ReMaSSing meets SCRaMbLE: using Cre/Lox recombination, selection, and backcrossing to evolve the synthetic yeast Sc2.0

• Grant number: 23/06203-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: March 01, 2024
• End date: February 28, 2025
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Ana Paula Jacobus
• Grantee: Lucas Souza de Bem
• Supervisor: Patrick Yizhi Cai
• Host Institution: Instituto de Pesquisa em Bioenergia (IPBEN). Universidade Estadual Paulista (UNESP). Campus de Rio Claro. Rio Claro , SP, Brazil
• Institution abroad: University of Manchester, England
• Associated to the scholarship: 21/13906-4 - Protocols for quantitative trait loci mapping in saccharomyces cerevisiae based on mass selections and backcrossings, BP.DD

Abstract

The Sc2.0 project is an international endeavor to create a fully synthetic Saccharomyces cerevisiae genome. A key aspect of the synthetic design is the inclusion of LoxPsym sites, which allow for the induction of structural genome variations through SCRaMbLE (Synthetic Chromosome Recombination and Modification by Lox-mediated Evolution). This remarkable mutagenic approach can be used to improve yeast phenotypes of industrial interest. With a similar goal, we have been mapping stress-tolerance quantitative trait loci (QTL) in recombinant yeasts through ReMaSSing (Reiterated Mass Selection and Backcrossing). Selecting populations under stress enriches tolerance QTLs, while backcrossing disrupts their genetic linkage to non-adaptive alleles, refining the mapping resolution. Here we propose to combine ReMaSSing with SCRaMbLE in a simple evolution protocol to improve yeast tolerance to lignocellulosic inhibitors. Following Cre/Lox-mediated chromosomal rearrangements, ReMaSSing will be applied to select adaptive variants and for purging deleterious mutations caused by pervasive SCRaMbLE recombination. Interestingly, the Brazilian bioethanol yeast PE-2 will be crossed with the synthetic yeast syn6.5, leading to the transfer of industrial relevant traits into the syn6.5 genome. Conversely, the introgression of synthetic chromosomes into the PE-2 background may transform the bioethanol yeast into a SCRaMbLE suitable strain. Nanopore sequencing will uncover the newly-formed chromosomal variants. The project will be conducted at the University of Manchester, UK, in the Patrick Cai Lab, a leading group of the Syn2.0 consortium. Besides its high impact and international visibility, the project will disseminate the synthetic genome technology in Brazil and foster its application in the development of industrial strains.