, chorismate, in E. coli not only serves for production of aromatic amino acids but can also be used for high-titer production of non-aromatic compounds, e.g., cyclohexadiene-transdiols. In an approach to diversity-oriented metabolic engineering (metabolic grafting), platform strains with increased flux through the general aromatic pathway were created by suitable gene deletions, additions, or rearrangements. Examples for rational strain constructions for l-phenylalanine and chorismate derivatives are given with emphasis on genetic engineering. As a result, l-phenylalanine producers are available, which were derived through several defined steps from E. coli K-12 wild type. These mutant strains showed l-phenylalanine titers of up to 38 g/l of l-phenylalanine (and up to 45.5 g/l using in situ product recovery). Likewise, two cyclohexadiene-transdiols could be recovered.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arabinose pathway engineering for arabitol-free xylitol production in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Xylose reductase (XR) is a key enzyme in biological xylitol production, and most XRs have broad substrate specificities. During xylitol production from biomass hydrolysate, non-specific XRs can reduce l-arabinose, which is the second-most abundant hemicellulosic sugar, to the undesirable byproduct arabitol, which interferes with xylitol crystallization in downstream processing. To minimize the flux from l-arabinose to arabitol, the l-arabinose-preferring, endogenous XR was replaced by a d-xylose-preferring heterologous XR in Candida tropicalis. Then, Bacillus licheniformis araA and Escherichia coli araB and araD were codon-optimized and expressed functionally in C. tropicalis for the efficient assimilation of l-arabinose. During xylitol fermentation, the control strains BSXDH-3 and KNV converted 9.9 g l-arabinose l⁻¹ into 9.5 and 8.3 g arabitol l⁻¹, respectively, whereas the recombinant strain JY consumed 10.5 g l-arabinose l⁻¹ for cell growth without forming arabitol. Moreover, JY produced xylitol with 42 and 16% higher productivity than BSXDH-3 and KNV, respectively.     

Two novel cyclic hexapeptides from the genetically engineered Actinosynnema pretiosum

Two novel cyclic hexapeptides designated actinosynneptides A (1) and B (2), together with three tryptophan containing diketopiperazines, namely cyclo(L-Trp-L-Trp) (3), cyclo(L-Trp-N-MeL-Trp) (4), and cyclo(N-MeL-Trp-N-MeL-Trp) (5), were isolated from the culture of the genetically engineered strain HGF052::asm18 derived from Actinosynnema pretiosum ATCC31565. Their structures were elucidated on the basis of spectroscopic analyses and single-crystal X-ray diffractions. Compound 1 is the first example of 3-amino-6-hydroxy-2-piperidone-containing cyclic peptides, and 1 and 2 showed moderate cytotoxic activities against HeLa and PC3 cell lines.

     

<Emphasis Type="SmallCaps">l</Emphasis>-Methioninase Production by Filamentous Fungi: I-Screening and Optimization Under Submerged Conditions

Findings show 21 fungal isolates belonging to eight genera recovered from Egyptian soils that have the potential to attack l-methionine under submerged conditions. Aspergillus flavipes had the most methioninolytic activity, giving the highest yield of l-methioninase (10.78 U/mg protein), rate of methionine uptake (93.0%), and growth rate (5.0 g/l), followed by Scopulariopsis brevicaulis and A. carneus. The maximum l-methioninase productivity (11.60 U/mg protein) by A. flavipes was observed using l-methionine (0.8%) as an enzyme-inductive agent and glucose (1%) as a co-dissimilated carbon source. A significant reduction in l-methioninase biosynthesis by A. flavipes was detected using carbon-free medium, suggesting the lack of ability to use l-methionine as a carbon and nitrogen source. Potassium dihydrogen phosphate (0.25%), the best source of phosphorus, favors enzyme biosynthesis and enhances the level of methionine uptake by A. flavipes. The maximum l-methioninase productivity (12.58 U/mg protein) and substrate uptake (95.6%) were measured at an initial pH of 7.0.     

Biological acidification during grape must fermentation using mixed cultures of <Emphasis Type="Italic">Kluyveromyces thermotolerans</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Four mixed culture fermentations of grape must were carried out with Kluyveromyces thermotolerans strain TH941 and Saccharomyces cerevisiae strain SCM952. In the first culture, both yeasts were added together, whereas in the remaining three cultures S. cerevisiae was added 1, 2, and 3 days after the inoculation of K. thermotolerans. The growth and survival of the K. thermotolerans strain and the amount of the produced l-lactic acid depend on the time of inoculation of the S. cerevisiae strain and provided an effective acidification during alcoholic fermentation. The four cultures contained, respectively, at the end of fermentation 0.18, 1.80, 4.28, and 5.13 g l-lactic acid l⁻¹. The grape must with an initial pH of 3.50 was effectively acidified (70% increase in titratable acidity, 0.30 pH unit decrease) by the production of 5.13 g l-lactic acid l⁻¹.     

Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood

Trametes versicolor was shown to produce extracellular laccase during surface cultivation on glucose, wheat straw and beech wood. Growth on both wheat straw and beech wood led to an increase as high as 3.5-fold in extracellular laccase activity, in comparison with growth on glucose. The corresponding yields in fungal biomass reached only about 20% of the value obtained on glucose. Manganese peroxidase activity␣appeared during growth on wheat straw and beech wood. Mycelia grown on glucose, wheat straw and beech wood also showed intracellular laccase activities, monitored with 2,6-dimethoxyphenol, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 4-hydroxy-3,5-dimethoxybenzaldehyde azine (syringaldazine) and 3,4-dihydroxyphenylalanine (l-DOPA). Assaying intracellular laccase with 2,6-dimethoxyphenol, syringaldazine and l-DOPA showed the maximum oxidation rates to be at pH values different from those producing maximum oxidation rates with extracellular laccase. In each case most of the total laccase activity was recovered from the culture filtrates. Growth on wheat straw and beech wood led to increased values for both extra- and intracellular laccase activities, based on fungal dry weight, in comparison with growth on glucose. Received: 18 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996     

l-Phenylalanine dehydrogenase from Brevibacterium sp. for production of l-phenylalanine by reductive amination of phenylpyruvate

Summary For production of l-phenylalanine the reductive amination of phenylpyruvate, catalyzed by phenylalanine-dehydrogenase was examined. To reach high levels and a sufficient stability of the inducible intracellular enzyme, growth conditions of Brevibacterium sp. are optimized. For continuous production of l-phenylalanine in an enzyme membrane reactor, the kinetic parameters of the partially purified enzyme are determined.In continuous production a space time yield of 37.4 g l-Phe l^-1 d^-1 can be reached.By means of the measured kinetic parameters and simultaneous calculation of the mass balances of all reaction components the behaviour of the reactor can be simulated. For certain conditions the multi-enzyme-system shows multiple steady-states.Abbreviations l-phe l-phenylalanine - phepy phenylpyruvate - PEG polyethylenglycol - pheDH l-phenylalanine dehydrogenase     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.     

Inhibition of cellulose saccharification and glycolignin-attacking enzymes of five lignocellulose-degrading fungi by ferulic acid

At 5 g/l, ferulic acid, a plant cell-wall phenolic, severely repressed growth of the lignocellulose-degrading fungi Trichoderma harzianum, Chaetomium cellulolyticum, Phanaerochaete chrysosporium, Trametes versicolor and Pleurotus sajor-caju. At 0.5 g/l, howerver, it slightly stimulated growth of the latter two organisms. Two classes of extracellular enzymes involved in cellulose and glycolignin breakdown were assayed: cellulases; and phenol oxidases as laccases. All of the strains depolymerized cellulose but two (T. versicolor and P. sajor-caju) also secreted laccases. Laccase-secreting fungal species had normal levels of cellulose saccharification except in the presence of 5 g ferulic acid/l, whereas saccharification by the other strains was suppressed at all concentrations of the phenolic tested.     

Growth and viability of mycelial fragments of white-rot fungi on some hydrogels

The viability of mycelial fragments of Trametes versicolor and Irpex lacteus and their growth on selected hydrogels are described. The size of mycelial fragments of the fungi did not significantly influence their viability. Alginate hydrogel films supported fungal growth better than agarose, carrageenan, chitosan and gelatin films, and had the highest mechanical strength but were less hydrophilic than the other hydrogels. All commercial alginates that were tested supported aseptic growth of fungal fragments without prior sterilization of the hydrogel solution. The viability of mycelial fragments in the hydrogel solutions was higher for some commercial alginates than that in laboratory grade alginate. The mechanical strength and hydrophilicity of hydrogels from alginate type Sobalg FD 155 and Meer HV were comparable to that of laboratory grade alginate. Sterilization and pH of the alginate hydrogel did not significantly influence the growth of T. versicolor mycelial fragments but affected the growth of I. lacteus. Concentrations of alginate in the range of 1–2% in the hydrogel did not affect the growth of entrapped mycelial fragments of these fungi. Received 25 June 1997/ Accepted in revised form 07 March 1998     

