Purification, molecular cloning, and antimicrobial activity of peptides from the skin secretion of the black-spotted frog, Rana nigromaculata

Antimicrobial peptides from a wide range of amphibian species, especially frogs of the genus Rana, have been characterised and are potential therapeutic agents. Here we describe the isolation, purification, and structural and biological characterisation of three novel antimicrobial peptides from the skin secretions of the black spotted frog, Rana nigromaculata, from Northeastern China. The peptides were identified as belonging to two known families: the temporin, which was first identified in R. nigromaculata from China, and the brevinin-2. Temporin-1RNa and temporin-1RNb both containing three positive charges and have a high potency against microorganisms (MIC: 3.13–8.3 μM against Gram-positive bacteria, 12.5–25.0 μM against Gram-negative bacteria, and 6.25–12.5 μM against Candida albicans) and a high haemolytic activity against human erythrocytes (HC₅₀: 100–150 μM). Brevinin-2RNa contains a single intra-disulphide bridge at the C-terminus that is active towards the tested Gram-positive bacteria but is not active against E. coli and P. aeruginosa. The cDNAs encoding three novel peptide precursors were also subsequently cloned from an R. nigromaculata skin cDNA library and sequenced. The precursors contain 58–72 amino acid residues, which include a conserved signal peptide, acidic propeptide, and the mature temporin-1RNa, temporin-1RNb and brevinin-2RNa. The CD spectra of temporin-1RNa and temporin-1RNb in water, 30 mM SDS and 50 % trifluoroethanol (TFE) indicated that both peptides adopted an aperiodic structure in water and an organised structure with an α-helical conformation in TFE and SDS solution. The conformational transition induced by TFE or SDS reflects the potential ability of temporin-1RNa and temporin-1RNb to interact with anionic membranes.     

Effects of acute and chronic hypoxia on the locomotion and enzyme of energy metabolism in Chinese shrimp Fenneropenaeus chinensis

ABSTRACT

To characterize the locomotor behaviors and their relation with physiological regulation in Chinese shrimp Fenneropenaeus chinensis, animals were held at approximately 6.0 (normoxia), 4.5, and 3.0 mg L^-1 dissolved oxygen (DO) for 1 day (acute) and 15 days (chronic), after which the swimming and tail-flipping abilities, and the activities of key enzymes involved in anaerobic and aerobic metabolism in hepatopancreas and pleopod and abdominal muscles were determined. Results showed that hepatopancreas was preferentially powered compared with pleopod and abdominal muscles during hypoxia. Physiological differences in muscles resulted in locomotion differences. F. chinensis presented reduced reliance on anaerobic glycolysis to conserve energy during chronic hypoxia at 3.0 mg L^-1 DO, but this physiological regulation reduce the survival of shrimp in the wild due to a reduction in tail-flipping. These findings suggested that when assessing the survival strategy of shrimp during hypoxia, both physiological regulation and behavioral changes should be considered.     

Molecular Cloning of Hemocyanin cDNA from Fenneropenaeus chinensis and Antimicrobial Analysis of Two C-terminal Fragments

Peptides derived from shrimp hemocyanin have antimicrobial properties. This is the first report of hemocyanin cDNA (FCHc) cloned from Fenneropenaeus chinensis and recombinant expression of two C-terminal fragments. Based on sequence analysis of Fenneropenaeus chinensis hemocyanin FCHc, we subcloned two FCHc fragments by designing special primers. Two antimicrobial peptides (AMPs) were derived from FCHc (FCHc-C1 and FCHc-C2). The recombinant sequence of FCHc-C1 consisted of 207 bp encoding 69 amino acids and the recombinant sequence of FCHc-C2 consisted of 120 bp encoding 40 amino acids. The results of Tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that recombinant FCHc-C1 and FCHc-C2 peptides (rFCHc-C1 and rFCHc-C2) were expressed successfully. An inhibition assay showed that FCHc-C1 and FCHc-C2 were anionic AMPs with antifungal and antibacterial activities.     

