Transformation of Penicillium chrysogenum with selection for increased resistance to benomyl

Incubation of protoplasts of Penicillium chrysogenum with the plasmid pBT-3 permitted selection of transformants on the basis of increased resistance to the anti-fungal agent benomyl. Transformants were obtained at a frequency of 1-2 per microgram of DNA. Southern analysis revealed that transformation had occurred by integration of vector sequences into the host genome.     

Transformation of Penicillium expansum with a heterologous gene which confers resistance to benomyl

Transformation of Penicillium expansum with the pBT6 vector yielded 8 to 34 transformants per g of DNA. Hybridization analyses revealed that homologous recombination occurred in most of the transformants. Twenty-one out of 25 transformants analysed showed hybridization patterns which were indistinguishable from that of the wild type.     

Transformation of the entomopathogenic fungus, Metarhizium flavoviride strain CG423 to benomyl resistance

Metarhizium flavoviride strain CG423 is being developed as a mycoinsecticide against grasshoppers. This strain has been transformed to resistance to the fungicide benomyl by a polyethylene glycol (PEG)-mediated procedure using a mutant tubulin gene from Neurospora crassa . Transformation frequencies of up to 84 transformants per microgram of transforming DNA were achieved. Benomyl-resistant transformants were obtained that could tolerate greater than 30 μg ml⁻¹ benomyl. Southern blot analysis of genomic DNA reveals that the mechanism of genetic transformation of all transformants was by homologous gene replacement of the β-tubulin allele.     

Transformation of the rice blast fungus Magnaporthe grisea to benomyl resistance

A transformation method based on a dominant selectable marker (benomyl resistance) was developed for the rice blast fungus Magnaporthe grisea. The heterologous gene for -tubulin from Neurospora crassa (pBT3) was used to obtain benomyl-resistant M. grisea transformants at a frequency of 20 to 30/g of DNA. Control transformations carried out with a plasmid conferring hygromycin resistance or a derivative of pBT3 containing a repetitive DNA sequence, yielded the same frequency of transformation as that of pBT3. Molecular analysis of the transformants indicated multiple integration of the vector DNA.     

Transformation of Metarhizium anisopliae with benomyl resistance and green fluorescent protein genes provides a tag for genetically engineered strains

A plasmid, pBGFP, carrying green fluorescent protein (gfp) and benomyl-resistance genes was constructed and transformed into Metarhizium anisopliae. The transformants grew normally and GFP fluorescence was detected. No change was found in virulence for the transformants. Fluorescence was detected in hyphae from the haemolymph of the infected locust, and the benomyl-resistance was maintained. Results suggested that the two markers provided a useful tool for screening and monitoring the engineered strains even after infection.     

Transformation of the entomopathogenic fungus Metarhizium anisopliae to benomyl resistance

Abstract Protoplasts of the entomopathogenic fungus Metarhizium anisopliae were transformed to benomyl resistance using cosmid pSV50 which harbours a β-tubulin gene cloned from a Neurospora crassa benomyl-resistant mutant. Transformant colonies, which appeared at a frequency of 4 per 50 μg DNA, grew and sporulated on 10 μg/ml benomyl, whereas the wild type was inhibited by 3 μg/ml. Southern blot hybridization of DNA from transformants showed that, in each case, tandem repeats of the cosmid had integrated at several chromosomal loci. The transformants were mitotically stable when subcultured on non-selective agar and retained the ability to infect and kill larvae of Manduca sexta . Two transformants were less virulent than the wild type and one of them showed slower in vitro spore germination. The benomyl-resistant phenotype persisted in reisolates from insect cadavers.     

产黄青霉菌响应苯氧乙酸的Ca2+信号转导机制

【目的】研究青霉素V生产过程中—Ca~(2+)信号转导途径参与产黄青霉菌对外源侧链前体苯氧乙酸的应答机制。【方法】考察4种不同机制的Ca~(2+)信号干扰剂[利心平、乙二醇双(2-氨基乙基醚)四乙酸、苏拉明和硫酸新霉素]对青霉素V产量和产黄青霉菌生物量的影响。运用Fluo-3/AM荧光染料对细胞进行染色,通过荧光显微镜成像和酶标仪定量检测两种方法监测胞内Ca~(2+)浓度的变化。【结果】苯氧乙酸添加后胞内Ca~(2+)相对含量高于对照组49.86%,而1 mmol/L磷酸酯酶C底物抑制剂硫酸新霉素的添加使得胞内Ca~(2+)相对含量降低了53.31%,同时青霉素V产量降低78.71%,表明产黄青霉菌可通过肌醇1,4,5-三磷酸信号途径调节胞内Ca~(2+)浓度来响应苯氧乙酸的胁迫。【结论】首次探究了Ca~(2+)信号转导途径在产黄青霉菌对苯氧乙酸应答中的作用,为丝状真菌中Ins(1,4,5)P3-Ca~(2+)信号转导途径的研究提供理论依据。     

