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Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts.
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The methylotrophic yeast Komagataella phaffii is one of the most important microbial platforms to produce recombinant proteins. Despite its importance in the context of industrial biotechnology, the use of synthetic biology approaches in K. phaffii is hampered by the fact that few genetic tools are available for precise control of gene expression in this system. In this work, we used an RNA aptamer activated by tetracycline to modulate protein production at the translational level. Using lacZ as gene reporter, we have demonstrated significant reduction of the heterologous protein upon addition of tetracycline. Furthermore, this genetic control device was applied for the control of Ku70p. This protein is involved in non-homologous recombination and the control of its production paves the way for the development of strains exhibiting higher rates of homologous recombination.
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The use of nucleotides for biomedical applications is an old desire in the scientific community. As we will present here, there are references published over the past 40 years with this intended use. The main problem is that, as unstable molecules, nucleotides require some additional protection to extend their shelf life in the biological environment. Among the different nucleotide carriers, the nano-sized liposomes proved to be an effective strategic tool to overcome all these drawbacks related to the nucleotide high instability. Moreover, due to their low immunogenicity and easy preparation, the liposomes were selected as the main strategy for delivery of the mRNA developed for COVID-19 immunization. For sure this is the most important and relevant example of nucleotide application for human biomedical conditions. In addition, the use of mRNA vaccines for COVID-19 has increased interest in the application of this type of technology to other health conditions. For this review article, we will present some of these examples, especially focused on the use of liposomes to protect and deliver nucleotides for cancer therapy, immunostimulatory activities, enzymatic diagnostic applications, some examples for veterinarian use, and the treatment of neglected tropical disease.
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As one of synthetic biology's foundations, biocircuits are a strategy of genetic parts assembling to recognize a signal and to produce a desirable output to interfere with a biological function. In this review, we revisited the progress in the biocircuits technology basis and its mandatory elements, such as the characterization and assembly of functional parts. Furthermore, for a successful implementation, the transcriptional control systems are a relevant point, and the computational tools help to predict the best combinations among the biological parts planned to be used to achieve the desirable phenotype. However, many challenges are involved in delivering and stabilizing the synthetic structures. Some research experiences, such as the golden crops, biosensors, and artificial photosynthetic structures, can indicate the positive and limiting aspects of the practice. Finally, we envision that the modulatory structural feature and the possibility of finer gene regulation through biocircuits can contribute to the complex design of synthetic chromosomes aiming to develop plants and algae with new or improved functions.
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Cryptococcus neoformans is the etiologic agent of cryptococcosis, a lethal worldwide disease. Synthetic biology could contribute to its better understanding through engineering genetic networks. However, its major challenge is the requirement of accessible genetic parts. The database presented here provides 23 biological parts for this organism in Synthetic Biology Open Language.
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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.
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Optogenética , Leveduras , Expressão Gênica , Proteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Leveduras/genéticaRESUMO
Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by ß-1,4 and ß-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors' ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production.
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Recently, new serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. Here, we describe the use of unidirectional genetic switches to evaluate the functionality of six serine integrases in different eukaryotic systems: the HEK 293T cell lineage, bovine fibroblasts and plant protoplasts. Moreover, integrase activity was also tested in human cell types of therapeutic interest: peripheral blood mononuclear cells (PBMCs), neural stem cells (NSCs) and undifferentiated embryonic stem (ES) cells. The switches were composed of plasmids designed to flip two different genetic parts driven by serine integrases. Cell-based assays were evaluated by measurement of EGFP fluorescence and by molecular analysis of attL/attR sites formation after integrase functionality. Our results demonstrate that all the integrases were capable of inverting the targeted DNA sequences, exhibiting distinct performances based on the cell type or the switchable genetic sequence. These results should support the development of tunable genetic circuits to regulate eukaryotic gene expression.
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Arabidopsis/enzimologia , Fibroblastos/enzimologia , Integrases/genética , Plasmídeos/genética , Protoplastos/enzimologia , Recombinação Genética , Serina/genética , Animais , Bovinos , Humanos , Integrases/metabolismo , Leucócitos Mononucleares/enzimologia , Regiões Promotoras Genéticas , Serina/metabolismoRESUMO
OBJECTIVE: This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p≤0.05. RESULTS: The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. CONCLUSION: There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population.
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Proteínas Reguladoras de Apoptose/genética , Artrite Reumatoide/genética , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/genética , Brasil , Estudos de Casos e Controles , Frequência do Gene , Humanos , Polimorfismo de Fragmento de Restrição , Receptor de Morte Celular Programada 1RESUMO
Abstract Rivers and streams are important reservoirs of freshwater for human consumption. These ecosystems are threatened by increasing urbanization, because raw sewage discharged into them alters their nutrient content and may affect the composition of their microbial community. In the present study, we investigate the taxonomic and functional profile of the microbial community in an urban lotic environment. Samples of running water were collected at two points in the São Pedro stream: an upstream preserved and non-urbanized area, and a polluted urbanized area with discharged sewage. The metagenomic DNA was sequenced by pyrosequencing. Differences were observed in the community composition at the two sites. The non-urbanized area was overrepresented by genera of ubiquitous microbes that act in the maintenance of environments. In contrast, the urbanized metagenome was rich in genera pathogenic to humans. The functional profile indicated that the microbes act on the metabolism of methane, nitrogen and sulfur, especially in the urbanized area. It was also found that virulence/defense (antibiotic resistance and metal resistance) and stress response-related genes were disseminated in the urbanized environment. The structure of the microbial community was altered by uncontrolled anthropic interference, highlighting the selective pressure imposed by high loads of urban sewage discharged into freshwater environments.
