Biogenesis of antibiotics-viewing its history and glimpses of the future.

This review aims at comparing some historical data with the current situation in the study of biogenesis of natural compounds, antibiotics in the first place. Biogenesis of tetracyclines and cycloheximide and related compounds serves as example. Examples of molecular biological and bioinformatics methods used in the study of antibiotic biogenesis are described both in terms of its historical aspects and the current knowledge.

Antimicrobials, drug discovery, and genome mining.

Over the years, antibiotics have provided an effective treatment for a number of microbial diseases. However recently, there has been an increase in resistant microorganisms that have adapted to our current antibiotics. One of the most dangerous pathogens is methicillin-resistant Staphylococcus aureus (MRSA). With the rise in the cases of MRSA and other resistant pathogens such as vancomycin-resistant Staphylococcus aureus, the need for new antibiotics increases every day. Many challenges face the discovery and development of new antibiotics, making it difficult for these new drugs to reach the market, especially since many of the pharmaceutical companies have stopped searching for antibiotics. With the advent of genome sequencing, new antibiotics are being found by the techniques of genome mining, offering hope for the future.

Tetanus toxin production in soy-based medium: nutritional studies and scale-up into small fermentors.

AIMS: To further improve the soy-based medium, devoid of animal and dairy products, for a production of tetanus toxin by nutritional studies and to scale-up the Clostridium tetani process into small fermentors. METHODS AND RESULTS: Optimum production of tetanus toxin did not require addition of pantothenic acid, thiamine, riboflavin, pyridoxine, biotin and uracil, growth factors used by previous investigators. Furthermore, l-tyrosine and l-cysteine could be eliminated from our soy-based medium without effect. Seven carbon sources were compared with glucose in the soy-based medium, but none was found to be superior to glucose. The process was successfully scaled-up into 250-ml bottles, 1-l bottles and 1-l fermentors. CONCLUSIONS: Quite remarkably, when comparing the tetanus production process in our soy-based medium with the traditional animal/dairy-containing media, our medium does not require addition of expensive vitamins, uracil or carbon sources other than glucose. Furthermore, the l-tyrosine and l-cysteine components could be eliminated, making the medium (Hy-Soy, glucose, powdered iron and inorganic salts) much more simple and economical. The successful scale-up from test tubes into 1-l fermentors allows us to predict that further scale-up into large fermentors will be successful. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (BSE; mad cow's disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. Our vegetable-based process avoids such unfortunate possibilities. The medium, having been made simpler and less expensive, and shown to be scaleable from test tubes into small fermentors, should be excellent for large scale production of tetanus toxin.

Menstrum for culture preservation and medium for seed preparation in a tetanus toxin production process containing no animal or dairy products.

AIMS: To completely eliminate animal and dairy products from the lyophilization menstrum and the seed medium used to produce tetanus toxin with Clostridium tetani. METHODS AND RESULTS: Tetanus toxin production in a recently developed fermentation medium lacking animal and dairy products was studied with different seed media. It was found that soy peptone could completely replace the beef heart infusion plus animal peptone previously used as seed medium. In addition, we found that cells lyophilized in soy milk could replace the usual type of cells lyophilized in cow's milk. CONCLUSIONS: We have now developed a complete tetanus toxin production process containing no animal and dairy products. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (Mad Cow's Disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. The new vegetable-based process described here avoids such unfortunate possibilities.

Performance of a recombinant strain of Streptomyces lividans for bioconversion of penicillin G to deacetoxycephalosporin G.

We examined the performance of Streptomyces lividans strain W25 containing a hybrid expandase (deacetoxycephalosporin C synthase; DAOCS) gene, obtained by in vivo recombination between the expandase genes of S. clavuligerus and Nocardia lactamdurans for resting-cell bioconversion of penicillin G to deacetoxycephalosporin G. Strain W25 carried out a much more effective level of bioconversion than the previously used strain, S. clavuligerus NP1. The two strains also differed in the concentrations of FeSO(4) and alpha-ketoglutarate giving maximal activity. Whereas NP1 preferred 1.8 mM FeSO(4 )and 1.3 mM alpha-ketoglutarate, recombinant W25 performed best at 0.45 mM FeSO(4) and 1.9 mM alpha-ketoglutarate.

Improvement in the resting-cell bioconversion of penicillin G to deacetoxycephalosporin G by addition of catalase.

AIMS: To improve the resting cell bioconversion of penicillin G to deacetoxycephalosporin G (DAOG) by elimination of an oxidizing intermediate which inactivates the enzyme during the reaction. METHODS AND RESULTS: Resting cells of Streptomyces clavuligerus strain NP1 were incubated with penicillin G, required co-factors and decane in the presence of catalase or superoxide dismutase, and production of DAOG was measured. Catalase stimulated the bioconversion but superoxide dismutase did not. CONCLUSIONS: Production of hydrogen peroxide during the ring expansion reaction is at least partially responsible for enzyme inactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Catalase addition improves the bioconversion and will contribute to the eventual replacement of the current multi-step, expensive and environmentally-unfriendly chemical ring expansion by a biological route.

Induction of microcin B17 formation in Escherichia coli ZK650 by limitation of oxygen and glucose is independent of glucose consumption rate.

We examined the consumption of glucose from the media in which Escherichia coli ZK650 was grown. This organism, which produces the polypeptide antibiotic microcin B17 best under conditions of limiting supplies of glucose and air, was grown with a low level of glucose (0.5 mg/ml) as well as a high level (5.0 mg/ml) under both high and low aeration. Glucose consumption rates were virtually identical under both high and low aeration. Thus, glucose consumption rate is not a regulating factor in microcin B17 formation.

Shear stress enhances microcin B17 production in a rotating wall bioreactor, but ethanol stress does not.

Stress, including that caused by ethanol, has been shown to induce or promote secondary metabolism in a number of microbial systems. Rotating-wall bioreactors provide a low stress and simulated microgravity environment which, however, supports only poor production of microcin B17 by Escherichia coli ZK650, as compared to production in agitated flasks. We wondered whether the poor production is due to the low level of stress and whether increasing stress in the bioreactors would raise the amount of microcin B17 formed. We found that applying shear stress by addition of a single Teflon bead to a rotating wall bioreactor improved microcin B17 production. By contrast, addition of various concentrations of ethanol to such bioreactors (or to shaken flasks) failed to increase microcin B17 production. Ethanol stress merely decreased production and, at higher concentrations, inhibited growth. Interestingly, cells growing in the bioreactor were much more resistant to the growth-inhibitory and production-inhibitory effects of ethanol than cells growing in shaken flasks.

Effect of solvents on bioconversion of penicillin G to deacetoxycephalosporin G.

The bioconversion of penicillin G, an inexpensive substrate, to the valuable intermediate for semisynthetic cephalosporin production, deacetoxycephalosporin G (DAOG), had been recently shown to be increased by eliminating agitation and adding decane. The present work examining other solvents shows that all alkanes tested are equivalent to decane in activity but that other solvents are either inhibitory or less active than alkanes. Optimum conditions of pH and temperature for the alkane system are not very different from the previously used aqueous system.

Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904.

Rapamycin (RPM) is produced by Streptomyces hygroscopicus FC904 isolated from soil in Fuzhou, China. It is a triene macrolide antibiotic with potential application as an immunosuppressant and drug for human gene therapy. In an attempt to improve rapamycin production, mutation and screening of the parent culture have been carried out. Thousands of survivors were obtained after mutagenesis by NTG (3 mg/ml) and UV (30 W, 15 cm, 30 seconds) of spore suspensions. None showed improved production of RPM. We determined the susceptibility to antibiotics of S. hygroscopicus FC904 by two fold dilutions of antibiotics in oatmeal agar plates. It was found that the strain was resistant to penicillin, erythromycin, RPM, tetracycline and chloramphenicol, but susceptible to mitomycin C (MIC, 10 microg/ml) and aminoglycosides such as gentamicin (MIC, 0.1 microg/ml), kanamycin (MIC, 0.1 microg/ml) and streptomycin (MIC, 0.3 microg/ml). Protoplasts of strain FC904 were prepared after finding the best conditions for their formation. They were treated with gentamicin, erythromycin, mitomycin C and NTG. Surprisingly, gentamicin was especially effective for obtaining higher RPM-producing mutants. Mutant C14 was selected by exposing the protoplasts of the parent strain FC904 to 1 microg/ml of gentamicin at 28 degrees C for 2 hours. A higher RPM-producing mutant (C14-1) was obtained from the protoplasts of mutant C14 treated with gentamicin, and its titer was 60% higher than that of the parent strain FC904 by HPLC analysis. Another improved mutant (C14-2) was obtained from the spores of mutant C 14 treated with 1 microg/ml of gentamicin plus 2 mg/ml of NTG at 28 degrees C for 2 hours. Mutant C14-2 had a titer 124% higher than FC904. The possible mechanism for the effect of gentamicin by using protoplasts or spore suspensions will be discussed, i.e. the possibility of gentamicin being a mutagen or a selective agent.

