Ana Lúcia Leitão

Potential of Penicillium species in the bioremediation field.

The effects on the environment of pollution, particularly that caused by various industrial activities, have been responsible for the accelerated fluxes of organic and inorganic matter in the ecosphere. Xenobiotics such as phenol, phenolic compounds, polycyclic aromatic hydrocarbons (PAHs), and heavy metals, even at low concentrations, can be toxic to humans and other forms of life. Many of the remediation technologies currently being used for contaminated soil and water involve not only physical and chemical treatment, but also biological processes, where microbial activity is the responsible for pollutant removal and/or recovery. Fungi are present in aquatic sediments, terrestrial habitats and water surfaces and play a significant part in natural remediation of metal and aromatic compounds. Fungi also have advantages over bacteria since fungal hyphae can penetrate contaminated soil, reaching not only heavy metals but also xenobiotic compounds. Despite of the abundance of such fungi in wastes, penicillia in particular have received little attention in bioremediation and biodegradation studies. Additionally, several studies conducted with different strains of imperfecti fungi, Penicillium spp. have demonstrated their ability to degrade different xenobiotic compounds with low co-substrate requirements, and could be potentially interesting for the development of economically feasible processes for pollutant transformation.

Hydroquinone: Environmental Pollution, Toxicity, and Microbial Answers

Hydroquinone is a major benzene metabolite, which is a well-known haematotoxic and carcinogenic agent associated with malignancy in occupational environments. Human exposure to hydroquinone can occur by dietary, occupational, and environmental sources. In the environment, hydroquinone showed increased toxicity for aquatic organisms, being less harmful for bacteria and fungi. Recent pieces of evidence showed that hydroquinone is able to enhance carcinogenic risk by generating DNA damage and also to compromise the general immune responses which may contribute to the impaired triggering of the host immune reaction. Hydroquinone bioremediation from natural and contaminated sources can be achieved by the use of a diverse group of microorganisms, ranging from bacteria to fungi, which harbor very complex enzymatic systems able to metabolize hydroquinone either under aerobic or anaerobic conditions. Due to the recent research development on hydroquinone, this review underscores not only the mechanisms of hydroquinone biotransformation and the role of microorganisms and their enzymes in this process, but also its toxicity.

Overexpression of the lat gene in Nocardia lactamdurans from strong heterologous promoters results in very high levels of lysine-6-aminotransferase and up to two-fold increase in cephamycin C production

Abstract The level of lysine-6-aminotransferase (encoded by the lat gene), an enzyme that commits lysine to the cephamycin biosynthesis pathway, is very low in wild type Nocardia lactamdurans. Two lat overexpression systems (pAMEXlat and pSAFlat) were constructed to express the promoterless lat gene of N. lactamdurans from the strong promoters amyP (of the α-amylase gene) and safP (of the secretion activating factor gene) of Streptomyces griseus. Both constructions led to very high levels of lysine-6-aminotransferase (between 8- and 15-fold) in the cells. Expression of lat from the amy promoter was optimal in glycerol-containing medium and was negatively regulated by glucose. The high levels of lysine-6-aminotransferase resulted in a 50–200% increase in cephamycin C production in the standard fermentation conditions. Onset of cephamycin C biosynthesis occurred at the same time in control and in lat-overexpressing strains, but the cephamycin production rate was clearly higher in transformants overexpressing the lat gene. Furthermore, HPLC analysis of cephamycin C in the culture broths revealed an early depletion of biosynthetic intermediates and an accumulation of cephamycin C when the lat gene was overexpressed. These results indicate that lysine-6-aminotransferase activity is limiting for cephamycin C biosynthesis under some culture conditions.