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Humanos , Urbanização , Microbiologia da Água , Rios/microbiologia , Metagenoma , Microbiota , Filogenia , RNA Ribossômico 16S/genética , Ecossistema , Metabolismo Energético , Redes e Vias Metabólicas , Metagenômica , Código de Barras de DNA TaxonômicoRESUMO
ABSTRACT Objective: This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p ≤ 0.05. Results: The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. Conclusion: There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population.
RESUMO Objetivo: Este estudo teve como objetivo analisar a relação entre o polimorfismo do gene PDCD1 (programmed cell death 1) (PD1.3G/A - rs11568821) com características do lúpus eritematoso sistêmico (LES) e da artrite reumatoide (AR) em uma população do sul do Brasil. Métodos: A técnica de PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Lenght Polymorphism) foi utilizada para analisar amostras de 95 pacientes com LES e 87 com AR, assim como em 128 indivíduos do grupo controle de Santa Catarina, sul do Brasil. Foi analisada a probabilidade de equilíbrio de Hardy-Weinberg (EHW) e a odds ratio (OR), considerando um IC 95% e p ≤ 0,05. Resultados: As frequências alélicas PD1.3 A foram de 0,095 (LES), 0,115 (AR) e 0,078 (controles). Os genótipos do grupo controle estavam em EHW, enquanto aqueles dos pacientes com LES e AR não estavam. No entanto, não foi encontrada associação entre o polimorfismo PD1.3 e a suscetibilidade ao LES ou à AR, nem com dados clínicos ou epidemiológicos. Conclusão: Não foi encontrada associação significativa entre o polimorfismo PD1.3 e a susceptibilidade ao LES ou à AR nessa população do sul do Brasil.
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Humanos , Artrite Reumatoide/genética , Predisposição Genética para Doença , Proteínas Reguladoras de Apoptose/genética , Lúpus Eritematoso Sistêmico/genética , Polimorfismo de Fragmento de Restrição , Brasil , Estudos de Casos e Controles , Receptor de Morte Celular Programada 1 , Frequência do GeneRESUMO
ABSTRACT In spite of advances in colorectal cancer treatments, approximately 1.4 million new global cases are estimated for 2015. In this sense, Brazilian plant diversity offers a multiplicity of essential oils as prospective novel anticancer compounds. This study aimed to evaluate the antiproliferative effect of the essential oils from four Lippia species in CT26.WT colon tumor cells, as a measurement of cell cycle phase distribution and microRNA expression. CT26.WT showed cell cycle arrest at G2/M phase after treatment with 100 µg/ml of Lippia alba (Mill.) N.E.Br. ex Britton & P. Wilson, Lippia sidoides Cham., and Lippia lacunosa Mart. & Schauer, Verbenaceae, essential oils and, at the same concentration, Lippia rotundifolia Cham. essential oil caused an augment of G0/G1 phase. The miRNA expression profiling shows change of expression in key oncogenic miRNAs genes after treatment. Our findings suggest growth inhibition mechanisms for all four essential oils on CT26.WT cells involving direct or indirect interference on cell cycle arrest and/or oncogenic miRNAs expression.
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Rivers and streams are important reservoirs of freshwater for human consumption. These ecosystems are threatened by increasing urbanization, because raw sewage discharged into them alters their nutrient content and may affect the composition of their microbial community. In the present study, we investigate the taxonomic and functional profile of the microbial community in an urban lotic environment. Samples of running water were collected at two points in the São Pedro stream: an upstream preserved and non-urbanized area, and a polluted urbanized area with discharged sewage. The metagenomic DNA was sequenced by pyrosequencing. Differences were observed in the community composition at the two sites. The non-urbanized area was overrepresented by genera of ubiquitous microbes that act in the maintenance of environments. In contrast, the urbanized metagenome was rich in genera pathogenic to humans. The functional profile indicated that the microbes act on the metabolism of methane, nitrogen and sulfur, especially in the urbanized area. It was also found that virulence/defense (antibiotic resistance and metal resistance) and stress response-related genes were disseminated in the urbanized environment. The structure of the microbial community was altered by uncontrolled anthropic interference, highlighting the selective pressure imposed by high loads of urban sewage discharged into freshwater environments.
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Metagenoma , Microbiota , Rios/microbiologia , Urbanização , Microbiologia da Água , Código de Barras de DNA Taxonômico , Ecossistema , Metabolismo Energético , Humanos , Redes e Vias Metabólicas , Metagenômica , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
OBJECTIVE: This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. METHODS: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg Equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p≤0.05. RESULTS: The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. CONCLUSION: There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population.