Catesbeianin-1, a novel antimicrobial peptide isolated from the skin of <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> (American bullfrog)

Objectives

To identify and characterize a novel antimicrobial peptide, catesbeianin-1.

Results

Catesbeianin-1 is 25 amino acids long and is α-helical, cationic and amphipathic. It had antimicrobial activity against Gram-positive and Gram-negative bacteria. It was resistant against trypsin and pepsin. Catesbeianin-1 exhibited moderate hemolytic activity (approx 8%) at 100 μg/ml, and its HC₅₀ (50% hemolytic concentration) was 300 μg/ml. Its cytotoxicity was approx 10–20% at 100 μg/ml, and its CC₅₀ (50% cytotoxic concentration) was >100 μg/ml. The LD₅₀ of catesbeianin-1 in mice was 80 mg/kg. At 3.1 µg/ml, catesbeianin-1 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus.

Conclusions

A new antimicrobial peptide from the skin of Lithobates catesbeianus (American bullfrog) may represent a template for the development of novel antimicrobial agents.

     

cDNA cloning, characterization and expression analysis of peroxiredoxin 5 gene in the ridgetail white prawn Exopalaemon carinicauda

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5′ untranslated region (UTR) of 14 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda.     

Chemistry and Antimicrobial Potential of the Buffalo Myeloid Cathelicidin,BuMAP-34

A putative cathelicidin antimicrobial peptide of 34 amino acid residues was deduced from buffalo myeloid gene sequences and named as Buffalo myeloid antimicrobial peptide-34 (BuMAP-34). Structure–function relationship of the custom synthesized peptide was evaluated in vitro. Highly cationic, amphipathic peptide showed a net charge of +6 and predicted hydrophobic ratio of 38 %. Phylogenetic analysis revealed an evolutionary relationship with Bovine myeloid antimicrobial peptide-34 (BMAP-34) of cattle, myeloid antimicrobial peptide-34 (MAP-34) of Goat and Sheep myeloid antimicrobial peptide-34 (SMAP-34). Peptide showed potent antimicrobial activity against a wide spectrum of microorganisms including Gram-negative and Gram-positive bacteria and fungi. Minimum inhibitory concentration (MIC) on various strains of bacteria, and fungus ranged from 1.1 to 1.5 µM except for P. multocida multocida (HS), which was >100 µM. Scanning electron microscopic (SEM) analysis of the peptide treated E. coli, S. aureus and C. albicans indicated cell lysis. Peptide also showed its ability to bind with anionic components of the cells which was confirmed by DNA binding assay. Haemolytic activity assay revealed absence of haemolysis in human RBCs at 12.5 µM and in sheep RBCs even at 100 µM concentration of the peptide. The present study suggests that the cathelicidin, BuMAP-34 has strong antimicrobial activity and could be developed as a promising broad spectrum antimicrobial agent.     

An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript> in the hepatopancreas of coastal lagoon wild and farmed shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

This study aimed to establish the combined effect of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p?<?0.05). However, AP activity from both wild and farmed shrimp was inhibited when incubated with AFB₁ and FB₁. The greatest inhibition occurred when AP was incubated with a mixture of AFB₁ and FB₁. The IC₅₀ for AFB₁ on AP activity of wild and farmed shrimp hepatopancreases was 0.790 and 0.398 μg/mL, respectively. The IC₅₀ of FB₁ was 0.87 μg/mL for wild shrimp and 0.69 μg/mL for farmed shrimp. These results suggest that, at the mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB₁?+?FB₁ suggesting a possible synergistic or potentiating inhibitory effect.     

