The Biosynthesis of Penicillin and Cephalosporin C are Regulated by ROS at Transcriptional Level.

In a recent work we showed that, besides lovastatin, ROS also accumulate during the production phase in Pencillium chrysogenum and in Acremonium chrysogenum, and that these ROS regulate the biosynthesis of penicillin and cephalosporin C. In the present study, we investigated the level at which this positive regulation is exerted. Internal ROS levels were manipulated, i.e., increased or decreased, in the production phase of the respective fermentations. Penicillin production decreased by 51.2% when internal ROS concentration was diminished by 50%, while a 62% production increase was observed when ROS were increased (62%). Similarly, Cephalosporin production decreased (35%) with antioxidants and increased (54.1%) with exogenous ROS. Expression analysis of the respective pcbAB genes, encoding the non-ribosomal peptide synthetase enzymes, was performed. Results showed down regulation of these genes in fermentations with lower ROS content, and upregulation in the cultures with higher ROS content, in both species. This showed that ROS regulation of penicillin in P. chrysogenum and of cephalosporin C in A. chrysogenum, is exerted at transcriptional level. In silico analysis of the pcbAB gene promoters in both species, suggested that this regulation could be mediated by stress-response transcription factors like Yap1, SrrA and/or MsnA, and/or by the Hap complex.

bZIP transcription factors PcYap1 and PcRsmA link oxidative stress response to secondary metabolism and development in Penicillium chrysogenum.

BACKGROUND: Reactive oxygen species (ROS) trigger different morphogenic processes in filamentous fungi and have been shown to play a role in the regulation of the biosynthesis of some secondary metabolites. Some bZIP transcription factors, such as Yap1, AtfA and AtfB, mediate resistance to oxidative stress and have a role in secondary metabolism regulation. In this work we aimed to get insight into the molecular basis of this regulation in the industrially important fungus Penicillium chrysogenum through the characterization of the role played by two effectors that mediate the oxidative stress response in development and secondary metabolism. RESULTS: In P. chrysogenum, penicillin biosynthesis and conidiation are stimulated by the addition of H2O2 to the culture medium, and this effect is mediated by the bZIP transcription factors PcYap1 and PcRsmA. Silencing of expression of both proteins by RNAi resulted in similar phenotypes, characterized by increased levels of ROS in the cell, reduced conidiation, higher sensitivity of conidia to H2O2 and a decrease in penicillin production. Both PcYap1 and PcRsmA are able to sense H2O2-generated ROS in vitro and change its conformation in response to this stimulus. PcYap1 and PcRsmA positively regulate the expression of brlA, the first gene of the conidiation central regulatory pathway. PcYap1 binds in vitro to a previously identified regulatory sequence in the promoter of the penicillin gene pcbAB: TTAGTAA, and to a TTACTAA sequence in the promoter of the brlA gene, whereas PcRsmA binds to the sequences TGAGACA and TTACGTAA (CRE motif) in the promoters of the pcbAB and penDE genes, respectively. CONCLUSIONS: bZIP transcription factors PcYap1 and PcRsmA respond to the presence of H2O2-generated ROS and regulate oxidative stress response in the cell. Both proteins mediate ROS regulation of penicillin biosynthesis and conidiation by binding to specific regulatory elements in the promoters of key genes. PcYap1 is identified as the previously proposed transcription factor PTA1 (Penicillin Transcriptional Activator 1), which binds to the regulatory sequence TTAGTAA in the pcbAB gene promoter. This is the first report of a Yap1 protein directly regulating transcription of a secondary metabolism gene. A model describing the regulatory network mediated by PcYap1 and PcRsmA is proposed.

Environmental cues that induce the physiology of solid medium: a study on lovastatin production by Aspergillus terreus.

AIMS: The aim of this work was to identify the main environmental factors that induce the special physiology displayed by fungi growing in solid culture-that is, higher secondary metabolite (SM) production-compared with those in submerged culture. METHODS AND RESULTS: Lovastatin-specific production (SP) was used as an indicator of the physiological status, and different model culture systems were used to evaluate the impact of potential solid-state fermentation (SSF) environmental stimuli. Direct contact with air was identified as an important stimulus. Cultures with two or more hours of exposure to air showed typical SSF lovastatin SP (1462% higher than cultures exposed for 0·08 h). Intermediate times of exposure generated intermediate physiological states. Support-related stimuli also induced higher lovastatin SP, even in a liquid environment (679% increase). CONCLUSIONS: Direct contact with air, as well as support-related stimuli, are major environmental cues that induce the physiology of solid medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This knowledge is the starting point to investigate how these environmental cues are sensed and transduced, impacting SM and enzyme production. These results have important applied potential in new strategies to generate overproducing strains, as well as application in the design of novel production systems.

