Optogenetic strategies for the control of gene expression in yeasts.

Optogenetics involves the use of light to control cellular functions and has become increasingly popular in various areas of research, especially in the precise control of gene expression. While this technology is already well established in neurobiology and basic research, its use in bioprocess development is still emerging. Some optogenetic switches have been implemented in yeasts for different purposes, taking advantage of a wide repertoire of biological parts and relatively easy genetic manipulation. In this review, we cover the current strategies used for the construction of yeast strains to be used in optogenetically controlled protein or metabolite production, as well as the operational aspects to be considered for the scale-up of this type of process. Finally, we discuss the main applications of optogenetic switches in yeast systems and highlight the main advantages and challenges of bioprocess development considering future directions for this field.

A Custom-Designed Recombinant Multiepitope Protein for Human Cytomegalovirus Diagnosis.

BACKGROUND: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. OBJECTIVE: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. METHODS: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). RESULTS: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. CONCLUSION: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.

Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756.

The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-directed mutagenesis using XynA as a template. XynA and its mutants were successfully overexpressed in Escherichia coli Rosetta-gami DE3 and purified, exhibiting maximum xylanolytic activity at pH 5 and 65°C. Three of the eleven mutants, Q158R, H209N, and N257D, demonstrated increased thermostability relative to the wild type at 70°C and 75°C.Q158R and N257D were stable in the pH range 5.0-10.0, while WT and H209N were stable from pH 8-10. CD analysis demonstrated that the WT and the three mutant enzymes were expressed in a folded form. H209N was the most thermostable mutant, showing a Tm of 71.3°C. Molecular dynamics modeling analyses suggest that the increase in H209N thermostability may beattributed to a higher number of short helices and salt bridges, which displayed a positive charge in the catalytic core, stabilizing its tertiary structure.

A Novel Structurally Stable Multiepitope Protein for Detection of HCV.

Hepatitis C virus (HCV) has emerged as the major pathogen of liver diseases in recent years leading to worldwide blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Accurate diagnosis for differentiation of hepatitis C from other viruses is thus of pivotal importance for proper treatment. In this work we developed a recombinant multiepitope protein (rMEHCV) for hepatitis C diagnostic purposes based on conserved and immunodominant epitopes from core, NS3, NS4A, NS4B, and NS5 regions of the virus polyprotein of genotypes 1a, 1b, and 3a, the most prevalent genotypes in South America (especially in Brazil). A synthetic gene was designed to encode eight epitopes in tandem separated by a flexible linker and bearing a his-tag at the C-terminal end. The recombinant protein was produced in Escherichia coli and purified in a single affinity chromatographic step with >95% purity. Purified rMEHCV was used to perform an ELISA which showed that the recombinant protein was recognized by IgG and IgM from human serum samples. The structural data obtained by circular dichroism (CD) spectroscopy showed that rMEHCV is a highly thermal stable protein at neutral and alkaline conditions. Together, these results show that rMEHCV should be considered an alternative antigen for hepatitis C diagnosis.

Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis.

Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and ß-sheet, similar to α/ß hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.

Draft Genome Sequence of FT9, a Novel Bacillus cereus Strain Isolated from a Brazilian Thermal Spring.

A Bacillus cereus strain, FT9, isolated from a hot spring in the midwest region of Brazil, had its entire genome sequenced.

Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii.

Saccharomyces boulardii (S. boulardii) is a probiotic yeast related to Saccharomyces cerevisiae (S. cerevisiae) but with distinct genetic, taxonomic and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. Here we show that, despite the similarity of the two yeasts, not all genetic tools used in S. cerevisiae can be applied in S. boulardii. While transformation of the latter could be obtained using a commercial kit developed for the former, consequent screening of successful transformants had to be optimized. We also show that several genes frequently used in genetic manipulation of S. cerevisiae (e.g., promoters and resistance markers) are present in S. boulardii. Sequencing revealed a high rate of homology (> 96%) between the orthologs of the two yeasts. However, we also observed some of them are not eligible to be targeted for transformation of S. boulardii. This work has important applications toward the potential of this probiotic yeast as an expression system for genes of interest.