Effects of Dietary <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> on Growth Performance,Digestive Enzymes and Gut Morphology of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

A 15-day feeding trial was conducted to investigate the effect of dietary Lactobacillus plantarum on growth performance, digestive enzyme activities and gut morphology of juvenile Pacific white shrimp, Litopenaeus vannamei (initial body weight = 7.96 ± 0.59 g). Four microbound diets were formulated to contain fermentation supernatant (FS), live bacteria (LB), dead bacteria (DB), and cell-free extract (CE) of L. plantarum. Results indicated that final weight was significantly higher in FS, DB, and CE group in comparison to the control group (P < 0.05). The maximum weight gain rate (WGR) and specific growth rate (SGR) of the CE diet group were significantly higher than that of other groups (P < 0.05). The FCR of CE diet group was lower than that of the control, LB, DB, and FS diets groups (P < 0.05). The highest digestive enzyme activities (amylase, lipase, and pepsin activity) in the hepatopancreas and gut of shrimp were observed in the CE diet group. Histological study revealed that dietary CE diet could significantly increase the enterocytes height of shrimp. The administration of cell-free extract of L. plantarum could effectively improve the growth performance of L. vannamei via the improvement of digestive enzyme activities and the enterocytes height of shrimp. The results of this study will be essential to promote application of probiotics in shrimp aquaculture.     

Antifungal activity of alkanols: inhibition of growth of spoilage yeasts

Primary aliphatic alkanols from C₆ to C₁₃ were tested for their antifungal activity against Saccharomyces cerevisiae using a broth dilution method. Undecanol (C₁₁) was found to be the most potent fungicide against this yeast with the minimum fungicidal concentration (MFC) of 25 μg/ml (0.14 mM), followed by decanol (C₁₀) with the minimum inhibitory concentration (MIC) of 50 μg/ml (0.31 mM). The time-kill curve study showed that undecanol was fungicidal against S. cerevisiae at any growth stages. This fungicidal activity was not influenced by pH values. Dodecanol (C₁₂) was the most effective fungistatic but did not show any fungicidal activity up to 1600 μg/mL. Fungistatic dodecanol quickly reduced cell viability, but the cell viability recovered shortly after and then finally became no longer different from the control indicating that the effect of dodecanol on S. cerevisiae was classified as a sublethal damage. However, fungistatic dodecanol combined with sublethal amount of anethole showed a fungicidal activity against this yeast. Anethole completely restricted the recovery of cell viability. Therefore expression of the synergistic effect was probably due to the blockade of the recovering process from dodecanol induced-stress. The alkanols tested inhibited glucose-induced acidification by inhibiting the plasma membrane H⁺-ATPase. Octanol (C₈) increased plasma membrane fluidity in the spheroplast cells of S. cerevisiae. The same series of aliphatic primary alkanols was also tested against a food spoilage fungus Zygosaccharomyces bailii and compared with their effects against S. cerevisiae. Decanol was found to be the most potent fungicide against Z. bailii with an MFC of 50 μg/ml (0.31 mM), whereas undecanol was found to be the most potent fungistatic with an MIC of 25 μg/ml (0.14 mM). The time-kill curve study showed that decanol was fungicidal against Z. bailii at any growth stage. This antifungal activity was slightly enhanced in combination with anethole. The primary antifungal action of medium-chain (C₉–C₁₂) alkanols comes from their ability as nonionic surfactants to disrupt the native membrane-associated function of the integral proteins. Hence, the antifungal activity of alkanols is mediated by biophysical process, and the maximum activity can be obtained when balance between hydrophilic and hydrophobic portions becomes the most appropriate.     