Improvement in the bioconversion of penicillin G to deacetoxycephalosporin G by elimination of agitation and addition of decane.

The bioconversion of penicillin G to deacetoxycephalosporin G (DAOG) using resting cells of Streptomyces clavuligerus could be a very valuable step in the economical production of semisynthetic cephalosporin antibiotics. The extent of the reaction, however, is very low due to inactivation of the ring expansion enzyme deacetoxycephalosporin C synthetase ("expandase") by reaction components. We show that elimination of agitation during the reaction lowers the rate but increases the amount of DAOG produced, presumably because the inactivation requires high levels of oxygen. Many additives to the medium were examined for their effect on the reaction. Clearly, the most effective compound was the organic solvent, decane.

Molecular genetics and industrial microbiology--30 years of marriage.

Thirty years ago, molecular genetics and industrial microbiology joined their hands in marriage. The event took place in Prague at the first Symposium on the Genetics of Industrial Microorganisms. My closing plenary lecture, titled "The Marriage of Genetics and Industrial Microbiology--After a long Engagement, a Bright Future," dealt with industrial uses of mutants, the lack of success with genetic recombination, control of branched and unbranched pathways and thoughts about the future, e.g., identifying the biochemical sites of beneficial mutations, exploitation of recombination and genetic means to increase production of enzymes. It is quite amazing that the Symposium was held 3 years before the advent of recombinant DNA technology. This important meeting was followed in 1976 by the first Genetics and Molecular Biology of Industrial Microorganisms (GMBIM) meeting in Orlando, all of the six subsequent GMBIM meetings being held in Bloomington, Indiana. Today, thousands of biotechnology companies are in existence making great progress in the pharmaceutical and agricultural sectors. Hundreds of new genetically engineered compounds, produced in microbial, mammalian or insect cells, are in clinical trails and many are already being marketed. The field is booming with new technologies such as transgenic animals and plants, site-directed mutagenesis, combinatorial biosynthesis, gene therapy, antisense, abzymes, high-throughput screening, monoclonal antibodies, PCR and many more. Agricultural biotechnology has made great strides but unfortunately its progress is being delayed by political controversy.

Secondary metabolism in simulated microgravity.

We have studied microbial secondary metabolism in a simulated microgravity (SMG) environment provided by NASA rotating-wall bioreactors (RWBs). These reactors were designed to simulate some aspects of actual microgravity that occur in space. Growth and product formation were observed in SMG in all cases studied, i.e., Bacillus brevis produced gramicidin S (GS), Streptomyces clavuligerus made beta-lactam antibiotics, Streptomyces hygroscopicus produced rapamycin, and Escherichia coli produced microcin B17 (MccB17). Of these processes, only GS production was unaffected by SMG; production of the other three products was inhibited. This was determined by comparison with performance in an RWB positioned in a different mode to provide a normal gravity (NG) environment. Carbon source repression by glycerol of the GS process, as observed in shaken flasks, was not observed in the RWBs, whether operated in the SMG or NG mode. The same phenomenon occurred in the case of MccB17 production, with respect to glucose repression. Thus, the negative effects of carbon source on GS and beta-lactam formation are presumably dependent on shear, turbulence, and/or vessel geometry, but not on gravity. Stimulatory effects of phosphate and the precursor L-lysine on beta-lactam antibiotic production, as observed in flasks, also occurred in SMG. An almost complete shift in the localization of produced MccB17 from cells to extracellular medium was observed when E. coli was grown in the RWB under SMG or NG. If a plastic bead was placed in the RWB, accumulation became cellular, as it is in shaken flasks, indicating that sheer stress favors a cellular location. In the case of rapamycin, the same type of shift was observed, but it was less dramatic, i.e., growth in the RWB under SMG shifted the distribution of produced rapamycin from 2/3 cellular:1/3 extracellular to 1/3 cellular:2/3 extracellular. Stress has been shown to induce or promote secondary metabolism in a number of other microbial systems. RWBs provide a low stress SMG environment, which, however, supports only poor production of MccB17, as compared to production in shaken flasks. We wondered whether the poor production in RWBs under SMG is due to the low level of stress, and whether increasing stress in the RWBs would raise the amount of MccB17 formed. We found that increasing shear stress by adding a single Teflon bead to the RWB improved MccB17 production. Although shear stress seems to have a marked positive effect on MccB17 production in SMG, addition of various concentrations of ethanol to RWBs (or to shaken flasks) failed to increase MccB17 production. Ethanol stress merely decreased production and, at higher concentrations, inhibited growth. Interestingly, cells growing in the RWB were much more resistant to the growth- and production-inhibitory effects of ethanol than cells growing in shaken flasks. With respect to S. hygroscopicus, addition of Teflon beads to the RWB reversed the inhibition of growth, but rapamycin production was still markedly inhibited, and the distribution did not revert back to a preferential cellular site.

