Armando Mejía

Production of Secondary Metabolites by Solid-State Fermentation

Microbial secondary metabolites are useful high value products that are normally produced by liquid culture; but could be advantageously produced by solid-state fermentation (SSF). Particularly if SSF could benefit from a deeper understanding of microbial physiology in a solid environment. Recent research indicates that different kind of secondary metabolites can be produced by SSF: antibiotics, phytohormones, food grade pigments, alkaloids, etc. Physiology in SSF shows several similarities with physiology in liquid medium, so similar strategies must be adapted for efficient processes. However, there are certain particularities of idiophase in solid medium which dictate the need for special strains.

Effect of particle size, packing density and agitation on penicillin production in solid state fermentation.

The use of a large particle size (14 mm) support (sugar cane bagasse) increased penicillin production by solid state fermentation by 37 %, however this effect was due to a higher sugar concentration in this bagasse fraction. Cultures with closer packing densities (0.35) produced 20 % more penicillin. Agitation did not have a negative effect on production if moisture loss during the operation is restituted.

Development of high penicillin producing strains for solid state fermentation

Penicillin production with an industrial strain and 4 strains of P. chrysogenum, in solid state fermentation (SSF) and liquid submerged fermentation (LSF), was determined. Their ability to produce the antibiotic in SSF in relation to their capacity to do so in LSF was evaluated. this was done by calculating the ratio PS/PL (production in SSF/production in LSF), which was called relative production. Clones were isolated from each strain and evaluated in a similar way. The strains presented different relative productions (from 1.4 to 2.5). Within the clones, a much wider range of relative productions was observed (0.6 to 16.7). On the other hand, the highest-producing strains in LSF were also the highest producers in SSF. This indicates that the production potential of a strain is an important factor in its production level in SSF. Moreover, the highest penicillin producing ciones (9,500 to 10,500 microg of penicillin/g were generated from high-yielding strains (P2 and ASP-78). However, the higher-producing strains (in LSF) showed lower relative performance, suggesting that higher producing strains tend to express less efficiently their potential in SSF. In this study, several overproducing clones, particularly suited for SSF, were obtained by the procedures followed. Production increases of 500 to 600 %, in this culture system, were achieved.

Oxidative state in idiophase links reactive oxygen species (ROS) and lovastatin biosynthesis: differences and similarities in submerged- and solid-state fermentations.

The present work was focused on finding a relationship between reactive oxygen species (ROS) and lovastatin biosynthesis (secondary metabolism) in Aspergillus terreus. In addition, an effort was made to find differences in accumulation and control of ROS in submerged (SmF) and solid-state fermentation (SSF), which could help explain higher metabolite production in the latter. sod1 expression, ROS content, and redox balance kinetics were measured during SmF and SSF. Results showed that A. terreus sod1 gene (oxidative stress defence enzyme) was intensely expressed during rapid growth phase (trophophase) of lovastatin fermentations. This high expression decreased abruptly, just before the onset of production (idiophase). However, ROS measurements detected high concentrations only in idiophase, suggesting a link between ROS and lovastatin biosynthesis. Apparently sod1 down regulation promotes the rise of ROS during idiophase. This oxidative state in idiophase was further supported by a high redox balance observed in trophophase that changed to a low value in idiophase (around six-fold lower). The patterns of ROS accumulation, sod1 expression, and redox balance behaviour were similar in SmF and SSF. However, sod1 expression and ROS concentration (ten-fold), were higher in SmF. Our results indicate a link between ROS and lovastatin biosynthesis. Also, showed differences of physiology in SSF that yield lower but more steady ROS concentrations, which could be associated to higher lovastatin production.

Respiration studies of penicillin solid-state fermentation.

An earlier work showed that when the bagasse content (BC) of the solid medium was decreased within a wide range of values, penicillin production by solid-state fermentation was always increased. Respiration studies were performed to understand how BC controls the secondary metabolism in this culture system. CO2 production of solid cultures with different compositions was monitored. In cultures of series A, the initial moisture content was increased and this variation was compensated by decreasing the nutrient and BC of the medium. In series B the initial moisture content was increased while BC was decreased and the nutrient content increased. In addition, penicillin production and respiration was also studied in extreme media (dry and concentrated and humid and diluted), with high and low BC. Criteria for the interpretation of respiration kinetics of the idiophase were established for the first time in this work. For the cumulative form (total CO2/g dry matter vs t) as well as for derivative (CO2/g dm/h vs t) respiration kinetics, the CO2 production rate (Q(CO2)) was determined by calculating the slope of the cumulative curve. Results indicate that Q(CO2) of the tropho- and idiophases was directly related to the BC of the solid medium (and inversely related to penicillin yields). These conclusions were confirmed by analysis of the derivative form, the results of which indicate that a lower but stable metabolic activity is essential for obtaining high penicillin yields in solid-state fermentation (SSF). The results indicate that the derivative CO2 production kinetics proved to be a more precise and sensitive indicator of the culture metabolic activity during idiophase than the cumulative respiration kinetics.

