Fermentative production of self-toxic fungal secondary metabolites

Fungi are well known for their vast diversity of secondary metabolites that include many life-saving drugs and highly toxic mycotoxins. In general, fungal cultures producing such metabolites are immune to their toxic effects. However, some are known to produce self-toxic compounds that can pose production optimization challenges if the metabolites are needed in large amounts for chemical modification. One such culture, LV-2841, was identified as the lead for one of our exploratory projects. This culture was found to be a slow grower that produced trace amounts of a known metabolite, cercosporamide, under the standard flask fermentation conditions, and extensive medium optimization studies failed to yield higher titers. Poor growth of the culture in liquid media was attributed to the self-toxicity of cercosporamide to the producing organism, and the minimum inhibitory concentration (MIC) of cercosporamide was estimated to be in the range of 8–16 μg/ml. Fermentations carried out in media containing Diaion^® HP20 resin afforded significantly higher titers of the desired compound. While several examples of resin-based fermentations of soil streptomyces have been published, this approach has rarely been used for fungal fermentations. Over a 100-fold increase in the production titer of cercosporamide, a self-toxic secondary metabolite, was achieved by supplementing the production medium with a commercially available neutral adsorbent resin.     

Biofilm-specific cross-species induction of antimicrobial compounds in bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10(T) and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10(T) and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

Bench-top fermentative production of plant benzylisoquinoline alkaloids using a bacterial platform

The plant secondary metabolites benzylisoquinoline alkaloids (BIAs) have diverse pharmaceutical activities, and some are used medicinally (e.g., morphine, codeine, berberine). Recently, we constructed a platform to produce BIAs using bioengineered Escherichia coli, which could be useful for bulk production. The E. coli strain used in this system produces the important intermediate (S)-reticuline from glucose or glycerol. Although the amount produced (40 mg/L) exceeded the amount that can be purified from plants, the conversion efficiency from glycerol was only 0.15%; thus, there was much room for improvement. Our production system was developed in a jar fermenter but it is difficult to work with multiple samples using this system. In contrast, many samples can be cultured in parallel using shake flask cultures, allowing optimization of production conditions. Here, we describe bench-top production of (S)-reticuline and optimization of culture conditions using shake flask cultures. The production of (S)-reticuline reached 33.9 mg/L.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10^T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10^T and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

Elevated 3-hydroxypropionaldehyde production from glycerol using a Citrobacter freundii mutant

A mutant strain of Citrobacter freundii capable of elevated 3-hydroxypropionaldehyde production from glycerol was isolated using chemical mutagenesis and a screening protocol. The protocol involved screening mutagenized bacterial cells on solid minimal medium containing 5 % (v/v) glycerol. Colonies were picked onto duplicate solid minimal medium plates and one plate was stained with 1 % (w/v) phloroglucinol. Those colonies staining red were further screened and a mutant, HPAO-1, was identified. The mutant strain produced a several-fold higher 3-hydroxypropionaldehyde concentration than did the parent strain when grown on 5 % (v/v) glycerol. The ratio of culture volume to flask volume influenced 3-hydroxypropionaldehyde production by the mutant cells compared to the parent cells. Aldehyde production was highest when the mutant strain was grown on 5 % (v/v) glycerol at a ratio of culture volume to flask volume of 1:3 or 1:12.5.     

Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed-batch cultivation of Taxus chinensis cells

A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 microM DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L(-1) sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 microM DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 +/- 29 mg L(-1), which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 +/- 41 mg L(-1) and 33.2 +/- 1.9 mg L(-1) d(-1), respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites.     

