Combinatorial Gene Overexpression and Recessive Mutant Gene Introduction in Sake Yeast

Abstract

Industrial yeast strains are generally diploid and are often defective in sporulation. Such strains are hence thought to be less tractable for manipulation by genetic engineering. To facilitate more reliable genetic manipulation of the diploid yeast Japanese sake, we constructed variants of this strain that were homozygous for a URA3 deletion, homozygous for either MATa or MATalpha, and homozygous for either the his3 or the lys4 mutation. A ura3-null genotype enabled gene targeting to be undertaken more easily. The TDH3 promoter was inserted upstream of six yeast genes that have been implicated in flavor control to drive their constitutive overexpression. The homozygous MAT alleles, combined with the non-complementary auxotrophic mutations in the targeted transformants, allowed for tetraploid selection through mating. This resulted in the combinatorial construction of tetraploid strains that overexpress two different genes simultaneously. In addition, a recessive mutant gene, sah1-1, that is known to overproduce S-adenosylmethionine, was introduced into the diploid sake strain by the replacement of one wild-type allele and subsequent disruption of the other. The resulting sah1-1/sah1Delta::URA3 strain produced higher amounts of S-adenosylmethionine than the wild type. The novel sake yeast diploid strains we generated in this study can thus undergo simple PCR-mediated gene manipulation and mating in a manner analogous to established laboratory strains. Moreover, none of these sake strains had extraneous sequences, and they are thus suitable for use in commercial applications.

YHp as a highly stable, hyper‐copy, hyper‐expression plasmid constructed using a full 2‐μm circle sequence in cir0 strains of Saccharomyces cerevisiae

“…Using loss of heterozygosity (Hashimoto et al, ), MAT a / MAT a homozygous strain was isolated from the MAT a / MATα RAK1546 strain, resulting in RAK1563. Then, we deleted URA3 gene using LYS4 in RAK1563, resulting in RAK2359 strain ( MAT a /MAT a ura3Δ::LYS4/ura3Δ::LYS4 his3/his3 lys4/lys4 ; Ano et al, ). When we transformed sake yeast RAK2359 mutant strain with YEp, the plasmid was highly unstable, and the expression was much lower than in transformed laboratory strains (data not shown).…”

“…The DNA fragment containing the TDH3 promoter‐driven yeast‐codon‐optimized red fluorescent protein ( TDH3 p‐yEmRFP‐ URA3 ) was amplified from YEpGAP‐cherry (Keppler‐Ross et al, ) using the primers D+547(+40)c‐YEpURA3‐230c and D+546(‐40)‐TDH3‐698, which contained additional 40‐base sequences for recombination with the 2‐μm plasmid. The three amplified fragments mentioned above were mixed and used to transform the sake yeast strain RAK2359 ura3 ‐ cir 0 lacking a natural 2‐μm plasmid (Ano et al, ). The transformants were selected on SD‐Ura plates, and the resulting colonies were checked by colony PCR using the primers 15C‐YEpREP1+1122c and TDH3‐545c.…”

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

“…The LYS2 , TRP1 , Pp HIS3 , HIS3 and LEU2 genes were amplified by PCR, with the primers containing 40 bp homologous sequences for targeting, and inserted at the ura3Δ0 locus in BY4705 (for LYS2 and TRP1 ) and BY4704 (for the others) to generate the marker strains RAK4002 ( LYS2 ), RAK4003 ( TRP1 ), RAK3600 (Pp HIS3 ), RAK3613 ( HIS3 ) and RAK3614 ( LEU2 ) (Figure ). To generate promoter strains, the promoters (indicated by ‘p’) used were TDH3 p, CWP2 p, ILV5 p, CUP1 p and GAL10 p. TDH3 p, CWP2 p and ILV5 p are constitutive overexpression promoters (Ano et al ., ; Ghaemmaghami et al ., ; Kuroda et al ., ; Petersen and Holmberg, ) and CUP1 p is a copper‐inducible promoter (Mascorro‐Gallardo et al ., ), while GAL10 p is a galactose‐inducible promoter containing four Gal4 transcription factor binding sites (Johnston and Davis, ). We also constructed shorter GAL10 p promoters by deleting one ( GAL10 p3) and two ( GAL10 p2) Gal4 binding sites from the 5′ region.…”

Characterization and designing of an SAM riboswitch to establish a high‐throughput screening platform for SAM overproduction in Saccharomyces cerevisiae

“…The limiting factor of SAM synthesis is l ‐methionine, which is generally added directly to the fermentation medium (Hu et al, 2009; Kanai et al, 2017; Zhao, Shi, et al, 2016). In addition to the overexpression of S ‐adenosylmethionine synthetase, previous studies investigated various other strategies to improve the production of SAM, such as adding more l ‐methionine in the fermentation process (Huang et al, 2012; Kamarthapu et al, 2013; Zhang et al, 2008; Zhao, Hang, et al, 2016; Zhao, Shi, et al, 2016), regulating the intracellular ATP concentration (Chen & Tan, 2018; Chen et al, 2017), or deleting the SPE2 gene (encoding SAM decarboxylase) (Balasundaram et al, 1994), GLC3 gene (encoding a glycogen branching enzyme) (Rowen et al, 1992; Zhao, Hang, et al, 2016) and SAH1 gene (encoding S ‐adenosyl‐ l ‐homocysteine hydrolase) (Ano et al, 2009; Mizunuma et al, 2004) to minimize SAM degradation and consumption (Chen et al, 2016; He et al, 2006; Zhao, Hang, et al, 2016; Zhao, Shi, et al, 2016). These strategies have achieved good results, but excessive supply of the precursors l ‐methionine and ATP, as well as the knockout of bypass pathway genes, will also inhibit cell growth.…”