Optimization of culture conditions for glucose oxidase production by a Penicillium chrysogenum SRT 19 strain

The enzyme glucose oxidase (GOD) has been used for a variety of biotechnological applications in food and pharmaceutical industries. In this study, the optimization of extracellular GOD production was carried out in a Penicillium chrysogenum SRT 19 strain isolated from contaminated and decaying cheese samples. Maximum GOD production was attained at pH 6 and 20°C in fermentation broth after 72 h of incubation. The effects of metal ions and sugars were screened for the induction of higher GOD production. The results revealed that glucose and lactose give the highest production of enzyme (0.670 and 0.552 U/mL, respectively) as compared with other sugars (sucrose, cellulose, mannitol and fructose). Out of the seven metal ions studied, CaCO₃ (1.123 U/mL) and FeSO₄ (0.822 U/mL) act as modulators, while MgSO₄ (0.535 U/mL), CuSO₄ (0.498 U/mL), HgCl₂ (0.476 U/mL), ZnSO₄ (0.457 U/mL) and BaSO₄ (0.422 U/mL) yield lower production. The study therefore suggests that a strain of P. chrysogenum SRT 19 can be used as a new strain for GOD production.     

Characterization of Penicillium chrysogenum physiology in submerged cultures by color and monochrome image analysis

Although filamentous microorganisms are widely used in industrial fermentation processes, their growth and differentation are not yet fully understood, because their biomass is structured, and therefore difficult to descrbie and to quantify. This lack of appropriate tools can hinder the optimization and control of the fermentation. A quantitative image analysis method was therefore developed for characterizing the physiology of the penicillin-producing mold Penicillium chrysogenum. This method is based on a differntial staining procedure showing six physiological states: growing material, three differentiated states characterzied by an increasing granulation, a highly vacuolized state, and dead segments having lost their cytoplasm. The image analysis software, with versions written for monochrome and color images, consisted of a semiautomatic binary mask computation step and a fully automatic segmentation step based on a fuzzy classification. (c) 1995 John Wiley & Sons, Inc.     

Changes in nucleotide pools during conidia formation of Penicillium chrysogenum in solid culture

Abstract Using Cellophane-sheet techniques, changes in nucleotide pool size during conidia formation of Penicillium chrysogenum on agar plates were studied. On conidia-forming medium (nutritionally poor medium), the pool size of nucleoside triphosphates, especially GTP, decreased dramatically at mid-growth phase, concomitantly with the exhaustion of extracellular phosphate. Onset of conidia formation was observed just after the GTP pool decrease.     

Use of the Weibull model to describe inactivation of dry‐harvested conidia of different Penicillium species by ethanol vapours

Aims: This study aimed at modelling the effect of ethanol vapours, in the range 0·7–7·5 kPa, on the inactivation of dry‐harvested conidia of Penicillium chrysogenum, Penicillium digitatum and Penicillium italicum. Methods and Results: Survival curves were modelled by a Weibull model: log (N/N₀) = ?1/2·303 (t/α)^β. The shape parameter β was different from one in all cases, indicating that the classical first‐order kinetics approach is the exception rather than the rule. Survival curves exhibited upward concavity (β < 1) with the notable exception of P. chrysogenum at ethanol vapour pressures 0·7 and 1·5 kPa. The scale parameter α (h) varied greatly depending on the ethanol vapour pressure and on the species. Conclusions: For safety reasons, it is recommended not to exceed an ethanol vapour pressure of 3·3 kPa. At 2·8 kPa, more than 4 log₁₀ reductions in viable conidia were achieved for all the species after 24‐h exposure. Significance and Impact of the Study: Ethanol has GRAS status in the USA and represents an interesting alternative to fungicides. The effectiveness of ethanol vapours to inactivate dry‐harvested conidia of some Penicillium was demonstrated in this study.     

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

Abstract

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55?°C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72?h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.     

Rheological characterization of media containing Penicillium chrysogenum

Samples from fed-batch fermentations of Penicillium chrysogenum on complex medium are rheologically characterized. The behavior is well described by a power law model for which the parameters are estimates. Furthermore, two types of model media are characterized and compared with the real fermentation samples. Xanthan solutions are found to mimic the rheological properties of the filamentous fungi much better than carboxymethyl cellulose (CMC) solutions. (c) 1993 John Wiley & Sons, Inc.     