Mutagens manufactured in fungal culture may affect DNA/RNA of producing fungi

Self-produced mutagens in culture by fungi may affect DNA analysis of the same fungi. This has not been considered previously. Many fungi produce numerous mutagenic secondary metabolites (SM) in culture. There is a paradox of growing fungi in media to produce representative DNA which also support mutagenic SM. This is a crucial issue in developing diagnostic and phylogenetic methods, especially for closely-related fungi. For example, idh gene analysis of the patulin metabolic pathway in fungi can be interpreted as producing some false negative and positive results in terms of possession, or nonpossession, of the gene from mutated strains. The most obvious mycotoxins and fungi to consider in this regard are aflatoxins and Aspergillus , as aflatoxins are the most mutagenic natural compounds. Many other fungi and SM are relevant. Conditions to grow fungi have not been selected to inhibit SM production although relevant data exist. In fact, fungi repair damaged nucleic acid (NA) and are capable of removing toxins by employing transporter proteins. These and NA repair mechanisms could be inhibited by secondary metabolites. Mutagenic effects may involve inhibition of DNA stabilizing enzymes. There may be an equivalent situation for bacteria. Researchers need to devise methods to reduce SM for valid protocols. More work on how mutagens affect the NA of producing fungus in vitro is required. The current review assesses the potential seriousness of the situation with selected papers.     

Establishment of adventitious root culture of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni in a roller bottle system

Cultures of adventitious roots of Stevia rebaudiana (Bert.) Bertoni were performed in a roller bottle system for the production of both primary and secondary metabolites. Adventitious roots were induced from 1-cm-long root tip explants derived from in vitro regenerated plantlets on solid Murashige and Skoog (MS 1962) media supplemented with 10.7 μM of α-naphthaleneacetic acid. These cultures were successfully maintained in the same medium for 6 months with regular subcultures after 4 weeks. Thereafter, the roots were cut into 1.0- to 1.5-cm-long segments and transferred to the roller bottle system containing a fresh root tissue culture on liquid MS medium supplemented with 10.7 μM NAA. The apparatus consisted of a flask rolling system adjusted to 4g, and 3° of flask inclination. The roots were allowed to grow in the absence of light for adaptation and adventitious root formation. The best conditions for cultivation were investigated, considering culture volume (25 ml), culture period (4 weeks), salt concentrations in the nutrient medium (33%) and optimal initial inoculum (0.2 g) of S. rebaudiana roots. These results could give important information on how to improve the development of adventitious roots of S. rebaudiana for the production of primary and secondary metabolites.     

Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.     

Gifted microbes for genome mining and natural product discovery

Actinomycetes are historically important sources for secondary metabolites (SMs) with applications in human medicine, animal health, and plant crop protection. It is now clear that actinomycetes and other microorganisms with large genomes have the capacity to produce many more SMs than was anticipated from standard fermentation studies. Indeed ~90 % of SM gene clusters (SMGCs) predicted from genome sequencing are cryptic under conventional fermentation and analytical analyses. Previous studies have suggested that among the actinomycetes with large genomes, some have the coding capacity to produce many more SMs than others, and that strains with the largest genomes tend to be the most gifted. These contentions have been evaluated more quantitatively by antiSMASH 3.0 analyses of microbial genomes, and the results indicate that many actinomycetes with large genomes are gifted for SM production, encoding 20–50 SMGCs, and devoting 0.8–3.0 Mb of coding capacity to SM production. Several Proteobacteria and Firmacutes with large genomes encode 20–30 SMGCs and devote 0.8–1.3 Mb of DNA to SM production, whereas cultured bacteria and archaea with small genomes devote insignificant coding capacity to SM production. Fully sequenced genomes of uncultured bacteria and archaea have small genomes nearly devoid of SMGCs.     

Solid-state fermentation: a promising microbial technology for secondary metabolite production

Solid state (substrate) fermentation (SSF) has been used successfully for the production of enzymes and secondary metabolites. These products are associated with the stationary phase of microbial growth and are produced on an industrial scale for use in agriculture and the treatment of disease. Many of these secondary metabolites are still produced by submerged liquid fermentations (SmF) even though production by this method has been shown to be less efficient than SSF. As large-scale production increases further, so do the costs and energy demands. SSF has been shown to produce a more stable product, requiring less energy, in smaller fermenters, with easier downstream processing measures. In this article we review an important area of biotechnology, since the recent evidence indicates that bacteria and fungi, growing under SSF conditions, are more than capable of supplying the growing global demand for secondary metabolites.     