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As antimicrobials are introduced into the environment, microorganisms may respond in different ways, sometimes displaying alterations in cellular physiology. Considering the clinical relevance of the Bacteroides fragilis, strains were selected to investigate bacterial response after exposure to subinhibitory concentrations (SIC) of ampicillin (AMP), ampicillin-sulbactam (AMS), clindamycin (CLI), chloramphenicol (CHL), and its relationship to a host model (BALB/c mice) after experimental challenge. Morphological alterations, and biochemical-physiological and genetic profiles were evaluated among drug-selected bacteria. Histopathological evaluation of the liver and spleen, and inflammatory cytokines were determined after bacterial infection in mice. AMP and AMS exposure were related to most significant cellular alterations. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria. Down regulation in adherence properties were also observed. Spleen and liver alterations were observed in different patterns. Increased levels of TNF-α, IL-6 and IFN-γ were also observed. Our results show that SICs of AMP, AMS, CLI and CHL may be related to alterations in cell physiology in B. fragilis with implications to the host-bacteria relationship. The data emphasizes the risks of inappropriate chemotherapy, and the concerns regarding ecological consequences lead by SICs of antimicrobials in resident microbiota.
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Antibacterianos/farmacologia , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/efeitos dos fármacos , Bacteroides fragilis/crescimento & desenvolvimento , Animais , Infecções por Bacteroides/genética , Infecções por Bacteroides/metabolismo , Bacteroides fragilis/patogenicidade , Feminino , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Virulência/efeitos dos fármacosRESUMO
Improving the quality and performance of soybean oil as biodiesel depends on the chemical composition of its fatty acids and requires an increase in monounsaturated acids and a reduction in polyunsaturated acids. Despite its current use as a source of biofuel, soybean oil contains an average of 25 % oleic acid and 13 % palmitic acid, which negatively impacts its oxidative stability and freezing point, causing a high rate of nitrogen oxide emission. Gas chromatography and ion mobility mass spectrometry were conducted on soybean fatty acids from metabolically engineered seed extracts to determine the nature of the structural oleic and palmitic acids. The soybean genes FAD2-1 and FatB were placed under the control of the 35SCaMV constitutive promoter, introduced to soybean embryonic axes by particle bombardment and down-regulated using RNA interference technology. Results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 94.58 %) and a reduction in palmitic acid (to <3 %) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and non-transgenic oil extracts.
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Ácidos Graxos/química , Glycine max/química , Plantas Geneticamente Modificadas/química , Sementes/química , Engenharia Metabólica , Plantas Geneticamente Modificadas/genética , Sementes/genética , Óleo de Soja/química , Óleo de Soja/genética , Óleo de Soja/metabolismo , Glycine max/genéticaRESUMO
Several Lippia species have been used in folk medicine mainly for gastrointestinal and respiratory diseases. Their biological properties have been partially associated to the terpenoids found in their essential oils. According to the World Health Organization, cancer is the leading cause of death worldwide and is described as a complex group of diseases with several hallmarks. One of its acceptable defining features is the cell proliferation beyond their boundaries forming the tumors. Importantly, some drugs currently available were discovered by the investigation of plant secondary metabolites. Thus, this study aimed to evaluate in vitro cytotoxic effect of the essential oils extracted from five Lippia species against tumor cell lines. The results indicated that mouse colon carcinoma CT26.WT cell line viability was significantly reduced showing an IC50 of 19.05, 30.20 and 36.30 µg/ml when treated with the essential oils of L. sidoides, L. salviifolia and L. rotundifolia, respectively. Human lung carcinoma A549 cell line also had a compromised viability to the action of L. alba carvone chemotype essential oil. The tested essential oils did not compromise viability of the normal cell line CHO. These finds suggest that the studied Lippia essential oils might be good candidates for further in-depth studies.
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Seeds are organs specialised in accumulating proteins, and they may provide a potential economically viable platform for the large-scale production and storage of many molecules for pharmaceutical and other productive sectors. Soybean [Glycine max (L.) Merrill] has a high seed protein content and represents an excellent source of abundant and cheap biomass. Under greenhouse conditions and a daily photoperiod of 23 h of light, the soybean plant's vegetative growth can be significantly extended by inducing more than a tenfold increase in seed production when compared with plants cultivated under field conditions. Some factors involved in the production of different recombinant proteins in soybean seeds are discussed in this review. These include transgenic system, regulatory sequences and the use of Mass Spectrometry as a new tool for molecular characterisation of seed produced recombinant proteins. The important intrinsic characteristics and possibility of genetically engineering soybean seeds, using current advances in recombinant DNA technology including metabolic engineering and synthetic biology, should form the foundation for large-scale and more precise genome modification, making this crop an important candidate as bioreactor for production of recombinant molecules.
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Glycine max/genética , Proteínas Recombinantes/genética , Proteínas de Soja/genética , Animais , Engenharia Genética/métodos , Genoma de Planta , Humanos , Espectrometria de Massas , Agricultura Molecular/métodos , Sementes , Glycine max/crescimento & desenvolvimento , Glycine max/metabolismo , TransgenesRESUMO
Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of ß-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host-bacteria relationship.