Interaction of the Two Proteins of the Methoxylation System Involved in Cephamycin C Biosynthesis IMMUNOAFFINITY, PROTEIN CROSS-LINKING, AND FLUORESCENCE SPECTROSCOPY STUDIES

Cephamycin C-producing microorganisms contain a two-protein enzyme system that converts cephalosporins to 7-methoxycephalosporins. Interaction between the two component proteins P7 (Mr 27,000) and P8 (Mr 32,000) has been studied by immunoaffinity chromatography using anti-P7 and anti-P8 antibodies, cross-linking with glutaraldehyde, and fluorescence spectroscopy analysis. Co-renaturation of the P7 and P8 polypeptides resulted in the formation of a protein complex with a molecular mass of 59 kDa, which corresponds to a heterodimer of P7 and P8. Glutaraldehyde cross-linking of the polypeptides after assembly of the protein complex showed the presence of a single heterodimer form that reacted with antibodies against P7 and P8. Each separate protein did not associate with itself into multimers. The P7·P8 complex co-purified by immunoaffinity chromatography from extracts of Nocardia lactamdurans and Streptomyces clavuligerus, suggesting that both proteins are present as an aggregate in vivo. Fluorescence spectroscopy studies of 5-methylaminonaphthalene-1-sulfonyl-P7 in response to increasing concentrations of P8 showed a blue shift in the fluorophore emission, indicating a conformational change of P7 in response to the interaction of P8 with an apparent dissociation constant of 47 μM. NADH showed affinity for the P7 component. The P7·P8 complex interacted strongly with the substrates S-adenosylmethionine and cephalosporin C, differently from that occurring with the separate P7 or P8 components, resulting in a strong blue shift in the fluorescence emission spectra of the complex.

Effect of amplification or targeted disruption of the β-lactamase gene of Nocardia lactamdurans on cephamycin biosynthesis

Abstract The bla gene of the cephamycin cluster of Nocardia lactamdurans has been subcloned in the shuttle plasmids pULVK2 and pULVK2A and amplified in N. lactamdurans LC411. The transformants showed two- to threefold higher β-lactamase activity. Formation of β-lactamase preceded the onset of cephamycin biosynthesis. The β-lactamase of N. lactamdurans inactivated penicillins and, to a lesser extent, cephalosporin C but did not hydrolyse cephamycin C. This β-lactamase was highly sensitive to clavulanic acid (50% inhibition was observed at 0.48 μg/ml clavulanic acid). The N. lactamdurans bla gene was disrupted in vivo by inertion of the kanamycin-resistance gene. Three bla-disrupted mutants, BD4, BD8 and BD12, were selected that lacked β-lactamase activity. Overexpresion of the bla gene resulted in N. lactamdurans transformants that were resistant to penicillin whereas mutants in which the bla gene was disrupted were supersensitive to this antibiotic. The three N. lactamdurans mutants with the bla gene disrupted showed a significant increase of cephamycin biosynthesis in solid medium, whereas transformants with the amplified bla gene produced reduced levels of cephamycin. The cephamycin-overproducing Merck strain N. lactamdurans MA4213 showed no detectable levels of β-lactamase activity. The β-lactamase plays a negative role in cephamycin biosynthesis in solid medium, but not in liquid medium.

Penicillium chrysogenum var. halophenolicum, a new halotolerant strain with potential in the remediation of aromatic compounds in high salt environments

A halotolerant phenylacetate-degrading fungus Penicillium CLONA2, previously isolated from a salt mine at Algarve (Portugal), was identified as a variant of P. chrysogenum using the ITS-5,8S rDNA and the D1/D2 domain of 28S rDNA sequences. The metabolic features and genetic characteristics suggest that this strain belongs to a subgroup of P. chrysogenum, named var. halophenolicum. The presence of the penicillin biosynthetic cluster was proven by Southern hybridizations using probes internal to the pcbAB and penDE genes and sequencing of the pcbAB-pcbC intergenic region. However the pcbAB-pcbC divergent promoter region contained 20 point modifications with respect to that of the wild type P. chrysogenum NRRL1951. The CLONA2 strain produced non-aromatic natural penicillins rather than benzylpenicillin in a medium containing potassium phenylacetate (the precursor of benzylpenicillin) and was able to grow well on phenylacetatic acid using it as sole carbon source. Due to the ability of P. chrysogenum CLONA2 to degrade aromatic compounds, this strain may be an interesting organism for aromatic compounds remediation in high salinity environments.