Cloning, molecular characterization and heterologous expression of AMY1, an alpha-amylase gene from Cryptococcus flavus.

A Cryptococcus flavus gene (AMY1) encoding an extracellular alpha-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) (144)DVVVNH(149), (II) (235)GLRIDSLQQ(243), (III) (263)GEVFN(267), (IV) (327)FLENQD(332), placed the enzyme in the GH13 alpha-amylase family. Furthermore, sequence comparison suggests that the C. flavusalpha-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae. The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL(-1)) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme (c. 67 kDa), part of which was due to N-glycosylation.

Transcriptional profile of ras1 and ras2 and the potential role of farnesylation in the dimorphism of the human pathogen Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes a human systemic mycosis with high incidence in Latin America. Owing to their participation in the control of pathogen morphogenesis, differentiation and virulence, it was decided to characterize ras genes in P. brasiliensis. ras1 and ras2 were identified to be coding for two different proteins with high identity. The ras transcriptional pattern was investigated by reverse transcription PCR (RT-PCR) during mycelium-to-yeast (M-->Y) transition, heat shock at 42 degrees C and after internalization of yeast cells by murine macrophages. Both genes were downregulated inside macrophages and ras1, at 42 degrees C. In contrast, ras genes did not show any transcriptional variation during the M-->Y transition. The fact that Ras proteins are attached to the membrane via farnesylation prompted the use of a farnesyltransferase inhibitor to investigate the importance of this process for vegetative growth and dimorphic transition. Farnesylation blockage interfered with the vegetative growth of yeast cells and stimulated germinative tube production even at 37 degrees C. During Y-->M transition, the inhibitor increased filamentation in a dose-dependent manner, indicating that impaired farnesylation favours the mycelium form of P. brasiliensis. The results suggest that ras genes might have a role in dimorphism, heat shock response and in host-pathogen interaction.

Hydrolytic enzymes in Paracoccidioides brasiliensis--ecological aspects.

Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis. The yeast form of this pathogen is found in the animal host whereas the mycelial form is recovered from living and non-living organic material. The sole carbon source available in these habitats is represented by polysaccharides from the plant cell wall. Hydrolytic enzymes are necessary to convert these polymers into simple sugars for fungal metabolism. We report on the presence of ortholog genes of hydrolytic enzymes identified in the P. brasiliensis transcriptome and on hydrolytic activities in supernatants of induced P. brasiliensis cultures of mycelium and yeast cells. Enzymatic assays have shown cellulase and xylanase activities, both being higher in mycelium than in the yeast form. Amylase and chitinase activities were detected only in mycelium. Data so far reinforce the idea that mycelial P. brasiliensis is a saprobe.

Functional genome of the human pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America. Pathogenicity is assumed to be a consequence of the cellular differentiation process that this fungus undergoes from mycelium to yeast cells during human infection. In an effort to elucidate the molecular mechanisms involved in this process a network of Brazilian laboratories carried out a transcriptome project for both cell types. This review focuses on the data analysis yielding a comprehensive view of the fungal metabolism and the molecular adaptations during dimorphism in P. brasiliensis from analysis of 6022 groups, related to expressed genes, which were generated from both mycelium and yeast phases.

Transcriptional profiles of the human pathogenic fungus Paracoccidioides brasiliensis in mycelium and yeast cells.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.

Overview and perspectives the transcriptome of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more comprehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis.

Biochemical characterization of alpha-amylase from the yeast Cryptococcus flavus.

During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The K(m) value for the pure enzyme was 0.056 mg ml(-1) with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50 degrees C. The enzyme retained 90% of the activity after incubation at 50 degrees C for 60 min and was inhibited by Cu(2+), Fe(2+) and Hg(2+).