Optimization of bagasse, nutrients and initial moisture ratios on the yield of penicillin in solid-state fermentation

An advanced solid-state fermentation (SSF) system (liquid medium absorbed on an inert support) has been applied to antibiotic production. The main components of this solid medium are: support (sugarcane bagasse), nutrients and water. The first two are solids and have to be considered to calculate the initial moisture content (IMC) of the medium. Earlier work indicated the importance of using high IMCs and concentrated media to obtain high penicillin yields in SSF. Nevertheless, the present work shows that high values of IMC or nutrients content can stimulate or inhibit penicillin production, depending on the strategy used to compensate this change (i.e. the proportions of the other two components). Conversely, increasing bagasse content always showed an inhibitory effect on the production. Since penicillin production depends on the combinations of these components, a global approach was used. The effect of the proportions of the three components on penicillin production was studied by means of a triangle of combinations and a 3D graph. It was possible to establish that high penicillin production is only obtained in a zone of low support content (10–12.5%). Surprisingly, one production maximum was observed in a zone of low moisture and high nutrients content (62 and 25.5% respectively); and another one in a zone of high moisture and a relatively low nutrients concentration (75.5 and 12.4% respectively).

The Streptomyces coelicolor genome encodes a type I ribosome-inactivating protein

Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in numerous plants, but also constitute a subunit of the bacterial Shiga toxin. Classification of plant RIPs is based on the absence (type I) or presence (type II) of an additional lectin module. In Shiga toxin, sugar binding is mediated by a distinct RIP-associated homopentamer. In the genome of two actinomycetes, we identified RIP-like proteins that resemble plant type I RIPs rather than the RIP subunit (StxA) of Shiga toxin. Some representatives of β- and γ-proteobacteria also contain genes encoding RIP-like proteins, but these are homologous to StxA. Here, we describe the isolation and initial characterization of the RIP-like gene product SCO7092 (RIPsc) from the Gram-positive soil bacterium Streptomyces coelicolor. The ripsc gene was expressed in Escherichia coli as a recombinant protein of about 30 kDa, and displayed the characteristic N-glycosidase activity causing specific rRNA depurination. In Streptomyces lividans and E. coli, RIPsc overproduction resulted in a dramatic decrease in the growth rate. In addition, intracellular production was deleterious for Saccharomyces cerevisiae. However, when applied externally to microbial cells, purified RIPsc did not display antibacterial or antifungal activity, suggesting that it cannot enter these cells. In a cell-free system, however, purified S. coelicolor RIPsc protein displayed strong inhibitory activity towards protein translation.

Ribosome-inactivating proteins with an emphasis on bacterial RIPs and their potential medical applications

Ribosome-inactivating proteins (RIPs) are toxic due to their N-glycosidase activity catalyzing depurination at the universally conserved α-sarcin loop of the 60S ribosomal subunit. In addition, RIPs have been shown to also have other enzymatic activities, including polynucleotide:adenosine glycosidase activity. RIPs are mainly produced by different plant species, but are additionally found in a number of bacteria, fungi, algae and some mammalian tissues. This review describes the occurrence of RIPs, with special emphasis on bacterial RIPs, including the Shiga toxin and RIP in Streptomyces coelicolor recently identified in S. coelicolor. The properties of RIPs, such as enzymatic activity and targeting specificity, and how their unique biological activity could be potentially turned into medical or agricultural tools to combat tumors, viruses and fungi, are highlighted.

Expression of the bacterial hemoglobin gene from Vitreoscilla stercoraria increases rifamycin B production in Amycolatopsis mediterranei.

It is well known that the culture for rifamycin B production by Amycolatopsis mediterranei requires high levels of dissolved oxygen, particularly in industrial processes. In this study, we report the construction of a vector for the expression of the bacterial hemoglobin gene (vhb) from Vitreoscilla stercoraria in a rifamycin B-overproducing strain of A. mediterranei. The effect was evaluated in the presence and absence of barbital. The vhb gene was cloned under the control of the PermE promoter, the Amycolatopsis lactamdurans plasmid pULVK2 origin of replication, the kanamycin-resistant gene (Km), the erythromycin-resistant gene (ermE) for selection, and ColE1. Industrial fermentation conditions were simulated in shake-flask cultures. Under low aeration, the transformed A. mediterranei strain with the vhb gene showed a 13.9% higher production of rifamycin B in a culture with barbital compared with the parental strain, and 29.5% higher production under the same conditions without barbital.

Biochemical mechanism of the effect of barbital on rifamycin B biosynthesis by Amycolatopsis mediterranei (M18 strain)

It is well known that 5,5-diethylbarbituric acid (barbital) in the culture medium can stimulate the production of rifamycin B by Amycolatopsis mediterranei, particularly in industrial processes. However, the mechanism by which barbital exerts this effect is unknown. Results in this work show that the barbital effect is only evident under low aeration conditions (50-ml microfermentors with 7 ml of medium, 0.08 l/h air flow). Under these conditions, cultures with barbital showed similar CO2 production (in relation to a control without barbital), but higher oxygen uptake indicated that the extra O2 consumed was used in the increased rifamycin biosynthesis. Moreover, using a resting cell system where no antibiotic is produced, it was possible to show that barbital inhibits the respiratory chain, since O2 uptake decreased by 30%. Finally, we present biochemical results that suggest that a cytochrome P450-type monoxygenase, which can use atmospheric oxygen, is induced by barbital in an industrial-type strain of A. mediterranei.