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.

Physiological effects of overexpressed sigma factors on fermentative stress response of Zymomonas mobilis.

Zymomonas mobilis is a bacterium of industrial interest due to its high ethanol productivity and high tolerance to stresses. Although the physiological parameters of fermentation are well characterized, there are few studies on the molecular mechanisms that regulate the response to fermentative stress. Z.

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.

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.

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.

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.

Genetic characterization and construction of an auxotrophic strain of Saccharomyces cerevisiae JP1, a Brazilian industrial yeast strain for bioethanol production.

Used for millennia to produce beverages and food, Saccharomyces cerevisiae also became a workhorse in the production of biofuels, most notably bioethanol. Yeast strains have acquired distinct characteristics that are the result of evolutionary adaptation to the stresses of industrial ethanol production. JP1 is a dominant industrial S.

Deciphering the magainin resistance process of Escherichia coli strains in light of the cytosolic proteome.

Antimicrobial peptides (AMPs) are effective antibiotic agents commonly found in plants, animals, and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. It is vital to understand the molecular details that define the mechanism of action of resistance to AMPs for a rational planning of the next antibiotic generation and also to shed some light on the complex AMP mechanism of action. Here, the antibiotic resistance of Escherichia coli ATCC 8739 to magainin I was evaluated in the cytosolic subproteome.

Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae.

An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH(4) (+) and inhibited by Cu(+2) and Hg(+2). Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to K(m) values, enzyme specificity, and secondary structure content were found.

Cell density-dependent linoleic acid toxicity to Saccharomyces cerevisiae.

Since the discovery of the apoptotic pathway in Saccharomyces cerevisiae, several compounds have been shown to cause apoptosis in this organism. While the toxicity of polyunsaturated fatty acids (PUFA) peroxides towards S. cerevisiae has been known for a long time, studies on the effect of nonoxidized PUFA are scarce.

Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli.

Urate oxidase (EC 1.7.3.

Bioethanol from lignocelluloses: Status and perspectives in Brazil.

The National Alcohol Program--PróAlcool, created by the government of Brazil in 1975 resulted less dependency on fossil fuels. The addition of 25% ethanol to gasoline reduced the import of 550 million barrels oil and also reduced the emission CO(2) by 110 million tons. Today, 44% of the Brazilian energy matrix is renewable and 13.

Expression of a glucose-tolerant beta-glucosidase from Humicola grisea var. thermoidea in Saccharomyces cerevisiae.

A beta-glucosidase gene (bgl4) from Humicola grisea var thermoidea was successfully expressed in Saccharomyces cerevisiae. The recombinant protein (BGL4(Sc)) was initially detected associated with yeast cells and later in the culture medium. BGL4(Sc) showed optimal pH and temperature of 6.

The influence of N-glycosylation on biochemical properties of Amy1, an alpha-amylase from the yeast Cryptococcus flavus.

The yeast Cryptococcus flavus secretes a glycosylated alpha-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.

Xylitol dehydrogenase from Candida tropicalis: molecular cloning of the gene and structural analysis of the protein.

Yeasts can metabolize xylose by the action of two key enzymes: xylose reductase and xylitol dehydrogenase. In this work, we present data concerning the cloning of the XYL2 gene encoding xylitol dehydrogenase from the yeast Candida tropicalis. The gene is present as a single copy in the genome and is controlled at the transcriptional level by the presence of the inducer xylose.

Hygromycin B-resistance phenotype acquired in Paracoccidioides brasiliensis via plasmid DNA integration.

Yeast cells of the human pathogenic fungus Paracoccidioides brasiliensis strain Pb01 were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved by electroporation, with intact or linearized plasmid DNA.

Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris.

We report the cloning of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris by a PCR approach. The coding sequence of the PGK1 gene comprises 1251 bp with the potential to encode a polypeptide of 416 amino acid residues, which shows high identity to homologous proteins from other yeasts. The promoter region of this gene (P(PGK1)) contains regulatory cis-elements found in other PGK1 genes, such as TATA box, CT-rich block and a heat shock element.