Novel cold-adaptive Penicillium strain FS010 secreting thermo-labile xylanase isolated from Yellow Sea

A novel cold-adaptive xylanolytic Penicillium strain FS010 was isolated from Yellow Sea sediments. The marine fungus grew well from 4 to 20 ℃; a lower （0 ℃） or higher （37 ℃） temperature limits its growth. The strain was identified as Penicillium chrysogenum. Compared with mesophilic P. chrysogenum, the cold-adaptive fungus secreted the cold-active xylanase （XYL） showing high hydrolytic activities at low temperature （2-15 ℃） and high sensitivity to high temperature （〉50 ℃）. The XYL gene was isolated from the cold-adaptive P. chrysogenum FS010 and designated as xyl. The deduced amino acid sequence of the protein encoded by xyl showed high homology with the sequence of glycoside hydrolase family 10. The gene was subcloned into an expression vector pGEX-4T- 1 and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase in Escherichia coli BL21. The expression product was purified and subjected to enzymatic characterization. The optimal temperature and pH for recombinant XYL was 25 ℃ and 5.5, respectively. Recombinant XYL showed nearly 80% of its maximal activity at 4 ℃ and was active in the pH range 3.0-9.5.     

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

During glucose‐limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β‐lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady‐state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose‐limited chemostat cultures at the dilution rates (D) of both 0.03 h^?1 and 0.05 h^?1 are determined to evaluate the quantitative impact on the physiology of a high‐yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h^?1, while this is not the case at D = 0.05 h^?1. In addition, a higher cytosolic redox status (NADH/NAD⁺), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h^?1, which are virtually absent at D = 0.03 h^?1. In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose‐limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial‐scale scenario.     

Novel Antifungal and Antifeedant Metabolites from Penicillium chrysogenum Co-Cultured with Nemania primolutea and Aspergillus fumigatus

The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A–B ( 1–2 ), and penigenumin ( 3 ) from N. primolutea, penemin ( 4 ) from P. chrysogenum were isolated from the coculture. On the other hand, P. chrysogenum inhibited the Aspergillus fumigatus in the coculture. Induced metabolites ( 13–16 ) with monasone naphthoquinone scaffolds including a new one from P. chrysogenum were produced by the coculture of P. chrysogenum, and A. fumigatus. Interesting, cryptic metabolites penicichrins A–B isolated from wild P. chrysogenum induced by host Ziziphus jujuba medium were also found in induced P. chrysogenum cultured in PDB ordinary medium. So the induction of penicichrin production by supplementing with host extract occurred in the fungus P. chrysogenum not the host medium. The productions of penicichrins were the spontaneous metabolism, and the metabolites ( 13–16 ) were the culture driven. Compounds 4 , 6 , 8 , 10 , 11 , 14 , and 15 showed significant antifungal activities against the phytopathogen Alternaria alternata with MIC_S of 1–8 μg/mL, and compounds 7 , 9 , and 12 indicated significant antifeedant activities against silkworms with feeding deterrence indexes (FDIs) of 92 %, 66 %, and 64 %. The carboxy group in 4-(2-hydroxybutynoxy)benzoic acid derivatives, and xylabisboeins; the hydroxy group in mellein derivatives; and the quinoid in monasone naphthoquinone increased the antifungal activities.     

Induced production of halogenated diphenyl ethers from the marine-derived fungus Penicillium chrysogenum

Manipulation of the fermentation of the marine‐derived fungus Penicillium chrysogenum by addition of CaBr₂ resulted in induced production of bromodiphenyl ether analogs. Two new free‐radical‐scavenging polybrominated diphenyl ethers, 1 and 2 , and three known diphenyl ethers, 3,3′‐dihydroxy‐5,5′‐dimethyldiphenyl ether ( 3 ), and an inseparable mixture of violacerol‐I ( 4 ) and violacerol‐II ( 5 ) were isolated. The structures of the two new polybromodiphenyl ethers 1 and 2 were assigned by combined spectroscopic‐data analysis, including deuterium‐induced isotope effect. Compounds 1 – 3 , and a mixture of 4 and 5 exhibited radical‐scavenging activities against 1,1‐diphenyl‐2‐picrylhydrazyl with IC₅₀ values of 18, 15, 42, and 6 μM , respectively. With the exception of 3 , the compounds were, therefore, more active than the positive control, ascorbic acid (IC₅₀ 20 μM ).     

Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate

Summary The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including -tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.