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

(formerly ) 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 as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products.

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.

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.

Engineering for the Production of Xylonic Acid from Sugarcane Bagasse Hydrolysate.

Sugarcane bagasse is an agricultural residue rich in xylose, which may be used as a feedstock for the production of high-value-added chemicals, such as xylonic acid, an organic acid listed as one of the top 30 value-added chemicals on a NREL report. Here, was engineered for the first time to produce xylonic acid from sugarcane bagasse hydrolysate. Seven coding genes for xylose dehydrogenase (XDH) were tested.

Construction and characterization of centromeric plasmids for Komagataella phaffii using a color-based plasmid stability assay.

The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K.

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.

Expression, purification, and characterization of asparaginase II from Saccharomyces cerevisiae in Escherichia coli.

l-asparaginase catalyzes the conversion of l-asparagine to l-aspartate and ammonium. This protein is an important therapeutic enzyme used for the treatment of acute lymphoblastic leukemia. In this study, the asparaginase II-encoding gene ASP3 from Saccharomyces cerevisiae was cloned into the expression vector pET28a in-fusion with a 6x histidine tag and was expressed in Escherichia coli BL21 (DE3) cells.

Increase of Candida antarctica lipase B production under PGK promoter in Pichia pastoris: effect of multicopies.

The effect of gene dosage on the production of Candida antarctica lipase B (CalB) in the methylotrophic yeast Komagataella phaffii, at high densities in a simple medium containing crude glycerin as the sole carbon source, is described. The use of crude glycerin, the main by-product of biodiesel production from vegetable oils, will reduce the production cost of the bioprocess. Two K.

A study on the use of strain-specific and homologous promoters for heterologous expression in industrial Saccharomyces cerevisiae strains.

Polymorphism is well known in Saccharomyces cerevisiae strains used for different industrial applications, however little is known about its effects on promoter efficiency. In order to test this, five different promoters derived from an industrial and a laboratory (S288c) strain were used to drive the expression of eGFP reporter gene in both cells. The ADH1 promoter (P ) in particular, which showed more polymorphism among the promoters analyzed, also exhibited the highest differences in intracellular fluorescence production.

Acetamidase as a dominant recyclable marker for Komagataella phaffii strain engineering.

We have investigated the use of the gene coding for acetamidase (amdS) as a recyclable dominant marker for the methylotrophic yeast Komagataella phaffii in order to broaden its genetic toolbox. First, the endogenous constitutive AMD2 gene (a putative acetamidase) was deleted generating strain LA1. A cassette (amdSloxP) was constructed bearing a codon-optimized version of the Aspergillus nidulans amdS gene flanked by loxP sites for marker excision with Cre recombinase.

Effects of PI3K and FSH on steroidogenesis, viability and embryo development of the cumulus-oocyte complex after in vitro culture.

The purpose of this study was to evaluate the effects of FSH and PI3K on the nuclear maturation, viability, steroidogenesis and embryo development of bovine cumulus-oocyte complexes (COCs). Oocyte maturation was achieved with MIV B, MIV B+100 µM LY294002, MIV B+10 ng/mL follicle stimulating hormone (FSH), or MIV B+10 ng/mL FSH+100 µM LY294002 treatments for 22-24 h. After the cultured COCs were denuded, oocytes were separated into those that extruded polar bodies (mature) and those that did not, and real-time polymerase chain reaction (PCR) for BAX, BCL2, LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1 genes was performed.

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker.

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug-resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose.

Molecular strategies to increase the levels of heterologous transcripts in Komagataella phaffii for protein production.

Komagataella phaffii (formerly Pichia pastoris) is a well-known fungal system for heterologous protein production in the context of modern biotechnology. To obtain higher protein titers in this system many researchers have sought to optimize gene expression by increasing the levels of transcription of the heterologous gene. This has been typically achieved by manipulating promoter sequences or by generating clones bearing multiple copies of the desired gene.

Engineering increased thermostability in the GH-10 endo-1,4-β-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.

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).

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects.

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application.

Coexpression of cellulases in Pichia pastoris as a self-processing protein fusion.

The term cellulase refers to any component of the enzymatic complex produced by some fungi, bacteria and protozoans which act serially or synergistically to catalyze the cleavage of cellulosic materials. Cellulases have been widely used in many industrial applications ranging from food industry to the production of second generation ethanol. In an effort to develop new strategies to minimize the costs of enzyme production we describe the development of a Pichia pastoris strain able to coproduce two different cellulases.

A constitutive expression system for Pichia pastoris based on the PGK1 promoter.

Objectives: To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results: P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth.

Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst.

Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach.

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

Lipases (EC 3.1.1.

Enhanced xylose fermentation and ethanol production by engineered Saccharomyces cerevisiae strain.

We have recently demonstrated that heterologous expression of a bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia enabled a laboratorial Saccharomyces cerevisiae strain to ferment xylose anaerobically, without xylitol accumulation. However, the recombinant yeast fermented xylose slowly. In this study, an evolutionary engineering strategy was applied to improve xylose fermentation by the xylA-expressing yeast strain, which involved sequential batch cultivation on xylose.

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.

Lutzomyia whitmani is the main vector of American Cutaneous Leishmaniasis in the Brazilian Federal District and the most prevalent species in residential areas of the Administrative Region of Sobradinho.

Although cases of cutaneous Leishmaniasis have been reported in Brasilia - DF, its mode of transmission is still unknown. Center of Disease Control traps (CDC trap) placed around Sobradinho, a periurban area in the Brazilian Federal District, were able to capture a sample of phlebotomines composed of 89% Lutzomyia whitmani, 7% Lu. bacula, and 3% Lu.

A novel promising Trichoderma harzianum strain for the production of a cellulolytic complex using sugarcane bagasse in natura.

Brazil is a major producer of agro-industrial residues, such as sugarcane bagasse, which could be used as raw material for microbial production of cellulases as an important strategy for the development of sustainable processes of second generation ethanol production. For this purpose, this work aimed at screening for glycosyl hydrolase activities of fungal strains isolated from the Brazilian Cerrado. Among 13 isolates, a Trichoderma harzianum strain (L04) was identified as a promising candidate for cellulase production when cultured on in natura sugarcane bagasse.