Transformation of the Yeast,<i>Saccharomyces carlsbergensis</i>, Using an Antibiotic Resistance Marker

Sakai, Kazuhisa; Yamamoto, Masayuki

doi:10.1080/00021369.1986.10867557

Cited by 5 publications

(5 citation statements)

References 6 publications

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“…701 and probably to other strains of S. cerevisiae , and that selection on a medium containing higher concentrations of AbA increases the copy number of the AUR1 ‐C gene integrated. This result is consistent with previous reports that the phenotype of resistance against antibiotics shows a dosage effect, and that transformation efficiency by the gene correlates well with copy number in the yeast cell [2, 3, 17].…”

Section: Resultssupporting

confidence: 93%

“…A dominant selective marker, such as resistance against a drug, is thought to be useful for recombinant DNA technology for industrial yeast strains because these cells are diploid or aneuploid, lack a sexual cycle, and are prototrophic. Several dominant selection markers in Saccharomyces have been established, including the G418‐resistant gene of Tn 903 [2], a PDR4 gene imparting cerulenin resistance [3], and a SMR1 gene conferring resistance to sulfometuron methyl [4]. Each of these markers, however, has limitations, in terms of suitable hosts and transformation efficiency.…”

Section: Introductionmentioning

confidence: 99%

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Transformation system for prototrophic industrial yeasts using the AUR1 gene as a dominant selection marker

et al. 1998

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We show a new transformation system for prototrophic yeast strains including those of Saccharomyces cerevisiae, Kluyveromyces lactis, K. marxianus, and Candida glabrata. This system is composed of an antibiotic, aureobasidin A (AbA), and its resistance gene AUR1-C as a selection marker. Southern analysis of genomic DNAs of the transformants indicated that the copy number of the plasmid increased from one to more than four, depending on the concentration of AbA used for selection of the transformants. The AUR1-C gene was also effective as a selection marker for gene disruption, and was able to disrupt both copies of the gene on homologous chromosomes of diploid cells by a single round of transformation. This system has a broad application in the transformation and gene disruption of prototrophic strains of a variety of yeast species.z 1998 Federation of European Biochemical Societies.

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Section: Resultssupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Transformation system for prototrophic industrial yeasts using the AUR1 gene as a dominant selection marker

et al. 1998

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“…An alternative explanation for the difference between the plasmid stabilities of the transformed hybrid and the SHU32a transformant could be that fewer generations occurred in the culture of the latter strain, which grew more slowly than the transformed hybrid. Further improvement in strains possessing both glucoamylase genes and ot-amylase genes could be made by incorporating the ot-amylase gene into a centromere vector (23) or by integrating the gene, together with the attached ADCJ promoter, into a chromosome of the host cell. Although Filho et al.…”

Section: Resultsmentioning

confidence: 99%

High-efficiency, one-step starch utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse alpha-amylase

Kim

Park

Mattoon

1988

Appl Environ Microbiol

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Transformed, hybrid Saccharomyces strains capable of simultaneous secretion of glucoamylase and e-amylase have been produced. These strains could carry out direct, one-step assimilation of starch, with conversion efficiency greater than 93% during a 5-day growth period. One of the transformants converted 92.8% of available starch into reducing sugars in only 2 days. Glucoamylase secretion by these strains resulted from expression of one or more chromosomal STA genes derived from Saccharomyces diastaticus. The strains were transformed by a plasmid (pMS12) containing mouse salivary at-amylase cDNA in an expression vector containing yeast alcohol dehydrogenase promoter and a segment of yeast 2,um plasmid. The major starch hydrolysis product produced by crude amylases found in culture broths was glucose, indicating that a-amylase and glucoamylase acted cooperatively.

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“…The transformation of H. uvarum DSM2768 with the linearised DNA containing the hygromycin selection cassette resulted in a highly efficient deletion of HuATF1. Previous reports have suggested that the phenotype of antibiotic resistance is dose dependent and that transformation efficiency correlates with the copy number in the yeast cell [44][45][46]. Hashida-Okado et al [47] succeeded in generating a null mutant of a diploid S. cerevisiae strain by transforming with only one selection marker.…”

Section: Discussionmentioning

confidence: 99%

Development of Genetic Modification Tools for Hanseniasporauvarum

Badura

Wyk

Brezina

et al. 2021

IJMS

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Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

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Transformation of the Yeast,Saccharomyces carlsbergensis, Using an Antibiotic Resistance Marker

Cited by 5 publications

References 6 publications

Transformation system for prototrophic industrial yeasts using the AUR1 gene as a dominant selection marker

Transformation system for prototrophic industrial yeasts using the AUR1 gene as a dominant selection marker

High-efficiency, one-step starch utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse alpha-amylase

Development of Genetic Modification Tools for Hanseniasporauvarum

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