Expression Cassettes for Formaldehyde and Fluoroacetate Resistance, Two Dominant Markers inSaccharomyces cerevisiae

Berg, M. A.; Steensma, H. Yde

doi:10.1002/(sici)1097-0061(199705)13:6<551::aid-yea113>3.0.co;2-0

Cited by 52 publications

(32 citation statements)

References 48 publications

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“…Effective selection without interference from background colonies required non-selective growth prior to plating on selective media with high concentrations of drug, which eliminated any background cells (Hadfield et al, 1986(Hadfield et al, , 1990Kunze et al, 1989;Del Pozo et al, 1991;Wenzel et al, 1992;Wach et al, 1994;Van den Berg et al, 1997;Park et al, 1999;Lacková and Subík, 1999). However, as shown in this work and that of others (Del Pozo et al, 1991;Shimura et al, 1993), the higher the drug concentration used, the lower the number of transformants obtained.…”

Section: Discussionmentioning

confidence: 60%

“…7 because of its strong resistance to geneticin, although high concentrations of geneticin have not been tested with the kanMX marker. In addition, the use of the kanMX marker (Wach et al, 1994) or resistance genes derived from other organisms (Gritz and Davies, 1983;Hadfield et al, 1986Hadfield et al, , 1990Zhu et al, 1986;Kunze et al, 1989;Wenzel et al, 1992;Van den Berg and Steensma, 1997) may not be suitable for industrial yeast manipulations because introduction of foreign genes into yeast used in the food and beverage industries will not be readily accepted by the public. From this point of view, genes derived from yeast origin might be more acceptable for commercial use.…”

Section: Discussionmentioning

confidence: 99%

“…However, these drug-resistance markers have several disadvantages: (a) transformation efficiencies of the drug-resistance markers were usually low compared to those of auxotrophic markers (Zhu et al, 1986;Kunze et al, 1989;Van den Berg et al, 1997;Shimura et al, 1993;Park et al, 1999); (b) transformant colonies selected by drug resistance were usually contaminated with non-transformed colonies that were a result of spontaneous drugresistance mutation or cells that were more resistant than the majority of the population (Casay et al, 1988;Shimura et al, 1993;Del Pozo et al, 1991); and (c) chromosomal integration was thought to be rare, due to its low transformation efficiency and/or its weakly resistant phenotype when integrated (Shimura et al, 1993;Gritz and Davies, 1983;Kunze et al, 1989;Inoue et al, 1993;Doignon et al, 1993;Van den Berg et al, 1997;Nakazawa et al, 1993). The reason for the poor transformation efficiency of drug-resistance markers may be explained in part by the fact that the drugs kill transformed yeast cells prematurely during the selection.…”

Section: Introductionmentioning

confidence: 99%

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Use of a YAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeast Saccharomyces cerevisiae

Akada

Shimizu

Matsushita

et al. 2001

Yeast

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Drug-resistance markers for yeast transformation are useful because they can be applied to strains without auxotrophic mutations. However, they are susceptible to technical difficulties, namely lower transformation efficiency and the appearance of drug-resistant mutants without the marker. To avoid these problems, we have constructed a phosphoglycerate kinase (PGK) promoter-driven YAP1 expression cassette, called PGKp-YAP1. Yeast cells containing PGKp-YAP1 were resistant to cycloheximide, a protein synthesis inhibitor, and also to cerulenin, a fatty acid synthesis inhibitor, but not to other drugs tested. The transformation efficiency of PGKp-YAP1 using cerulenin selection was comparable to that using a URA3 auxotrophic marker when low concentrations of cerulenin were used. Non-transformed drug-resistant colonies did appear on the low-concentration cerulenin plates. However, these non-transformed colonies could easily be identified, based on their cycloheximide sensitivity and/or their resistance to aureobasidin A to which the transformants were sensitive. Therefore, the dual drug resistance of PGKp-YAP1 could be used as an effective selection for PGKp-YAP1 recipient cells. The PGKp-YAP1 marker was used to disrupt the LYS2 gene and to transform an industrial yeast strain, indicating that this marker can be used for efficient and reliable gene manipulations in any Saccharomyces cerevisiae strain.