Transcriptomic Crosstalk between Fungal Invasive Pathogens and Their Host Cells: Opportunities and Challenges for Next-Generation Sequencing Methods

Fungal invasive infections are an increasing health problem. The intrinsic complexity of pathogenic fungi and the unmet clinical need for new and more effective treatments requires a detailed knowledge of the infection process. During infection, fungal pathogens are able to trigger a specific transcriptional program in their host cells. The detailed knowledge of this transcriptional program will allow for a better understanding of the infection process and consequently will help in the future design of more efficient therapeutic strategies. Simultaneous transcriptomic studies of pathogen and host by high-throughput sequencing (dual RNA-seq) is an unbiased protocol to understand the intricate regulatory networks underlying the infectious process. This protocol is starting to be applied to the study of the interactions between fungal pathogens and their hosts. To date, our knowledge of the molecular basis of infection for fungal pathogens is still very limited, and the putative role of regulatory players such as non-coding RNAs or epigenetic factors remains elusive. The wider application of high-throughput transcriptomics in the near future will help to understand the fungal mechanisms for colonization and survival, as well as to characterize the molecular responses of the host cell against a fungal infection.

Fungal extrolites as a new source for therapeutic compounds and as building blocks for applications in synthetic biology.

Secondary metabolic pathways of fungal origin provide an almost unlimited resource of new compounds for medical applications, which can fulfill some of the, currently unmet, needs for therapeutic alternatives for the treatment of a number of diseases. Secondary metabolites secreted to the extracellular medium (extrolites) belong to diverse chemical and structural families, but the majority of them are synthesized by the condensation of a limited number of precursor building blocks including amino acids, sugars, lipids and low molecular weight compounds also employed in anabolic processes. In fungi, genes related to secondary metabolic pathways are frequently clustered together and show a modular organization within fungal genomes. The majority of fungal gene clusters responsible for the biosynthesis of secondary metabolites contain genes encoding a high molecular weight condensing enzyme which is responsible for the assembly of the precursor units of the metabolite. They also contain other auxiliary genes which encode enzymes involved in subsequent chemical modification of the metabolite core. Synthetic biology is a branch of molecular biology whose main objective is the manipulation of cellular components and processes in order to perform logically connected metabolic functions. In synthetic biology applications, biosynthetic modules from secondary metabolic processes can be rationally engineered and combined to produce either new compounds, or to improve the activities and/or the bioavailability of the already known ones. Recently, advanced genome editing techniques based on guided DNA endonucleases have shown potential for the manipulation of eukaryotic and bacterial genomes. This review discusses the potential application of genetic engineering and genome editing tools in the rational design of fungal secondary metabolite pathways by taking advantage of the increasing availability of genomic and biochemical data.

Effect of exogenous lysine on the expression of early cephamycin C biosynthetic genes and antibiotic production in Nocardia lactamdurans MA4213

Abstract. In β-lactam producing microorganisms, the first step in the biosynthesis of the β-lactam ring is the condensation of three amino acid precursors: α-aminoadipate, L-cysteine and D-valine. In Nocardia lactamdurans and other cephamycin-producing actinomycetes, α-aminoadipate is generated from L-lysine by two sequential enzymatic steps. The first step involves a lysine-6-aminotransferase activity (LAT), considered to be one of the rate-limiting steps for antibiotic biosynthesis. Here, we report the effect of exogenous lysine on antibiotic production by N. lactamdurans MA4213. Lysine-supplemented cultures showed higher titers of cephamycin C, an effect that was more significant at early fermentation times. The increase in cephamycin C production was not quantitatively correlated with specific LAT activity in lysine-supplemented cultures. Observation of a positive effect of lysine on cephamycin C production by N. lactamdurans was dependent on carbon source availability in the culture media. Supplementation of the culture media with exogenous lysine did not affect the mRNA levels of the early biosynthetic genes controlled by the bidirectional promoter. These results indicate that L-lysine is required not only for antibiotic biosynthesis, but particularly as carbon or nitrogen source.

Unzippers, resolvers and sensors: a structural and functional biochemistry tale of RNA helicases.

The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.