Antibacterial Activity of Long-Chain Primary Alcohols from <Emphasis Type="Italic">Solena amplexicaulis</Emphasis> Leaves

Extraction, thin layer chromatography and gas chromatography–mass spectrometry of Solena amplexicaulis (Lam.) Gandhi, commonly known as creeping cucumber, (Cucurbitaceae) leaves revealed 21 long-chain primary alcohols, and 100 g leaves indicated presence of 3651.59 ± 327.18 SE µg long-chain primary alcohols. 1-Heptadecanol and 1-triacontanol were the predominant and least abundant primary alcohols, representing for 780.44 ± 42.59 and 3.28 ± 0.55 SE μg, respectively. Antibacterial property of the complete synthetic blend (0.1%), comparable to long-chain alcohols as detected by GC-FID of 100 g S. amplexicaulis leaf extracts was evaluated on the pathogenic bacteria Salmonella gallinarum by agar well diffusion method, and exhibited 20.4, 26.7 and 38.2 mm zone of inhibition at 25, 50 and 100 μl doses, respectively. One hundred µl dose of 6 individual pure synthetic compounds, 1-tridecanol, 1-pentadecanol, 1-heptadecanol, 1-nonadecanol, 1-eicosanol and 1-tricosanol comparable to the amounts present in 0.1% solution of pure isolated alcohols from S. amplexicaulis leaves displayed 16.2, 17.7, 18.6, 22.8, 15.8 and 14.5 mm zone of inhibition against this bacterium, respectively. Hundred µl dose from a synthetic blend of above 6 compounds (comparable to the proportions as present in 0.1% solution of pure isolated alcohols from 100 g S. amplexicaulis leaves) exhibited 38.1 mm zone of inhibition against this bacterium. Furthermore, 100 μl dose from a mixture (1:1) comprising of chloramphenicol (1 µg/ml) and a synthetic blend of above 6 compounds displayed 38.8 mm inhibition zone against S. gallinarum, and hence, this combination might be used against this pathogenic bacteria.     

Potential cytotoxic and mutagenic effect of Pinus wallichiana,Daphne oleiodes and Bidens chinensis

The Pinus wallichiana, Daphne oleiodes and Bidens chinensis have a long history of being used traditionally for treatment of various types of disorders. Most of the uses have been without any scientific evidence and toxicological assessment. We evaluated the mutagenic and cytotoxic capabilities of various parts of P. wallichiana, D. oleoides and B. chinensis. Ames Salmonella mutagenicity assay determined the mutagenicity activity against TA 98 and TA 100 bacterial strains of Salmonella typhimurium without metabolic activator S9 system. The number of mutant colonies in negative control was considered as limit to determine the mutagenicity effects of every extract. Brine shrimps lethality bioassay was used to determine the cytotoxic capabilities of the selected plants. The P. wallichiana, D. oleiodes and B. chinensis did not showed any mutagenic activity both for frameshift mutation (TA98) and base-pair substitution (TA100) without S9 mixture. The crude methanolic extract of P. wallichiana stem showed moderate cytotoxicity (53.33%) at 1000 μg/ml with LD₅₀ value 599.634. The D. oleoides fruit showed a toxicity of 60% at 1000 μg/ml with LD₅₀ value 367.730. The B. chinensis (whole plant) showed lethality of 63.3% at 1000 μg/ml, with LD₅₀ 204.833. The absence of any mutagenic activity of crude extract of the tested plants in both bacteria strains, TA 98 and TA 100 without the S9 mix confirms the safety of these plants to the consumers.     

Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein from oriental river prawn,Macrobrachium nipponense

The lipopolysaccharide and β-1,3-glucan binding protein (LGBP), one of the pattern recognition proteins, plays an important role in the innate immune response of invertebrates. A 1,506 bp full-length cDNA of a LGBP gene was cloned and characterized from the oriental river prawn Macrobrachium nipponense (named as MnLGBP). Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,119 bp, encoding a protein of 372 amino acids including a 21-aa signal peptide. The calculated molecular mass of the mature protein (351 aa) was 39.9 kDa with an estimated pI of 4.63. The MnLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. Sequence comparison based on the deduced amino acid sequence of MnLGBP showed varied identity of 89, 76 and 74 % with those of Macrobrachium rosenbergii LGBP, Marsupenaeus japonicus β-1,3-glucan binding proteins, and Fenneropenaeus chinensis LGBP, respectively. Quantitative RT-PCR results showed that MnLGBP was expressed in nerve, intestine, muscle, gill, heart, haemocytes and at the highest level in hepatopancreas. After challenge with the pathogen, Aeromonas hydrophila and Vibrio parahaemolyticus, the expression of MnLGBP mRNA was significantly upregulated in the hepatopancreas compared to the control group. At the same time, the mRNA level of MnproPO increased dramatically at 48 h after injection of bacteria. These data should be helpful to better understand the function of MnLGBP in the prawn immune system.     

