An improved procedure for root surface disinfection suitable for observations of proliferation of intraradical hyphae of arbuscular mycorrhizal fungusGlomus fistulosum

A disinfection procedure for mycorrhizal root segment surface was improved using neomycin and polymyxin B besides sodium hypochlorite, streptomycin and penicillin G, to obtain clean material for observation of proliferation of hyphae of arbuscular mycorrhizal (AM) fungi. The procedure of surface disinfection is more efficient, in terms of incidence of contamination, compared to the original procedure involving only sodium hypochlorite, streptomycin and penicillin G. A further decrease of visually detectable contamination can be achieved using bacteriostatic concentrations of rolitetracycline which cause a decrease of hyphal growth of the AM fungusGlomus fistulosum. The technique was used in experiment studying the uptake ofU-¹⁴C-glucose by proliferating hyphae. An active uptake of glucose occurred even in the presence of all four antibiotics at concentrations of 500 mg/L.     

Magnesium ions alleviate the negative effect of manganese on Glomus claroideum BEG23

The phytotoxicity of excessive levels of manganese (Mn), an essential micronutrient, can be alleviated significantly by a high supply of magnesium (Mg) ions to plants. A similar interaction of these two elements in the development of arbuscular mycorrhizal (AM) fungi has been verified in two experimental systems. In in vitro experiments, an outgrowth of hyphae from excised, surface-disinfected root segments colonised with Glomus claroideum BEG23 was measured after 5 days incubation in liquid media. When only Mn ions were present in the media at higher concentrations (>0.05 mM), the growth of hyphae from root segments was reduced significantly. Addition of magnesium sulphate to the incubation solution reduced the inhibitory effects of Mn on hyphal growth. Alleviation of Mn toxicity by Mg ions observed in in vitro experiments was verified also for the symbiotic association between G. claroideum and maize as a host plant in a hydroponics sand culture experiment.     

The response of Glomus fistulosum–maize mycorrhiza to treatments with culture fractions from Pseudomonas putida

 This study examined which culture fraction of the plant-growth-promoting bacterium Pseudomonas putida (Trevisan) Migula has an effect on growth and mycorrhiza formation of maize (Zea mays L.). Shoot dry weight and total leaf area of plants did not increase after inoculation with Glomus fistulosum but were significantly higher than the controls when the plants were dualinoculated with G. fistulosum and living cells of P. putida. Mycorrhizal infection of the roots was significantly higher when plants were inoculated with G. fistulosum together with living cells of P. putida or with G. fistulosum and dialysed cell extracts of P. putida than with G. fistulosum alone. Development of arbuscular mycorrhizal (AM) extraradical hyphae and the proportion of extraradical hyphae showing NADH diaphorase activity were significantly enhanced by inoculation of plants with living cells of P. putida or dialysed cell extracts of P. putida. No stimulation of extraradical hyphae proliferation from in vitro incubated mycorrhizal root segments was observed after application of culture fractions of P. putida. However, the percentage contamination of the root segments by extraneous filamentous fungi significantly decreased in the presence of livingcells of P. putida. Accepted: 12 January 1996     

Effect of forest fire on number, viability and post-fire re-establishment of arbuscular mycorrhizae

 Forest fire can affect arbuscular mycorrhizal (AM) fungi by changing the soil conditions and by directly altering AM proliferation. We studied the effects of a severe forest fire at Margalla Hills near Islamabad on the number and viability of AM fungal propagules in the burnt soil and their role in the re-establishment of post-fire infection in colonized plants. Compared with a nearby control area, the burnt site had a similar number of total spores but a lower number of viable AM fungal propagules. The roots of the two most frequent species at the burnt site, Dodonaea viscosa and Aristida adscensionis, showed a gradual increase in percentage root length colonized by AM fungi in general and hyphal infection in particular. Our results indicate resumption of mycorrhizal activity following the fire, probably from AM hyphae in the roots of these dominant shrubs. Accepted: 18 July 1997     

Diversity of arbuscular mycorrhizal fungi colonising roots of the grass species<Emphasis Type="Italic"> Agrostis capillaris</Emphasis> and<Emphasis Type="Italic"> Lolium perenne</Emphasis> in a field experiment

