Development of a sexually evolving approach in Saccharomyces cerevisiae based on selection of recombinant diploid using flow cytometry

Orientador: Gonçalo Amarante Guimarães PereiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A conscientização acerca de questões ambientais aumenta a demanda por combustíveis renováveis. No cenário da indústria energética brasileira, o bioetanol tem grande destaque e sua demanda cresce anualmente. A produção de etanol depende das leveduras industriais, capazes de manter altos níveis de produtividade e rendimento sob as condições de estresse inerentes ao processo. Neste contexto, o desenvolvimento de linhagens mais adaptadas é uma necessidade contínua para a otimização do processo convencional, bem como para a viabilização econômica de tecnologias emergentes, como o etanol hemicelulósico. Para isto, a evolução adaptativa por reprodução sexuada figura como uma alternativa promissora e complementar à engenharia genética, que acelera a adaptação e permite a combinação de caracteres responsáveis pela robustez observada em diferentes linhagens industriais. Tipicamente, este processo consiste na dissecção de um grande número de tétrades e na análise de muitos segregantes, o que torna o processo extremamente laborioso. Sabe-se que Saccharomyces cerevisiae tem a capacidade de evadir o ciclo meiótico após a ocorrência de eventos de recombinação homóloga, e retomar o ciclo mitótico, gerando células com genoma altamente recombinado, denominada RTG (do inglês, Return to Growth). Por esta razão, este trabalho teve como principal objetivo desenvolver uma abordagem que produza diplóides recombinantes sem a necessidade de esporulação; propondo uma metodologia rápida e facilmente aplicada ao estudo e desenvolvimento de leveduras dedicadas à processos industriais. Neste escopo, a expressão transiente de uma versão da proteína repórter GFP foi utilizada como marcador do ciclo celular para seleção de possíveis diploides recombinantes por citometria de fluxo. No período do presente projeto foi possível definir um bom sistema de expressão composto de uma GFP e uma OFP, bem como determinar os FACS gating a serem utilizados na abordagem proposta. No entanto ainda se faz necessário ajustes no que tange a meia vida do GFPAbstract: The awareness of environmental issues increases the demand for renewable fuels. In the scenario of the Brazilian energy industry, bioethanol stands out and its demand grows annually. The production of ethanol depends on industrial yeasts, capable of maintaining high levels of productivity and yield under the conditions of stress inherent to the process. In this context, the development of more adapted lineages is a continuous demand for the optimization of the conventional process, as well as for the economic viability of emerging technologies, such as hemicellulosic ethanol. For this, the adaptive evolution through sexual reproduction is a promising and complementary alternative to genetic engineering. This accelerates the adaptation and allows the combination of characters responsible for the robustness observed in different industrial strains. Typically, this process consists of the dissection of a large number of tetrads and the analysis of many segregants, which makes the process extremely laborious. It is known that Saccharomyces cerevisiae has the ability to evade the meiotic cycle after the occurrence of homologous recombination events, and to resume the mitotic cycle, generating cells with highly recombined genomes, called RTG (Return to Growth). For this reason, this work had as main objective to develop an approach that produces recombinant diploids without the need of sporulation; proposing a methodology that is quickly and easily applied to the study and development of yeasts dedicated to industrial processes. In this scope, the transient expression of a GFP was used as a cell cycle marker for the selection of possible recombinant diploids by flow cytometry. During the present work it was possible to define both a good expression system composed of a GFP and an OFP; and the FACS gating to be used on the approach. However adjustments are still necessary regarding GFP¿s half-lifeMestradoGenética de MicroorganismosMestra em Genética e Biologia Molecular132983/2017-62016/02506-7CNPQFAPES

Conversion of an inactive xylose isomerase into a functional enzyme by co-expression of GroEL-GroES chaperonins in Saccharomyces cerevisiae

Abstract Background Second-generation ethanol production is a clean bioenergy source with potential to mitigate fossil fuel emissions. The engineering of Saccharomyces cerevisiae for xylose utilization is an essential step towards the production of this biofuel. Though xylose isomerase (XI) is the key enzyme for xylose conversion, almost half of the XI genes are not functional when expressed in S. cerevisiae. To date, protein misfolding is the most plausible hypothesis to explain this phenomenon. Results This study demonstrated that XI from the bacterium Propionibacterium acidipropionici becomes functional in S. cerevisiae when co-expressed with GroEL-GroES chaperonin complex from Escherichia coli. The developed strain BTY34, harboring the chaperonin complex, is able to efficiently convert xylose to ethanol with a yield of 0.44 g ethanol/g xylose. Furthermore, the BTY34 strain presents a xylose consumption rate similar to those observed for strains carrying the widely used XI from the fungus Orpinomyces sp. In addition, the tetrameric XI structure from P. acidipropionici showed an elevated number of hydrophobic amino acid residues on the surface of protein when compared to XI commonly expressed in S. cerevisiae. Conclusions Based on our results, we elaborate an extensive discussion concerning the uncertainties that surround heterologous expression of xylose isomerases in S. cerevisiae. Probably, a correct folding promoted by GroEL-GroES could solve some issues regarding a limited or absent XI activity in S. cerevisiae. The strains developed in this work have promising industrial characteristics, and the designed strategy could be an interesting approach to overcome the non-functionality of bacterial protein expression in yeasts

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Núcleos de Ensino da Unesp: artigos 2008

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq