Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

Abstract

e Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNAguided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

“…The strain was constructed previously (31) through heterologous expression of XYL1 (coding for xylose reductase [XR]), XYL2 (coding for xylitol dehydrogenase [XDH]), and XYL3 (coding for xylulokinase [XK]) from Scheffersomyces stipitis in S. cerevisiae D452-2 (MAT␣ leu2 ura3 his3 can1) and optimization of the expression levels of XR, XDH, and XK, laboratory evolution on xylose, and deletion of ALD6, coding for acetaldehyde dehydrogenase. The auxotrophic marker genes in the strains SR8-trp and SR8-4 (Table 1) were recovered by the CRISPR-cas9 method (32,33). These strains were kindly provided by Yong-Su Jin's lab.…”

Metabolic Engineering of Probiotic Saccharomyces boulardii

“…The resulting PCR product was digested by SacI-NotI and ligated to pRS42H, resulting in intermediate plasmid pRS42H-gURA3 ( Table 1). The gBlocks gene fragments of TRP1 (29) were amplified by using primer pair gTRP1-U and gTRP1-D (Table 1), and the resulting PCR product was doubly digested by NotI-SpeI and then ligated to pRS42H-gURA3, forming tandem guide RNA plasmid p42H-gURA3-gTRP1 (Table 1) designed for simultaneous URA3 and TRP1 inactivation.…”

“…As for the construction of tandem guide RNA plasmid p42H-gURA3-gTRP1, the gBlocks gene fragments of URA3 (29) were amplified by using primer pair gURA3-U and gURA3-D (Table 1). The resulting PCR product was digested by SacI-NotI and ligated to pRS42H, resulting in intermediate plasmid pRS42H-gURA3 ( Table 1).…”

Methods of yeast genome editing

“…Наиболее часто используемый белок в генетической инженерии S. cerevisiae -Cas9 из Streptococcus pyogenes. Последовательность гена белка может быть природной Bao et al, 2015), оптимизи-рованной для экспрессии в клетках человека Gao, Zhao, 2014;Zhang et al, 2014;Jakočiūnas et al, 2015;Mans et al, 2015;Stovicek et al, 2015) и дрожжей (Horwitz et al, 2015;Generoso et al, 2016). Ген белка Cas9 экспрессировали под контролем конститутивных промо-торов разной силы.…”

“…Ген белка Cas9 экспрессировали под контролем конститутивных промо-торов разной силы. Наиболее часто используемый про-мотор -TEF1 Zhang et al, 2014;Bao et al, 2015;Jakočiūnas et al, 2015;Mans et al, 2015;Shi et al, 2016;Jensen et al, 2017;Liu et al, 2017;Ng, Dean, 2017), второй по частоте использования промотор -ADH1 (Gao, Zhao, 2014;Jacobs et al, 2014;Fernandez, Berro, 2016;Reider Apel et al, 2017), далее следуют RNR2 , FBA1 (Horwitz et al, 2015), TDH3 (Laughery et al, 2015), PGK1 (Lee et al, 2015) и др.…”