Analysis of arbuscular mycorrhizal (AM) fungal diversity through morphological characters of spores and intraradicular hyphae has suggested previously that preferential associations occur between plants and AM fungi. A field experiment was established to investigate whether AM fungal diversity is affected by different host plants in upland grasslands. Indigenous vegetation from plots in an unimproved pasture was replaced with monocultures of either Agrostis capillaris or Lolium perenne. Modification of the diversity of AM fungi in these plots was evaluated by analysis of partial sequences in the large subunit (LSU) ribosomal RNA (rDNA) genes. General primers for AM fungi were designed for the PCR amplification of partial sequences using DNA extracted from root tissues of A. capillaris and L. perenne. PCR products were used to construct LSU rDNA libraries. Sequencing of randomly selected clones indicated that plant roots were colonised by AM fungi belonging to the genera Glomus, Acaulospora and Scutellospora. There was a difference in the diversity of AM fungi colonising roots of A. capillaris and L. perenne that was confirmed by PCR using primers specific for each sequence group. These molecular data suggest the existence of a selection pressure of plants on AM fungal communities.     

Alleviation of Cadmium Toxicity to <i>Medicago Truncatula</i> by AMF Involves the Changes of Cd Speciation in Rhizosphere Soil and Subcellular Distribution

Arbuscular mycorrhizal fungi (AMF) can improve plant tolerance to several abiotic stresses, including heavy metals, drought or salinity exposure. However, the role of AMF in alleviation of soil cadmium (Cd)-induced toxicity to plants is still largely unknown. In this study, Cd speciation in soil and subcellular distribution of Cd were used to characterize the roles of application AM fungi in the alleviation of Cd toxicity in alfalfa plants. Our results showed that the addition of Glomus mosseae in Cd contaminated soil (10 mg/Kg) significantly increased soil pH, cation exchange capacity (CEC) and organic matter in rhizosphere soil with Medicago truncatula L., and then account for significantly decreased contents of exchangeable and carbonate-bounded Cd speciation in rhizosphere soil, indicating alleviation of plant toxicity by reduction of bioavailable fractions of Cd. Although there is no significant difference found in Cd accumulation by roots and shoots respectively between Cd and AM-Cd treatments, more portion of Cd was recorded compartmentalization in cell wall fraction of both root and shoot in treatment of Cd with AM application, indicating alleviation of Cd toxicity to plant cell. Herein, application of AM fungi in Cd treatments performed to inhibit the appearance of Cd toxicity symptoms, including the improvement of leaf electrolyte leakage, root elongation, seedling growth and biomass. This information provides a clearer understanding of detoxification strategy of AM fungi on Cd behavior with development and stabilization of soil structure and subcellular distribution of plant.     

Colonization potential of in vitro-produced arbuscular mycorrhizal fungus spores compared with a root-segment inoculum from open pot culture

 A reliable inoculum, free from other microorganisms, to produce arbuscular mycorhizal (AM) plants is of the greatest importance when studying the interaction between AM plants and soil microorganisms. We investigated the colonization of leeks from monoxenic in vitro-produced Glomus intraradices spores. The isolated spores were produced using a two-compartment in vitro growth system previously described. A spore suspension was used as inoculum and was compared to the inoculum potential of endomycorrhizal root segments of pot-grown leek (Allium porrum L.) plants. The leeks were grown in a controlled environment and two types of sterilized growth media were tested: calcined montmorillonite clay and a soil mix. Root colonization progressed faster in the soil mix than in the clay. However, in this medium, after an initial delay, root colonization from in vitro-produced spores was essentially the same as that observed with the root-segment inoculum, reaching 44% and 58% respectively, after 16 weeks. Leek roots colonized by the monoxenically-produced spores harbored only the studied AMF fungi while the roots colonized from the root segments were substantially contaminated by other fungi. Accepted: 25 December 1998     

Terpenes from the Red Alga <Emphasis Type="Italic">Sphaerococcus coronopifolius</Emphasis> Inhibit the Settlement of Barnacles

In this study, we screened eight terpenes isolated from the organic extract of Sphaerococcus coronopifolius for their antifouling activity in order to find possible new sources of non-toxic or less toxic bioactive antifoulants. The anti-settlement activity (EC₅₀) and the degree of toxicity (LC₅₀) of S. coronopifolius metabolites was evaluated using larvae of the cirriped crustacean Amphibalanus (Balanus) amphitrite (cyprids and nauplii) as model organism. For five of eight tested metabolites EC₅₀ was lower than 5 mg/L. The most promising results were observed for bromosphaerol (3), which expressed an EC₅₀ value of 0.23 mg/L, in combination with low toxicity levels (LC₅₀ > 100 mg/L). The therapeutic ratio—an index used to estimate whether settlement inhibition is due to toxicity or other mechanisms—is also calculated and discussed.     

