Applications of the Methylotrophic Yeast Komagataella phaffii in the Context of Modern Biotechnology.

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.

Use of an on/off tetracycline riboswitch to control protein production in Komagataella phaffii.

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.

Nucleotides Entrapped in Liposome Nanovesicles as Tools for Therapeutic and Diagnostic Use in Biomedical Applications.

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.

Biocircuits in plants and eukaryotic algae.

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.

Cryptococcus neoformans Database in Synthetic Biology Open Language.

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.

Comparative metagenome of a stream impacted by the urbanization phenomenon

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.

Identification of dysregulated microRNA expression and their potential role in the antiproliferative effect of the essential oils from four different Lippia species against the CT26.WT colon tumor cell line

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.

Comparative metagenome of a stream impacted by the urbanization phenomenon.

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.

Bacteroides fragilis response to subinhibitory concentrations of antimicrobials includes different morphological, physiological and virulence patterns after in vitro selection.

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.

Morphological, biochemical, physiological and molecular aspects of the response of Fusobacterium nucleatum exposed to subinhibitory concentrations of antimicrobials.

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.

Multidrug-resistance and toxic metal tolerance of medically important bacteria isolated from an aquaculture system.

The use of antimicrobials and toxic metals should be considered carefully in aquaculture and surrounding environments. We aimed to evaluate medically relevant bacteria in an aquaculture system and their susceptibility to antimicrobials and toxic metals. Selective cultures for enterobacteria (ENT), non-fermenting Gram-negative rods (NFR) and Gram-positive cocci (GPC) were obtained from water samples collected in two different year seasons. The isolated bacteria were biochemically identified and antimicrobial and toxic metal susceptibility patterns were determined. Overall, 407 representative strains were recovered. In general, bacteria isolated from fish ponds showed higher multiple antibiotic resistance indices when compared to those isolated from a water-fed canal. Resistance to penicillin and azithromycin was observed more frequently in the GPC group, whereas resistance to ampicillin and ampicillin/sulbactam or gentamicin was observed more frequently in the ENT and NFR groups, respectively. All the isolated bacteria were tolerant to nickel, zinc, chromium and copper at high levels (≥1,024 µg mL(-1)), whereas tolerance to cadmium and mercury varied among the isolated bacteria (2-1,024 µg mL(-1)). Multidrug-resistant bacteria were more frequent and diverse in fish ponds than in the water-fed canal. A positive correlation was observed between antimicrobial resistance and metal tolerance. The data point out the need for water treatment associated with the aquaculture system.

Distribution, detection of enterotoxigenic strains and antimicrobial drug susceptibility patterns of Bacteroides fragilis group in diarrheic and non-diarrheic feces from Brazilian infants

Despite the importance of gastrointestinal diseases and their global distribution, affecting millions of individuals around the world, the role and antimicrobial susceptibility patterns of anaerobic bacteria such as those in the Bacteroides fragilis group (BFG) are still unclear in young children. This study investigated the occurrence and distribution of species in the BFG and enterotoxigenic strains in the fecal microbiota of children and their antimicrobial susceptibility patterns. Diarrheic (n=110) and non-diarrheic (n=65) fecal samples from children aged 0-5 years old were evaluated. BFG strains were isolated and identified by conventional biochemical, physiological and molecular approaches. Alternatively, bacteria and enterotoxigenic strains were detected directly from feces by molecular biology. Antimicrobial drug susceptibility patterns were determined by the agar dilution method according to the guidelines for isolated bacteria. BFG was detected in 64.3 percent of the fecal samples (55 percent diarrheic and 80.4 percent non-diarrheic), and 4.6 percent were enterotoxigenic. Antimicrobial resistance was observed against ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, meropenem, ceftriaxone, clindamycin and chloramphenicol. The data show that these bacteria are prevalent in fecal microbiota at higher levels in healthy children. The molecular methodology was more effective in identifying the B. fragilis group when compared to the biochemical and physiological techniques. The observation of high resistance levels stimulates thoughts about the indiscriminate use of antimicrobial drugs in early infancy. Further quantitative studies are needed to gain a better understanding of the role of these bacteria in acute diarrhea in children.

Distribution, detection of enterotoxigenic strains and antimicrobial drug susceptibility patterns of bacteroides fragilis group in diarrheic and non-diarrheic feces from brazilian infants.

Despite the importance of gastrointestinal diseases and their global distribution, affecting millions of individuals around the world, the role and antimicrobial susceptibility patterns of anaerobic bacteria such as those in the Bacteroides fragilis group (BFG) are still unclear in young children. This study investigated the occurrence and distribution of species in the BFG and enterotoxigenic strains in the fecal microbiota of children and their antimicrobial susceptibility patterns. Diarrheic (n=110) and non-diarrheic (n=65) fecal samples from children aged 0-5 years old were evaluated. BFG strains were isolated and identified by conventional biochemical, physiological and molecular approaches. Alternatively, bacteria and enterotoxigenic strains were detected directly from feces by molecular biology. Antimicrobial drug susceptibility patterns were determined by the agar dilution method according to the guidelines for isolated bacteria. BFG was detected in 64.3% of the fecal samples (55% diarrheic and 80.4% non-diarrheic), and 4.6% were enterotoxigenic. Antimicrobial resistance was observed against ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, meropenem, ceftriaxone, clindamycin and chloramphenicol. The data show that these bacteria are prevalent in fecal microbiota at higher levels in healthy children. The molecular methodology was more effective in identifying the B. fragilis group when compared to the biochemical and physiological techniques. The observation of high resistance levels stimulates thoughts about the indiscriminate use of antimicrobial drugs in early infancy. Further quantitative studies are needed to gain a better understanding of the role of these bacteria in acute diarrhea in children.