The natural functions of secondary metabolites.

Secondary metabolites, including antibiotics, are produced in nature and serve survival functions for the organisms producing them. The antibiotics are a heterogeneous group, the functions of some being related to and others being unrelated to their antimicrobial activities. Secondary metabolites serve: (i) as competitive weapons used against other bacteria, fungi, amoebae, plants, insects, and large animals; (ii) as metal transporting agents; (iii) as agents of symbiosis between microbes and plants, nematodes, insects, and higher animals; (iv) as sexual hormones; and (v) as differentiation effectors. Although antibiotics are not obligatory for sporulation, some secondary metabolites (including antibiotics) stimulate spore formation and inhibit or stimulate germination. Formation of secondary metabolites and spores are regulated by similar factors. This similarity could insure secondary metabolite production during sporulation. Thus the secondary metabolite can: (i) slow down germination of spores until a less competitive environment and more favorable conditions for growth exist; (ii) protect the dormant or initiated spore from consumption by amoebae; or (iii) cleanse the immediate environment of competing microorganisms during germination.

Methionine interference in rapamycin production involves repression of demethylrapamycin methyltransferase and S-adenosylmethionine synthetase.

In a chemically defined medium, L-methionine decreased production of rapamycin and increased that of demethylrapamycin. Growth with L-methionine yielded cells with a lower ability to convert demethylrapamycin to rapamycin and decreased the level of S-adenosylmethionine synthetase and S-adenosylmethionine. Thus, methionine represses at least one methyltransferase of rapamycin biosynthesis and S-adenosylmethionine synthetase.

Immobilized Streptomyces clavuligerus NP1 cells for biotransformation of penicillin G into deacetoxycephalosporin G.

An investigation was conducted to determine whether immobilized resting cells of Streptomyces clavuligerus NP1, entrapped on a polymeric matrix, are able to perform oxidative ring expansion of benzylpenicillin into deacetoxycephalosporin G by virtue of their deacetoxycephalosporin C synthase ("expandase") activity. Cells entrapped in polyethyleneimine-barium alginate (1.5%) were able to sustain activity for at least four 2-h cycles, whereas free resting cells were inactive after the second cycle. Although entrapped cells exhibited lower oxidative ring expansion activity than free resting cells, immobilization may offer storage stability, recyclability, and operational stability for biotransformation of penicillins to cephalosporins, thus contributing to the development of a biological means for the production of the important industrial intermediate 7-aminodeacetoxycephalosporanic acid.

Growth of Steptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production.

Growth of Streptomyces hygroscopicus under conditions of simulated microgravity in a rotating-wall bioreactor resulted in a pellet form of growth, lowered dry cell weight, and inhibition of rapamycin production. With the addition of Teflon beads to the bioreactor, growth became much less pelleted, dry cell weight increased but rapamycin production was still markedly inhibited. Growth under simulated microgravity favored extracellular production of rapamycin, in contrast to a greater percentage of cell-bound rapamycin observed under normal gravity conditions.

Relief from glucose interference in microcin B17 biosynthesis by growth in a rotating-wall bioreactor.

Glucose interference in production of microcin B17 by Escherichia coli ZK650 was decreased sevenfold by growth in a ground-based rotating-wall bioreactor operated in the simulated microgravity mode as compared with growth in flasks. When cells were grown in the bioreactor in the normal gravity mode, relief from glucose interference was even more dramatic, amounting to a decrease in glucose interference of over 100-fold.

Enhancement of the antifungal activity of rapamycin by the coproduced elaiophylin and nigericin.

Streptomyces hygroscopicus ATCC 29253 produces rapamycin, elaiophylin and nigericin. Although elaiophylin has no activity against Candida albicans ATCC 11651, it markedly enhances rapamycin's antifungal activity. Nigericin has only weak activity on its own but it also enhances rapamycin action. Surprisingly, elaiophylin does not enhance nigericin activity on C. albicans.