Effects of arbuscular mycorrhiza and phosphorus application on arsenic toxicity in sunflower (Helianthus annuus L.) and on the transformation of arsenic in the rhizosphere

Effects of arbuscular mycorrhiza (AM) and phosphorus (P) application on arsenic (As) toxicity were studied in a rhizobox system with As-contaminated soil collected from Shimane Prefecture, Japan. The treatments consisted of a combination of two levels of AM (Glomus aggregatum) inoculation (−AM and +AM) and two levels of P application (−P and +P at 30 mg P kg⁻¹). Sunflower (Helianthus annuus L.) seedlings were cultured in rhizoboxes for 6 weeks. Rates of root AM infection in +AM treatments were about 40% regardless of P application. AM inoculation as well as P application reduced As toxicity symptoms, most clearly so in the +AM−P treatment. Plant growth was highest in the +AM + P treatment. Shoot As concentrations were slightly reduced by AM inoculation but enhanced by P application. Shoot P concentration in the +AM−P treatment was similar to that of +P treatments and was higher than in −AM−P. Analyses of rhizosphere soils at the end of the cultivation period indicated that P application increased water-soluble As (WS−As) in all compartments while AM inoculation increased WS−As in the central compartment only. Both the WS−arsenite [WS−As^(III)] and the dominant form, arsenate [WS−As^(V)], showed gradients toward the root surface. Dimethylarsine (DMAA) was detected in the +AM treatments only. To our knowledge, this is the first report of the occurrence of DMAA in the mycorrhizosphere. AM inoculation increased WS−P similarly as +P treatments did and promoted acid phosphatase activity in the soil. In conclusion, AM inoculation alleviated the effects of As toxicity by improving P nutrition without increasing As concentrations in the shoots. Moreover, AM appeared to be involved in the transformation of soil inorganic As into less toxic organic forms.     

Antimicrobial activity of the extracts from fruits of <Emphasis Type="Italic">Rumex</Emphasis> L. species

This study was designed primarily to investigate the antibacterial and antifungal activity of the extracts from fruits of six Rumex L. species: R. acetosa L., R. acetosella L., R. confertus Willd., R. crispus L., R. hydrolapathum Huds. and R. obtusifolius L. The 7 Grampositive and 7 Gram-negative bacteria strains and 5 fungal ones were tested by agar and broth dilution method. Determination of minimal inhibitory concentration (MIC) revealed that the extracts from R. confertus, R. crispus, R. hydrolapathum and R. obtusifolius exerted differential inhibitory effect on the growth of Gram-positive bacteria — staphylococci (MIC=62.5–125 μg/mL) and Gramnegative bacteria — Escherichia coli ATCC 3521, Proteus mirabilis, Pseudomonas aeruginosa (MIC=125→500 μg/mL); MIC values determined by agar dilution method were somewhat higher. The same extracts inhibited also the growth of fungi — Candida spp. or Trichophyton mentagrophytes ATCC 9533 (MIC=250–500 μg/mL), as found by agar dilution method. The total content of polyphenols (11.66–78.36 mg/g), anthracene derivatives (0.26–12.93 mg/g) and tannins (4.00–11.16%) was also determined.     

Application of in vitro methods to study carbon uptake and transport by AM fungi

Just as multi-compartmented root chambers have advantages over standard plastic pots for the study of nutrient uptake by arbuscular mycorrhizal [AM] fungi in soil, so the split-plate in vitro system has advantages over the standard dual culture system for the study of the physiology of AM fungi. We used the split-plate culture system of Ri T-DNA transformed Daucus carota L. roots and Glomus intraradices Schenck & Smith, in which only the fungus has access to the distal compartment, to study the ability of germ tubes and extraradical and intraradical hyphae to take up ¹³C-labeled substrates. Labeled substrates were added to one side of the plate divider and plates were incubated for 8 weeks while the fungus proliferated on the side from which the root was excluded. Tissues then were recovered from the plate and examined via NMR spectroscopy. Results showed that the morphological phases of the fungus differed in their ability to take up these substrates, most notably that intraradical hyphae take up hexose while extraradical hyphae cannot. In addition, NMR studies indicated that intraradical hyphae actively synthesized lipids while extraradical hyphae did not. These data show that eventual axenic culture of AM fungi is more than a matter of finding the proper substrate for growth. Genetic regulation must be overcome to make extraradical hyphae behave like intraradical hyphae in terms of C uptake and metabolism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Beyond the rhizosphere: growth and function of arbuscular mycorrhizal external hyphae in sands of varying pore sizes