A smaller particle size improved the oral bioavailability of monkey head mushroom, Hericium erinaceum, powder resulting in enhancement of the immune response and disease resistance of white shrimp, Litopenaeus vannamei

The effects of different particle sizes (100–150, 74–100, and <74 μm) of powder of the dried and ground stipe from the monkey head mushroom, Hericium erinaceum, on the immune response and disease resistance of white shrimp, Litopenaeus vannamei, against the pathogen, Vibrio alginolyticus, were examined. Mushroom powder with a particle size of <74 μm had a significantly higher effect on the disease resistance of shrimp compared to particle sizes of >74 μm. Mortality of shrimp after being injected with V. alginolyticus was particle size-dependent, increasing from 66.7% ± 3.3%–93.3% ± 3.3% with diets containing stipe particle sizes of <74 and 100–150 μm, respectively. The mortality of shrimp fed the diet containing <74-μm stipe powder for 28 days was significant lower than that of shrimp fed with the control diet and the diet containing 74–100-μm stipe powder after being challenged by V. alginolyticus. The optimal concentration of the <74-μm mushroom powder for enhancing the immune response and disease resistance of shrimp was 0.2 μg (g shrimp)^?1 day^?1. No significant change in the total hemocyte count, differential hemocyte count, glutathione reductase, or phagocytic activity was found in shrimp fed the control diet and mushroom powder-containing diet at a level of up to 0.2 μg (g shrimp)^?1 day^?1. Shrimp fed 0.2 μg (g shrimp)^?1 day^?1 of a mushroom-containing diet had a significantly higher disease resistance to V. alginolyticus via an increase in phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and glutathione peroxidase activity. Therefore, a diet containing the stipe powder of monkey head mushroom with a particle size <74 μm at a level of 0.2 μg (g shrimp)^?1 day^?1 was found to enhance the immunity and disease resistance of shrimp.     

Contact and fumigant activities of Citrullus colocynthis formulations against Callosobruchus chinensis (Coleoptera: Bruchidae)

The efficacy of two different formulations of Citrullus colocynthis extracts (emulsifiable and powder) were tested as contact and fumigant toxicants against cowpea weevil adults, Callosobruchus chinensis. The emulsifiable concentrate showed repellent activity against the adults. No eggs were laid by females in the choice test at 1.0% concentration compared with 150.0 eggs in control. Using the same concentration, the females deposited 1.4 eggs in the non-choice test compared with 78.2 eggs in control. The concentration of 2.5 μl/38.5 ml air of citrullus emulsifiable caused 100% mortality to adults during one day in the fumigation test. Also, the vapor of citrullus emulsifiable was highly effective against eggs of cowpea weevil, where, at 2.5 μl/38.5 ml air, no eggs hatched. Both formulations affect the different biological aspects of cowpea weevil, however, citrullus emulsifiable concentrate was more potent than the citrullus powder.     