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Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Use of a YAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeast Saccharomyces cerevisiae

Akada

Shimizu

Matsushita

et al. 2001

Yeast

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“…Several marker genes used in yeast genetics confer resistance against antibiotics or other toxic compounds (42). Selection for strains that carry such marker genes requires the addition of these toxic compounds to the growth media.…”

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confidence: 99%

Auxotrophic Yeast Strains in Fundamental and Applied Research

Pronk

2002

Appl Environ Microbiol

262

224

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“…The overexpression of the endogenous S. cerevisiae gene encoding formaldehyde dehydrogenase (SFA1) was previously shown to result in enhanced formaldehyde resistance, presumably via formaldehyde oxidation (41). The aim of the present study was to investigate whether high-level expression of heterologous Fld and of Fmd would enable the use of formaldehyde as an auxiliary substrate by aerobic, glucose-limited chemostat cultures of S. cerevisiae.…”

mentioning

confidence: 99%

Engineering and Analysis of a Saccharomyces cerevisiae Strain That Uses Formaldehyde as an Auxiliary Substrate

Baerends

Hulster

Geertman

et al. 2008

Appl Environ Microbiol

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We demonstrated that formaldehyde can be efficiently coutilized by an engineered Saccharomyces cerevisiae strain that expresses Hansenula polymorpha genes encoding formaldehyde dehydrogenase (FLD1) and formate dehydrogenase (FMD), in contrast to wild-type strains. Initial chemostat experiments showed that the engineered strain coutilized formaldehyde with glucose, but these mixed-substrate cultures failed to reach steadystate conditions and did not exhibit an increased biomass yield on glucose. Subsequent transcriptome analyses of chemostat cultures of the engineered strain, grown on glucose-formaldehyde mixtures, indicated that the presence of formaldehyde in the feed caused biotin limitations. Further transcriptome analysis demonstrated that this biotin inactivation was prevented by using separate formaldehyde and vitamin feeds. Using this approach, steady-state glucose-limited chemostat cultures were obtained that coutilized glucose and formaldehyde. Coutilization of formaldehyde under these conditions resulted in an enhanced biomass yield of the glucose-limited cultures. The biomass yield was quantitatively consistent with the use of formaldehyde as an auxiliary substrate that generates NADH and subsequently, via oxidative phosphorylation, ATP. On an electron pair basis, the biomass yield increase observed with formaldehyde was larger than that observed previously for formate, which is tentatively explained by different modes of formate and formaldehyde transport in S. cerevisiae.In industrial biotechnology, the carbon feedstock is usually a sugar or sugar-rich substrate such as molasses, which often represents an important cost factor. In many processes, a large portion of the sugar feed is not converted into product but is instead dissimilated to provide free energy (e.g., in the form of ATP equivalents or proton motive force) and/or reducing power [in the form of NAD(P)H]. The use of mixed substrates consisting of a sugar and an additional, inexpensive source of reducing power and/or free energy represents an interesting approach to reduce the costs of industrial fermentation. Ideally, this practice should lead to a situation in which the sugar is used exclusively as a carbon source for biomass and/or product formation. The validity of this "auxiliary substrate" concept has been demonstrated in many academic studies using compounds such as formate or thiosulfate, whose oxidation can provide free energy but which cannot be used as carbon sources (2,7,26).Methanol is a low-cost chemical which can be derived from fossil sources or from biomass (via syngas, a CO-and H 2 -containing gas mixture generated by gasification of organic matter) (9, 17) and is an interesting candidate for the role of "auxiliary substrate" in industrial biotechnology. However, only a few industrial microorganisms are capable of utilizing methanol, including several yeast species that can grow on methanol as the sole carbon and energy source (35,42,46). In these methylotrophic yeasts, like Hansenula polymorpha and Pichia pastoris, meth...

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Expression Cassettes for Formaldehyde and Fluoroacetate Resistance, Two Dominant Markers inSaccharomyces cerevisiae

Cited by 52 publications

References 48 publications

Use of a YAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeast Saccharomyces cerevisiae

Use of a YAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeast Saccharomyces cerevisiae

Auxotrophic Yeast Strains in Fundamental and Applied Research

Engineering and Analysis of a Saccharomyces cerevisiae Strain That Uses Formaldehyde as an Auxiliary Substrate

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