Research on nutrient acquisition by symbiotic arbuscular mycorrhizal (AM) fungi has mainly focused on the root–fungus interface and less attention has been given to the growth and functioning of external hyphae in the bulk soil. The growth and function of external hyphae may be affected by unfavourable soil environments, such as compacted soils in which pores may be narrow. The effects of pore size on the growth of two AM fungi (Glomus intraradices and G. mosseae) and their ability to transport ³³P from the bulk soil to the host were investigated. Trifolium subterraneum L. plants were grown individually in `single arm cross-pots' with and without AM fungi. The side arm was separated from the main compartment by nylon mesh to prevent root penetration. It contained three zones: 5 mm of soil:sand mix (HC1); 25 mm of media treatment (HC2); and 20 mm of ³³P-labelled soil (HC3). There were four media treatments; soil and three types of quartz sand with most common continuous pore diameters of 100, 38 and 26 m. AM plants had similar growth and total P uptake in all treatments. However, plants grown with G. intraradices contained almost three times more ³³P than those grown with G. mosseae, indicating G. intraradices obtained a greater proportion of P at a distance from the host roots. Differences in P acquisition were not correlated with production of external hyphae in the four media zones and changes in sand pore size did not affect the ability of the fungi studied to acquire P at a distance from the host roots. Production of external hyphae in HC2 was influenced by fungal species and media treatment. Both fungi produced maximum amounts of external hyphae in the soil medium. Sand pore size affected growth of G. intraradices (but not G. mosseae) and hyphal diameter distributions of both fungi. The results suggest that not only are G. mosseae and G. intraradices functionally complementary in terms of spatial phosphorus acquisition, they are also capable of altering their morphology in response to the soil environment.     

Influence of arbuscular mycorrhizal fungi on soil structure and aggregate stability of a vertisol

The influence of arbuscular mycorrhizal (AM) fungi on aggregate stability of a semi-arid Indian vertisol was studied in a pot experiment in which Sorghum bicolor (L.) was grown as test plant for 10 weeks. Pasteurized soil inoculated with AM fungi was studied with pasteurized and unpasteurized soils as references. A part of the soil in each pot was placed in nylon mesh bags to separate effects of roots and hyphae. The sorghum plants were planted outside the mesh bags which permitted AM hyphae to enter while excluding roots. Aggregate stability of the soil was determined by wet-sieving and turbidimetric measurements. Development of the AM fungi was quantified as colonized root length and external hyphal length. Soil exposed to growth of roots and hyphae (outside mesh bags) showed aggregates with larger geometric mean diameter (GMD) in pasteurized soil inoculated with AM fungi than in pasteurized uninoculated soil. There was no significant difference in GMD of the inoculated, pasteurized soil and the unpasteurized soil. No significant effects of inoculation or plant growth were found in pasteurized soil exposed to hyphal growth only (inside the mesh bags). However, the unpasteurized soil had significantly higher GMD than the pasteurized soil, irrespective of plants and inoculum. Turbidimetric measurements of soil exposed to roots and hyphae (outside mesh bags) showed the highest aggregate stability for the inoculated pasteurized soil. These results demonstrate that AM fungi contribute to the stabilization of soil aggregates in a vertisol, and that the effect is significant after only one growing season. The effect was associated with both AM hyphae and the stimulation of root growth by AM fungi. The contribution from plant roots and AM hyphae to aggregate stability of different size fractions is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

