Role of AtYap1 in the reactive oxygen species regulation of lovastatin production in Aspergillus terreus.

Lovastatin has great medical and economic importance, and its production in Aspergillus terreus is positively regulated at transcriptional level, by reactive oxygen species (ROS) generated during idiophase. To investigate the role of the transcription factor Yap1 in the regulation of lovastatin biosynthesis by ROS, an orthologue of yap1 was identified in A. terreus TUB F-514 and knocked down (silenced) by RNAi. Results confirmed that the selected knockdown strain (Siyap1) showed decreased yap1 expression in both culture systems (submerged and solid-state fermentation). Transformants showed higher sensitivity to oxidative stress. Interestingly, knockdown mutant showed higher ROS levels in idiophase and an important increase in lovastatin production in submerged and solid-state fermentations: 60 and 70% increase, respectively. Furthermore, sporulation also increased by 600%. This suggested that AtYap1 was functioning as a negative regulator of the biosynthetic genes, and that lack of AtYap1 in the mutants would be derepressing these genes and could explain increased production. However, we have shown that lovastatin production is proportional to ROS levels, so ROS increase in the mutants alone could also be the cause of production increase. In this work, when ROS levels were decreased with antioxidant, to the levels shown by the parental strain, the lovastatin production and kinetics were similar to the ones of the parental strain. This means that AtYap1 does not regulate lovastatin biosynthetic genes, and that production increase observed in the knockdown strain was an indirect effect caused by ROS increase. This conclusion is compared with studies on other secondary metabolites produced by other fungal species. KEY POINTS: • ROS regulates lovastatin biosynthesis at transcriptional level, in solid-state, and in submerged fermentations. • ATyap1 knockdown mutants showed important lovastatin production increases (60 and 70%) and higher ROS levels. • When ROS were decreased in the silenced mutant to the parental strain's level, lovastatin kinetics were identical to the parental strain's.

Novel antimicrobial activity of protein produced by Streptomyces lividans TK24 against the phytopathogen Clavibacter michiganensis.

Antimicrobial proteins and peptides are an alternative to current antibiotics. Here, we report an antimicrobial activity in a low-molecular-weight protein secreted naturally by Streptomyces lividans TK24 when glucose or glycerol were used as carbon sources. The antimicrobial activity was demonstrated against Bacillus subtilis, Bacillus cereus, Kokuria rhizophila, Clostridium sporogenes and Clavibacter michiganensis, causal pathogen of tomato bacterial canker; one of the most destructive bacterial diseases of this crop. The protein fraction with antimicrobial activity was identified and quantified by LC-MS/MS. From a total of 155 proteins, 11 were found to be within the range of 11.3-13.9 kDa of which four proteins were selected by functional analysis as possibly responsible for the antimicrobial activity. Protein fractionation, correlation analysis between antimicrobial activity and abundance of selected proteins, as well as transcriptional expression analysis, indicate that 50S ribosomal protein L19 is the main candidate responsible for antimicrobial activity.

New knowledge about the biosynthesis of lovastatin and its production by fermentation of Aspergillus terreus.

Lovastatin, and its semisynthetic derivative simvastatine, has great medical and economic importance, besides great potential for other uses. In the last years, a deeper and more complex view of secondary metabolism regulation has emerged, with the incorporation of cluster-specific and global transcription factors, and their relation to signaling cascades, as well as the new level of epigenetic regulation. Recently, a new mechanism, which regulates lovastatin biosynthesis, at transcriptional level, has been discovered: reactive oxygen species (ROS) regulation; also new unexpected environmental stimuli have been identified, which induce the synthesis of lovastatin, like quorum sensing-type molecules and support stimuli. The present review describes this new panorama and uses this information, together with the knowledge on lovastatin biosynthesis and genomics, as the foundation to analyze literature on optimization of fermentation parameters and medium composition, and also to fully understand new strategies for strain genetic improvement. This new knowledge has been applied to the development of more effective culture media, with the addition of molecules like butyrolactone I, oxylipins, and spermidine, or with addition of ROS-generating molecules to increase internal ROS levels in the cell. It has also been applied to the development of new strategies to generate overproducing strains of Aspergillus terreus, including engineering of the cluster-specific transcription factor (lovE), global transcription factors like the ones implicated in ROS regulation (or even mitochondrial alternative respiration aox gen), or the global regulator LaeA. Moreover, there is potential to apply some of these findings to the development of novel unconventional production systems. KEY POINTS: • New findings in regulation of lovastatin biosynthesis, like ROS regulation. • Induction by unexpected stimuli: autoinducer molecules and support stimuli. • Recent reports on culture medium and process optimization from this stand point. • Applications to molecular genetic strain improvement methods and production systems.

Penicillin and cephalosporin biosyntheses are also regulated by reactive oxygen species.

