Aiqi Fang

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.

Effect of amino acids on rapamycin biosynthesis by Streptomyces hygroscopicus

In a chemically defined medium containing aspartate, arginine and histidine to support good growth, addition of L-lysine stimulated rapamyycin production by 150%. This was probably due to its conversion to pipecolic acid, a rapamycin precursor. Phenylalanine and methionine interfered in rapamycin production by unknown mechanisms.

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

Abstract. 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.

Gramicidin S Production by Bacillus brevis in Simulated Microgravity

Abstract. In a continuing study of microbial secondary metabolism in simulated microgravity, we have examined gramicidin S (GS) production by Bacillus brevis strain Nagano in NASA High Aspect Rotating Vessels (HARVs), which are designed to simulate some aspects of microgravity. Growth and GS production were found to occur under simulated microgravity. When performance under simulated microgravity was compared with that under normal gravity conditions in the bioreactors, GS production was found to be unaffected by simulated microgravity. The repressive effect of glycerol in flask fermentations was not observed in the HARV. Thus the negative effect of glycerol on specific GS formation is dependent on shear and/or vessel geometry, not gravity.

Secondary metabolism in simulated microgravity: beta-lactam production by Streptomyces clavuligerus

Rotating bioreactors designed at NASA’s Johnson Space Center were used to simulate a microgravity environment in which to study secondary metabolism. The system examined was β-lactam antibiotic production by Streptomyces clavuligerus. Both growth and β-lactam production occurred in simulated microgravity. Stimulatory effects of phosphate and L-lysine, previously detected in normal gravity, also occurred in simulated microgravity. The degree of β-lactam antibiotic production was markedly inhibited by simulated microgravity.

Growth of Streptomyces Hygroscopicus in Rotating-Wall Bioreactor Under Simulated Microgravity Inhibits Rapamycin Production

Abstract 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.

The substrate specificity of deacetoxycephalosporin C synthase (“expandase”) of Streptomyces clavuligerus is extremely narrow

Abstract Cell-free extracts of Streptomyces clavuligerus contain the enzyme deacetoxycephalosporin C synthase (“expandase”), which catalyzes the oxidative ring expansion of the natural substrate δ- d -α-aminoadipyl-6-APA (penicillin N) into the primary cephalosporin, deacetoxycephalosporin C in the presence of magnesium ions, ferrous ions, ascorbate, and α-ketoglutarate. Eighteen unnatural side chain analogues of penicillin N were exposed to these reaction conditions. Only d -carboxymethylcysteinyl-6-APA was found to undergo ring expansion. Of special interest is the observation that adipyl-6-APA and m -carboxyphenylacetyl-6-APA were not expanded.

Production of Clostridium difficile toxin in a medium totally free of both animal and dairy proteins or digests

In the hope of developing a vaccine against Clostridium difficile based on its toxin(s), we have developed a fermentation medium for the bacterium that results in the formation of Toxin A and contains no meat or dairy products, thus obviating the problem of possible prion diseases. Particular preparations of hydrolyzed soy proteins, especially Soy Peptone A3, have been found to replace both the meat/dairy product tryptone in the preparation of working cell banks and seed media, and NZ-Soy BL4 does the same in the fermentation medium. These replacements yield even higher toxin titers.

Unexpected enhancement of β-lactam antibiotic formation inStreptomyces clavuligerus by very high concentrations of exogenous lysine

Abstractl-Lysine is known to stimulate production of β-lactam antibiotics byStreptomyces clavuligerus via provision of the lysine breakdown product,l-α-aminoadipic acid, which is a limiting precursor. Previous investigations utilized levels of 10–20 mMl-lysine as an addition to chemically-defined media resulting in 50–100% improvement in antibiotic production. We were surprised to note that as the concentration was further increased, the organism responded by producing even higher titers of antibiotics. The optimum concentration of 100 mMl-lysine yielded an approximate 500% increase in production with only minor effects on growth.dl- andd-lysine also exerted enhancements suggesting the presence of a lysine racemase or some other route fromd-lysine tol-α-aminoadipate in this organism;d-lysine was considerably less potent thandl- orl-lysine.

Methionine Interference in Rapamycin Production Involves Repression of Demethylrapamycin Methyltransferase and S-Adenosylmethionine Synthetase

ABSTRACT 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 ofS-adenosylmethionine synthetase andS-adenosylmethionine. Thus, methionine represses at least one methyltransferase of rapamycin biosynthesis andS-adenosylmethionine synthetase.