AM真菌对煤矿沉陷区不同复合基质上 紫花苜蓿生长和生理特性的影响

该研究采用盆栽试验法,以紫花苜蓿(Medicago sativa)为材料,将煤矸石(CG)、粉煤灰(FA)和沙土(SS)按不同质量配比设置T1(CG∶FA∶SS=75%∶25%∶0%)、T2(CG∶FA∶SS=60%∶25%∶15%)、T3(CG∶FA∶SS=45%∶25%∶30%)、T4(CG∶FA∶SS=30%∶25%∶45%)四组混合基质以模拟,并以T5(CG∶FA∶SS=0%∶0%∶100%)为对照,AM真菌选用摩西斗管囊霉(F.m)和幼套近明球囊霉(C.e),通过单接菌和混合接菌,探索其对紫花苜蓿根系的侵染率、幼苗生长、抗氧化物酶活性和渗透调节物质变化的影响。结果表明:(1)在五组基质上,接种AM真菌显著提高了紫花苜蓿根系的菌根侵染率和菌根依赖性,且基质T4接种F.m+C.e[F.m∶C.e=1∶1(W/W)]的值最大(64.31%和86.24%)。(2)接种AM真菌不同程度提高了紫花苜蓿株高、基径、叶面积和生物量,且混合接菌的效果优于单接菌。(3)基质中填加过量煤矿废弃物抑制了植株根系的生长,接种AM真菌后显著提高了紫花苜蓿总根长、根表面积、根体积,降低了根平均直径。(4)不同接菌处理的紫花苜蓿叶片POD、SOD、CAT活性以及可溶性糖、可溶性蛋白含量总体表现为F.m+C.eC.eF.mnon-AMF,且接种F.m+C.e的增长幅度最大。研究表明煤矿废弃物复合逆境抑制紫花苜蓿的生长,接种AM真菌显著提高了幼苗生长、抗氧化物酶活性和渗透调节物质,提高了植物抗逆性能,且以基质T4接种F.m+C.e的效果最佳。     

Influences of Arbuscular Mycorrhizal Fungus Glomus mosseae on Growth and Nutrition of Lentil Irrigated with Arsenic Contaminated Water

Arsenic (As) contamination of irrigation water represents a major constraint to Bangladesh agriculture. While arbuscular mycorrhizal (AM) fungi have their most significant effect on P uptake, they have also been shown to alleviate metal toxicity to the host plant. This study examined the effects of As and inoculation with an AM fungus, Glomus mosseae, on lentil (Lens culinaris L. cv. Titore). Plants were grown with and without AM inoculum for 9 weeks in a sand and terra green mixture 50:50 v/v and watered with five levels of As (0, 1, 2, 5, 10 mg As L⁻¹ arsenate). Inoculum of Rhizobium leguminosarum b.v. Viceae strain 3841 was applied to all plants. Plants were fed with modified Hoagland solution (1/10 N of a full-strength solution and without P). Plant height, leaf number, pod number, plant biomass and shoot and root P concentration/offtake increased significantly due to mycorrhizal infection. Plant height, leaf/ pod number, plant biomass, root length, shoot P concentration/offtake, root P offtake and mycorrhizal infection decreased significantly with increasing As concentration. However, mycorrhizal inoculation reduced As concentration in roots and shoots. This study shows that growing lentil with compatible AM inoculum can minimise As toxicity and increase growth and P uptake.     

Modulation of biocidal activity of <Emphasis Type="Italic">Calothrix</Emphasis> sp. and <Emphasis Type="Italic">Anabaena</Emphasis> sp. by environmental factors

An investigation was undertaken to evaluate a set of cyanobacterial strains in terms of production of biocidal compounds exhibiting allelochemical and fungicidal properties. Two cyanobacterial strains — Anabaena sp. and Calothrix sp. were selected for further investigation, on the basis of their larger inhibition zones on the lawn of Synechocystis and Synechococcus sp. and two phytopathogenic fungi — Rhizoctonia bataticola and Pythium debaryanum. The diameter of the inhibition zone was largest when extracellular filtrates of the two cultures incubated at high light intensity (90–100 μmol photons m⁻² s⁻¹) and temperature (40 ± 2 °C) or grown in medium containing two-folds higher P (1.4 mg/L, as compared to 0.7 mg/L in BG 11 medium) were taken. A pH of 8 was the most optimal for both strains, in terms of growth and biocidal activity. Partial purification of ethyl acetate extract using TLC, followed by GLC revealed a single peak. This study highlights the importance of environmental factors in aggravating or reducing the toxic effects of these harmful cyanobacteria and their potential as a biocontrol agent.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of Persian poppy (<Emphasis Type="Italic">Papaver bracteatum</Emphasis>)