Solid-state and submerged fermentations show different gene expression profiles in cephalosporin C production by Acremonium chrysogenum.

Despite the importance of Acremonium chrysogenum as the only cephalosporin C (CPC) producer, there is still a limited understanding about the molecular mechanisms regulating antibiotic biosynthesis in this fungus. Based on the previously described relationship between environmental pH and antibiotic production in numerous filamentous fungi, we studied the expression of genes related to CPC production in A. chrysogenum. We report for the first time similarities and differences, characterizing CPC production by A. chrysogenum under a variable pH environment, in submerged and solid-state fermentation. This characterization is supported by measurements of parameters, like CPC production, pH, growth, and expression levels of several genes involved, directly or indirectly, in CPC production. Interesting differences in intermediate (Pen N) and certain biosynthetic gene expression levels were observed. Our results point out some relationships between physiological features and gene expression that open important improvement perspectives for both culture systems.

Lovastatin biosynthetic genes of Aspergillus terreus are expressed differentially in solid-state and in liquid submerged fermentation.

Molecular studies were performed to establish the causes of the superior lovastatin productivity of a novel solid-state fermentation (SSF) process, in relation with liquid submerged fermentation (SmF; 20 mg/g vs. 0.65 mg/ml). In SSF, biosynthetic genes lovE and lovF transcripts accumulated to high levels from day 1 to day 7. In this period, lovE transcript showed 4.6-fold higher accumulation levels (transcription) than the highest level detected in SmF (day 5). lovF transcript showed two-fold higher expression than the highest point in SmF. In SmF, the expression was only detected clearly on day 5 and, showing a 50% decrease, on day 7. These results show that the higher lovastatin production in SSF is related to a more intense transcription of these biosynthetic genes. A strong expression of gldB gene in lovastatin SSF indicated that Aspergillus terreus senses osmotic stress during the course of SSF, but not in SmF. However, when a liquid medium of identical concentration was used in SmF, lovastatin production decreased in SSF.

Influence of pH regulation and nutrient content on cephalosporin C production in solid-state fermentation by Acremonium chrysogenum C10.

AIMS: To investigate the effect of pH regulation and nutrient concentration on cephalosporin C (CPC) production in solid-state fermentation (SSF), using sugarcane bagasse as inert support, impregnated with liquid medium. METHODS AND RESULTS: Solid-state fermentation using different initial pH values, buffer and nutrient concentrations were performed. Results revealed pH as a key parameter in CPC SSF, as it hampered the antibiotic production not only above 7.8, but also under 6.4. Using initial pH lower than 6.8 and PB in the solid medium, it was possible to keep pH within the production range, increase the production period (from 1 to 3 days) and hence the CPC yield from 468 to 3200 microg gdm(-1) (g(-1) of dry matter). CONCLUSION: Parameters that help to keep pH in adequate values for CPC production in SSF, such as initial pH, buffering system and nutrient concentration, can greatly increase the production time and CPC yields in this fermentation technique. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on CPC production on impregnated support, and the only one revealing pH as a key parameter; it is also shown that high nutrient concentration can improve CPC yields in SSF as long as pH is kept under control.

A fungal phenoloxidase (tyrosinase) involved in pentachlorophenol degradation.

Amylomyces rouxii eliminated 85% of initial pentachlorophenol (PCP) at 12.5 mg l(-1) when grown with 0.1 g tyrosine l(-1), but only 55% without tyrosine. Addition of tyrosine in the culture medium increased the monophenolase activity by 1.8-fold. Tyrosinase is thus indicated to be the phenoloxidase involved in PCP degradation by A. rouxii .

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.

Overproduction of rifamycin B by Amycolatopsis mediterranei and its relationship with the toxic effect of barbital on growth.

A novel method for selecting overproducing strains of rifamycin B was developed. This technique involves the use of lysozyme and the effect of barbital on the growth of A. mediterranei. Complete medium added with glycine and barbital was inoculated with mutagenized mycelium, incubated for 48 hours and treated with lysozyme. The lysozyme resistant mycelium was washed with dilute detergent. Complete medium with glycine but without barbital was inoculated with the washed mycelium. Protoplasts were obtained and regenerated and the colonies were picked and seeded on Bennet agar plates with and without barbital. Two selected mutants were sensitive to 0.5% barbital producing 200% more rifamycin than the parental strain. In addition, 30 barbital resistant mutants were isolated and their production level was lower than the one observed with the parental strain. These results suggest that the effect of barbital on secondary metabolism (rifamycin production) is related to its effect on primary metabolism.

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.

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.

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.