Introduction of a stress-responsive gene, yggG, enhances the yield of l-phenylalanine with decreased acetic acid production in a recombinant Escherichia coli

A stress-responsive gene, yggG, was introduced into an l-phenylalanine producer, Escherichia coli AJ12741. In shake-flask culture, the yggG-containing recombinant strain (named AJ12741/pHYGG) produced 6.4 g l-phenylalanine l⁻¹ at the end of culture and its yield on glucose was 0.16 g l-phenylalanine g glucose⁻¹. These values are much higher than those of the original AJ12741 strain (3.7 g l-phenylalanine l⁻¹ and 0.09 g l-phenylalanine g glucose⁻¹, respectively). On the other hand, AJ12741/pHYGG strain produced only 4.5 g acetic acid l⁻¹ and its yield on glucose was about a half of that of the AJ12741 culture. Analysis of gene expression revealed that in late growth phase, the expression levels of genes involved in acetic acid production (pta, ackA, and poxB) were relatively low in AJ12741/pHYGG cells. In particular, the level of poxB expression in AJ12741/pHYGG strains was one-seventh of that of the original strain. These results suggest that the formation of a bottleneck for acetic acid production brings about a metabolic flow favorable to l-phenylalanine synthesis in the recombinant strain over-expressing the yggG gene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of five serine proteases with trypsin-, chymotrypsin- and elastase-like characteristics from the gut of the lugworm Arenicola marina (L.) (Polychaeta)

Summary Five proteases were isolated from the digestive fluid of the lugworm, Arenicola marina L. The enzymes (molecular weight 24.0–24.6 kDa) were classified as serine proteases. Three enzymes showed a cleavage specificity corresponding to mammalian trypsin (E.C. 3.4.21.4). One protease possessed a chymotrypsin-like cleavage pattern (E.C. 3.4.21.1), and the fifth preferred cleavage behind short-chain amino acids like an elastase (E.C. 3.4.21.36). Detailed investigations revealed differences in molecular characteristics and cleavage patterns compared to mammalian proteases, especially in the chymotrypsin- and the elastase-like enzymes.Abbreviations APNE N-acetyl-d/l-Phe -naphthyl ester - BANA N-benzoyl-d/l-Arg -naphthylamide - BAPNA N-benzoyl-d/l-Arg-4-nitroanilide - BIGGANA N-benzoyl-l-Ile-l-Glu-Gly-l-Arg-4-nitroanilide - BLPNA N-benzoyl-d/l-Lys-4-nitroanilide - BTEE N-benzoyl-l-Tyr ethyl ester - enzyme T1/T2/T3 trypsin-like enzyme - enzyme ChT chymotrypsin-like enzyme - enzyme E elastase-like enzyme - GPANA N-glutaryl-l-Phe-4-nitroanilide - MUF 4-methylumbelliferryl - MW molecular weight - PMSF phenylmethylsulphonyl fluoride - SAAPPNA N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-4-nitroanilide - SBTI soybean trypsin inhibitor - SPPNA N-succinyl-l-Phe-4-nitroanilide - TAME N-tosyl-l-Arg methyl ester - TFA trifluoracetic acid - TLCK N-tosyl-l-Lys chloromethyl ketone - TPCK N-tosyl-l-Phe chloromethyl ketone - TRIS tris(hydroxymethyl)aminomethane     

Supramolecular-mediated immobilization of l-phenylalanine dehydrogenase on cyclodextrin-coated Au electrodes for biosensor applications

A supramolecular approach was used for adsorbing a monolayer of adamantane-modified phenylalanine dehydrogenase on β-cyclodextrin-coated Au electrodes. The enzyme electrode (poised at +200 mV vs. Ag/AgCl) showed a linear amperometric response up to 3 mM l-phenylalanine (l-Phe) with a lower detection limit of 15 μM. The reversible nature of this immobilization approach was confirmed.     

The ability of white-rot fungi to degrade the endocrine-disrupting compound nonylphenol

Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l^–1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l^–1 and 99 mg l^–1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14°C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l^–1 and 79 mg l^–1 of nonylphenol, respectively. No pollutant removal was recorded at 4°C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg^–1 of nonylphenol. Only 1.3±0.1% of nonylphenol remained in the soil after 5 weeks of incubation.