ADDITIVE GENETIC EFFECTS OF SEA4 IN PHENOTYPES OF  5-(HYDROXYMETHYL)FURFURAL RESISTANCE AND THERMOTOLERANCE IN  INDUSTRIAL STRAINS OF SACCHAROMYCES CEREVISIAE

Julia Ferreira Oliveira; Fellipe da Silveira Bezerra de Mello; Gonçalo Amarante Guimarães Pereira

Título do Trabalho

ADDITIVE GENETIC EFFECTS OF SEA4 IN PHENOTYPES OF 5-(HYDROXYMETHYL)FURFURAL RESISTANCE AND THERMOTOLERANCE IN INDUSTRIAL STRAINS OF SACCHAROMYCES CEREVISIAE

Autores

Julia Ferreira Oliveira
Fellipe da Silveira Bezerra de Mello
Gonçalo Amarante Guimarães Pereira

Modalidade

Apresentação de Pôster

Área temática

Genética de Microrganismos

Data de Publicação

09/06/2023

País da Publicação

Brasil

Idioma da Publicação

Inglês

Página do Trabalho

https://www.even3.com.br/anais/v-gbmeeting/611410-additive-genetic-effects-of-sea4-in-phenotypes-of--5-(hydroxymethyl)furfural-resistance-and-thermotolerance-in--i

ISBN

978-85-5722-785-9

Palavras-Chave

Bioethanol, additive effects, industrial robustness, Saccharomyces cerevisiae, yeast genetic engineering

Resumo

The second generation ethanol industry is based on the fermentation of xylose present in lignocellulosic biomass. Genetically modified strains of Saccharomyces cerevisiae are capable of assimilating this monosaccharide, but face unfavorable conditions typical to the industrial scale of production. Pre-treatment and hydrolysis of the lignocellulosic material generates inhibitors that negatively affect yeast performance and the conversion of the sugars. Aldehyde inhibitory compounds, such as 5-(hydroxymethyl)furfural (HMF) and the occurrence of temperature fluctuations are two drawbacks for yeast metabolism. In this context, the recalcitrance of the industrial environment fuels the search for robust strains that present phenotypes resistant to those adversities, allowing byosynthesis to prevail. In a recently published study, new causative genes for HMF- and thermotolerance in a bioethanol yeast strain (FMY097) were identified, amongst them SEA4 - an upstream regulator of TORC1 (Target of Rapamycin Complex 1). Evidence suggests there may be additive genetic effects at play, pointing to a complex network of gene interactions that involves essential intracelullar signalling pathways such as cell wall integrity (CWI) and target of rampamycin (TOR). In this project, we sought to investigate the underlying genetic basis of this robust phenotype through the replacement of the alleles previously identified in pairs, to assess the presence of additive and/or epistatic effects. For that, alleles PKC1, SRO77, MIX23 and SEA2 and SEA3 (alleles involved in the SEACAT complex) - all of them with rare non-synonymous mutations found in the industrial strain - will be evaluated regarding its influence in the laboratory strain BY4742 SEA4FMY097. Yeast transformation procedures were accomplished through CRISPR/Cas9 in order to build double-mutants in two steps: deletion of the endogenous BY4742 allele through replacement with an hygromicin resistance cassete, followed by insertion of the FMY097 same allele. At every step, colonies were screened in selective medium and underwent molecular confirmation through PCR amplification and electrophoresisAt total, seven new S. cerevisiae strains were obtained. Three rounds of yeast transformation remain to be completed but the available strains were phenotyped in 10mM and 20mM HMF to assess growth and evaluate robustness. Preliminary results show a trend towards resistance in multiple strains, in particular BY4742 SEA4FMY097 SEA3?, which still has to undergo insertion of the FMY097 allele. Following through with this line of research brings about the possibility for continuous enhancement, with the discovery of more targets for the genetic and metabolic engineering of S. cerevisiae applied to xylose fermentation in industrial conditions, and also, greater understanding of the contribution of pathways CWI and TORC1 to cell robustness.

Título do Evento: V GBMeeting
Cidade do Evento: Campinas
Título dos Anais do Evento: Anais do GBMeeting: Encontro Anual da Pós Graduação em Genética e Biologia Molecular da UNICAMP
Nome da Editora: Even3
Meio de Divulgação: Meio Digital

Como citar

OLIVEIRA, Julia Ferreira; MELLO, Fellipe da Silveira Bezerra de; PEREIRA, Gonçalo Amarante Guimarães. ADDITIVE GENETIC EFFECTS OF SEA4 IN PHENOTYPES OF 5-(HYDROXYMETHYL)FURFURAL RESISTANCE AND THERMOTOLERANCE IN INDUSTRIAL STRAINS OF SACCHAROMYCES CEREVISIAE.. In: Anais do GBMeeting: Encontro Anual da Pós Graduação em Genética e Biologia Molecular da UNICAMP. Anais...Campinas(SP) Unicamp, 2023. Disponível em: https//www.even3.com.br/anais/v-gbmeeting/611410-ADDITIVE-GENETIC-EFFECTS-OF-SEA4-IN-PHENOTYPES-OF--5-(HYDROXYMETHYL)FURFURAL-RESISTANCE-AND-THERMOTOLERANCE-IN--I. Acesso em: 07/12/2024