In an earlier work on lovastatin production by Aspergillus terreus, we found that reactive oxygen species (ROS) concentration increased to high levels precisely at the start of the production phase (idiophase) and that these levels were sustained during all idiophase. Moreover, it was shown that ROS regulate lovastatin biosynthesis. ROS regulation has also been reported for aflatoxins. It has been suggested that, due to their antioxidant activity, aflatoxins are regulated and synthesized like a second line of defense against oxidative stress. To study the possible ROS regulation of other industrially important secondary metabolites, we analyzed the relationship between ROS and penicillin biosynthesis by Penicillium chrysogenum and cephalosporin biosynthesis by Acremonium chrysogenum. Results revealed a similar ROS accumulation in idiophase in penicillin and cephalosporin fermentations. Moreover, when intracellular ROS concentrations were decreased by the addition of antioxidants to the cultures, penicillin and cephalosporin production were drastically reduced. When intracellular ROS were increased by the addition of exogenous ROS (H2O2) to the cultures, proportional increments in penicillin and cephalosporin biosyntheses were obtained. It was also shown that lovastatin, penicillin, and cephalosporin are not antioxidants. Taken together, our results provide evidence that ROS regulation is a general mechanism controlling secondary metabolism in fungi.

Improving rifamycin production in Amycolatopsis mediterranei by expressing a Vitreoscilla hemoglobin (vhb) gene fused to a cytochrome P450 monooxygenase domain.

Expression of the vhb gene encoding hemoglobin from Vitreoscilla stercoraria in several organisms, clearly enhances oxygen-dependent product formation. In a previous work, we expressed the vhb gene that encodes hemoglobin from V. stercoraria in Amycolatopsis mediterranei, resulting in an increase (oxygen-dependent formation) in rifamycin B production. In the present work, we first confirm; by heterologous expression in Escherichia coli, that rif-orf5 from the rifamycin biosynthetic gene cluster, really encodes a cytochrome P450 enzyme, which is the key step for oxygen incorporation in the final biosynthetic product. Likewise, we fused rif-orf5 to the vhb gene, as part of a genetic engineering strategy. The fused genes were used to generate an Amycolatopsis mediterranei transformant (Msb-HbCYP5). Interestingly, the fermentation of Msb-HbCYP5 manifested 1.5-fold higher rifamicin B production than the transformant with only the hemoglobin gene, and 2.2-fold higher than the parental strain.

Key role of alternative oxidase in lovastatin solid-state fermentation.

Lovastatin is a commercially important secondary metabolite produced by Aspergillus terreus, either by solid-state fermentation or by submerged fermentation. In a previous work, we showed that reactive oxygen species (ROS) accumulation in idiophase positively regulates lovastatin biosynthetic genes. In addition, it has been found that lovastatin-specific production decreases with aeration in solid-state fermentation (SSF). To study this phenomenon, we determined ROS accumulation during lovastatin SSF, under high and low aeration conditions. Paradoxically, high aeration caused lower ROS accumulation, and this was the underlying reason of the aeration effect on lovastatin production. Looking for a mechanism that is lowering ROS production under those conditions, we studied alternative respiration. The alternative oxidase provides an alternative route for electrons passing through the electron transport chain to reduce oxygen. Here, we showed that an alternative oxidase (AOX) is expressed in SSF, and only during idiophase. It was shown that higher aeration induces higher alternative respiration (AOX activity), and this is a mechanism that limits ROS generation and keeps them within healthy limits and adequate signaling limits for lovastatin production. Indeed, the aox gene was induced in idiophase, i.e., at the time of ROS accumulation. Moreover, exogenous ROS (H2O2), added to lovastatin solid-state fermentation, induced higher AOX activity. This suggests that high O2 availability in SSF generates dangerously high ROS, so alternative respiration is induced in SSF, indirectly favoring lovastatin production. Conversely, alternative respiration was not detected in lovastatin-submerged fermentation (SmF), although exogenous ROS also induced relatively low AOX activity in SmF.

Streptomyces as Overexpression System for Heterologous Production of an Antimicrobial Peptide.

BACKGROUND: Antimicrobial peptides could be used in several fields of application, and large quantities of antimicrobial peptides would be required. However, their production is very expensive; this is why a suitable production method, alternative to traditional chemical synthesis is necessary. Production of recombinant antimicrobial peptides in prokaryotic systems has demonstrated the viability of this approach. Nevertheless, expression of antimicrobial peptides in Escherichia coli an others microorganisms is potentially limited due to their toxicity to host cells and susceptibility to proteolytic degradation. As an alternative, we describe a successful antimicrobial peptide production system in Streptomyces lividans which showed to be effective for the secretion of large quantities of cationic antimicrobial peptides. OBJECTIVE: Therefore, as a solution to the difficulties for heterologous expression of CAP we demonstrate efficient production by S. lividans. METHOD: In this study, a strategy for CAP overexpression is presented based on the construction of an expression cassette for Streptomyces lividans TK24. For the construction of this cassette, the peptide of interest was fused to the vsi promoter and signal sequence (vsi-ss) of the subtilisin inhibitor from Streptomyces venezuelae CBS762.70, which is a signal peptide with a proven high secretion efficiency. The cloning vector used was pIJ486, which includes a transcription terminator sequence and a thiostrepton resistance marker. This system contains elements that allow the increase of the efficiency of the peptide's expression. RESULTS: The production system allows the efficient secretion of the peptide to the growth medium, thereby simplifying its recovery and avoiding its toxic effect on the producing organism. The production obtained demonstrated the system's efficiency by achieving a peptide concentration of 11.61 mg/ml. This represents at least a 10-fold increase compared to previously established strategies. CONCLUSION: The expression system constructed may facilitate the production of large amounts of peptides with antimicrobial activity.

