Viviane Castelo Branco Reis

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. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.

Functional expression of Burkholderia cenocepacia xylose isomerase in yeast increases ethanol production from a glucose–xylose blend

This study presents results regarding the successful cloning of the bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia and its functional expression in Saccharomyces cerevisiae. The recombinant yeast showed to be competent to efficiently produce ethanol from both glucose and xylose, which are the main sugars in lignocellulosic hydrolysates. The heterologous expression of the gene xylA enabled a laboratorial yeast strain to ferment xylose anaerobically, improving ethanol production from a fermentation medium containing a glucose-xylose blend similar to that found in sugar cane bagasse hydrolysates. The insertion of xylA caused a 5-fold increase in xylose consumption, and over a 1.5-fold increase in ethanol production and yield, in comparison to that showed by the WT strain, in 24h fermentations, where it was not detected accumulation of xylitol. These findings are encouraging for further studies concerning the expression of B. cenocepacia xylA in an industrial yeast strain.

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. cerevisiae strain isolated from a sugarcane mill and is becoming increasingly popular for bioethanol production in Brazil. In this work, we carried out the genetic characterization of this strain and developed a set of tools to permit its genetic manipulation. Using flow cytometry, mating type, and sporulation analysis, we verified that JP1 is diploid and homothallic. Vectors with dominant selective markers for G418, hygromycin B, zeocin, and ρ-fluoro-dl-phenylalanine were used to successfully transform JP1 cells. Also, an auxotrophic ura3 mutant strain of JP1 was created by gene disruption using integration cassettes with dominant markers flanked by loxP sites. Marker excision was accomplished by the Cre/loxP system. The resulting auxotrophic strain was successfully transformed with an episomal vector that allowed green fluorescent protein expression.

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

Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of ∼60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37°C, respectively, and retained 90% of its activity after 72 hours of incubation at −20°C and 4°C.

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.

Expression of a kexin-like gene from the human pathogenic fungus Paracoccidioides brasiliensis in Saccharomyces cerevisiae

Kexin-like proteins are proteinases belonging to the subtilase family which are involved in the processing of pro-proteins to their active forms. In fungi, kexin-like proteins are involved in several important cellular processes, including mating and dimorphism. Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis undergoes a thermo-regulated dimorphic transition which is essential for the establishment of the infection. Although the molecular mechanisms which rule this process are still unknown, several genes identified in P. brasiliensis have been implicated in dimorphism, including kex2, a kexin-like protein. In this work we have used the bakers yeast Saccharomyces cerevisiae as a host to perform heterologous expression analysis of the P. brasiliensis kex2 gene. Our data shows that kex2 can complement the functions of a S. cerevisiae kex2 mutant strain and could therefore be considered its functional homologue.

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

BackgroundA 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. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele.ResultsA K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth.ConclusionsIn this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.

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

ABSTRACT 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. The aim of this work is to describe how these different molecular strategies have been applied in K. phaffii presenting their advantages and drawbacks.

Genetic analysis of the Komagataella phaffii centromeres by 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. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work we designed a color-based genetic assay to investigate genetic stability in K. phaffii. Plasmids bearing each centromere and the ADE3 marker were evaluated in terms of mitotic stability in an ade2/ade3 auxotrophic strain which allows plasmid screening through colony color. Plasmid copy number was verified through qPCR. Our results confirmed that the centromeric plasmids were maintained at low copy number as a result of typical chromosome-like segregation during cell division. These features, combined with high transformation efficiency and in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox. Author summary The methylotrophic yeast Komagataella phaffii is considered as one of the most important platforms for the production of proteins and metabolites. We sought in this study to develop a color-based genetic system widely used in other yeasts to assess mitotically stability of vectors carrying the proposed K. phaffii centromeres. First, we constructed a K. phaffii strain (LA3) mutant for ADE2 and ADE3; this resulted in a strain that forms white colonies and when transformed with a vector (pPICH-ADE3) carrying ADE3 turns red. Next, the four K. phaffii centromeres were cloned into pPICH-ADE3 and tested in LA3 for copy number and plasmid stability. Centromeres are responsible for proper chromosome segregation during cell division, hence guaranteeing that both daughter cells receive one copy of the duplicated DNA. Our results show that three K. phaffii centromeres behaved as expected conferring extra stability to the replicative plasmids and maintaining them at low copy number. Once characterized, centromeres can be used as parts in the construction of advanced genetic manipulation tools, thus allowing the construction of strains capable of expressing large metabolic pathways for the production of complex biochemicals.

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. This cassette was successfully tested as a dominant selection marker for transformation of the LA1 strain after selection on plates containing acetamide as a sole nitrogen source. Finally, amdSloxP was used to sequentially disrupt the K. phaffii ADE2 and URA5 genes. After each disruption event, a Cre-mediated marker recycling step was performed by plating cells on medium containing fluoroacetamide. In conclusion, amdS proved to be a suitable tool for K. phaffii transformation and marker recycling thus providing a new antibiotic-free system for genetic manipulation of this yeast.