Persian poppy (Papaver bracteatum Lindl.) is an important commercial source of medicinal opiates and related compounds. In this research, calli were induced from seeds, roots, cotyledons and hypocotyls of P. bracteatum at a high efficiency. The optimized callus induction media consisted of the Murashige and Skoog (MS) basic media supplemented with 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin and 15 mg/L ascorbic acid. The concentrations of 2,4-D and ascorbic acid were found critical to callus induction and proliferation. Subsequent subcultures resulted in excellent callus proliferation. Ascorbic acid at concentration 15 mg/L increased the callus proliferation significantly. Maximum callus growth was achieved when the explants were incubated at 25°C. MS salts at full strength were found inhibitory for callus induction, while ľ MS salts were found to favor callus induction. Shoot regeneration of calli in vitro was achieved on ľ MS medium containing 0.5 mg/L benzylamine purine and 1.0 mg/L naphthalene acetic acid. Analysis of alkaloid extracts from Persian poppy tissues by high-performance liquid chromatography showed that thebaine accumulated in the tissues of plants. The thebaine alkaloid profile of the Persian poppy is a well-defined model to evaluate the potential for metabolic engineering of thebaine production in P. bracteatum.     

Influence of two legume species on hyphal production and activity of two arbuscular mycorrhizal fungi

 Two arbuscular mycorrhizal (AM) fungi (Glomus mosseae and G. intraradices) were compared for abundance of intraradical and soil-borne hyphae in association with Astragalus sinicum, a small-seeded, and Glycine max, a large-seeded legume. A. sinicum was more responsive than G. max to mycorrhizal formation, especially at early growth stages. Biomass allocation was greater in roots than shoots for mycorrhizal A. sinicum, while the opposite was true for G. max. Hyphal development in root and soil compartments was estimated by trypan blue staining and after staining for succinate dehydrogenase (SDH) or alkaline phosphatase (ALP) activity. Total fungal abundance increased steadily in roots and soil with time to a maximum 8 weeks after planting. SDH- and ALP-active AM hyphae increased in roots during plant growth but decreased in soil at later harvests. Mycorrhizal root mass in A. sinicum and G. max increased about 14-fold and 2.5-fold, respectively, but total length of soil hyphae produced per plant differed little, so that the pattern of AM soil to root abundance of the two fungi varied considerably with the host plant. Accepted: 23 July 1997     

Size-related dietary changes observed in young-of-the-year pumpkinseed (<Emphasis Type="Italic">Lepomis gibbosus</Emphasis>): stomach contents and fatty acid analyses

Changes in dietary composition and its effects on the fatty acid (FA) composition of young-of-the-year (YOY) pumpkinseed (Lepomis gibbosus) from an artificial reservoir (Mirgenbach—northeastern France) were related to body size as shown by stomach content and FA analyses. Comparisons were made between three size classes of fishes: 25–35 mm total length (TL), 35–45 mm TL and 45–55 mm TL. Diets of the youngest L. gibbosus (TL ≤ 35 mm) consisted mainly of zooplanktonic microcrustaceans and Chironomidae. Ontogenetic development influenced the FA composition of L. gibbosus. Older YOY L. gibbosus showed an increase in proportions of monounsaturated FA proportions and a decrease in polyunsaturated FA and mainly essential FA (particularly docosahexaenoic acid). The low ω3/ω6 ratio and low PUFA content (mainly on DHA) suggested that L. gibbosus would not transfer the benefits of consuming ω3 PUFA up the food web.     

High-level production of a kringle domain variant by high-cell-density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human kringle domains (KDs) are ubiquitously expressed binding modulators that fold into seven flexible loops and it has been previously demonstrated that KDs can be engineered toward target-specific binding proteins as a non-antibody protein scaffold. Here, we report a method for efficient expression of a KD derivative (KD548)—a promising anti-cancer agent—by high-cell-density culture of Escherichia coli at a preparative scale production. The correct folding of KD548 requires three disulfide bonds. Nevertheless, cytoplasmic expression of KD548 in E. coli led to good yields of highly soluble proteins with high activity. For efficient expression, four sets of expression systems consisting of different promoters (lac or T7) and fusion tags (His or FLAG) were examined. Of these, the expression system using a combination of the T7 promoter with the FLAG tag resulted in the highest production in shake flask cultivation as well as in high-cell-density cultivation performed in a 6.6-L jar bioreactor. When protein expression was induced at high-cell density (optical density [OD] = 100) and when complex feeding solutions were supplemented, cell density (maximum OD = 184) and production yield (∼5.4 g/L) were significantly enhanced to values that were much higher than those found previously with Pichia cultivation (<8 mg/L).