Effects of neonatal treatment with two phytoestrogens on male rat sexual behavior and partner preference.

The aim of this work was to compare the effect of neonatal treatment with the phytoestrogens coumestrol (COU) and genistein (GEN), administered in equimolecular doses, on the sexual behavior and partner preference of male rats. Four groups of male rats were injected daily from day 1 to 5 with 150 µg of GEN, an equivalent amount of COU, 1 µg of ß-estradiol 3-benzoato (EB), or olive oil (VEH) (control). A fifth group remained intact. In the GEN group, intromission and ejaculation latencies decreased, whereas ejaculatory frequency increased. Contrasting results were observed in COU males. EB males could not ejaculate and their mount and intromission latencies increased significantly. To determine sexual-partner preferences, a multiple partner preference arena was used and two types of tests were performed, the first one without allowing contact test (CT) with the stimulus animals, followed by a CT. COU and GEN groups did not show preference for any stimulus animal, whereas the EB males preferred the expert male. When CT with the stimulus animals was allowed, GEN-males preferred the receptive female, unlike the COU and EB groups. It is concluded that neonatal treatment with COU and GEN induced opposite effects, the effects of COU being more estrogenic.

Targeting the Osmotic Stress Response for Strain Improvement of an Industrial Producer of Secondary Metabolites.

The transition from primary to secondary metabolism in antibiotic-producing Streptomyces correlates with expression of genes involved in stress responses. Consequently, regulatory pathways that regulate specific stress responses are potential targets to manipulate to increase antibiotic titers. In this study, genes encoding key proteins involved in regulation of the osmotic stress response in Streptomyces avermitilis, the industrial producer of avermectins, are investigated as targets. Disruption of either osaBSa, encoding a response regulator protein, or osaCSa, encoding a multidomain regulator of the alternative sigma factor SigB, led to increased production of both oligomycin, by up to 200%, and avermectin, by up to 37%. The mutations also conditionally affected morphological development; under osmotic stress, the mutants were unable to erect an aerial mycelium. In addition, we demonstrate the delivery of DNA into a streptomycete using biolistics. The data reveal that information on stress regulatory responses can be integrated in rational strain improvement to improve yields of bioactive secondary metabolites.

Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum.

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and ß-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of ß-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and ß-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.

Reactive oxygen species regulate lovastatin biosynthesis in Aspergillus terreus during submerged and solid-state fermentations.

In a previous work we detected an important increase in reactive oxygen species (ROS) concentrations during idiophase in lovastatin fermentations. Hence, the objective of the present work was to determine if ROS contributes to the regulation of lovastatin biosynthesis. Exogenous antioxidants were used to reduce ROS accumulation. The addition of N-Acetyl-L-cysteine (NAC) decreased ROS accumulation and concurrent lovastatin production. In solid-state fermentation (SSF), the addition of 100 mM of NAC lowered ROS accumulation by 53%, together with a 79% decrease in lovastatin biosynthesis. A similarly, situation was observed in submerged fermentation (SmF). Decreased lovastatin production was due to a lower expression of the regulatory gene lovE, and gene lovF. Moreover, the addition of H2O2 to the culture caused precocious gene expression and lovastatin biosynthesis. These results indicate that ROS accumulation in idiophase contributes to the regulation of the biosynthetic genes. It was considered that Yap1 (Atyap1) could be a transcription factor linking ROS with lovastatin biosynthesis. In a Northern analysis, Aspergillus terreus yap1 gene (Atyap1) was highly expressed during trophophase but down regulated during idiophase. Conversely, expression pattern of srrA gene, suggested that SrrA could positively control lovastatin biosynthesis, and also explaining the characteristics of the biosynthesis in SSF.

Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of β-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and β-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.

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.

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.

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 medium's 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.

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.

RifP; a membrane protein involved in rifamycin export in Amycolatopsis mediterranei.

The rifamycin gene cluster in Amycolatopsis mediterranei includes the gene rifP, whose role in antibiotic production has not yet been established. In this work, the rifP gene was silenced and the results indicated that it codes for a protein to export rifamycin, avoiding its accumulation inside the cell. An antisense cassette was constructed by inserting the rifP gene in an antisense orientation downstream from the modified ermE* promoter, and upstream of the Tasd terminator (aspartate semialdehyde dehydrogenase of A. lactamdurans). Partial silencing of the rifP gene by the use of the antisense cassette, cloned in the plasmid pUAMAE5, resulted in a 70% decrease in the extracellular rifamycin B. A protein of 53 kDa was absent in the membrane fraction of the silenced strain. This is the same size of the expected product from the rifP gene. The 2D structure analysis indicated it belongs to a Drug:H+ antiporter family which includes a wide number of membrane transport proteins.

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.