Antifouling chromanols isolated from brown alga Sargassum horneri

Biofouling in aquatic environments have a wide range of detrimental effects on man-made structures and cause economic loss. Current antifouling compounds including Diuron, dichlorofluanid, and Irgarol are toxic and can accumulate in marine environments. Thus, effective and environmentally friendly antifoulants are needed. Six structurally similar compounds were isolated from the brown alga, Sargassum horneri, based on bioactivity-guided isolation by reversed-phased liquid flash chromatography and high-performance liquid chromatography. Six chemical constituents possessing antifouling activities were identified as chromanols consisting of polyprenyl chain by nuclear magnetic resonance and mass spectroscopy. Antifouling activities of these six compounds were determined against representative fouling organisms including a hard fouling organism the mussel Mytilus edulis, a soft fouling macroalga Ulva pertusa, the biofouling diatom Navicula annexa, and the biofouling bacteria Pseudomonas aeruginosa KNP-3 and Alteromonas sp. KNS-8. The compounds could inhibit larvae settlement of mussel M. edulis with an EC₅₀ of 0.11–3.34 μg mL^?1, spore settlement of U. pertusa zoospores (EC₅₀ of 0.01–0.43 μg mL^?1), and the diatom N. annexa (EC₅₀ of 0.008–0.19 μg mL^?1). The two biofouling bacteria were sensitive to the tested compounds (minimum inhibitory concentration of 1.68–36.8 and 1.02–30.4 μg mL^?1, respectively). From toxicity tests on juvenile Sebastes schlegelii fish, brine shrimp Artemia salina, and microalga Tetraselmis suecica, S3 had the lowest LC₅₀ values of 60.2, 108, and 6.7 μg mL^?1 and exhibited no observed effect concentration at 24.5, 41.6, and 3.1 μg mL^?1 for these three tested marine organisms, respectively.     

Dodecyl sorbitan ethers as antimicrobials against Gram-positive bacteria

A range of amphiphilic sorbitan ethers has been synthesized in two steps from sorbitan following an acetalization/hydrogenolysis sequence. These sorbitan ethers and the acetal intermediates have been evaluated as antimicrobials against Gram-negative and Gram-positive bacteria. No antimicrobial activity was observed for Gram-negative bacteria. However, the compounds bearing a linear dodecyl chain exhibit antimicrobial activity (MIC as low as 8 μg/mL) against Gram-positive bacteria such as Listeria monocytogenes, Enterococcus faecalis and Staphylococcus aureus. Encouraged by these preliminary results, dodecyl sorbitan was tested against a range of resistant strains and was found to be active against vancomycin-, methicillin- and daptomycin-resistant strains (MIC = 32–64 μg/mL).     

Environmentally Safe Production of 7-ACA by Recombinant Acremonium chrysogenum

7-Amino cephalosporanic acid (7-ACA), which is currently obtained by chemical deacylation from cephalosporin C (CPC), is a major intermediate for industrial production of β-lactam antibiotics. 7-ACA can also be produced from CPC by enzymatic route including two-step and one-step procedures. In our research, an ecs gene coding for CPC acylase was synthesized and cloned into pET-28a(+) to construct an E. coli expression plasmid pYG232. E. coli BL21(DE3) bearing pYG232 was induced by IPTG and successfully expressed the recombinant ECS (88.9 kDa). Under the optimal conditions: 0.5 mg/ml purified ECS protein, 5 mg/ml CPC, 100 mM Tris–Cl (pH 9.6), supplement with 7 mM Zn²⁺, slightly shaking for 6 h at 25°C, the transformation productivity was 54.4%. Then, ecs was cloned downstream of an A. chrysogenum endogenous promotor, PpcbC, to construct pYG233 for expression in A. chrysogenum. pYG233 was introduced into a CPC high-producer via integrative transformation of protoplasts. Two independent bleomycin-resistant transformants were investigated by PCR, Southern blotting, quantitative RT–PCR, western blotting, and fermentation. Although these two transformants both have one copy of integrated ecs, they showed different expression level of ECS protein and 7-ACA production. When concentration of CaCO₃ was reduced to 50 mM, ZnSO₄ was increased to 7 mM, CuSO₄ was eliminated from the fermentation media, and the pH was adjusted to 8.0 at day 4 during fermentation, 7-ACA production of one of the transformants could reach 1701 μg/ml, indicated that more than 30% of CPC produced by this high-producer have been transformed into 7-ACA directly in vivo. This is the highest 7-ACA production by direct fermentation ever reported.