A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae

Acetic acid is inhibitory to the growth of the yeast Saccharomyces cerevisiae , causing ATP starvation and oxidative stress, which leads to the suboptimal production of fuels and chemicals from lignocellulosic biomass. In this study, where each strain of a CRISPRi library was characterized individually, many essential and respiratory growth-essential genes that regulate tolerance to acetic acid were identified, providing a new understanding of the stress response of yeast and new targets for the bioengineering of industrial yeast.

Analysis of fermentative parameters and the importance of Saccharomyces cerevisiae in the development of goods and services

In the fermentation process, yeasts need to adapt to the environmental changes that occur during the production process. Responses to these adjustments can alter biochemical routes and the amount of metabolites produced. Thus, the objective was to analyze the fermentative parameters of industrial yeast strains in different growing conditions, as well as to evaluate the its applicability in different sectors of goods and services. A pre-inoculum was performed with the YPSAC 5% medium for the activation of the yeasts Catanduva-1 and Fleischmann that remained incubated for 24 hours at 30 °C at 250 rpm. After the cells were recovered by centrifugation and inoculated in the fermentation medium based on sugarcane juice at 15 °Brix at temperatures of 30 and 40 °C. Aliquots were removed for the analysis of the fermentative parameters. Concomitantly, a survey was carried out regarding the use of yeasts in the process of preparing goods and services. The data show that the best yeast fermentation performance occurred at 30 °C in 10 hours. In addition, yeasts have the ability to produce, under ideal conditions, metabolites that can be used in different biotechnological processes.

Rapid Colorimetric Detection of Genome Evolution in SCRaMbLEd Synthetic Saccharomyces cerevisiae Strains

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

Rapid Colorimetric Detection of Genome Evolution in SCRaMbLEd Synthetic Saccharomyces cerevisiae Strains

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimise genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (Rapid Evolution Detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with haploid progeny of industrial yeast strains to produce stress tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate optimal SCRaMbLE induction times of different Cre-recombinse expression systems for the development of industrial strains.