Microbial growth on peptones from fish industrial wastes

Industrial fish peptone was an excellent substrate for biomass production in solid and submerged fermentations. The maximal growth rates of several microorganisms were two to three times higher than those grown on beef (bacto)peptones and the final biomass concentrations were almost twice as great as those grown on beef peptones. Fish peptones did not increase the production of secondary metabolites relative to those produced on beef peptones in non-optimized media. Fish peptone has promising potential as a substrate for biomass production.     

Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

Culture in the rotating-wall vessel affects recombinant protein production capability of two insect cell lines in different manners

Summary The production of recombinant secreted alkaline phosphatase protein in virally infected insect cells was studied in shaker flask and high aspect rotating-wall vessel (HARV) culture. Two commonly used cell lines, Spodoptera frugiperda Sf-9 (Sf-9) and a nonaggregating isolate of the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) cell line, Trichoplusia ni Tn-5B1-4-NA (TN-5B1-4-NA), were used and monitored for 120-h postinfection. Different responses to culture in the HARV were seen in the two cell lines. While the Sf-9 cell line was able to produce slightly greater amounts of recombinant protein in the HARV than in shaker flask controls, the Tn-5B1-4-NA cell line produced significantly lesser amounts in the HARV than in the shaker flasks. Both cell lines exhibited longer life spans and longer periods of protein production in HARV culture than in shaker flask culture, presumably due to lower levels of shear encountered in the HARV. The important difference was in the protein production rate responses of the two cell lines. While the protein production rates of Sf-9 cells were comparable in both HARV and shaker flask cultures, the protein production rates of Tn-5B1-4-NA cells were much lower in HARV culture than in shaker flask cultures. The conclusion is drawn that cell line-specific adaptation to the HARV strongly influences recombinant protein production.     

Isolation and production of artemisinin and stigmasterol in hairy root cultures of Artemisia annua

Scaled-up hairy root culture of Artemisia annua L. was established in three-liter Erlenmeyer flask. Both artemisinin and stigmasterol that derive from the common precursors of isopentenyl diphosphate and farnesyl pyrophosphate were isolated from hairy roots. The production rate of artemisinin isolated by column chromatography from hairy root cultures was 0.54% (mg.gDW⁻¹). Stigmasterol was identified by mass spectrometry and nuclear magnetic resonance analysis. The production of stigmasterol isolated by column chromatography from hairy root cultures was 108.3% (mg.gDW⁻¹). In hairy root cultures, the production rate of stigmasterol was estimated to be 201 times greater than that of artemisinin. Our results suggest that investigation of secondary metabolites may provide a new insight to study artemisinin production in hairy root cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Surface Attachment Induced Production of Antimicrobial Compounds by Marine Epiphytic Bacteria Using Modified Roller Bottle Cultivation

A modified roller bottle culture method elicited the production of antimicrobial compounds from 2 epibiotic marine bacterial strains, EI-34-6 and II-111-5, isolated from the surface of the marine alga Palmaria palmata. These isolates, tentatively identified as Bacillus species, were grown as a biofilm on the surface of nutrient glycerol ferric agar (NGFA) and marine Columbia glycerol agar (MCGA) on the inside of a rolling bottle. The biofilm was shown to be stable, and the cells were difficult to remove from the agar surface. The culture supernatant exhibited a different antibiotic spectrum when the strains were grown using the agar roller bottle method compared with shake flask cultures or nonagar roller bottle cultures. These results suggest that biofilm formation is an important factor in the production of antimicrobial compounds by these 2 strains, and roller bottle cultivation also allowed production of these compounds to be increased. The methodology used here has the potential to allow increased production of useful secondary metabolites such as antibiotics from marine epibiotic bacteria.