Javier Barrios-González

Biotechnological production and applications of statins

Statins are a group of extremely successful drugs that lower cholesterol levels in blood; decreasing the risk of heath attack or stroke. In recent years, statins have also been reported to have other biological activities and numerous potential therapeutic uses. Natural statins are lovastatin and compactin, while pravastatin is derived from the latter by biotransformation. Simvastatin, the second leading statin in the market, is a lovastatin semisynthetic derivative. Lovastatin is mainly produced by Aspergillus terreus strains, and compactin by Penicillium citrinum. Lovastatin and compactin are produced industrially by liquid submerged fermentation, but can also be produced by the emerging technology of solid-state fermentation, that displays some advantages. Advances in the biochemistry and genetics of lovastatin have allowed the development of new methods for the production of simvastatin. This lovastatin derivative can be efficiently synthesized from monacolin J (lovastatin without the side chain) by a process that uses the Aspergillus terreus enzyme acyltransferase LovD. In a different approach, A. terreus was engineered, using combinational biosynthesis on gene lovF, so that the resulting hybrid polyketide synthase is able to in vivo synthesize 2,2-dimethylbutyrate (the side chain of simvastatin). The resulting transformant strains can produce simvastatin (instead of lovastatin) by direct fermentation.

Penicillin production by solid state fermentation

SummaryPenicillin was produced by a non-sterile solid state fermentation (SSF) on bagasse impregnated with culture medium. The use of concentrated media greatly enhanced the antibiotic production in this system. It was observed that adequate initial moisture content (70%) of the impregnated solid medium results in higher production. A comparison between solid and liquid fermentation showed superior yield and productivity.

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.

High lovastatin production by Aspergillus terreus in solid-state fermentation on polyurethane foam: An artificial inert support

A novel solid-state fermentation (SSF) process, using high-density polyurethane foam (PUF) as an inert support, was developed for the production of lovastatin. Results indicated that forced aeration is not conducive to metabolite production since it reduces the solid mediums moisture content. The highest level of production was achieved in closed flasks (CF), in which 7.5 mg of lovastatin was generated per gram of dry culture, equivalent to 493 mug/mg dry mycelium. However, since mycelial growth is aeration-dependent, the CF cultures presented the lowest level of growth: 15.19 mg/gdc (milligrams per gram of dry culture). It was possible to increase the biomass concentration to 24.4 mg/gdc by increasing the culture medium concentration to 2.5x and the initial moisture content of the solid medium to 85%. Results also revealed that the density of the culture support is a key parameter in determining lovastatin production; high yields were only obtained on PUF at a density of 17 or 20 kg/m(3). SSF using the latter reached a lovastatin level of 19.95 mg/gdc, with specific production of 815 mug/mg dry mycelium. A comparative study showed that lovastatin production during PUF SSF was two-fold higher than that of the better-known system of bagasse SSF. Moreover, lovastatin yields on PUF were 30 times higher than those of liquid submerged fermentation (SmF; 0.57 mg/ml) and lovastatin biomass was almost 15 times more productive.

Germination of concentrated suspensions of spores from Aspergillus niger

SummaryGermination of condiospores fromA.niger in very concentrated suspension was required to inoculate solid fermentation media, but a germination self-inhibitor was detected in spores. It was found that the inhibitory effect depended on the composition of the medium and was reduced when glucose was used as a carbon source. The effect of the self-inhibitor was eliminated by washing the spores and using glucose and a protein nitrogen source in the germination medium. By this method it was possible to increase about 100 times (106 to 108) spore concentration, keeping more than 90% germination.

Elimination of the associated microbial community and bioencapsulation of bacteria in the rotifer Brachionus plicatilis

The bioencapsulation of live bacteria in the rotifer Brachionus plicatilis was determined under monoxenic conditions. The first objective was to evaluate the microbiota of the rotifer during intensive production and to obtain sterile rotifer cultures starting from adult females or amictic eggs using PVP-Iodine, Hydrogen peroxide or antibiotic mixtures. In the rotifers, the proportion of vibrios increased significantly during the mass production, displacing other unidentified marine bacteria. Rotifers, in the absence of culturable bacteria were obtained starting from amictic eggs and using Trimetroprim-sulfametoxasole (Bactrim Roche®) at 10 ml l−1. The effect of members of Vibrionaceae on the survival and growth rate of rotifers was determined under monoxenic conditions. The survival of rotifers was not affected in the presence of different isolates, while amictic egg formation occurred and the populations increased when the strains Vibrio proteolyticus C279 and Aeromonas media C226 were tested. All isolates were successfully incorporated in the rotifers, since there was no significant difference between the numbers of bioencapsulated cells of different strains of isolates. The results show that it is possible to replace the microbial community in rotifer cultures, started from disinfected amictic eggs, with selected bacterial strains. This could be used as a tool for future studies to reveal the role of specific bacteria on first larval stages of marine fish species.

An isolate of Rhizopus nigricans capable of tolerating and removing pentachlorophenol

Rhizopusnigricans, isolated from an industrial effluent (paper mill), was resistant to pentachlorophenol (PCP) in Petri dishes and in submerged cultures (100 and 25 mg l−1 respectively). It was shown that this strain of R. nigricans can remove PCP in submerged culture. When 12.5 mg of PCPl−1 were added at 48 h, this compound had been completely removed by 144h. Results indicated that the fungus did not produce extracellular lignin peroxidase (LiP) and laccase, but extracellular phenoloxidase production was observed. The synthesis of the latter enzyme was stimulated by the presence of PCP and/or tyrosine. These results indicate that this fungus, and probably other filamentous fungi, have an interesting potential to be used in processes for chlorophenol biodegradation.

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.