CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.

Molecular Tools for Leveraging the Potential of the Acid-Tolerant Yeast Zygosaccharomyces bailii as Cell Factory.

Microorganisms offer a tremendous potential as cell factories, and they are indeed been used by humans since the previous centuries for biotransformations. Among them, yeasts combine the advantage of a unicellular state with a eukaryotic organization. Moreover, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycete budding yeast, is widely known for its peculiar tolerance to different stresses, among which are organic acids. Moreover, the recent reclassification of the species, including diverse hybrids, is further expanding both fundamental and applied interests. It is therefore reasonable that despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here we describe in detail the methods for determining chromosome number, size, and aneuploidy, transformation, classical target gene disruption or gene integration, and designing of episomal expression plasmids helpful for engineering the yeast Z. bailii .

Insights on life cycle and cell identity regulatory circuits for unlocking genetic improvement in Zygosaccharomyces and Kluyveromyces yeasts.

Evolution has provided a vast diversity of yeasts that play fundamental roles in nature and society. This diversity is not limited to genotypically homogeneous species with natural interspecies hybrids and allodiploids that blur species boundaries frequently isolated. Thus, life cycle and the nature of breeding systems have profound effects on genome variation, shaping heterozygosity, genotype diversity and ploidy level. The apparent enrichment of hybrids in industry-related environments suggests that hybridization provides an adaptive route against stressors and creates interest in developing new hybrids for biotechnological uses. For example, in the Saccharomyces genus where regulatory circuits controlling cell identity, mating competence and meiosis commitment have been extensively studied, this body of knowledge is being used to combine interesting traits into synthetic F1 hybrids, to bypass F1 hybrid sterility and to dissect complex phenotypes by bulk segregant analysis. Although these aspects are less known in other industrially promising yeasts, advances in whole-genome sequencing and analysis are changing this and new insights are being gained, especially in the food-associated genera Zygosaccharomyces and Kluyveromyces. We discuss this new knowledge and highlight how deciphering cell identity circuits in these lineages will contribute significantly to identify the genetic determinants underpinning complex phenotypes and open new avenues for breeding programmes.

Lipoma de cólon ascendente / Lipoma of the ascending colon

Os lipomas do cólon são neoplasias benignas, ocupando o segundo lugar dentre os tumores benignos do cólon, com uma incidência de 0,2% dos casos. Frequentemente são assintomáticos, porém de acordo com o seu tamanho pode estar associados à dor abdominal, oclusão intestinal devido à intussuscepção, alterações no hábito intestinal e hematoquezia. Acometem uma faixa etária ao redor dos 70 anos com predominância do sexo feminino, sendo encontrada em cerca de 90% dos casos no cólon direito, como lesão única. Podem ser sésseis ou pediculados de coloração amarelada, recobertos por mucosa de aspecto normal, acometendo a submucosa, a mucosa ou a subserosa. Endoscopicamente têm o aspecto de coxim almofadado. O tratamento mais adequado para o caso de lesões volumosas é a sua ressecção por colotomia ou colectomia parcial, devendo ser indicado a aplicação do método endoscópico para os casos de lipomas com até 2,0 cm, observando-se uma alta taxa de complicações quando da ressecção por colonoscopia naqueles com tamanho superior, como p.ex. hemorragia e perfuração. Na opção pela ressecção endoscópica, deve-se iniciá-la pela injeção de solução salina com adrenalina na submucosa e na base da lesão, no volume de 5 a 10 ml, elevando-a, seguido da sua retirada por alça de polipectomia por múltiplos recortes ou pela dissecção no plano submucoso. Uma alternativa técnica que reduz o risco de complicações é o emprego do procedimento de unroofing, em que se promove a ressecção da metade superior da lesão, com a retirada do remanescente tecidual junto à parede cólica facilitada pela aspiração ou curetagem local.